TY - JOUR AB - Biogenesis of small interfering RNAs (siRNA) in Drosophila melanogaster involves the processing of double-stranded RNA (dsRNA) by Dcr-2 with Loqs-PD/R2D2 and Ago2. Here, we show that Loqs-PD and Ago2 are found in biomolecular condensates in vivo and display liquid-liquid phase separation in vitro. The phase separation of Loqs-PD depends on the RNA-binding capability of its double-stranded RNA-binding domains and is further modulated by the preceding N-terminal region. An intrinsically disordered region in Ago2 (Ago2IDR) forms condensates in the presence of RNA in vitro. Combining NMR spectroscopy and mutational analysis, we show that Ago2IDR/RNA condensates are fluid, with significant polypeptide backbone flexibility, and are stabilized by a dense network of interactions involving arginine and aromatic side chains. Co-partitioning of Loqs-PD into Ago2IDR/dsRNA condensates depends on its ability to bind RNA. An RNase III enzyme can act on Ago2IDR/dsRNA condensates and reduce phase separation. Our results indicate that the unique features of the Ago2 IDR, which are broadly conserved in arthropods, drive biomolecular condensate formation, suggesting that phase separation plays a role in siRNA processing in Drosophila, potentially tuning the efficiency of dsRNA-mediated antiviral defense. AU - Hipp, C. AU - Mussgnug, S.* AU - Choudhary, P.* AU - Kang, H.-S. AU - Asami, S. AU - Sastre, J.* AU - Donau, C.* AU - Böttcher, R.* AU - Gemmecker, G. AU - Boekhoven, J.* AU - Förstemann, K.* AU - Sattler, M. C1 - 75187 C2 - 57829 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - Molecular mechanisms of biomolecular condensate formation in Drosophila melanogaster siRNA biogenesis. JO - Nucleic Acids Res. VL - 53 IS - 14 PB - Oxford Univ Press PY - 2025 SN - 0305-1048 ER - TY - JOUR AB - Retinoic acid receptors (RARs) and the vitamin D receptor (VDR) regulate distinct but overlapping gene sets in multiple cell types. The abundance and characteristics of regulatory regions, occupied by both RARs and VDR are largely unexplored. We used global approaches (ChIP-seq, RNA-seq, and ATAC-seq) and bioinformatics tools to map and characterize common binding regions of RARα and VDR in differentiated human THP-1 cells. We found that the cistromes of ligand-activated RARα and VDR largely overlapped, and their agonists (AM580 and calcitriol) co-regulated several genes, often cooperatively. Common binding regions were frequently (but not exclusively) annotated with co-regulated genes and exhibited increased MED1 occupancy upon ligand stimulation, suggesting their involvement in gene regulation. Chromatin accessibility was typically higher in the common regions than in regions occupied exclusively by RARα or VDR. DNA response elements for RARα (DR1/2/5) and VDR (DR3) were enriched in the common regions, albeit the co-occurrence of the two types of canonical motifs was low (8.4%), suggesting that "degenerate" DR1/2/5 and DR3 motifs or other sequences could mediate the binding. In summary, common binding regions of RARα and VDR are at the crossroads of the retinoid and vitamin D pathways, playing important roles in their convergence and cooperation. AU - Mianesaz, H.* AU - Göczi, L.* AU - Nagy, G.* AU - Póliska, S.* AU - Fadel, L. AU - Bojcsuk, D.* AU - Penyige, A.* AU - Szirák, K.* AU - AlHaman, F.* AU - Nagy, L.* AU - Vámosi, G.* AU - Széles, L.* C1 - 73918 C2 - 56995 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - Genomic regions occupied by both RARα and VDR are involved in the convergence and cooperation of retinoid and vitamin D signaling pathways. JO - Nucleic Acids Res. VL - 53 IS - 6 PB - Oxford Univ Press PY - 2025 SN - 0305-1048 ER - TY - JOUR AB - Adenosine-to-inosine (A-to-I) editing is a highly abundant modification of double-stranded RNA (dsRNA) and plays an important role in posttranscriptional gene regulation. Editing of multiple inosines by the ADAR1 enzyme leads to A-to-I hyper-editing of non-coding dsRNA, such as 3'UTRs, transposable elements, or foreign pathogenic RNAs, and is implicated in immune response and human diseases including cancer. The structural consequences of hyper-editing and its role in protein binding are poorly understood. Here, we combine solution nuclear magnetic resonance spectroscopy (NMR), biophysical methods such as small-angle X-ray scattering, and molecular dynamics simulations to study the sequence-dependent effects on conformation and dynamics of A-to-I hyper-editing for a 20-mer dsRNA and recognition of such RNAs by Endonuclease V. By comparing non-edited, single-edited, and hyper-edited dsRNA, we identify unique conformational features and extensive dynamics associated with hyper-editing, resulting in significantly increased base-pair opening. Hyper-edited dsRNA is more extended and adopts a highly dynamic ensemble of canonical and non-canonical conformations, which lead to preferential binding by Endonuclease V. Our integrated experimental and computational analysis identifies unique structural and dynamic features that are likely linked to specific protein recognition and the unique biological consequences of hyper-editing. AU - Müller-Hermes, C. AU - Piomponi, V.* AU - Hilber, S.* AU - Asami, S.* AU - Kreutz, C.* AU - Bussi, G.* AU - Sattler, M. C1 - 75028 C2 - 57706 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - Unique conformational dynamics and protein recognition of A-to-I hyper-edited dsRNA. JO - Nucleic Acids Res. VL - 53 IS - 12 PB - Oxford Univ Press PY - 2025 SN - 0305-1048 ER - TY - JOUR AB - The Human Musashi-1 (MSI-1) is an RNA-binding protein that recognizes (G/A)U1-3AGU and UAG sequences in diverse RNAs through two RNA Recognition Motif (RRM) domains and regulates the fate of target RNA. Here, we have combined structural biology and computational approaches to analyse the binding of the RRM domains of human MSI-1 with single-stranded and structured RNA ligands. We have used our recently developed computational tool RRMScorer to design a set of substitutions in the MSI-1 protein and the investigated RNA strands to modulate the binding affinity and selectivity. The in silico predictions of the designed protein-RNA interactions are assessed by nuclear magnetic resonance and surface plasmon resonance. These experiments have also been used to study the competition of the two RRM domains of MSI-1 for the same binding site within linear and harpin RNA. Our experimental results shed light on MSI-RNA interactions, thus opening the way for the development of new biomolecules for in vitro and in vivo studies and downstream applications. AU - Pérez-Ràfols, A.* AU - Pérez-Ropero, G.* AU - Cerofolini, L.* AU - Sperotto, L. AU - Roca-Martínez, J.* AU - Higuera-Rodriguez, R.A.* AU - Russomanno, P.* AU - Kaiser, W.J.* AU - Vranken, W.F.* AU - Danielson, U.H.* AU - Provenzani, A.* AU - Martelli, T.* AU - Sattler, M. AU - Buijs, J.* AU - Fragai, M.* C1 - 75343 C2 - 57944 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - Deciphering the RNA recognition by Musashi-1 to design protein and RNA variants for in vitro and in vivo applications. JO - Nucleic Acids Res. VL - 53 IS - 15 PB - Oxford Univ Press PY - 2025 SN - 0305-1048 ER - TY - JOUR AB - For genome editing, the use of CRISPR ribonucleoprotein (RNP) complexes is well established and often the superior choice over plasmid-based or viral strategies. RNPs containing dCas9 fusion proteins, which enable the targeted manipulation of transcriptomes and epigenomes, remain significantly less accessible. Here, we describe the production, delivery, and optimization of second generation CRISPRa RNPs (dRNPs). We characterize the transcriptional and cellular consequences of dRNP treatments in a variety of human target cells and show that the uptake is very efficient. The targeted activation of genes demonstrates remarkable potency, even for genes that are strongly silenced, such as developmental master transcription factors. In contrast to DNA-based CRISPRa strategies, gene activation is immediate and characterized by a sharp temporal precision. We also show that dRNPs allow very high-target multiplexing, enabling undiminished gene activation of multiple genes simultaneously. Applying these insights, we find that intensive target multiplexing at single promoters synergistically elevates gene transcription. Finally, we demonstrate in human stem and differentiated cells that the preferable features of dRNPs allow to instruct and convert cell fates efficiently without the need for DNA delivery or viral vectors. AU - Schmdit, T. AU - Wiesbeck, M. AU - Egert, L. AU - Truong, T.-T. AU - Danese, A.* AU - Voshagen, L.* AU - Imhof, S.* AU - Iraci Borgia, M.* AU - Deeksha AU - Neuner, A.M. AU - Köferle, A. AU - Geerlof, A. AU - Mourao, A. AU - Stricker, S.H. C1 - 73804 C2 - 57108 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP) complexes. JO - Nucleic Acids Res. VL - 53 IS - 6 PB - Oxford Univ Press PY - 2025 SN - 0305-1048 ER - TY - JOUR AB - CORUM (https://mips.helmholtz-muenchen.de/corum/) is a public database that offers comprehensive information about mammalian protein complexes, including their subunits, functions and associations with human diseases. The newly released CORUM 5.0, encompassing 7193 protein complexes, is the largest dataset of manually curated mammalian protein complexes publicly available. This update represents the most significant upgrade to the database in >15 years. At present, the molecular processes in cells that are influenced by drugs are only incompletely understood. In this latest release, we have begun systematically investigating the impact of drugs on protein complexes. Our studies are based on a dataset from DrugCentral comprising 725 protein drug targets with approved drugs and known mechanisms of action. To date, we have identified 1975 instances from the literature where a drug affects the formation and/or function of a protein complex. Numerous examples highlight the crucial role of understanding drug-protein complex relationships in drug efficacy. The expanded dataset and the inclusion of drug effects on protein complexes are expected to significantly enhance the utility and application potential of CORUM 5.0 in fields such as network medicine and pharmacological research. AU - Steinkamp, R. AU - Tsitsiridis, G. AU - Brauner, B. AU - Montrone, C. AU - Fobo, G. AU - Frishman, G. AU - Avram, S.* AU - Oprea, T.I.* AU - Ruepp, A. C1 - 72306 C2 - 56555 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - D651-D657 TI - CORUM in 2024: Protein complexes as drug targets. JO - Nucleic Acids Res. VL - 53 IS - D1 PB - Oxford Univ Press PY - 2025 SN - 0305-1048 ER - TY - JOUR AB - Foundation models, such as DNABERT and Nucleotide Transformer, have recently shaped a new direction in DNA research. Trained in an unsupervised manner on a vast quantity of genomic data, they can be used for a variety of downstream tasks, such as promoter prediction, DNA methylation prediction, gene network prediction, or functional variant prioritization. However, these models are often trained and evaluated on entire genomes, neglecting genome partitioning into different functional regions. In our study, we investigate the efficacy of various unsupervised approaches, including genome-wide and 3' untranslated region (3'UTR)-specific foundation models on human 3'UTR regions. To this end, we train a set of popular transformer architectures on a 3'UTR-specific dataset comprising 3 783 714 3'UTR sequences (6.6B bp) of 241 Zoonomia species. Our evaluation includes downstream tasks specific for RNA biology, such as recognition of binding motifs of RNA-binding proteins, detection of functional genetic variants, prediction of expression levels in massively parallel reporter assays, and estimation of messenger RNA half-life. Remarkably, models specifically trained on 3'UTR sequences demonstrate superior performance when compared to established genome-wide foundation models in three out of four downstream tasks. Our results underscore the importance of considering genome partitioning into distinct functional regions when training and evaluating foundation models. In addition, the proposed set of 3'UTR-specific tasks can be used for benchmarking of future models. AU - Vilov, S. AU - Heinig, M. C1 - 75580 C2 - 58045 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - Investigating the performance of foundation models on human 3'UTR sequences. JO - Nucleic Acids Res. VL - 53 IS - 17 PB - Oxford Univ Press PY - 2025 SN - 0305-1048 ER - TY - JOUR AB - Progressing transcription and replication machineries profoundly impact their underlying chromatin template. Consequently, transcription-replication conflict (TRC) sites are vulnerable to chromatin and epigenome alterations, provoking genome instability. Here, we engineered an inducible TRC reporter system using a genome-integrated R-loop-prone sequence and characterized the dynamic changes of the local chromatin structure inflicted by TRCs, leading to reduced nucleosome occupancy and replication fork blockage. Strikingly, inducing a small number of TRCs on the genome results in a measurable global replication stress response. Furthermore, we find a TRC-dependent increase in H3K79 methylation specifically at the R-loop forming TRC site. Accordingly, inhibition of the H3K79 methyltransferase DOT1L leads to reduced transcriptional output and an exacerbated DNA damage response, suggesting that deposition of this mark is required for effective transcription recovery and resolution of TRCs. Our work shows the molecular dynamics and reveals a specific epigenetic modifier bookmarking TRC sites, relevant to cancer and other diseases. AU - Werner, M. AU - Trauner, M. AU - Schauer, T. AU - Ummethum, H. AU - Márquez-Gómez, E. AU - Lalonde, M. AU - Lee, C.S.K. AU - Tsirkas, I. AU - Sajid, A. AU - Murriello, A.C. AU - Längst, G.* AU - Hamperl, S. C1 - 73452 C2 - 56867 TI - Transcription-replication conflicts drive R-loop-dependent nucleosome eviction and require DOT1L activity for transcription recovery. JO - Nucleic Acids Res. VL - 53 IS - 4 PY - 2025 SN - 0305-1048 ER - TY - JOUR AB - Acetylation of lysine 16 of histone H4 (H4K16ac) stands out among the histone modifications, because it decompacts the chromatin fiber. The metazoan acetyltransferase MOF (KAT8) regulates transcription through H4K16 acetylation. Antibody-based studies had yielded inconclusive results about the selectivity of MOF to acetylate the H4 N-terminus. We used targeted mass spectrometry to examine the activity of MOF in the male-specific lethal core (4-MSL) complex on nucleosome array substrates. This complex is part of the Dosage Compensation Complex (DCC) that activates X-chromosomal genes in male Drosophila. During short reaction times, MOF acetylated H4K16 efficiently and with excellent selectivity. Upon longer incubation, the enzyme progressively acetylated lysines 12, 8 and 5, leading to a mixture of oligo-acetylated H4. Mathematical modeling suggests that MOF recognizes and acetylates H4K16 with high selectivity, but remains substrate-bound and continues to acetylate more N-terminal H4 lysines in a processive manner. The 4-MSL complex lacks non-coding roX RNA, a critical component of the DCC. Remarkably, addition of RNA to the reaction non-specifically suppressed H4 oligo-acetylation in favor of specific H4K16 acetylation. Because RNA destabilizes the MSL-nucleosome interaction in vitro we speculate that RNA accelerates enzyme-substrate turn-over in vivo, thus limiting the processivity of MOF, thereby increasing specific H4K16 acetylation. AU - Kiss, A.E.* AU - Venkatasubramani, A.V.* AU - Pathirana, D.* AU - Krause, S.* AU - Sparr, A.C.* AU - Hasenauer, J. AU - Imhof, A.* AU - Müller, M.* AU - Becker, P.B.* C1 - 70025 C2 - 55364 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 4889-4905 TI - Processivity and specificity of histone acetylation by the male-specific lethal complex. JO - Nucleic Acids Res. VL - 52 IS - 9 PB - Oxford Univ Press PY - 2024 SN - 0305-1048 ER - TY - JOUR AB - Alternative splicing and multiple transcription start and termination sites can produce a diverse repertoire of mRNA transcript variants from a given gene. While the full picture of the human transcriptome is still incomplete, publicly available RNA datasets have enabled the assembly of transcripts. Using publicly available deep sequencing data from 927 human samples across 48 tissues, we quantified known and new transcript variants, provide an interactive, browser-based application Splice-O-Mat and demonstrate its relevance using adhesion G protein-coupled receptors (aGPCRs) as an example. On average, 24 different transcript variants were detected for each of the 33 human aGPCR genes, and several dominant transcript variants were not yet annotated. Variable transcription starts and complex exon-intron structures encode a flexible protein domain architecture of the N- and C termini and the seven-transmembrane helix domain (7TMD). Notably, we discovered the first GPCR (ADGRG7/GPR128) with eight transmembrane helices. Both the N- and C terminus of this aGPCR were intracellularly oriented, anchoring the N terminus in the plasma membrane. Moreover, the assessment of tissue-specific transcript variants, also for other gene classes, in our application may change the evaluation of disease-causing mutations, as their position in different transcript variants may explain tissue-specific phenotypes. AU - Kuhn, C.K.* AU - Stenzel, U.* AU - Berndt, S.I* AU - Liebscher, I.* AU - Schöneberg, T.* AU - Horn, S. C1 - 70104 C2 - 55424 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 3823-3836 TI - The repertoire and structure of adhesion GPCR transcript variants assembled from publicly available deep-sequenced human samples. JO - Nucleic Acids Res. VL - 52 IS - 7 PB - Oxford Univ Press PY - 2024 SN - 0305-1048 ER - TY - JOUR AB - Gene expression is a multi-step process that converts DNA-encoded information into proteins, involving RNA transcription, maturation, degradation, and translation. While transcriptional control is a major regulator of protein levels, the role of post-transcriptional processes such as RNA processing and degradation is less well understood due to the challenge of measuring their contributions individually. To address this challenge, we investigated the control of gene expression in Trypanosoma brucei, a unicellular parasite assumed to lack transcriptional control. Instead, mRNA levels in T. brucei are controlled by post-transcriptional processes, which enabled us to disentangle the contribution of both processes to total mRNA levels. In this study, we developed an efficient metabolic RNA labeling approach and combined ultra-short metabolic labeling with transient transcriptome sequencing (TT-seq) to confirm the long-standing assumption that RNA polymerase II transcription is unregulated in T. brucei. In addition, we established thiol (SH)-linked alkylation for metabolic sequencing of RNA (SLAM-seq) to globally quantify RNA processing rates and half-lives. Our data, combined with scRNA-seq data, indicate that RNA processing and stability independently affect total mRNA levels and contribute to the variability seen between individual cells in African trypanosomes. AU - Luzak, V.* AU - Osses, E.* AU - Danese, A. AU - Odendaal, C.* AU - Cosentino, R.O.* AU - Stricker, S.H. AU - Haanstra, J.R.* AU - Erhard, F.* AU - Siegel, T.N.* C1 - 72761 C2 - 56737 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - SLAM-seq reveals independent contributions of RNA processing and stability to gene expression in African trypanosomes. JO - Nucleic Acids Res. PB - Oxford Univ Press PY - 2024 SN - 0305-1048 ER - TY - JOUR AB - Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches. AU - Matsuda, A.* AU - Plewka, J.* AU - Rawski, M.* AU - Mourao, A. AU - Zajko, W.* AU - Siebenmorgen, T. AU - Kresik, L.* AU - Lis, K.* AU - Jones, A. AU - Pachota, M.* AU - Karim, A.* AU - Hartman, K.* AU - Nirwal, S.* AU - Sonani, R.* AU - Chykunova, Y.* AU - Minia, I.* AU - Mak, P.* AU - Landthaler, M.* AU - Nowotny, M.* AU - Dubin, G.* AU - Sattler, M. AU - Suder, P.* AU - Popowicz, G.M. AU - Pyrc, K.* AU - Czarna, A.* C1 - 70235 C2 - 55452 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 6441-6458 TI - Despite the odds: Formation of the SARS-CoV-2 methylation complex. JO - Nucleic Acids Res. VL - 52 IS - 11 PB - Oxford Univ Press PY - 2024 SN - 0305-1048 ER - TY - JOUR AB - RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro- and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA- and DNA-related processes by concentrating them in membraneless organelles. The general function and wide distribution of PUN motifs across species suggest that in an ancient 'RNA world' PUN-like motifs may have supported the correct folding of early ribozymes. AU - Niedner, A. AU - Monecke, T.* AU - Hennig, J. AU - Klostermann, M.* AU - Hofweber, M.* AU - Davydova, E.-O. AU - Gerber, A.P.* AU - Anosova, I. AU - Mayer, W.* AU - Müller, M. AU - Heym, R.G. AU - Janowski, R. AU - Paillart, J.C.* AU - Dormann, D.* AU - Zarnack, K.* AU - Sattler, M. AU - Niessing, D. C1 - 72418 C2 - 56609 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 14205-14228 TI - Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation. JO - Nucleic Acids Res. VL - 52 IS - 22 PB - Oxford Univ Press PY - 2024 SN - 0305-1048 ER - TY - JOUR AB - The UCSC Genome Browser (https://genome.ucsc.edu) is a widely utilized web-based tool for visualization and analysis of genomic data, encompassing over 4000 assemblies from diverse organisms. Since its release in 2001, it has become an essential resource for genomics and bioinformatics research. Annotation data available on Genome Browser includes both internally created and maintained tracks as well as custom tracks and track hubs provided by the research community. This last year's updates include over 25 new annotation tracks such as the gnomAD 4.1 track on the human GRCh38/hg38 assembly, the addition of three new public hubs, and significant expansions to the Genome Archive[GenArk) system for interacting with the enormous variety of assemblies. We have also made improvements to our interface, including updates to the browser graphic page, such as a new popup dialog feature that now displays item details without requiring navigation away from the main Genome Browser page. GenePred tracks have been upgraded with right-click options for zooming and precise navigation, along with enhanced mouseOver functions. Additional improvements include a new grouping feature for track hubs and hub description info links. A new tutorial focusing on Clinical Genetics has also been added to the UCSC Genome Browser. AU - Perez, G.* AU - Barber, G.P.* AU - Benet-Pages, A. AU - Casper, J.* AU - Clawson, H.* AU - Diekhans, M.* AU - Fischer, C.* AU - Gonzalez, J.N.* AU - Hinrichs, A.S.* AU - Lee, C.M.* AU - Nassar, L.R.* AU - Raney, B.J.* AU - Speir, M.L.* AU - van Baren, M.J.* AU - Vaske, C.J.* AU - Haussler, D.* AU - Kent, W.J.* AU - Haeussler, M.* C1 - 72158 C2 - 56458 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - The UCSC Genome Browser database: 2025 update. JO - Nucleic Acids Res. PB - Oxford Univ Press PY - 2024 SN - 0305-1048 ER - TY - JOUR AB - The UCSC Genome Browser (https://genome.ucsc.edu) is a web-based genomic visualization and analysis tool that serves data to over 7,000 distinct users per day worldwide. It provides annotation data on thousands of genome assemblies, ranging from human to SARS-CoV2. This year, we have introduced new data from the Human Pangenome Reference Consortium and on viral genomes including SARS-CoV2. We have added 1,200 new genomes to our GenArk genome system, increasing the overall diversity of our genomic representation. We have added support for nine new user-contributed track hubs to our public hub system. Additionally, we have released 29 new tracks on the human genome and 11 new tracks on the mouse genome. Collectively, these new features expand both the breadth and depth of the genomic knowledge that we share publicly with users worldwide. AU - Raney, B.J.* AU - Barber, G.P.* AU - Benet-Pages, A. AU - Casper, J.* AU - Clawson, H.* AU - Cline, M.S.* AU - Diekhans, M.* AU - Fischer, C.* AU - Navarro Gonzalez, J.* AU - Hickey, G.* AU - Hinrichs, A.S.* AU - Kuhn, R.M.* AU - Lee, B.T.* AU - Lee, C.M.* AU - Le Mercier, P.* AU - Miga, K.H.* AU - Nassar, L.R.* AU - Nejad, P.* AU - Paten, B.* AU - Perez, G.* AU - Schmelter, D.* AU - Speir, M.L.* AU - Wick, B.D.* AU - Zweig, A.S.* AU - Haussler, D.* AU - Kent, W.J.* AU - Haeussler, M.* C1 - 69735 C2 - 54994 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - D1082-D1088 TI - The UCSC Genome Browser database: 2024 update. JO - Nucleic Acids Res. VL - 52 IS - D1 PB - Oxford Univ Press PY - 2024 SN - 0305-1048 ER - TY - JOUR AB - Chromatin, the nucleoprotein complex consisting of DNA and histone proteins, plays a crucial role in regulating gene expression by controlling access to DNA. Chromatin modifications are key players in this regulation, as they help to orchestrate DNA transcription, replication, and repair. These modifications recruit epigenetic 'reader' proteins, which mediate downstream events. Most modifications occur in distinctive combinations within a nucleosome, suggesting that epigenetic information can be encoded in combinatorial chromatin modifications. A detailed understanding of how multiple modifications cooperate in recruiting such proteins has, however, remained largely elusive. Here, we integrate nucleosome affinity purification data with high-throughput quantitative proteomics and hierarchical interaction modeling to estimate combinatorial effects of chromatin modifications on protein recruitment. This is facilitated by the computational workflow asteRIa which combines hierarchical interaction modeling, stability-based model selection, and replicate-consistency checks for a stable estimation of Robust Interactions among chromatin modifications. asteRIa identifies several epigenetic reader candidates responding to specific interactions between chromatin modifications. For the polycomb protein CBX8, we independently validate our results using genome-wide ChIP-Seq and bisulphite sequencing datasets. We provide the first quantitative framework for identifying cooperative effects of chromatin modifications on protein binding. AU - Stadler, M. AU - Lukauskas, S. AU - Bartke, T. AU - Müller, C.L. C1 - 70683 C2 - 55707 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 6129-6144 TI - asteRIa enables robust interaction modeling between chromatin modifications and epigenetic readers. JO - Nucleic Acids Res. VL - 52 IS - 11 PB - Oxford Univ Press PY - 2024 SN - 0305-1048 ER - TY - JOUR AB - Promoter-proximal Polymerase II (Pol II) pausing is a key rate-limiting step for gene expression. DNA and RNA-binding trans-acting factors regulating the extent of pausing have been identified. However, we lack a quantitative model of how interactions of these factors determine pausing, therefore the relative importance of implicated factors is unknown. Moreover, previously unknown regulators might exist. Here we address this gap with a machine learning model that accurately predicts the extent of promoter-proximal Pol II pausing from large-scale genome and transcriptome binding maps and gene annotation and sequence composition features. We demonstrate high accuracy and generalizability of the model by validation on an independent cell line which reveals the model's cell line agnostic character. Model interpretation in light of prior knowledge about molecular functions of regulatory factors confirms the interconnection of pausing with other RNA processing steps. Harnessing underlying feature contributions, we assess the relative importance of each factor, quantify their predictive effects and systematically identify previously unknown regulators of pausing. We additionally identify 16 previously unknown 7SK ncRNA interacting RNA-binding proteins predictive of pausing. Our work provides a framework to further our understanding of the regulation of the critical early steps in transcriptional elongation. AU - Akcan, T.S. AU - Vilov, S. AU - Heinig, M. C1 - 67491 C2 - 54167 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 1608-1624 TI - Predictive model of transcriptional elongation control identifies trans regulatory factors from chromatin signatures. JO - Nucleic Acids Res. VL - 51 IS - 4 PB - Oxford Univ Press PY - 2023 SN - 0305-1048 ER - TY - JOUR AU - Bannister, A.J.* AU - Schneider, R. AU - Varga-Weisz, P.* C1 - 68068 C2 - 54546 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 7709-7713 TI - Editorial: Colyn Crane-Robinson (1935-2023). JO - Nucleic Acids Res. VL - 51 IS - 15 PB - Oxford Univ Press PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - Transposon screens are powerful in vivo assays used to identify loci driving carcinogenesis. These loci are identified as Common Insertion Sites (CISs), i.e. regions with more transposon insertions than expected by chance. However, the identification of CISs is affected by biases in the insertion behaviour of transposon systems. Here, we introduce Transmicron, a novel method that differs from previous methods by (i) modelling neutral insertion rates based on chromatin accessibility, transcriptional activity and sequence context and (ii) estimating oncogenic selection for each genomic region using Poisson regression to model insertion counts while controlling for neutral insertion rates. To assess the benefits of our approach, we generated a dataset applying two different transposon systems under comparable conditions. Benchmarking for enrichment of known cancer genes showed improved performance of Transmicron against state-of-the-art methods. Modelling neutral insertion rates allowed for better control of false positives and stronger agreement of the results between transposon systems. Moreover, using Poisson regression to consider intra-sample and inter-sample information proved beneficial in small and moderately-sized datasets. Transmicron is open-source and freely available. Overall, this study contributes to the understanding of transposon biology and introduces a novel approach to use this knowledge for discovering cancer driver genes. AU - Bredthauer, C. AU - Fischer, A.* AU - Ahari, A.J.* AU - Cao, X.* AU - Weber, J.* AU - Rad, L.* AU - Rad, R.* AU - Wachutka, L.* AU - Gagneur, J. C1 - 67169 C2 - 54228 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - Transmicron: Accurate prediction of insertion probabilities improves detection of cancer driver genes from transposon mutagenesis screens. JO - Nucleic Acids Res. VL - 51 IS - 4 PB - Oxford Univ Press PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned +1 and +2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo. AU - Chanou, A. AU - Weiß, M. AU - Holler, K. AU - Sajid, A. AU - Straub, T.* AU - Krietsch, J.* AU - Sanchi, A.* AU - Ummethum, H. AU - Lee, C.S.K. AU - Kruse, E. AU - Trauner, M. AU - Werner, M. AU - Lalonde, M. AU - Lopes, M.* AU - Scialdone, A. AU - Hamperl, S. C1 - 68751 C2 - 54961 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 12303-12324 TI - Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency. JO - Nucleic Acids Res. VL - 51 IS - 22 PB - Oxford Univ Press PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process. AU - Keil, P.* AU - Wulf, A.* AU - Kachariya, N. AU - Reuscher, S.* AU - Huhn, K.* AU - Silbern, I.* AU - Altmüller, J.* AU - Keller, M.* AU - Stehle, R. AU - Zarnack, K.* AU - Sattler, M. AU - Urlaub, H.* AU - Sträßer, K.* C1 - 67122 C2 - 53429 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 831-851 TI - Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA. JO - Nucleic Acids Res. VL - 51 IS - 2 PB - Oxford Univ Press PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders. AU - Molitor, L. AU - Klostermann, M.* AU - Bacher, S. AU - Merl-Pham, J. AU - Spranger, N. AU - Burczyk, S.* AU - Ketteler, C. AU - Rusha, E. AU - Tews, D.* AU - Pertek, A. AU - Proske, M. AU - Busch, A.* AU - Reschke, S.* AU - Feederle, R. AU - Hauck, S.M. AU - Blum, H.* AU - Drukker, M. AU - Fischer-Posovszky, P.* AU - König, J.* AU - Zarnack, K.* AU - Niessing, D. C1 - 67275 C2 - 54209 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 1297-1316 TI - Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies. JO - Nucleic Acids Res. VL - 51 IS - 3 PB - Oxford Univ Press PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - The UCSC Genome Browser (https://genome.ucsc.edu) is an omics data consolidator, graphical viewer, and general bioinformatics resource that continues to serve the community as it enters its 23rd year. This year has seen an emphasis in clinical data, with new tracks and an expanded Recommended Track Sets feature on hg38 as well as the addition of a single cell track group. SARS-CoV-2 continues to remain a focus, with regular annotation updates to the browser and continued curation of our phylogenetic sequence placing tool, hgPhyloPlace, whose tree has now reached over 12M sequences. Our GenArk resource has also grown, offering over 2500 hubs and a system for users to request any absent assemblies. We have expanded our bigBarChart display type and created new ways to visualize data via bigRmsk and dynseq display. Displaying custom annotations is now easier due to our chromAlias system which eliminates the requirement for renaming sequence names to the UCSC standard. Users involved in data generation may also be interested in our new tools and trackDb settings which facilitate the creation and display of their custom annotations. AU - Nassar, L.R.* AU - Barber, G.P.* AU - Benet-Pages, A. AU - Casper, J.* AU - Clawson, H.* AU - Diekhans, M.* AU - Fischer, C.* AU - Gonzalez, J.N.* AU - Hinrichs, A.S.* AU - Lee, B.T.* AU - Lee, C.M.* AU - Muthuraman, P.* AU - Nguy, B.* AU - Pereira, T.* AU - Nejad, P.* AU - Perez, G.* AU - Raney, B.J.* AU - Schmelter, D.* AU - Speir, M.L.* AU - Wick, B.D.* AU - Zweig, A.S.* AU - Haussler, D.* AU - Kuhn, R.M.* AU - Haeussler, M.* AU - Kent, W.J.* C1 - 66861 C2 - 53332 SP - D1188–D1195 TI - The UCSC genome browser database: 2023 update. JO - Nucleic Acids Res. VL - 51 IS - D1 PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - Linker H1 histones play an important role in animal and human pathogenesis, but their function in plant immunity is poorly understood. Here, we analyzed mutants of the three canonical variants of Arabidopsis H1 histones, namely H1.1, H1.2 and H1.3. We observed that double h1.1h1.2 and triple h1.1h1.2h1.3 (3h1) mutants were resistant to Pseudomonas syringae and Botrytis cinerea infections. Transcriptome analysis of 3h1 mutant plants showed H1s play a key role in regulating the expression of early and late defense genes upon pathogen challenge. Moreover, 3h1 mutant plants showed enhanced production of reactive oxygen species and activation of mitogen activated protein kinases upon pathogen-associated molecular pattern (PAMP) treatment. However, 3h1 mutant plants were insensitive to priming with flg22, a well-known bacterial PAMP which induces enhanced resistance in WT plants. The defective defense response in 3h1 upon priming was correlated with altered DNA methylation and reduced global H3K56ac levels. Our data place H1 as a molecular gatekeeper in governing dynamic changes in the chromatin landscape of defense genes during plant pathogen interaction. AU - Sheikh, A.H.* AU - Nawaz, K.* AU - Tabassum, N.* AU - Almeida-Trapp, M.* AU - Mariappan, K.G.* AU - Alhoraibi, H.* AU - Rayapuram, N.* AU - Aranda, M.* AU - Groth, M. AU - Hirt, H.* C1 - 67519 C2 - 54083 SP - 4252-4265 TI - Linker histone H1 modulates defense priming and immunity in plants. JO - Nucleic Acids Res. VL - 51 IS - 9 PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - Ligand/protein molecular recognition involves a dynamic process, whereby both partners require a degree of structural plasticity to regulate the binding/unbinding event. Here, we present the characterization of the interaction between a highly dynamic G-rich oligonucleotide, M08s-1, and its target protein, human α-thrombin. M08s-1 is the most active anticoagulant aptamer selected thus far. Circular dichroism and gel electrophoresis analyses indicate that both intramolecular and intermolecular G-quadruplex structures are populated in solution. The presence of thrombin stabilises the antiparallel intramolecular chair-like G-quadruplex conformation, that provides by far the main contribution to the biological activity of the aptamer. The crystal structure of the thrombin-oligonucleotide complex reveals that M08s-1 adopts a kinked structural organization formed by a G-quadruplex domain and a long duplex module, linked by a stretch of five purine bases. The quadruplex motif hooks the exosite I region of thrombin and the duplex region is folded towards the surface of the protein. This structural feature, which has never been observed in other anti-exosite I aptamers with a shorter duplex motif, hinders the approach of a protein substrate to the active site region and may well explain the significant increase in the anticoagulant activity of M08s-1 compared to the other anti-exosite I aptamers. AU - Troisi, R.* AU - Napolitano, V. AU - Rossitto, E.* AU - Osman, W.* AU - Nagano, M.* AU - Wakui, K.* AU - Popowicz, G.M. AU - Yoshimoto, K.* AU - Sica, F.* C1 - 68060 C2 - 54538 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 8880-8890 TI - Steric hindrance and structural flexibility shape the functional properties of a guanine-rich oligonucleotide. JO - Nucleic Acids Res. VL - 51 IS - 16 PB - Oxford Univ Press PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - The establishment of cellular identity is driven by transcriptional and epigenetic regulators of the chromatin proteome - the chromatome. Comprehensive analyses of the chromatome composition and dynamics can therefore greatly improve our understanding of gene regulatory mechanisms. Here, we developed an accurate mass spectrometry (MS)-based proteomic method called Chromatin Aggregation Capture (ChAC) followed by Data-Independent Acquisition (DIA) and analyzed chromatome reorganizations during major phases of pluripotency. This enabled us to generate a comprehensive atlas of proteomes, chromatomes, and chromatin affinities for the ground, formative and primed pluripotency states, and to pinpoint the specific binding and rearrangement of regulatory components. These comprehensive datasets combined with extensive analyses identified phase-specific factors like QSER1 and JADE1/2/3 and provide a detailed foundation for an in-depth understanding of mechanisms that govern the phased progression of pluripotency. The technical advances reported here can be readily applied to other models in development and disease. AU - Ugur, E.* AU - De La Porte, A. AU - Qin, W.* AU - Bultmann, S.* AU - Ivanova, A.* AU - Drukker, M. AU - Mann, M.* AU - Wierer, M.* AU - Leonhardt, H.* C1 - 67455 C2 - 54112 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 2671-2690 TI - Comprehensive chromatin proteomics resolves functional phases of pluripotency and identifies changes in regulatory components. JO - Nucleic Acids Res. VL - 51 IS - 6 PB - Oxford Univ Press PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - 5-Methyluridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme for m5U formation at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by hTRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with cross-linking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that hTRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of hTRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation. AU - Witzenberger, M. AU - Burczyk, S.* AU - Settele, D. AU - Mayer, W.* AU - Welp, L.M.* AU - Heiss, M.* AU - Wagner, M.* AU - Monecke, T.* AU - Janowski, R. AU - Carell, T.* AU - Urlaub, H.* AU - Hauck, S.M. AU - Voigt, A.* AU - Niessing, D. C1 - 68295 C2 - 54638 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 8691-8710 TI - Human TRMT2A methylates tRNA and contributes to translation fidelity. JO - Nucleic Acids Res. VL - 51 IS - 16 PB - Oxford Univ Press PY - 2023 SN - 0305-1048 ER - TY - JOUR AB - A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5' and 3' end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy. AU - Ambike, S. AU - Cheng, C.-C. AU - Feuerherd, M. AU - Velkov, S. AU - Baldassi, D.* AU - Afridi, S.Q. AU - Porras-Gonzalez, D.L. AU - Wei, X. AU - Hagen, P. AU - Kneidinger, N.* AU - Stoleriu, M.G.* AU - Grass, V. AU - Burgstaller, G. AU - Pichlmair, A. AU - Merkel, O.M. AU - Ko, C. AU - Michler, T. C1 - 63899 C2 - 51736 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 333-349 TI - Targeting genomic SARS-CoV-2 RNA with siRNAs allows efficient inhibition of viral replication and spread. JO - Nucleic Acids Res. VL - 50 IS - 1 PB - Oxford Univ Press PY - 2022 SN - 0305-1048 ER - TY - JOUR AB - In infected cells, Epstein-Barr virus (EBV) alternates between latency and lytic replication. The viral bZIP transcription factor ZEBRA (Zta, BZLF1) regulates this cycle by binding to two classes of ZEBRA response elements (ZREs): CpG-free motifs resembling the consensus AP-1 site recognized by cellular bZIP proteins and CpG-containing motifs that are selectively bound by ZEBRA upon cytosine methylation. We report structural and mutational analysis of ZEBRA bound to a CpG-methylated ZRE (meZRE) from a viral lytic promoter. ZEBRA recognizes the CpG methylation marks through a ZEBRA-specific serine and a methylcytosine-arginine-guanine triad resembling that found in canonical methyl-CpG binding proteins. ZEBRA preferentially binds the meZRE over the AP-1 site but mutating the ZEBRA-specific serine to alanine inverts this selectivity and abrogates viral replication. Our findings elucidate a DNA methylation-dependent switch in ZEBRA's transactivation function that enables ZEBRA to bind AP-1 sites and promote viral latency early during infection and subsequently, under appropriate conditions, to trigger EBV lytic replication by binding meZREs. AU - Bernaudat, F.* AU - Gustems, M. AU - Günther, J. AU - Oliva, M.F.* AU - Buschle, A. AU - Göbel,C. AU - Pagniez, P.* AU - Lupo, J.* AU - Signor, L.* AU - Müller, C.W.* AU - Morand, P.* AU - Sattler, M. AU - Hammerschmidt, W. AU - Petosa, C.* C1 - 63778 C2 - 51610 SP - 490-511 TI - Structural basis of DNA methylation-dependent site selectivity of the Epstein-Barr virus lytic switch protein ZEBRA/Zta/BZLF1. JO - Nucleic Acids Res. VL - 50 IS - 1 PY - 2022 SN - 0305-1048 ER - TY - JOUR AB - The A-repeat region of the lncRNA Xist is critical for X inactivation and harbors several N6-methyladenosine (m6A) modifications. How the m6A modification affects the conformation of the conserved AUCG tetraloop hairpin of the A-repeats and how it can be recognized by the YTHDC1 reader protein is unknown. Here, we report the NMR solution structure of the (m6A)UCG hairpin, which reveals that the m6A base extends 5' stacking of the A-form helical stem, resembling the unmethylated AUCG tetraloop. A crystal structure of YTHDC1 bound to the (m6A)UCG tetraloop shows that the (m6A)UC nucleotides are recognized by the YTH domain of YTHDC1 in a single-stranded conformation. The m6A base inserts into the aromatic cage and the U and C bases interact with a flanking charged surface region, resembling the recognition of single-stranded m6A RNA ligands. Notably, NMR and fluorescence quenching experiments show that the binding requires local unfolding of the upper stem region of the (m6A)UCG hairpin. Our data show that m6A can be readily accommodated in hairpin loop regions, but recognition by YTH readers requires local unfolding of flanking stem regions. This suggests how m6A modifications may regulate lncRNA function by modulating RNA structure. AU - Jones, A. AU - Tikhaia, E. AU - Mourao, A. AU - Sattler, M. C1 - 64364 C2 - 51912 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 2350-2362 TI - Structural effects of m6A modification of the Xist A-repeat AUCG tetraloop and its recognition by YTHDC1. JO - Nucleic Acids Res. VL - 50 IS - 4 PB - Oxford Univ Press PY - 2022 SN - 0305-1048 ER - TY - JOUR AB - Computational models have great potential to accelerate bioscience, bioengineering, and medicine. However, it remains challenging to reproduce and reuse simulations, in part, because the numerous formats and methods for simulating various subsystems and scales remain siloed by different software tools. For example, each tool must be executed through a distinct interface. To help investigators find and use simulation tools, we developed BioSimulators (https://biosimulators.org), a central registry of the capabilities of simulation tools and consistent Python, command-line and containerized interfaces to each version of each tool. The foundation of BioSimulators is standards, such as CellML, SBML, SED-ML and the COMBINE archive format, and validation tools for simulation projects and simulation tools that ensure these standards are used consistently. To help modelers find tools for particular projects, we have also used the registry to develop recommendation services. We anticipate that BioSimulators will help modelers exchange, reproduce, and combine simulations. AU - Shaikh, B.* AU - Smith, L.P.* AU - Vasilescu, D.* AU - Marupilla, G.* AU - Wilson, M.* AU - Agmon, E.* AU - Agnew, H.* AU - Andrews, S.S.* AU - Anwar, A.* AU - Beber, M.E.* AU - Bergmann, F.T.* AU - Brooks, D.* AU - Brusch, L.* AU - Calzone, L.* AU - Choi, K.* AU - Cooper, J.* AU - Detloff, J.* AU - Drawert, B.* AU - Dumontier, M.* AU - Ermentrout, G.B.* AU - Faeder, J.R.* AU - Freiburger, A.P.* AU - Fröhlich, F.* AU - Funahashi, A.* AU - Garny, A.* AU - Gennari, J.H.* AU - Gleeson, P.* AU - Goelzer, A.* AU - Haiman, Z.* AU - Hasenauer, J.* AU - Hellerstein, J.L.* AU - Hermjakob, H.* AU - Hoops, S.* AU - Ison, J.C.* AU - Jahn, D.* AU - Jakubowski, H.V.* AU - Jordan, R.* AU - Kalaš, M.* AU - König, M.* AU - Liebermeister, W.* AU - Sheriff, R.S.M.* AU - Mandal, S.* AU - McDougal, R.* AU - Medley, J.K.* AU - Mendes, P.* AU - Müller, R.* AU - Myers, C.J.* AU - Naldi, A.* AU - Nguyen, T.V.N.* AU - Nickerson, D.P.* AU - Olivier, B.G.* AU - Patoliya, D.* AU - Paulevé, L.* AU - Petzold, L.R.* AU - Priya, A.* AU - Rampadarath, A.K.* AU - Rohwer, J.M.* AU - Saglam, A.S.* AU - Singh, D.* AU - Sinha, A.* AU - Snoep, J.D.* AU - Sorby, H.* AU - Spangler, R.* AU - Starruß, J.* AU - Thomas, P.J.* AU - van Niekerk, D.* AU - Weindl, D. AU - Zhang, F.* AU - Zhukova, A.* AU - Goldberg, A.P.* AU - Schaff, J.C.* AU - Blinov, M.L.* AU - Sauro, H.M.* AU - Moraru, I.I.* AU - Karr, J.R.* C1 - 65019 C2 - 52147 SP - W108-W114 TI - BioSimulators: A central registry of simulation engines and services for recommending specific tools. JO - Nucleic Acids Res. VL - 50 IS - W1 PY - 2022 SN - 0305-1048 ER - TY - JOUR AB - DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs. AU - Stolz, P.* AU - Mantero, A.S.* AU - Tvardovskiy, A. AU - Ugur, E.* AU - Wange, L.E.* AU - Mulholland, C.B.* AU - Cheng, Y.* AU - Wierer, M.* AU - Enard, W.* AU - Schneider, R. AU - Bartke, T. AU - Leonhardt, H.* AU - Elsässer, S.J.* AU - Bultmann, S.* C1 - 65868 C2 - 52556 SP - 8491-8511 TI - TET1 regulates gene expression and repression of endogenous retroviruses independent of DNA demethylation. JO - Nucleic Acids Res. VL - 50 IS - 15 PY - 2022 SN - 0305-1048 ER - TY - JOUR AB - The CORUM database has been providing comprehensive reference information about experimentally characterized, mammalian protein complexes and their associated biological and biomedical properties since 2007. Given that most catalytic and regulatory functions of the cell are carried out by protein complexes, their composition and characterization is of greatest importance in basic and disease biology. The new CORUM 4.0 release encompasses 5204 protein complexes offering the largest and most comprehensive publicly available dataset of manually curated mammalian protein complexes. The CORUM dataset is built from 5299 different genes, representing 26% of the protein coding genes in humans. Complex information from 3354 scientific articles is mainly obtained from human (70%), mouse (16%) and rat (9%) cells and tissues. Recent curation work includes sets of protein complexes, Functional Complex Groups, that offer comprehensive collections of published data in specific biological processes and molecular functions. In addition, a new graphical analysis tool was implemented that displays co-expression data from the subunits of protein complexes. CORUM is freely accessible at http://mips.helmholtz-muenchen.de/corum/. AU - Tsitsiridis, G. AU - Steinkamp, R. AU - Giurgiu, M.* AU - Brauner, B. AU - Fobo, G. AU - Frishman, G. AU - Montrone, C. AU - Ruepp, A. C1 - 66821 C2 - 53227 SP - D539-D545 TI - CORUM: The comprehensive resource of mammalian protein complexes-2022. JO - Nucleic Acids Res. VL - 51 IS - D1 PY - 2022 SN - 0305-1048 ER - TY - JOUR AB - Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV's lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV's lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV's lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus. AU - Buschle, A. AU - Mrozek-Gorska, P. AU - Cernilogar, F.M.* AU - Ettinger, A. AU - Pich, D. AU - Krebs, S.* AU - Mocanu, B. AU - Blum, H.* AU - Schotta, G.* AU - Straub, T.* AU - Hammerschmidt, W. C1 - 61443 C2 - 50252 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 3217-3241 TI - Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation. JO - Nucleic Acids Res. VL - 49 IS - 6 PB - Oxford Univ Press PY - 2021 SN - 0305-1048 ER - TY - JOUR AB - The EDGAR platform, a web server providing databases of precomputed orthology data for thousands of microbial genomes, is one of the most established tools in the field of comparative genomics and phylogenomics. Based on precomputed gene alignments, EDGAR allows quick identification of the differential gene content, i.e. the pan genome, the core genome, or singleton genes. Furthermore, EDGAR features a wide range of analyses and visualizations like Venn diagrams, synteny plots, phylogenetic trees, as well as Amino Acid Identity (AAI) and Average Nucleotide Identity (ANI) matrices. During the last few years, the average number of genomes analyzed in an EDGAR project increased by two orders of magnitude. To handle this massive increase, a completely new technical backend infrastructure for the EDGAR platform was designed and launched as EDGAR3.0. For the calculation of new EDGAR3.0 projects, we are now using a scalable Kubernetes cluster running in a cloud environment. A new storage infrastructure was developed using a file-based high-performance storage backend which ensures timely data handling and efficient access. The new data backend guarantees a memory efficient calculation of orthologs, and parallelization has led to drastically reduced processing times. Based on the advanced technical infrastructure new analysis features could be implemented including POCP and FastANI genomes similarity indices, UpSet intersecting set visualization, and circular genome plots. Also the public database section of EDGAR was largely updated and now offers access to 24,317 genomes in 749 free-to-use projects. In summary, EDGAR 3.0 provides a new, scalable infrastructure for comprehensive microbial comparative gene content analysis. The web server is accessible at http://edgar3.computational.bio. AU - Dieckmann, M.A.* AU - Beyvers, S.* AU - Nkouamedjo-Fankep, R.C.* AU - Hanel, P. AU - Jelonek, L.* AU - Blom, J.* AU - Goesmann, A.* C1 - 62024 C2 - 50574 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - W185-W192 TI - EDGAR3.0: Comparative genomics and phylogenomics on a scalable infrastructure. JO - Nucleic Acids Res. VL - 49 IS - W1 PB - Oxford Univ Press PY - 2021 SN - 0305-1048 ER - TY - JOUR AB - Epstein-Barr virus (EBV) is a human herpesvirus associated with human cancers worldwide. Ex vivo, the virus efficiently infects resting human B lymphocytes and induces their continuous proliferation. This process is accompanied by a global reprogramming of cellular gene transcription. However, very little is known on the impact of EBV infection on the regulation of alternative splicing, a pivotal mechanism that plays an essential role in cell fate determination and is often deregulated in cancer. In this study, we have developed a systematic time-resolved analysis of cellular mRNA splice variant expression during EBV infection of resting B lymphocytes. Our results reveal that major modifications of alternative splice variant expression appear as early as day 1 post-infection and suggest that splicing regulation provides-besides transcription-an additional mechanism of gene expression regulation at the onset of B cell activation and proliferation. We also report a role for the viral proteins, EBNA2 and EBNA-LP, in the modulation of specific alternative splicing events and reveal a previously unknown function for EBNA-LP-together with the RBM4 splicing factor-in the alternative splicing regulation of two important modulators of cell proliferation and apoptosis respectively, NUMB and BCL-X. AU - Manet, E.* AU - Polvèche, H.* AU - Mure, F.* AU - Mrozek-Gorska, P. AU - Roisné-Hamelin, F.* AU - Hammerschmidt, W. AU - Auboeuf, D.* AU - Gruffat, H.* C1 - 63049 C2 - 51243 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 10657–10676 TI - Modulation of alternative splicing during early infection of human primary B lymphocytes with Epstein-Barr virus (EBV): A novel function for the viral EBNA-LP protein. JO - Nucleic Acids Res. VL - 49 IS - 18 PB - Oxford Univ Press PY - 2021 SN - 0305-1048 ER - TY - JOUR AB - Post-transcriptional control is essential to safeguard structural and metabolic changes in enucleated reticulocytes during their terminal maturation to functional erythrocytes. The timely synthesis of arachidonate 15-lipoxygenase (ALOX15), which initiates mitochondria degradation at the final stage of reticulocyte maturation is regulated by the multifunctional protein HNRNPK. It constitutes a silencing complex at the ALOX15 mRNA 3' untranslated region that inhibits translation initiation at the AUG by impeding the joining of ribosomal 60S subunits to 40S subunits. To elucidate how HNRNPK interferes with 80S ribosome assembly, three independent screens were applied. They consistently demonstrated a differential interaction of HNRNPK with RPS19, which is localized at the head of the 40S subunit and extends into its functional center. During induced erythroid maturation of K562 cells, decreasing arginine dimethylation of HNRNPK is linked to a reduced interaction with RPS19 in vitro and in vivo. Dimethylation of residues R256, R258 and R268 in HNRNPK affects its interaction with RPS19. In noninduced K562 cells, RPS19 depletion results in the induction of ALOX15 synthesis and mitochondria degradation. Interestingly, residue W52 in RPS19, which is frequently mutated in Diamond-Blackfan Anemia (DBA), participates in specific HNRNPK binding and is an integral part of a putative aromatic cage. AU - Naarmann-de Vries, I.S.* AU - Senatore, R.* AU - Moritz, B.* AU - Marx, G.* AU - Urlaub, H.* AU - Niessing, D. AU - Ostareck, D.H.* AU - Ostareck-Lederer, A.* C1 - 61452 C2 - 50260 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 3507-3523 TI - Methylated HNRNPK acts on RPS19 to regulate ALOX15 synthesis in erythropoiesis. JO - Nucleic Acids Res. VL - 49 IS - 6 PB - Oxford Univ Press PY - 2021 SN - 0305-1048 ER - TY - JOUR AB - DNA methylation is essential to development and cellular physiology in mammals. Faulty DNA methylation is frequently observed in human diseases like cancer and neurological disorders. Molecularly, this epigenetic mark is linked to other chromatin modifications and it regulates key genomic processes, including transcription and splicing. Each round of DNA replication generates two hemi-methylated copies of the genome. These must be converted back to symmetrically methylated DNA before the next S-phase, or the mark will fade away; therefore the maintenance of DNA methylation is essential. Mechanistically, the maintenance of this epigenetic modification takes place during and after DNA replication, and occurs within the very dynamic context of chromatin re-assembly. Here, we review recent discoveries and unresolved questions regarding the mechanisms, dynamics and fidelity of DNA methylation maintenance in mammals. We also discuss how it could be regulated in normal development and misregulated in disease. AU - Petryk, N.* AU - Bultmann, S.* AU - Bartke, T. AU - Defossez, P.-A.* C1 - 60738 C2 - 49548 SP - 3020–3032 TI - Staying true to yourself: Mechanisms of DNA methylation maintenance in mammals JO - Nucleic Acids Res. VL - 49 IS - 6 PY - 2021 SN - 0305-1048 ER - TY - JOUR AB - Repair of covalent DNA-protein crosslinks (DPCs) by the metalloprotease SPRTN prevents genome instability, premature aging and carcinogenesis. SPRTN is specifically activated by DNA structures containing single- and double-stranded features, but degrades the protein components of DPCs promiscuously and independent of amino acid sequence. This lack of specificity is useful to target diverse protein adducts, however, it requires tight control in return, in order to prohibit uncontrolled proteolysis of chromatin proteins. Here, we discover the components and principles of a ubiquitin switch, which negatively regulates SPRTN. We demonstrate that monoubiquitylation is induced in an E3 ligase-independent manner and, in contrast to previous assumptions, does not control chromatin access of the enzyme. Data obtained in cells and in vitro reveal that monoubiquitylation induces inactivation of the enzyme by triggering autocatalytic cleavage in trans while also priming SPRTN for proteasomal degradation in cis. Finally, we show that the deubiquitylating enzyme USP7 antagonizes this negative control of SPRTN in the presence of DPCs. AU - Zhao, S.* AU - Kieser, A.* AU - Li, H.Y.* AU - Reinking, H.K.* AU - Weickert, P.* AU - Euteneuer, S.* AU - Yaneva, D.* AU - Acampora, A.C.* AU - Götz, M.J.* AU - Feederle, R. AU - Stingele, J.* C1 - 61249 C2 - 49786 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 902-915 TI - A ubiquitin switch controls autocatalytic inactivation of the DNA-protein crosslink repair protease SPRTN. JO - Nucleic Acids Res. VL - 49 IS - 2 PB - Oxford Univ Press PY - 2021 SN - 0305-1048 ER - TY - JOUR AB - The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wetlab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www. crispr-clue.de. All in all, CLUE represents a resourcesaving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences. AU - Becker, M. AU - Noll-Puchta, H.* AU - Amend, D. AU - Nolte, F.* AU - Fuchs, C. AU - Jeremias, I. AU - Braun, C.J.* C1 - 59254 C2 - 48689 CY - Great Clarendon St, Oxford Ox2 6dp, England TI - CLUE: A bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries. JO - Nucleic Acids Res. VL - 48 IS - 13 PB - Oxford Univ Press PY - 2020 SN - 0305-1048 ER - TY - JOUR AB - Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3'-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3'-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs. AU - Binas, O.* AU - Tants, J.N.* AU - Peter, S.A.* AU - Janowski, R. AU - Davydova, E.-O. AU - Braun, J.* AU - Niessing, D. AU - Schwalbe, H.* AU - Weigand, J.E.* AU - Schlundt, A.* C1 - 59272 C2 - 48707 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 7385-7403 TI - Structural basis for the recognition of transiently structured AU-rich elements by Roquin. JO - Nucleic Acids Res. VL - 48 IS - 13 PB - Oxford Univ Press PY - 2020 SN - 0305-1048 ER - TY - JOUR AB - High-content imaging and single-cell genomics are two of the most prominent high-throughput technologies for studying cellular properties and functions at scale. Recent studies have demonstrated that information in large imaging datasets can be used to estimate gene mutations and to predict the cell-cycle state and the cellular decision making directly from cellular morphology. Thus, high-throughput imaging methodologies, such as imaging flow cytometry can potentially aim beyond simple sorting of cellpopulations. We introduce IFC-seq, a machine learning methodology for predicting the expression profile of every cell in an imaging flow cytometry experiment. Since it is to-date unfeasible to observe singlecell gene expression and morphology in flow, we integrate uncoupled imaging data with an independent transcriptomics dataset by leveraging common surface markers. We demonstrate that IFC-seq successfully models gene expression of a moderate number of key gene-markers for two independent imaging flow cytometry datasets: (i) human blood mononuclear cells and (ii) mouse myeloid progenitor cells. In the case of mouse myeloid progenitor cells IFC-seq can predict gene expression directly from brightfield images in a label-free manner, using a convolutional neural network. The proposed method promises to add gene expression information to existing and new imaging flow cytometry datasets, at no additional cost. AU - Chlis, N.-K. AU - Rausch, L.* AU - Brocker, T.* AU - Kranich, J.* AU - Theis, F.J. C1 - 61223 C2 - 49761 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 11335-11346 TI - Predicting single-cell gene expression profiles of imaging flow cytometry data with machine learning. JO - Nucleic Acids Res. VL - 48 IS - 20 PB - Oxford Univ Press PY - 2020 SN - 0305-1048 ER - TY - JOUR AB - The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR's anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription. Usingmice with a point mutation in GR's zinc finger, that still tether via protein-protein interactions while being unable to recognize DNA, we demonstrate that DNA binding is essential for both transcriptional activation and repression. Performing ChIP-Seq, RNA-Seq and proteomics under inflammatory conditions, we show that DNA recognition is required for the assembly of a functional co-regulator complex to mediate glucocorticoid responses. Our findings may contribute to the development of safer immunomodulators with fewer side effects. AU - Escoter Torres, L. AU - Greulich, F. AU - Quagliarini, F. AU - Wierer, M.* AU - Uhlenhaut, N.H. C1 - 59586 C2 - 48857 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 8393-8407 TI - Anti-inflammatory functions of the glucocorticoid receptor require DNA binding. JO - Nucleic Acids Res. VL - 48 IS - 15 PB - Oxford Univ Press PY - 2020 SN - 0305-1048 ER - TY - JOUR AB - The RNA helicase RIG-I plays a key role in sensing pathogen-derived RNA. Double-stranded RNA structures bearing 5'-tri- or diphosphates are commonly referred to as activating RIG-I ligands. However, endogenous RNA fragments generated during viral infection via RNase L also activate RIG-I. Of note, RNase-digested RNA fragments bear a 5'-hydroxyl group and a 2',3'-cyclic phosphate. How endogenous RNA fragments activate RIG-I despite the lack of 5'-phosphorylation has not been elucidated. Here we describe an endogenous RIG-I ligand (eRL) that is derived from the internal transcribed spacer 2 region (ITS2) of the 45S ribosomal RNA after partial RNase A digestion in vitro, RNase A protein transfection or RNase L activation. The immunostimulatory property of the eRL is dependent on 2',3'-cyclic phosphate and its sequence is characterized by a G-quadruplex containing sequence motif mediating guanosine-5'-triphosphate (GTP) binding. In summary, RNase generated self-RNA fragments with 2',3'-cyclic phosphate function as nucleotide-5'-triphosphate binding aptamers activating RIG-I. AU - Jung, S. AU - von Thülen, T.* AU - Yang, I.* AU - Laukemper, V.* AU - Rupf, B.* AU - Janga, H.* AU - Panagiotidis, G.-D.* AU - Schoen, A.* AU - Nicolai, M.* AU - Schulte, L.N.* AU - Obermann, H.-L.* AU - Weber, F.* AU - Kaufmann, A.* AU - Bauer, S.* C1 - 63336 C2 - 51269 SP - 10397-10412 TI - A ribosomal RNA fragment with 2',3'-cyclic phosphate and GTP-binding activity acts as RIG-I ligand. JO - Nucleic Acids Res. VL - 48 IS - 18 PY - 2020 SN - 0305-1048 ER - TY - JOUR AB - During interphase centromeres often coalesce into a small number of chromocenters, which can be visualized as distinct, DAPI dense nuclear domains. Intact chromocenters play a major role in maintaining genome stability as they stabilize the transcriptionally silent state of repetitive DNA while ensuring centromere function. Despite its biological importance, relatively little is known about the molecular composition of the chromocenter or the processes that mediate chromocenter formation and maintenance. To provide a deeper molecular insight into the composition of the chromocenter and to demonstrate the usefulness of proximity-based biotinylation as a tool to investigate those questions, we performed super resolution microscopy and proximity-based biotinylation experiments of three distinct proteins associated with the chromocenter in Drosophila. Our work revealed an intricate internal architecture of the chromocenter suggesting a complex multilayered structure of this intranuclear domain. AU - Kochanova, N.Y.* AU - Schauer, T.* AU - Mathias, G.P.* AU - Lukacs, A.* AU - Schmidt, A.* AU - Flatley, A. AU - Schepers, A. AU - Thomae, A.W.* AU - Imhof, A.* C1 - 59098 C2 - 48699 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 4161-4178 TI - A multi-layered structure of the interphase chromocenter revealed by proximity-based biotinylation. JO - Nucleic Acids Res. VL - 48 IS - 8 PB - Oxford Univ Press PY - 2020 SN - 0305-1048 ER - TY - JOUR AB - Genomic imprinting is an epigenetic process regulated by germline-derived DNA methylation that is resistant to embryonic reprogramming, resulting in parental origin-specific monoallelic gene expression. A subset of individuals affected by imprinting disorders (IDs) displays multi-locus imprinting disturbances (MLID), which may result from aberrant establishment of imprinted differentially methylated regions (DMRs) in gametes or their maintenance in early embryogenesis. Here we investigated the extent of MLID in a family harbouring a ZFP57 truncating variant and characterize the interactions between human ZFP57 and the KAP1 co-repressor complex. By ectopically targeting ZFP57 to reprogrammed loci in mouse embryos using a dCas9 approach, we confirm that ZFP57 recruitment is sufficient to protect oocyte-derived methylation from reprogramming. Expression profiling in human pre-implantation embryos and oocytes reveals that unlike in mice, ZFP57 is only expressed following embryonic-genome activation, implying that other KRAB-zinc finger proteins (KZNFs) recruit KAP1 prior to blastocyst formation. Furthermore, we uncover ZNF202 and ZNF445 as additional KZNFs likely to recruit KAP1 to imprinted loci during reprogramming in the absence of ZFP57. Together, these data confirm the perplexing link between KZFPs and imprint maintenance and highlight the differences between mouse and humans in this respect. AU - Monteagudo-Sánchez, A.* AU - Hernandez Mora, J.R.* AU - Simon, C.* AU - Burton, A. AU - Tenorio, J.* AU - Lapunzina, P.* AU - Clark, S.* AU - Esteller, M.* AU - Kelsey, G.* AU - López-Siguero, J.P.* AU - de Nanclares, G.P.* AU - Torres-Padilla, M.E. AU - Monk, D.* C1 - 60583 C2 - 49409 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 11394-11407 TI - The role of ZFP57 and additional KRAB-zinc finger proteins in the maintenance of human imprinted methylation and multi-locus imprinting disturbances. JO - Nucleic Acids Res. VL - 48 IS - 20 PB - Oxford Univ Press PY - 2020 SN - 0305-1048 ER - TY - JOUR AB - In recent years, hundreds of novel RNA-binding proteins (RBPs) have been identified, leading to the discovery of novel RNA-binding domains. Furthermore, unstructured or disordered low-complexity regions of RBPs have been identified to play an important role in interactions with nucleic acids. However, these advances in understanding RBPs are limited mainly to eukaryotic species and we only have limited tools to faithfully predict RNA-binders in bacteria. Here, we describe a support vector machine-based method, called TriPepSVM, for the prediction of RNA-binding proteins. TriPepSVM applies string kernels to directly handle protein sequences using tri-peptide frequencies. Testing the method in human and bacteria, we find that several RBP-enriched tri-peptides occur more often in structurally disordered regions of RBPs. TriPepSVM outperforms existing applications, which consider classical structural features of RNA-binding or homology, in the task of RBP prediction in both human and bacteria. Finally, we predict 66 novel RBPs in Salmonella Typhimurium and validate the bacterial proteins ClpX, DnaJ and UbiG to associate with RNA in vivo. AU - Bressin, A.* AU - Schulte-Sasse, R.* AU - Figini, D.* AU - Urdaneta, E.C.* AU - Beckmann, B.M.* AU - Marsico, A. C1 - 55922 C2 - 46722 SP - 4406-4417 TI - TriPepSVM: De novo prediction of RNA-binding proteins based on short amino acid motifs. JO - Nucleic Acids Res. VL - 47 IS - 9 PY - 2019 SN - 0305-1048 ER - TY - JOUR AB - Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other nonpioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors. AU - Cernilogar, F.M.* AU - Hasenöder AU - Wang, Z.* AU - Scheibner, K. AU - Burtscher, I. AU - Sterr, M. AU - Smialowski, P. AU - Groh, S.* AU - Evenroed, I.M.* AU - Gilfillan, G.D.* AU - Lickert, H. AU - Schotta, G.* C1 - 56647 C2 - 47211 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 9069-9086 TI - Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2. JO - Nucleic Acids Res. VL - 47 IS - 17 PB - Oxford Univ Press PY - 2019 SN - 0305-1048 ER - TY - JOUR AB - CORUM is a database that provides a manually curated repository of experimentally characterized protein complexes from mammalian organisms, mainly human (67%), mouse (15%) and rat (10%). Given the vital functions of these macromolecular machines, their identification and functional characterization is foundational to our understanding of normal and disease biology. The new CORUM 3.0 release encompasses 4274 protein complexes offering the largest and most comprehensive publicly available dataset of mammalian protein complexes. The CORUM dataset is built from 4473 different genes, representing 22% of the protein coding genes in humans. Protein complexes are described by a protein complex name, subunit composition, cellular functions as well as the literature references. Information about stoichiometry of subunits depends on availability of experimental data. Recent developments include a graphical tool displaying known interactions between subunits. This allows the prediction of structural interconnections within protein complexes of unknown structure. In addition, we present a set of 58 protein complexes with alternatively spliced subunits. Those were found to affect cellular functions such as regulation of apoptotic activity, protein complex assembly or define cellular localization. CORUM is freely accessible at http://mips.helmholtz-muenchen.de/corum/. AU - Giurgiu, M. AU - Reinhard, J. AU - Brauner, B. AU - Dunger-Kaltenbach, I. AU - Fobo, G. AU - Frishman, D. AU - Montrone, C. AU - Ruepp, A. C1 - 54591 C2 - 45697 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - D559-D563 TI - CORUM: The comprehensive resource of mammalian protein complexes2019. JO - Nucleic Acids Res. VL - 47 IS - D1 PB - Oxford Univ Press PY - 2019 SN - 0305-1048 ER - TY - JOUR AB - Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to gamma H2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The gamma H2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range gamma H2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei. AU - Harpprecht, L.* AU - Baldi, S.* AU - Schauer, T.* AU - Schmidt, A.* AU - Bange, T.* AU - Robles, M.S.* AU - Kremmer, E. AU - Imhof, A.* AU - Becker, P.B.* C1 - 56878 C2 - 47340 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 7444-7459 TI - A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling. JO - Nucleic Acids Res. VL - 47 IS - 14 PB - Oxford Univ Press PY - 2019 SN - 0305-1048 ER - TY - JOUR AB - Next-generation sequencing technologies have made it possible to carry out transcriptome analysis at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) data provide insights into cellular dynamics, including intercellular heterogeneity as well as inter- and intra-cellular fluctuations in gene expression that cannot be studied using populations of cells. The utilization of scRNA-seq is, however, restricted to cell types that can be isolated from their original tissues, and it can be difficult to obtain precise positional information for these cells in situ. Here, we established single cell-digital gene expression (1cell-DGE), a method of scRNA-seq that uses micromanipulation to extract the contents of individual living cells in intact tissue while recording their positional information. With 1cell-DGE, we could detect differentially expressed genes (DEGs) during the reprogramming of leaf cells of the moss Physcomitrella patens, identifying 6382 DEGs between cells at 0 and 24 h after excision. Furthermore, we identified a subpopulation of reprogramming cells based on their pseudotimes, which were calculated using transcriptome profiles at 24 h. 1cell-DGE with microcapillary manipulation can be used to analyze the gene expression of individual cells without detaching them from their tightly associated tissues, enabling us to retain positional information and investigate cell-cell interactions. AU - Kubo, M.* AU - Nishiyama, T.* AU - Tamada, Y.* AU - Sano, R.* AU - Ishikawa, M.* AU - Murata, T.* AU - Imai, A.* AU - Lang, D. C1 - 58013 C2 - 48024 SP - 4539-4553 TI - Single-cell transcriptome analysis of Physcomitrella leaf cells during reprogramming using microcapillary manipulation. JO - Nucleic Acids Res. VL - 47 IS - 9 PY - 2019 SN - 0305-1048 ER - TY - JOUR AB - HuR/ELAVL1 is an RNA-binding protein involved in differentiation and stress response that acts primarily by stabilizing messenger RNA (mRNA) targets. HuR comprises three RNA recognition motifs (RRMs) where the structure and RNA binding of RRM3 and of full-length HuR remain poorly understood. Here, we report crystal structures of RRM3 free and bound to cognate RNAs. Our structural, NMR and biochemical data show that RRM3 mediates canonical RNA interactions and reveal molecular details of a dimerization interface localized on the -helical face of RRM3. NMR and SAXS analyses indicate that the three RRMs in full-length HuR are flexibly connected in the absence of RNA, while they adopt a more compact arrangement when bound to RNA. Based on these data and crystal structures of tandem RRM1,2-RNA and our RRM3-RNA complexes, we present a structural model of RNA recognition involving all three RRM domains of full-length HuR. Mutational analysis demonstrates that RRM3 dimerization and RNA binding is required for functional activity of full-length HuR in vitro and to regulate target mRNAs levels in human cells, thus providing a fine-tuning for HuR activity in vivo. AU - Pabis, M. AU - Popowicz, G.M. AU - Stehle, R. AU - Fernández-Ramos, D.* AU - Asami, S. AU - Warner, L. AU - García-Mauriño, S.M.* AU - Schlundt, A. AU - Martínez-Chantar, M.L.* AU - Díaz-Moreno, I.* AU - Sattler, M. C1 - 55415 C2 - 46114 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 1011-1029 TI - HuR biological function involves RRM3-mediated dimerization and RNA binding by all three RRMs. JO - Nucleic Acids Res. VL - 47 IS - 2 PB - Oxford Univ Press PY - 2019 SN - 0305-1048 ER - TY - JOUR AB - The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery. AU - Pabis, M. AU - Corsini, L. AU - Vincendeau, M. AU - Tripsianes, K.* AU - Gibson, T.J.* AU - Brack-Werner, R. AU - Sattler, M. C1 - 55715 C2 - 46471 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 4859-4871 TI - Modulation of HIV-1 gene expression by binding of a ULM motif in the Rev protein to UHM-containing splicing factors. JO - Nucleic Acids Res. VL - 47 IS - 9 PB - Oxford Univ Press PY - 2019 SN - 0305-1048 ER - TY - JOUR AB - Mammalian-wide interspersed repeats (MIRs) are retrotransposed elements of mammalian genomes. Here, we report the specific binding of zinc finger protein ZNF768 to the sequence motif GCTGTGTG (N-20) CCTCTCTG in the core region of MIRs. ZNF768 binding is preferentially associated with euchromatin and promoter regions of genes. Binding was observed for genes expressed in a cell type-specific manner in human B cell line Raji and osteosarcoma U2OS cells. Mass spectrometric analysis revealed binding of ZNF768 to Elongator components Elp1, Elp2 and Elp3 and other nuclear factors. The N-terminus of ZNF768 contains a heptad repeat array structurally related to the C-terminal domain (CTD) of RNA polymerase II. This array evolved in placental animals but not marsupials and monotreme species, displays species-specific length variations, and possibly fulfills CTD related functions in gene regulation. We propose that the evolution of MIRs and ZNF768 has extended the repertoire of gene regulatory mechanisms in mammals and that ZNF768 binding is associated with cell type-specific gene expression. AU - Rohrmoser, M. AU - Kluge, M.* AU - Yahia, Y.* AU - Gruber-Eber, A. AU - Maqbool, M.A.* AU - Forné, I.* AU - Krebs, S.* AU - Blum, H.* AU - Greifenberg, A.K.* AU - Geyer, M.* AU - Descostes, N.* AU - Imhof, A.* AU - Andrau, J.C.* AU - Friedel, C.C.* AU - Eick, D. C1 - 54798 C2 - 45912 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 700-715 TI - MIR sequences recruit zinc finger protein ZNF768 to expressed genes. JO - Nucleic Acids Res. VL - 47 IS - 2 PB - Oxford Univ Press PY - 2019 SN - 0305-1048 ER - TY - JOUR AB - Affinity maturation of the humoral immune response depends on somatic hypermutation (SHM) of immunoglobulin (Ig) genes, which is initiated by targeted lesion introduction by activation-induced deaminase (AID), followed by error-prone DNA repair. Stringent regulation of this process is essential to prevent genetic instability, but no negative feedback control has been identified to date. Here we show that poly(ADP-ribose) polymerase-1 (PARP-1) is a key factor restricting AID activity during somatic hypermutation. Poly(ADP-ribose) (PAR) chains formed at DNA breaks trigger AID-PAR association, thus preventing excessive DNA damage induction at sites of AID action. Accordingly, AID activity and somatic hypermutation at the Ig variable region is decreased by PARP-1 activity. In addition, PARP-1 regulates DNA lesion processing by affecting strand biased A:T mutagenesis. Our study establishes a novel function of the ancestral genome maintenance factor PARP-1 as a critical local feedback regulator of both AID activity and DNA repair during Ig gene diversification. AU - Tepper, S.* AU - Mortusewicz, O.* AU - Członka, E.* AU - Bello, A.* AU - Schmidt, A.* AU - Jeschke, J.* AU - Fischbach, A.* AU - Pfeil, I. AU - Petersen-Mahrt, S.K.* AU - Mangerich, A.* AU - Helleday, T.* AU - Leonhardt, H.* AU - Jungnickel, B.* C1 - 56161 C2 - 46870 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 7418-7429 TI - Restriction of AID activity and somatic hypermutation by PARP-1. JO - Nucleic Acids Res. VL - 47 IS - 14 PB - Oxford Univ Press PY - 2019 SN - 0305-1048 ER - TY - JOUR AB - With the availability of deep RNA sequencing, model organisms such as Xenopus offer an outstanding opportunity to investigate the genetic basis of vertebrate organ formation from its embryonic beginnings. Here we investigate dynamics of the RNA landscape during formation of the Xenopus tropicalis larval epidermis. Differentiation of non-neural ectoderm starts at gastrulation and takes about one day to produce a functional mucociliary epithelium, highly related to the one in human airways. To obtain RNA expression data, uncontaminated by nonepidermal tissues of the embryo, we use prospective ectodermal explants called Animal Caps (ACs), which differentiate autonomously into a ciliated epidermis. Their global transcriptome is investigated at three key timepoints, with a cumulative sequencing depth of similar to 10(8) reads per developmental stage. This database is provided as online Web Tool to the scientific community. In this paper, we report on global changes in gene expression, an unanticipated diversity of mRNA splicing isoforms, expression patterns of repetitive DNA Elements, and the complexity of circular RNAs during this process. Computationally we derive transcription factor hubs from this data set, which may help in the future to define novel genetic drivers of epidermal differentiation in vertebrates. AU - Angerilli, A.* AU - Smialowski, P. AU - Rupp, R.A.* C1 - 54509 C2 - 45648 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 8772-8787 TI - The Xenopus animal cap transcriptome: Building a mucociliary epithelium. JO - Nucleic Acids Res. VL - 46 IS - 17 PB - Oxford Univ Press PY - 2018 SN - 0305-1048 ER - TY - JOUR AB - Aromatic hydrocarbons belong to the most abundant contaminants in groundwater systems. They can serve as carbon and energy source for a multitude of indigenous microorganisms. Predictions of contaminant biodegradation and microbial growth in contaminated aquifers are often vague because the parameters of microbial activity in the mathematical models used for predictions are typically derived from batch experiments, which don't represent conditions in the field. In order to improve our understanding of key drivers of natural attenuation and the accuracy of predictive models, we conducted comparative experiments in batch and sediment flow-through systems with varying concentrations of contaminant in the inflow and flow velocities applying the aerobic Pseudomonas putida strain F1 and the denitrifying Aromatoleum aromaticum strain EbN1. We followed toluene degradation and bacterial growth by measuring toluene and oxygen concentrations and by direct cell counts. In the sediment columns, the total amount of toluene degraded by P. putida F1 increased with increasing source concentration and flow velocity, while toluene removal efficiency gradually decreased. Results point at mass transfer limitation being an important process controlling toluene biodegradation that cannot be assessed with batch experiments. We also observed a decrease in the maximum specific growth rate with increasing source concentration and flow velocity. At low toluene concentrations, the efficiencies in carbon assimilation within the flow-through systems exceeded those in the batch systems. In all column experiments the number of attached cells plateaued after an initial growth phase indicating a specific "carrying capacity" depending on contaminant concentration and flow velocity. Moreover, in all cases, cells attached to the sediment dominated over those in suspension, and toluene degradation was performed practically by attached cells only. The observed effects of varying contaminant inflow concentration and flow velocity on biodegradation could be captured by a reactive-transport model. By monitoring both attached and suspended cells we could quantify the release of new-grown cells from the sediments to the mobile aqueous phase. Studying flow velocity and contaminant concentrations as key drivers of contaminant transformation in sediment flow-through microcosms improves our system understanding and eventually the prediction of microbial biodegradation at contaminated sites. AU - Behrens, G.* AU - Winzen, R.* AU - Rehage, N.* AU - Doerrie, A.* AU - Barsch, M.* AU - Hoffmann, A.L.* AU - Hackermueller, J.* AU - Tiedje, C.* AU - Heissmeyer, V. AU - Holtmann, H.* C1 - 53806 C2 - 45038 CY - 233 Spring St, New York, Ny 10013 Usa SP - 4256-4270 TI - A translational silencing function of MCPIP1/Regnase-1 specified by the target site context. JO - Nucleic Acids Res. VL - 46 IS - 8 PB - Springer PY - 2018 SN - 0305-1048 ER - TY - JOUR AB - Temporal changes to the concentration of molecular species such as mRNA, which take place in response to various environmental cues, can often be modeled as simple continuous functions such as a single pulse (impulse) model. The simplicity of such functional representations can provide an improved performance on fundamental tasks such as noise reduction, imputation and differential expression analysis. However, temporal gene expression profiles are often studied with models that treat time as a categorical variable, neglecting the dependence between time points. Here, we present ImpulseDE2, a framework for differential expression analysis that combines the power of the impulse model as a continuous representation of temporal responses along with a noise model tailored specifically to sequencing data. We compare the simple categorical models to ImpulseDE2 and to other continuous models based on natural cubic splines and demonstrate the utility of the continuous approach for studying differential expression in time course sequencing experiments. A unique feature of ImpulseDE2 is the ability to distinguish permanently from transiently up- or down-regulated genes. Using an in vitro differentiation dataset, we demonstrate that this gene classification scheme can be used to highlight distinct transcriptional programs that are associated with different phases of the differentiation process. AU - Fischer, D.S. AU - Theis, F.J. AU - Yosef, N.* C1 - 54788 C2 - 45856 TI - Impulse model-based differential expression analysis of time course sequencing data. JO - Nucleic Acids Res. VL - 46 IS - 20 PY - 2018 SN - 0305-1048 ER - TY - JOUR AB - DNA is the carrier of all cellular genetic information and increasingly used in nanotechnology. Quantitative understanding and optimization of its functions requires precise experimental characterization and accurate modeling of DNA properties. A defining feature of DNA is its helicity. DNA unwinds with increasing temperature, even for temperatures well below the melting temperature. However, accurate quanti-tation of DNA unwinding under external forces and a microscopic understanding of the corresponding structural changes are currently lacking. Here we combine single-molecule magnetic tweezers measurements with atomistic molecular dynamics and coarse-grained simulations to obtain a comprehensive view of the temperature dependence of DNA twist. Experimentally, we find that DNA twist changes by Tw(T) = (−11.0 ± 1.2)◦/(◦C·kbp), independent of applied force, in the range of forces where torque-induced melting is negligible. Our atomistic simulations predict Tw(T) = (−11.1 ± 0.3)◦/(◦C·kbp), in quantitative agreement with experiments, and suggest that the untwisting of DNA with temperature is predominantly due to changes in DNA structure for defined backbone substates, while the effects of changes in substate populations are minor. Coarse-grained simulations using the oxDNA framework yield a value of Tw(T) = (−6.4 ± 0.2)◦/(◦C·kbp) in semi-quantitative agreement with experiments. AU - Kriegel, F.* AU - Matek, C. AU - Dršata, T.* AU - Kulenkampff, K.* AU - Tschirpke, S.* AU - Zacharias, M.* AU - Lankaš, F.* AU - Lipfert, J.* C1 - 54016 C2 - 45197 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 7998-8009 TI - The temperature dependence of the helical twist of DNA. JO - Nucleic Acids Res. VL - 46 IS - 15 PB - Oxford Univ Press PY - 2018 SN - 0305-1048 ER - TY - JOUR AB - Nonsense-mediated mRNA decay (NMD) is important for RNA quality control and gene regulation in eukaryotes. NMD targets aberrant transcripts for decay and also directly influences the abundance of non-aberrant transcripts. In animals, the SMG1 kinase plays an essential role in NMD by phosphorylating the core NMD factor UPF1. Despite SMG1 being ubiquitous throughout the plant kingdom, little is known about its function, probably because SMG1 is atypically absent from the genome of the model plant, Arabidopsis thaliana. By combining our previously established SMG1 knockout in moss with transcriptome-wide analysis, we reveal the range of processes involving SMG1 in plants. Machine learning assisted analysis suggests that 32% of multi-isoform genes produce NMD-targeted transcripts and that splice junctions downstream of a stop codon act as the major determinant of NMD targeting. Furthermore, we suggest that SMG1 is involved in other quality control pathways, affecting DNA repair and the unfolded protein response, in addition to its role in mRNA quality control. Consistent with this, smg1 plants have increased susceptibility to DNA damage, but increased tolerance to unfolded protein inducing agents. The potential involvement of SMG1 in RNA, DNA and protein quality control has major implications for the study of these processes in plants. AU - Lloyd, J.P.B.* AU - Lang, D. AU - Zimmer, A.D.* AU - Causier, B.* AU - Reski, R.* AU - Davies, B.* C1 - 54061 C2 - 45260 CY - Great Clarendon St, Oxford Ox2 6dp, England SP - 5822-5836 TI - The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens. JO - Nucleic Acids Res. VL - 46 IS - 11 PB - Oxford Univ Press PY - 2018 SN - 0305-1048 ER - TY - JOUR AB - Gene expression profiles have been extensively discussed as an aid to guide the therapy by predicting disease outcome for the patients suffering from complex diseases, such as cancer. However, prediction models built upon single-gene (SG) features show poor stability and performance on independent datasets. Attempts to mitigate these drawbacks have led to the development of network-based approaches that integrate pathway information to produce meta-gene (MG) features. Also, MG approaches have only dealt with the two-class problem of good versus poor outcome prediction. Stratifying patients based on their molecular subtypes can provide a detailed view of the disease and lead to more personalized therapies. We propose and discuss a novel MG approach based on de novo pathways, which for the first time have been used as features in a multi-class setting to predict cancer subtypes. Comprehensive evaluation in a large cohort of breast cancer samples from The Cancer Genome Atlas (TCGA) revealed that MGs are considerably more stable than SG models, while also providing valuable insight into the cancer hallmarks that drive them. In addition, when tested on an independent benchmark non-TCGA dataset, MG features consistently outperformed SG models. We provide an easy-touse web service at http:// pathclass. compbio. sdu. dk where users can upload their own gene expression datasets from breast cancer studies and obtain the subtype predictions from all the classifiers. AU - Alcaraz, N.* AU - List, M.* AU - Batra, R. AU - Vandin, F.* AU - Ditzel, H.J.* AU - Baumbach, J.* C1 - 52040 C2 - 43675 CY - Oxford TI - De novo pathway-based biomarker identification. JO - Nucleic Acids Res. VL - 45 IS - 16 PB - Oxford Univ Press PY - 2017 SN - 0305-1048 ER - TY - JOUR AB - Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. AU - Clauß, K.* AU - Popp, A.P.* AU - Schulze, L.* AU - Hettich, J.* AU - Reisser, M.* AU - Escoter Torres, L. AU - Uhlenhaut, N.H. AU - Gebhardt, J.C.M.* C1 - 52044 C2 - 43687 CY - Oxford SP - 11121-11130 TI - DNA residence time is a regulatory factor of transcription repression. JO - Nucleic Acids Res. VL - 45 IS - 19 PB - Oxford Univ Press PY - 2017 SN - 0305-1048 ER - TY - JOUR AB - An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior. AU - Deng, T.* AU - Postnikov, Y.* AU - Zhang, S.* AU - Garrett, L. AU - Becker, L. AU - Rácz, I. AU - Hölter, S.M. AU - Wurst, W. AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Bustin, M.* C1 - 50068 C2 - 42051 SP - 3031-3045 TI - Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation. JO - Nucleic Acids Res. VL - 45 IS - 6 PY - 2017 SN - 0305-1048 ER - TY - JOUR AB - CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. AU - He, F.* AU - Vestergaard, G. AU - Peng, W.M.* AU - She, Q.* AU - Peng, X.* C1 - 50812 C2 - 42890 CY - Oxford SP - 1902-1913 TI - CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b. JO - Nucleic Acids Res. VL - 45 IS - 4 PB - Oxford Univ Press PY - 2017 SN - 0305-1048 ER - TY - JOUR AB - The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine(376) and Serine(389) by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially colocalized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2(S4-S8) and CHK1(S345) phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE(-/-)), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis. AU - Kumar, V.* AU - Fleming, T.* AU - Terjung, S.* AU - Gorzelanny, C.* AU - Gebhardt, C.* AU - Agrawal, R.* AU - Mall, M.A.* AU - Ranzinger, J.* AU - Zeier, M.* AU - Madhusudhan, T.* AU - Ranjan, S.* AU - Isermann, B.* AU - Liesz, A.* AU - Deshpande, D.* AU - Häring, H.-U. AU - Biswas, S.K.* AU - Reynolds, P.R.* AU - Hammes, H.P.* AU - Peperkok, R.* AU - Angel, P.* AU - Herzig, S. AU - Nawroth, P.P. C1 - 52253 C2 - 43835 CY - Oxford SP - 10595-10613 TI - Homeostatic nuclear RAGE-ATM interaction is essential for efficient DNA repair. JO - Nucleic Acids Res. VL - 45 IS - 18 PB - Oxford Univ Press PY - 2017 SN - 0305-1048 ER - TY - JOUR AB - Genome-wide association studies identified numerous disease risk loci. Delineating molecular mechanisms influenced by cis-regulatory variants is essential to understand gene regulation and ultimately disease pathophysiology. Combining bioinformatics and public domain chromatin information with quantitative proteomics supports prediction of cis-regulatory variants and enabled identification of allele-dependent binding of both, transcription factors and coregulators at the type 2 diabetes associated PPARG locus. We found rs7647481A nonrisk allele binding of Yin Yang 1 (YY1), confirmed by allele-specific chromatin immunoprecipitation in primary adipocytes. Quantitative proteomics also found the coregulator RING1 and YY1 binding protein (RYBP) whose mRNA levels correlate with improved insulin sensitivity in primary adipose cells carrying the rs7647481A nonrisk allele. Our findings support a concept with diverse cis-regulatory variants contributing to disease pathophysiology at one locus. Proteome-wide identification of both, transcription factors and coregulators, can profoundly improve understanding of mechanisms underlying genetic associations. AU - Lee, H.K. AU - Qian, K. AU - von Toerne, C. AU - Hoerburger, L.* AU - Claussnitzer, M. AU - Hoffmann, C.* AU - Glunk, V. AU - Wahl, S. AU - Breier, M. AU - Eck, F.* AU - Jafari, L.* AU - Molnos, S. AU - Grallert, H. AU - Dahlman, I.* AU - Arner, P.* AU - Brunner, C.* AU - Hauner, H. AU - Hauck, S.M. AU - Laumen, H. C1 - 50783 C2 - 42541 SP - 3266-3279 TI - Allele-specific quantitative proteomics unravels molecular mechanisms modulated by cis-regulatory PPARG locus variation. JO - Nucleic Acids Res. VL - 45 IS - 6 PB - Oxford Academic PY - 2017 SN - 0305-1048 ER - TY - JOUR AB - Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy. AU - Senís, E.* AU - Mockenhaupt, S.* AU - Rupp, D.* AU - Bauer, T.* AU - Paramasivam, N.* AU - Knapp, B. AU - Gronych, J.* AU - Grosse, S.D.* AU - Windisch, M.P.* AU - Schmidt, F.* AU - Theis, F.J. AU - Eils, R.* AU - Lichter, P.* AU - Schlesner, M.* AU - Bartenschlager, R.* AU - Grimm, D.* C1 - 49456 C2 - 32474 CY - Oxford TI - TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus. JO - Nucleic Acids Res. VL - 45 IS - 1 PB - Oxford Univ Press PY - 2017 SN - 0305-1048 ER - TY - JOUR AB - RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC. AU - Tants, J.-N. AU - Fesser, S.* AU - Kern, T. AU - Stehle, R. AU - Geerlof, A. AU - Wunderlich, C.* AU - Juen, M.* AU - Hartlmüller, C. AU - Böttcher, R.* AU - Kunzelmann, S.* AU - Lange, O.L.* AU - Kreutz, C.* AU - Förstemann, K.* AU - Sattler, M. C1 - 52158 C2 - 43781 CY - Oxford SP - 12536-12550 TI - Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference. JO - Nucleic Acids Res. VL - 45 IS - 21 PB - Oxford Univ Press PY - 2017 SN - 0305-1048 ER - TY - JOUR AB - The dynamic interaction of DNA methylation and transcription factor binding in regulating spatiotemporal gene expression is essential for embryogenesis, but the underlying mechanisms remain understudied. In this study, using mouse models and integration of in vitro and in vivo genetic and epigenetic analyses, we show that the binding of REST (repressor element 1 (RE1) silencing transcription factor; also known as NRSF) to its cognate RE1 sequences is temporally regulated by non-CpGmethylation. This process is dependent on DNA methyltransferase 3B (DNMT3B) and leads to suppression of adult cardiac genes in developing hearts. We demonstrate that DNMT3B preferentially mediates non-CpG methylation of REST-targeted genes in the developing heart. Downregulation of DNMT3B results in decreased non-CpG methylation of RE1 sequences, reduced REST occupancy, and consequently release of the transcription suppression during later cardiac development. Together, these findings reveal a critical gene silencingmechanism in developing mammalian hearts that is regulated by the dynamic interaction of DNMT3B-mediated non-CpG methylation and REST binding. AU - Zhang, D.* AU - Wu, B.L.* AU - Wang, P.* AU - Wang, Y.* AU - Lu, P.* AU - Nechiporuk, T.* AU - Floß, T. AU - Greally, J.M.* AU - Zheng, D.* AU - Zhou, B.* C1 - 51283 C2 - 43166 SP - 3102-3115 TI - Non-CpG methylation by DNMT3B facilitates REST binding and gene silencing in developing mouse hearts. JO - Nucleic Acids Res. VL - 45 IS - 6 PY - 2017 SN - 0305-1048 ER - TY - JOUR AB - In B cells infected by the cancer-associated Epstein-Barr virus (EBV), RUNX3 and RUNX1 transcription is manipulated to control cell growth. The EBV-encoded EBNA2 transcription factor (TF) activates RUNX3 transcription leading to RUNX3-mediated repression of the RUNX1 promoter and the relief of RUNX1-directed growth repression. We show that EBNA2 activates RUNX3 through a specific element within a -97 kb super-enhancer in a manner dependent on the expression of the Notch DNA-binding partner RBP-J. We also reveal that the EBV TFs EBNA3B and EBNA3C contribute to RUNX3 activation in EBV-infected cells by targeting the same element. Uncovering a counter-regulatory feed-forward step, we demonstrate EBNA2 activation of a RUNX1 super-enhancer (-139 to -250 kb) that results in low-level RUNX1 expression in cells refractory to RUNX1-mediated growth inhibition. EBNA2 activation of the RUNX1 super-enhancer is also dependent on RBP-J. Consistent with the context-dependent roles of EBNA3B and EBNA3C as activators or repressors, we find that these proteins negatively regulate the RUNX1 super-enhancer, curbing EBNA2 activation. Taken together our results reveal cell-type-specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth. AU - Gunnell, A.* AU - Webb, H.M.* AU - Wood, C.D.* AU - McClellan, M.J.* AU - Wichaidit, B.* AU - Kempkes, B. AU - Jenner, R.G.* AU - Osborne, C.* AU - Farrell, P.J.* AU - West, M.J.* C1 - 47910 C2 - 39753 CY - Oxford SP - 4636-4650 TI - RUNX super-enhancer control through the Notch pathway by Epstein-Barr virus transcription factors regulates B cell growth. JO - Nucleic Acids Res. VL - 44 IS - 10 PB - Oxford Univ Press PY - 2016 SN - 0305-1048 ER - TY - JOUR AB - The Epstein-Barr virus (EBV) transforms B cells by expressing latent proteins and the BHRF1 microRNA cluster. MiR-BHRF1-3, its most transforming member, belongs to the recently identified group of weakly expressed microRNAs. We show here that miR-BHRF1-3 displays an unusually low propensity to form a stem-loop structure, an effect potentiated by miR-BHRF1-3's proximity to the BHRF1 polyA site. Cloning miR-BHRF1-2 or a cellular microRNA, but not a ribozyme, 5' of miR-BHRF1-3 markedly enhanced its expression. However, a virus carrying mutated miR-BHRF1-2 seed regions expressed miR-BHRF1-3 at normal levels and was fully transforming. Therefore, miR-BHRF1-2's role during transformation is independent of its seed regions, revealing a new microRNA function. Increasing the distance between miR-BHRF1-2 and miR-BHRF1-3 in EBV enhanced miR-BHRF1-3's expression but decreased its transforming potential. Thus, the expression of some microRNAs must be restricted to a narrow range, as achieved by placing miR-BHRF1-3 under the control of miR-BHRF1-2. AU - Haar, J.* AU - Contrant, M.* AU - Bernhardt, K.* AU - Feederle, R. AU - Diederichs, S.* AU - Pfeffer, S.* AU - Delecluse, H.J.* C1 - 47650 C2 - 39376 CY - Oxford SP - 1326-1341 TI - The expression of a viral microRNA is regulated by clustering to allow optimal B cell transformation. JO - Nucleic Acids Res. VL - 44 IS - 3 PB - Oxford Univ Press PY - 2016 SN - 0305-1048 ER - TY - JOUR AB - eggNOG is a public resource that provides Orthologous Groups (OGs) of proteins at different taxonomic levels, each with integrated and summarized functional annotations. Developments since the latest public release include changes to the algorithm for creating OGs across taxonomic levels, making nested groups hierarchically consistent. This allows for a better propagation of functional terms across nested OGs and led to the novel annotation of 95 890 previously uncharacterized OGs, increasing overall annotation coverage from 67% to 72%. The functional annotations of OGs have been expanded to also provide Gene Ontology terms, KEGG pathways and SMART/Pfam domains for each group. Moreover, eggNOG now provides pairwise orthology relationships within OGs based on analysis of phylogenetic trees. We have also incorporated a framework for quickly mapping novel sequences to OGs based on precomputed HMM profiles. Finally, eggNOG version 4.5 incorporates a novel data set spanning 2605 viral OGs, covering 5228 proteins from 352 viral proteomes. All data are accessible for bulk downloading, as a web-service, and through a completely redesigned web interface. The new access points provide faster searches and a number of new browsing and visualization capabilities, facilitating the needs of both experts and less experienced users. AU - Huerta-Cepas, J.* AU - Szklarczyk, D.* AU - Forslund, K.* AU - Cook, H.* AU - Heller, D.* AU - Walter, M.C. AU - Rattei, T.* AU - Mende, D.R.* AU - Sunagawa, S.* AU - Kuhn, M.* AU - Jensen, L.J.* AU - von Mering, C.* AU - Bork, P.* C1 - 47344 C2 - 40585 CY - Oxford SP - D286-D293 TI - eggNOG 4.5: A hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. JO - Nucleic Acids Res. VL - 44 IS - D1 PB - Oxford Univ Press PY - 2016 SN - 0305-1048 ER - TY - JOUR AB - Bromodomain-containing protein 4 (BRD4) is amember of the bromo-and extraterminal (BET) domaincontaining family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelialspecific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXOtranscription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression. AU - Nagarajan, S.* AU - Bedi, U.* AU - Budida, A.* AU - Hamdan, F.H.* AU - Mishra, V.K.* AU - Najafova, Z.* AU - Xie, W.* AU - Alawi, M.* AU - Indenbirken, D.* AU - Knapp, S.* AU - Chiang, C.M.* AU - Grundhoff, A.* AU - Kari, V.* AU - Scheel, C. AU - Wegwitz, F.* AU - Johnsen, S.A.* C1 - 51287 C2 - 43164 SP - 3130-3145 TI - BRD4 promotes p63 and GRHL3 expression downstream of FOXO in mammary epithelial cells. JO - Nucleic Acids Res. VL - 45 IS - 6 PY - 2016 SN - 0305-1048 ER - TY - JOUR AB - PGSB (Plant Genome and Systems Biology: formerly MIPS) PlantsDB (http://pgsb.helmholtz-muenchen.de/plant/index.jsp) is a database framework for the comparative analysis and visualization of plant genome data. The resource has been updated with new data sets and types as well as specialized tools and interfaces to address user demands for intuitive access to complex plant genome data. In its latest incarnation, we have re-worked both the layout and navigation structure and implemented new keyword search options and a new BLAST sequence search functionality. Actively involved in corresponding sequencing consortia, PlantsDB has dedicated special efforts to the integration and visualization of complex triticeae genome data, especially for barley, wheat and rye. We enhanced CrowsNest, a tool to visualize syntenic relationships between genomes, with data from the wheat sub-genome progenitor Aegilops tauschii and added functionality to the PGSB RNASeqExpressionBrowser. GenomeZipper results were integrated for the genomes of barley, rye, wheat and perennial ryegrass and interactive access is granted through PlantsDB interfaces. Data exchange and cross-linking between PlantsDB and other plant genome databases is stimulated by the transPLANT project (http://transplantdb.eu/). AU - Spannagl, M. AU - Nussbaumer, T. AU - Bader, K.C. AU - Martis, M.M. AU - Seidel, M. AU - Kugler, K.G. AU - Gundlach, H. AU - Mayer, K.F.X. C1 - 47184 C2 - 39162 CY - Oxford SP - D1141-D1147 TI - PGSB PlantsDB: Updates to the database framework for comparative plant genome research. JO - Nucleic Acids Res. VL - 44 IS - D1 PB - Oxford Univ Press PY - 2016 SN - 0305-1048 ER - TY - JOUR AB - The chromatin remodeling complex NoRC, comprising the subunits SNF2h and TIP5/BAZ2A, mediates heterochromatin formation at major clusters of repetitive elements, including rRNA genes, centromeres and telomeres. Association with chromatin requires the interaction of the TAM (TIP5/ARBP/MBD) domain of TIP5 with noncoding RNA, which targets NoRC to specific genomic loci. Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins. However, the TAM domain exhibits an extended MBD fold with unique C-terminal extensions that constitute a novel surface for RNA binding. Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo. Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA. AU - Anosova, I. AU - Melnik, S.* AU - Tripsianes, K. AU - Kateb, F. AU - Grummt, I.* AU - Sattler, M. C1 - 44553 C2 - 36942 CY - Oxford SP - 5208-5220 TI - A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes. JO - Nucleic Acids Res. VL - 43 IS - 10 PB - Oxford Univ Press PY - 2015 SN - 0305-1048 ER - TY - JOUR AB - ProtPhylo is a web-based tool to identify proteins that are functionally linked to either a phenotype or a protein of interest based on co-evolution. ProtPhylo infers functional associations by comparing protein phylogenetic profiles (co-occurrence patterns of orthology relationships) for more than 9.7 million non-redundant protein sequences from all three domains of life. Users can query any of 2048 fully sequenced organisms, including 1678 bacteria, 255 eukaryotes and 115 archaea. In addition, they can tailor ProtPhylo to a particular kind of biological question by choosing among four main orthology inference methods based either on pair-wise sequence comparisons (One-way Best Hits and Best Reciprocal Hits) or clustering of orthologous proteins across multiple species (OrthoMCL and eggNOG). Next, ProtPhylo ranks phylogenetic neighbors of query proteins or phenotypic properties using the Hamming distance as a measure of similarity between pairs of phylogenetic profiles. Candidate hits can be easily and flexibly prioritized by complementary clues on subcellular localization, known protein-protein interactions, membrane spanning regions and protein domains. The resulting protein list can be quickly exported into a csv text file for further analyses. ProtPhylo is freely available at http://www.protphylo.org. AU - Cheng, Y. AU - Perocchi, F. C1 - 44813 C2 - 37061 CY - Oxford SP - W160-W168 TI - ProtPhylo: Identification of protein-phenotype and protein-protein functional associations via phylogenetic profiling. JO - Nucleic Acids Res. VL - 41 IS - W1 PB - Oxford Univ Press PY - 2015 SN - 0305-1048 ER - TY - JOUR AB - The laboratory mouse is a key model organism to investigate mechanism and therapeutics of human disease. The number of targeted genetic mouse models of disease is growing rapidly due to high-throughput production strategies employed by the International Mouse Phenotyping Consortium (IMPC) and the development of new, more efficient genome engineering techniques such as CRISPR based systems. We have previously described the European Mouse Mutant Archive (EMMA) resource and how this international infrastructure provides archiving and distribution worldwide for mutant mouse strains. EMMA has since evolved into INFRAFRONTIER (http://www.infrafrontier.eu), the pan-European research infrastructure for the systemic phenotyping, archiving and distribution of mouse disease models. Here we describe new features including improved search for mouse strains, support for new embryonic stem cell resources, access to training materials via a comprehensive knowledgebase and the promotion of innovative analytical and diagnostic techniques. AU - INFRAFRONTIER Consortium (de Castro, A. AU - Fessele, S. AU - Steinkamp, R. AU - Hagn, M. AU - Raess, M. AU - Hrabě de Angelis, M. AU - Meehan, T.F.*) C1 - 42916 C2 - 35866 CY - Oxford SP - D1171-D1175 TI - INFRAFRONTIER - providing mutant mouse resources as research tools for the international scientific community. JO - Nucleic Acids Res. VL - 53 IS - D1 PB - Oxford Univ Press PY - 2015 SN - 0305-1048 ER - TY - JOUR AB - Perturbations of mammalian organisms including diseases, drug treatments and gene perturbations in mice affect organ systems differently. Some perturbations impair relatively few organ systems while others lead to highly heterogeneous or systemic effects. Organ System Heterogeneity DB (http://mips.helmholtz-muenchen.de/Organ_System_Heterogeneity/) provides information on the phenotypic effects of 4865 human diseases, 1667 drugs and 5361 genetically modified mouse models on 26 different organ systems. Disease symptoms, drug side effects and mouse phenotypes are mapped to the System Organ Class (SOC) level of the Medical Dictionary of Regulatory Activities (MedDRA). Then, the organ system heterogeneity value, a measurement of the systemic impact of a perturbation, is calculated from the relative frequency of phenotypic features across all SOCs. For perturbations of interest, the database displays the distribution of phenotypic effects across organ systems along with the heterogeneity value and the distance between organ system distributions. In this way, it allows, in an easy and comprehensible fashion, the comparison of the phenotypic organ system distributions of diseases, drugs and their corresponding genetically modified mouse models of associated disease genes and drug targets. The Organ System Heterogeneity DB is thus a platform for the visualization and comparison of organ system level phenotypic effects of drugs, diseases and genes. AU - Mannil, D. AU - Vogt, I. AU - Prinz, J. AU - Campillos, M. C1 - 32554 C2 - 35142 CY - Oxford SP - D900-D906 TI - Organ system heterogeneity DB: A database for the visualization of phenotypes at the organ system level. JO - Nucleic Acids Res. VL - 43 IS - D1 PB - Oxford Univ Press PY - 2015 SN - 0305-1048 ER - TY - JOUR AB - Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions. AU - Mulholland, C.B.* AU - Smets, M.* AU - Schmidtmann, E.* AU - Leidescher, S.* AU - Markaki, Y.* AU - Hofweber, M.* AU - Qin, W.* AU - Manzo, M.* AU - Kremmer, E. AU - Thanisch, K.* AU - Bauer, C.* AU - Rombaut, P.* AU - Herzog, F.* AU - Leonhardt, H.* AU - Bultmann, S.* C1 - 44980 C2 - 37131 TI - A modular open platform for systematic functional studies under physiological conditions. JO - Nucleic Acids Res. VL - 43 IS - 17 PY - 2015 SN - 0305-1048 ER - TY - JOUR AB - MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to distinct target mRNAs leading to translational repression and mRNA decay. Ago proteins interact with a member of the GW protein family, referred to as TNRC6A-C in mammals, which coordinate downstream gene-silencing processes. The cytoplasmic functions of TNRC6 and Ago proteins are reasonably well established. Both protein families are found in the nucleus as well. Their detailed nuclear functions, however, remain elusive. Furthermore, it is not clear which import routes Ago and TNRC6 proteins take into the nucleus. Using different nuclear transport assays, we find that Ago as well as TNRC6 proteins shuttle between the cytoplasm and the nucleus. While import receptors might function redundantly to transport Ago2, we demonstrate that TNRC6 proteins are imported by the Importin-β pathway. Finally, we show that nuclear localization of both Ago2 and TNRC6 proteins can depend on each other suggesting actively balanced cytoplasmic Ago - TNRC6 levels. AU - Schraivogel, D.* AU - Schindler, S.G.* AU - Danner, J.* AU - Kremmer, E. AU - Pfaff, J.* AU - Hannus, S.* AU - Depping, R.* AU - Meister, G.* C1 - 46322 C2 - 37595 SP - 7447-7461 TI - Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels. JO - Nucleic Acids Res. VL - 43 IS - 15 PY - 2015 SN - 0305-1048 ER - TY - JOUR AB - Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV. AU - Truong, D.J.J. AU - Kühner, K. AU - Kühn, R. AU - Werfel, S.* AU - Engelhardt, S.* AU - Wurst, W. AU - Ortiz, O. C1 - 45333 C2 - 37284 CY - Oxford SP - 6450-6458 TI - Development of an intein-mediated split-Cas9 system for gene therapy. JO - Nucleic Acids Res. VL - 43 IS - 13 PB - Oxford Univ Press PY - 2015 SN - 0305-1048 ER - TY - JOUR AB - The Similarity Matrix of Proteins (SIMAP, http://mips.gsf.de/simap/) database has been designed to massively accelerate computationally expensive protein sequence analysis tasks in bioinformatics. It provides pre-calculated sequence similarities interconnecting the entire known protein sequence universe, complemented by pre-calculated protein features and domains, similarity clusters and functional annotations. SIMAP covers all major public protein databases as well as many consistently re-annotated metagenomes from different repositories. As of September 2013, SIMAP contains >163 million proteins corresponding to similar to 70 million non-redundant sequences. SIMAP uses the sensitive FASTA search heuristics, the Smith-Waterman alignment algorithm, the InterPro database of protein domain models and the BLAST2GO functional annotation algorithm. SIMAP assists biologists by facilitating the interactive exploration of the protein sequence universe. Web-Service and DAS interfaces allow connecting SIMAP with any other bioinformatic tool and resource. All-against-all protein sequence similarity matrices of project-specific protein collections are generated on request. Recent improvements allow SIMAP to cover the rapidly growing sequenced protein sequence universe. New Web-Service interfaces enhance the connectivity of SIMAP. Novel tools for interactive extraction of protein similarity networks have been added. Open access to SIMAP is provided through the web portal; the portal also contains instructions and links for software access and flat file downloads. AU - Arnold, R.* AU - Goldenberg, F.* AU - Mewes, H.-W. AU - Rattei, T.* C1 - 30822 C2 - 33944 CY - Oxford SP - D279-D284 TI - SIMAP - the database of all-against-all protein sequence similarities and annotations with new interfaces and increased coverage. JO - Nucleic Acids Res. VL - 42 IS - D1 PB - Oxford Univ Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1. AU - Bazot, Q.* AU - Deschamps, T.* AU - Tafforeau, L.* AU - Siouda, M.* AU - Leblanc, P.* AU - Harth-Hertle, M.L. AU - Rabourdin-Combe, C.* AU - Lotteau, V.* AU - Kempkes, B. AU - Tommasino, M.* AU - Gruffat, H.* AU - Manet, E.* C1 - 31879 C2 - 34847 CY - Oxford SP - 9700-9716 TI - Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1. JO - Nucleic Acids Res. VL - 42 IS - 15 PB - Oxford Univ Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - Bacterial infectious diseases are the result of multifactorial processes affected by the interplay between virulence factors and host targets. The host-Pseudomonas and Coxiella interaction database (HoPaCI-DB) is a publicly available manually curated integrative database (http://mips.helmholtz-muenchen.de/HoPaCI/) of host-pathogen interaction data from Pseudomonas aeruginosa and Coxiella burnetii. The resource provides structured information on 3585 experimentally validated interactions between molecules, bioprocesses and cellular structures extracted from the scientific literature. Systematic annotation and interactive graphical representation of disease networks make HoPaCI-DB a versatile knowledge base for biologists and network biology approaches. AU - Bleves, S.* AU - Dunger, I. AU - Walter, M.C. AU - Frangoulidis, D.* AU - Kastenmüller, G. AU - Voulhoux, R.* AU - Ruepp, A. C1 - 28104 C2 - 32938 CY - Oxford SP - D671-D676 TI - HoPaCI-DB: Host-Pseudomonas and Coxiella interaction database. JO - Nucleic Acids Res. VL - 42 IS - D1 PB - Oxford Univ. Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - Knowledge about non-interacting proteins (NIPs) is important for training the algorithms to predict protein-protein interactions (PPIs) and for assessing the false positive rates of PPI detection efforts. We present the second version of Negatome, a database of proteins and protein domains that are unlikely to engage in physical interactions (available online at http://mips.helmholtz-muenchen.de/proj/ppi/negatome). Negatome is derived by manual curation of literature and by analyzing three-dimensional structures of protein complexes. The main methodological innovation in Negatome 2.0 is the utilization of an advanced text mining procedure to guide the manual annotation process. Potential non-interactions were identified by a modified version of Excerbt, a text mining tool based on semantic sentence analysis. Manual verification shows that nearly a half of the text mining results with the highest confidence values correspond to NIP pairs. Compared to the first version the contents of the database have grown by over 300%. AU - Blohm, P. AU - Frishman, G. AU - Smialowski, P. AU - Goebels, F.* AU - Wachinger, B. AU - Ruepp, A. AU - Frishman, D. C1 - 28253 C2 - 33033 CY - Oxford SP - D396-D400 TI - Negatome 2.0: A database of non-interacting proteins derived by literature mining, manual annotation and protein structure analysis. JO - Nucleic Acids Res. VL - 42 IS - D1 PB - Oxford Univ. Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - Understanding how regulatory networks globally coordinate the response of a cell to changing conditions, such as perturbations by shifting environments, is an elementary challenge in systems biology which has yet to be met. Genome-wide gene expression measurements are high dimensional as these are reflecting the condition-specific interplay of thousands of cellular components. The integration of prior biological knowledge into the modeling process of systems-wide gene regulation enables the large-scale interpretation of gene expression signals in the context of known regulatory relations. We developed COGERE (http://mips.helmholtz-muenchen.de/cogere), a method for the inference of condition-specific gene regulatory networks in human and mouse. We integrated existing knowledge of regulatory interactions from multiple sources to a comprehensive model of prior information. COGERE infers condition-specific regulation by evaluating the mutual dependency between regulator (transcription factor or miRNA) and target gene expression using prior information. This dependency is scored by the non-parametric, nonlinear correlation coefficient η(2) (eta squared) that is derived by a two-way analysis of variance. We show that COGERE significantly outperforms alternative methods in predicting condition-specific gene regulatory networks on simulated data sets. Furthermore, by inferring the cancer-specific gene regulatory network from the NCI-60 expression study, we demonstrate the utility of COGERE to promote hypothesis-driven clinical research. AU - Ellwanger, D.C. AU - Leonhardt, J. AU - Mewes, H.-W. C1 - 32507 C2 - 35109 CY - Oxford TI - Large-scale modeling of condition-specific gene regulatory networks by information integration and inference. JO - Nucleic Acids Res. VL - 42 IS - 21 PB - Oxford Univ Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis. AU - Frankenberger, S. AU - Davari, K.* AU - Fischer-Burkart, S. AU - Böttcher, K.* AU - Tomi, N.S.* AU - Zimber-Strobl, U. AU - Jungnickel, B. C1 - 29287 C2 - 33554 CY - Oxford SP - 3666-3674 TI - Checkpoint kinase 1 negatively regulates somatic hypermutation. JO - Nucleic Acids Res. VL - 42 IS - 6 PB - Oxford Univ Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - CpG methylation in mammalian DNA is known to interfere with gene expression by inhibiting the binding of transactivators to their cognate sequence motifs or recruiting proteins involved in gene repression. An Epstein-Barr virus-encoded transcription factor, Zta, was the first example of a sequence-specific transcription factor that preferentially recognizes and selectively binds DNA sequence motifs with methylated CpG residues, reverses epigenetic silencing and activates gene transcription. The DNA binding domain of Zta is homologous to c-Fos, a member of the cellular AP-1 (activator protein 1) transcription factor family, which regulates cell proliferation and survival, apoptosis, transformation and oncogenesis. We have identified a novel AP-1 binding site termed meAP-1, which contains a CpG dinucleotide. If methylated, meAP-1 sites are preferentially bound by the AP-1 heterodimer c-Jun/c-Fos in vitro and in cellular chromatin in vivo. In activated human primary B cells, c-Jun/c-Fos locates to these methylated elements in promoter regions of transcriptionally activated genes. Reminiscent of the viral Zta protein, c-Jun/c-Fos is the first identified cellular member of the AP-1 family of transactivators that can induce expression of genes with methylated, hence repressed promoters, reversing epigenetic silencing. AU - Gustems, M. AU - Woellmer, A. AU - Rothbauer, U.* AU - Eck, S.H. AU - Wieland, T. AU - Lutter, D. AU - Hammerschmidt, W. C1 - 29087 C2 - 33630 CY - Oxford SP - 3059-3072 TI - c-Jun/c-Fos heterodimers regulate cellular genes via a newly identified class of methylated DNA sequence motifs. JO - Nucleic Acids Res. VL - 42 IS - 5 PB - Oxford Univ Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - The Fe(II) and 2-oxoglutarate dependent oxygenase Jmjd6 has been shown to hydroxylate lysine residues in the essential splice factor U2 auxiliary factor 65 kDa subunit (U2AF65) and to act as a modulator of alternative splicing. We describe further evidence for the role of Jmjd6 in the regulation of pre-mRNA processing including interactions of Jmjd6 with multiple arginine-serine-rich (RS)-domains of SR- and SR-related proteins including U2AF65, Luc7-like protein 3 (Luc7L3), SRSF11 and Acinus S', but not with the bona fide RS-domain of SRSF1. The identified Jmjd6 target proteins are involved in different mRNA processing steps and play roles in exon dependent alternative splicing and exon definition. Moreover, we show that Jmjd6 modifies splicing of a constitutive splice reporter, binds RNA derived from the reporter plasmid and punctually co-localises with nascent RNA. We propose that Jmjd6 exerts its splice modulatory function by interacting with specific SR-related proteins during splicing in a RNA dependent manner. AU - Heim, A.* AU - Grimm, C.* AU - Müller, U.* AU - Häußler, S.* AU - Mackeen, M.M.* AU - Merl, J. AU - Hauck, S.M. AU - Kessler, B.M.* AU - Schofield, C.J.* AU - Wolf, A. AU - Böttger, A.* C1 - 31594 C2 - 34635 CY - Oxford SP - 7833-7850 TI - Jumonji domain containing protein 6 (Jmjd6) modulates splicing and specifically interacts with arginine-serine-rich (RS) domains of SR- and SR-like proteins. JO - Nucleic Acids Res. VL - 42 IS - 12 PB - Oxford Univ Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - The appropriate expression of the roughly 30,000 human genes requires multiple layers of control. The oncoprotein MYC, a transcriptional regulator, contributes to many of the identified control mechanisms, including the regulation of chromatin, RNA polymerases, and RNA processing. Moreover, MYC recruits core histone-modifying enzymes to DNA. We identified an additional transcriptional cofactor complex that interacts with MYC and that is important for gene transcription. We found that the trithorax protein ASH2L and MYC interact directly in vitro and co-localize in cells and on chromatin. ASH2L is a core subunit of KMT2 methyltransferase complexes that target histone H3 lysine 4 (H3K4), a mark associated with open chromatin. Indeed, MYC associates with H3K4 methyltransferase activity, dependent on the presence of ASH2L. MYC does not regulate this methyltransferase activity but stimulates demethylation and subsequently acetylation of H3K27. KMT2 complexes have been reported to associate with histone H3K27-specific demethylases, while CBP/p300, which interact with MYC, acetylate H3K27. Finally WDR5, another core subunit of KMT2 complexes, also binds directly to MYC and in genome-wide analyses MYC and WDR5 are associated with transcribed promoters. Thus, our findings suggest that MYC and ASH2L-KMT2 complexes cooperate in gene transcription by controlling H3K27 modifications and thereby regulate bivalent chromatin. AU - Ullius, A.* AU - Luscher-Firzlaff, J.* AU - Costa, I.G.* AU - Walsemann, G.* AU - Forst, A.H.* AU - Gusmao, E.G.* AU - Kapelle, K. AU - Kleine, H.* AU - Kremmer, E. AU - Vervoorts, J.* AU - Lüscher, B.* C1 - 31525 C2 - 34507 CY - Oxford SP - 6901-6920 TI - The interaction of MYC with the trithorax protein ASH2L promotes gene transcription by regulating H3K27 modification. JO - Nucleic Acids Res. VL - 42 IS - 11 PB - Oxford Univ Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5' splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2-RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. AU - Wang, I. AU - Hennig, J. AU - Jagtap, P.K. AU - Sonntag, M. AU - Valcárcel, J.* AU - Sattler, M. C1 - 30941 C2 - 34041 CY - Oxford SP - 5949-5966 TI - Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1. JO - Nucleic Acids Res. VL - 42 IS - 9 PB - Oxford Univ Press PY - 2014 SN - 0305-1048 ER - TY - JOUR AB - Although multiple biological phenomena are related to temperature (e. g. elevation of body temperature due to an illness, adaptation to environmental temperature conditions, biology of coldblooded versus warm-blooded organisms), the molecular mechanisms of these processes remain to be understood. Perturbations of secondary RNA structures may play an important role in an organism's reaction to temperature change-in all organisms from viruses and bacteria to humans. Here, we present RNAtips (temperature-induced perturbation of structure) web server, which can be used to predict regions of RNA secondary structures that are likely to undergo structural alterations prompted by temperature change. The server can also be used to: (i) detect those regions in two homologous RNA sequences that undergo different structural perturbations due to temperature change and (ii) test whether these differences are specific to the particular nucleotide substitutions distinguishing the sequences. The RNAtips web server is freely accessible without any login requirement at http://rnatips.org. AU - Chursov, A.* AU - Kopetzky, S.J.* AU - Bocharov, G.* AU - Frishman, D. AU - Shneider, A.* C1 - 27322 C2 - 32636 SP - W486-W491 TI - RNAtips: Analysis of temperature-induced changes of RNA secondary structure. JO - Nucleic Acids Res. VL - 41 IS - W1 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the 'long-term evolution experiment'. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins. AU - Chursov, A.* AU - Frishman, D. AU - Shneider, A.* C1 - 27893 C2 - 32845 SP - 7854-7860 TI - Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution. JO - Nucleic Acids Res. VL - 41 IS - 16 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis. AU - de la Vega, L.* AU - Hornung, J.* AU - Kremmer, E. AU - Milanovic, M.* AU - Schmitz, M.L.* C1 - 25739 C2 - 31918 SP - 5731-5745 TI - Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage. JO - Nucleic Acids Res. VL - 41 IS - 11 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA. AU - Garg, D. AU - Beribisky, A.V. AU - Ponterini, G.* AU - Ligabue, A.* AU - Marverti, G.* AU - Martello, A.* AU - Costi, M.P.* AU - Sattler, M. AU - Wade, R.C.* C1 - 24690 C2 - 31636 SP - 4159-4170 TI - Translational repression of thymidylate synthase by targeting its mRNA. JO - Nucleic Acids Res. VL - 41 IS - 7 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - Co-transcriptional pre-mRNA processing relies on reversible phosphorylation of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAP II). In this study, we replaced in live cells the endogenous Rpb1 by S2A Rpb1, where the second serines (Ser2) in the CTD heptapeptide repeats were switched to alanines, to prevent phosphorylation. Although slower, S2A RNAP II was able to transcribe. However, it failed to recruit splicing components such as U2AF65 and U2 snRNA to transcription sites, although the recruitment of U1 snRNA was not affected. As a consequence, co-transcriptional splicing was impaired. Interestingly, the magnitude of the S2A RNAP II splicing defect was promoter dependent. In addition, S2A RNAP II showed an impaired recruitment of the cleavage factor PCF11 to pre-mRNA and a defect in 3'-end RNA cleavage. These results suggest that CTD Ser2 plays critical roles in co-transcriptional pre-mRNA maturation in vivo: It likely recruits U2AF65 to ensure an efficient co-transcriptional splicing and facilitates the recruitment of pre-mRNA 3'-end processing factors to enhance 3'-end cleavage. AU - Gu, B.* AU - Eick, D. AU - Bensaude, O.* C1 - 23756 C2 - 31281 SP - 1591-1603 TI - CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo. JO - Nucleic Acids Res. VL - 41 IS - 3 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - Regulated intramembrane proteolysis (RIP) is a critical mechanism for intercellular communication and regulates the function of membrane proteins through sequential proteolysis. RIP typically starts with ectodomain shedding of membrane proteins by extracellular membrane-bound proteases followed by intramembrane proteolysis of the resulting membrane-tethered fragment. However, for the majority of RIP proteases the corresponding substrates and thus, their functions, remain unknown. Proteome-wide identification of RIP protease substrates is possible by mass spectrometry-based quantitative comparison of RIP substrates or their cleavage products between different biological states. However, this requires quantification of peptides from only the ectodomain or cytoplasmic domain. Current analysis software does not allow matching peptides to either domain. Here we present the QARIP (Quantitative Analysis of Regulated Intramembrane Proteolysis) web server which matches identified peptides to the protein transmembrane topology. QARIP allows determination of quantitative ratios separately for the topological domains (cytoplasmic, ectodomain) of a given protein and is thus a powerful tool for quality control, improvement of quantitative ratios and identification of novel substrates in proteomic RIP datasets. To our knowledge, the QARIP web server is the first tool directly addressing the phenomenon of RIP. The web server is available at http://webclu.bio.wzw.tum.de/qarip/. This website is free and open to all users and there is no login requirement. AU - Ivankov, D.N.* AU - Bogatyreva, N.S.* AU - Honigschmid, P.* AU - Dislich, B.* AU - Högl, S.* AU - Kuhn, P.H.* AU - Frishman, D. AU - Lichtenthaler, S.F.* C1 - 27325 C2 - 32635 SP - W459-W464 TI - QARIP: A web server for quantitative proteomic analysis of regulated intramembrane proteolysis. JO - Nucleic Acids Res. VL - 41 IS - W1 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - The rapidly increasing amount of plant genome (sequence) data enables powerful comparative analyses and integrative approaches and also requires structured and comprehensive information resources. Databases are needed for both model and crop plant organisms and both intuitive search/browse views and comparative genomics tools should communicate the data to researchers and help them interpret it. MIPS PlantsDB (http://mips.helmholtz-muenchen.de/plant/genomes.jsp) was initially described in NAR in 2007 [Spannagl,M., Noubibou,O., Haase,D., Yang,L., Gundlach,H., Hindemitt, T., Klee,K., Haberer,G., Schoof,H. and Mayer,K.F. (2007) MIPSPlantsDB-plant database resource for integrative and comparative plant genome research. Nucleic Acids Res., 35, D834-D840] and was set up from the start to provide data and information resources for individual plant species as well as a framework for integrative and comparative plant genome research. PlantsDB comprises database instances for tomato, Medicago, Arabidopsis, Brachypodium, Sorghum, maize, rice, barley and wheat. Building up on that, state-of-the-art comparative genomics tools such as CrowsNest are integrated to visualize and investigate syntenic relationships between monocot genomes. Results from novel genome analysis strategies targeting the complex and repetitive genomes of triticeae species (wheat and barley) are provided and cross-linked with model species. The MIPS Repeat Element Database (mips-REdat) and Catalog (mips-REcat) as well as tight connections to other databases, e.g. via web services, are further important components of PlantsDB. AU - Nussbaumer, T. AU - Martis, M.M. AU - Roessner, S.K. AU - Pfeifer, M. AU - Bader, K.C. AU - Sharma, S. AU - Gundlach, H. AU - Spannagl, M. C1 - 11721 C2 - 30769 SP - D1144-D1151 TI - MIPS PlantsDB: A database framework for comparative plant genome research. JO - Nucleic Acids Res. VL - 41 IS - D1 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - Modern high-throughput methods allow the investigation of biological functions across multiple 'omics' levels. Levels include mRNA and protein expression profiling as well as additional knowledge on, for example, DNA methylation and microRNA regulation. The reason for this interest in multi-omics is that actual cellular responses to different conditions are best explained mechanistically when taking all omics levels into account. To map gene products to their biological functions, public ontologies like Gene Ontology are commonly used. Many methods have been developed to identify terms in an ontology, overrepresented within a set of genes. However, these methods are not able to appropriately deal with any combination of several data types. Here, we propose a new method to analyse integrated data across multiple omics-levels to simultaneously assess their biological meaning. We developed a model-based Bayesian method for inferring interpretable term probabilities in a modular framework. Our Multi-level ONtology Analysis (MONA) algorithm performed significantly better than conventional analyses of individual levels and yields best results even for sophisticated models including mRNA fine-tuning by microRNAs. The MONA framework is flexible enough to allow for different underlying regulatory motifs or ontologies. It is ready-to-use for applied researchers and is available as a standalone application from http://icb.helmholtz-muenchen.de/mona. AU - Sass, S. AU - Buettner, F. AU - Müller, N.S. AU - Theis, F.J. C1 - 28223 C2 - 33012 SP - 9622-9633 TI - A modular framework for gene set analysis integrating multilevel omics data. JO - Nucleic Acids Res. VL - 41 IS - 21 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - DNA methyltransferase 1 (Dnmt1) reestablishes methylation of hemimethylated CpG sites generated during DNA replication in mammalian cells. Two subdomains, the proliferating cell nuclear antigen (PCNA)-binding domain (PBD) and the targeting sequence (TS) domain, target Dnmt1 to the replication sites in S phase. We aimed to dissect the details of the cell cycle-dependent coordinated activity of both domains. To that end, we combined super-resolution 3D-structured illumination microscopy and fluorescence recovery after photobleaching (FRAP) experiments of GFP-Dnmt1 wild type and mutant constructs in somatic mouse cells. To interpret the differences in FRAP kinetics, we refined existing data analysis and modeling approaches to (i) account for the heterogeneous and variable distribution of Dnmt1-binding sites in different cell cycle stages; (ii) allow diffusion-coupled dynamics; (iii) accommodate multiple binding classes. We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time T-res 10 s). In late S phase, this binding class is taken over by a substantially stronger (T-res similar to 22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions. We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions. AU - Schneider, K.* AU - Fuchs, C. AU - Dobay, A.* AU - Rottach, A.* AU - Qin, W.H.* AU - Wolf, P.* AU - Alvarez-Castro, J.M.* AU - Nalaskowski, M.M.* AU - Kremmer, E. AU - Schmid, V.* AU - Leonhardt, H.* AU - Schermelleh, L.* C1 - 24806 C2 - 31690 SP - 4860-4876 TI - Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling. JO - Nucleic Acids Res. VL - 41 IS - 9 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - Recognition of the 3'-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein-protein and protein-RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1(NTD)). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1(NTD) with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65(UHM)) reveals that, in addition to the known U2AF65(UHM)-SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65(UHM), which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1-U2AF65-RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1-U2AF65 or the SF1-U2AF65-RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3'-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1-U2AF65-RNA complex. AU - Zhang, Y.-Y. AU - Madl, T. AU - Bagdiul, I.* AU - Kern, T. AU - Kang, H.-S. AU - Zou, P. AU - Mäusbacher, N.* AU - Sieber, S.A.* AU - Krämer, A.* AU - Sattler, M. C1 - 22971 C2 - 30986 SP - 1343-1354 TI - Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3'-splice site recognition. JO - Nucleic Acids Res. VL - 41 IS - 2 PB - Oxford Univ. Press PY - 2013 SN - 0305-1048 ER - TY - JOUR AB - In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. AU - Arakawa, H. AU - Bednar, T.* AU - Wang, M.* AU - Paul, K.* AU - Mladenov, E.* AU - Bencsik-Theilen, A.A.* AU - Iliakis, G.* C1 - 6560 C2 - 29332 SP - 2599-2610 TI - Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells. JO - Nucleic Acids Res. VL - 40 IS - 6 PB - Oxford Univ. Press PY - 2012 SN - 0305-1048 ER - TY - JOUR AB - The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date. AU - Bönisch, C.* AU - Schneider, K.* AU - Pünzeler, S.* AU - Wiedemann, S.M.* AU - Bielmeier, C.* AU - Bocola, M.* AU - Eberl, H.C.* AU - Kuegel, W.* AU - Neumann, J.* AU - Kremmer, E. AU - Leonhardt, H.* AU - Mann, M.* AU - Michaelis, J.* AU - Schermelleh, L.* AU - Hake, S.B.* C1 - 8638 C2 - 30214 SP - 5951-5964 TI - H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization. JO - Nucleic Acids Res. VL - 40 IS - 13 PB - Oxford Univ. Press PY - 2012 SN - 0305-1048 ER - TY - JOUR AB - It is generally accepted that functionally important RNA structure is more conserved than sequence due to compensatory mutations that may alter the sequence without disrupting the structure. For small RNA molecules sequence-structure relationships are relatively well understood. However, structural bioinformatics of mRNAs is still in its infancy due to a virtual absence of experimental data. This report presents the first quantitative assessment of sequence-structure divergence in the coding regions of mRNA molecules based on recently published transcriptome-wide experimental determination of their base paring patterns. Structural resemblance in paralogous mRNA pairs quickly drops as sequence identity decreases from 100% to 85-90%. Structures of mRNAs sharing sequence identity below roughly 85% are essentially uncorrelated. This outcome is in dramatic contrast to small functional non-coding RNAs where sequence and structure divergence are correlated at very low levels of sequence similarity. The fact that very similar mRNA sequences can have vastly different secondary structures may imply that the particular global shape of base paired elements in coding regions does not play a major role in modulating gene expression and translation efficiency. Apparently, the need to maintain stable three-dimensional structures of encoded proteins places a much higher evolutionary pressure on mRNA sequences than on their RNA structures. AU - Chursov, A.* AU - Walter, M.C. AU - Schmidt, T. AU - Mironov, A.* AU - Shneider, A.* AU - Frishman, D. C1 - 7270 C2 - 29630 SP - 956-962 TI - Sequence-structure relationships in yeast mRNAs. JO - Nucleic Acids Res. VL - 40 IS - 3 PB - Oxford Univ Press PY - 2012 SN - 0305-1048 ER - TY - JOUR AB - A large amount of differentially expressed proteins (DEPs) have been identified in various cancer proteomics experiments, curation and annotation of these proteins are important in deciphering their roles in oncogenesis and tumor progression, and may further help to discover potential protein biomarkers for clinical applications. In 2009, we published the first database of DEPs in human cancers (dbDEPCs). In this updated version of 2011, dbDEPC 2.0 has more than doubly expanded to over 4000 protein entries, curated from 331 experiments across 20 types of human cancers. This resource allows researchers to search whether their interested proteins have been reported changing in certain cancers, to compare their own proteomic discovery with previous studies, to picture selected protein expression heatmap across multiple cancers and to relate protein expression changes with aberrance in other genetic level. New important developments include addition of experiment design information, advanced filter tools for customer-specified analysis and a network analysis tool. We expect dbDEPC 2.0 to be a much more powerful tool than it was in its first release and can serve as reference to both proteomics and cancer researchers. AU - He, Y.* AU - Zhang, M.* AU - Ju, Y.* AU - Yu, Z.* AU - Lv, D.* AU - Sun, H.* AU - Yuan, W.* AU - He, F.* AU - Zhang, J.* AU - Li, H.* AU - Li, J.* AU - Wang-Sattler, R. AU - Li, Y.* AU - Zhang, G.* AU - Xie, L.* C1 - 7183 C2 - 29528 SP - D964-D971 TI - dbDEPC 2.0: Updated database of differentially expressed proteins in human cancers. JO - Nucleic Acids Res. VL - 40 IS - DI PB - Oxford Univ. Press PY - 2012 SN - 0305-1048 ER - TY - JOUR AB - Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo. Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. Here, we show that a major modification of H2B in Drosophila melanogaster is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless of their cell cycle distribution. We identified dNTMT (CG1675) as the enzyme responsible for H2B methylation. We also found that the level of N-terminal methylation is regulated by dART8, an arginine methyltransferase that physically interacts with dNTMT and asymmetrically methylates H3R2. Our results demonstrate the existence of a complex containing two methyltransferases enzymes, which negatively influence each other's activity. AU - Villar-Garea, A.* AU - Forne, I.* AU - Vetter, I.* AU - Kremmer, E. AU - Thomae, A.* AU - Imhof, A.* C1 - 6061 C2 - 29119 SP - 1536-1549 TI - Developmental regulation of N-terminal H2B methylation in Drosophila melanogaster. JO - Nucleic Acids Res. VL - 40 IS - 4 PB - Oxford Univ. Press PY - 2012 SN - 0305-1048 ER - TY - JOUR AB - BioProfiling.de provides a comprehensive analytical toolkit for the interpretation gene/protein lists. As input, BioProfiling.de accepts a gene/protein list. As output, in one submission, the gene list is analyzed by a collection of tools which employs advanced enrichment or network-based statistical frameworks. The gene list is profiled with respect to the most information available regarding gene function, protein interactions, pathway relationships, in silico predicted microRNA to gene associations, as well as, information collected by text mining. BioProfiling.de provides a user friendly dialog-driven web interface for several model organisms and supports most available gene identifiers. AU - Antonov, A.V. C1 - 6258 C2 - 29081 SP - W323-W327 TI - BioProfiling.de: Analytical web portal for high-throughput cell biology. JO - Nucleic Acids Res. VL - 39 IS - SUPPL. 2 PB - Oxford Univ. Press PY - 2011 SN - 0305-1048 ER - TY - JOUR AB - Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo. AU - Felle, M.* AU - Joppien, S.* AU - Németh, A.* AU - Diermeier, S.* AU - Thalhammer, V.* AU - Dobner, T.* AU - Kremmer, E. AU - Kappler, R.* AU - Längst, G. C1 - 6235 C2 - 28650 SP - 8355-8365 TI - The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1. JO - Nucleic Acids Res. VL - 39 IS - 19 PB - Oxford Univ. Press PY - 2011 SN - 0305-1048 ER - TY - JOUR AB - In recent years, the field of epigenetics has grown dramatically and has become one of the most dynamic and fast-growing branches of molecular biology. The amount of diseases suspected of being influenced by DNA methylation is rising steadily and includes common diseases such as schizophrenia, bipolar disorder, Alzheimer's disease, diabetes, atherosclerosis, cancer, major psychosis, lupus and Parkinson's disease. Due to cellular heterogeneity of methylation patterns, epigenetic analyses of single cells become a necessity. One rationale is that DNA methylation profiles are highly variable across individual cells, even in the same organ, dependent on the function of the gene, disease state, exposure to environmental factors (e.g. radiation, drugs or nutrition), stochastic fluctuations and various other causes. Using a polymerase chain reaction (PCR)-slide microreaction system, we present here a methylation-sensitive PCR analysis, the restriction enzyme-based single-cell methylation assay (RSMA), in the analysis of DNA methylation patterns in single cells. This method addresses the problems of cell heterogeneity in epigenetics research; it is comparably affordable, avoids complicated microfluidic systems and offers the opportunity for high-throughput screening, as many single cells can be screened in parallel. In addition to this study, critical principles and caveats of single cell methylation analyses are discussed. AU - Kantlehner, M.* AU - Kirchner, R.* AU - Hartmann, P.* AU - Ellwart, J.W. AU - Alunni-Fabbroni, M.* AU - Schumacher, A.* C1 - 4215 C2 - 28793 SP - e44 TI - A high-throughput DNA methylation analysis of a single cell. JO - Nucleic Acids Res. VL - 39 IS - 7 PB - Oxford Univ Press PY - 2011 SN - 0305-1048 ER - TY - JOUR AB - Domain Interaction MAp (DIMA, available at http://webclu.bio.wzw.tum.de/dima) is a database of predicted and known interactions between protein domains. It integrates 5807 structurally known interactions imported from the iPfam and 3did databases and 46,900 domain interactions predicted by four computational methods: domain phylogenetic profiling, domain pair exclusion algorithm correlated mutations and domain interaction prediction in a discriminative way. Additionally predictions are filtered to exclude those domain pairs that are reported as non-interacting by the Negatome database. The DIMA Web site allows to calculate domain interaction networks either for a domain of interest or for entire organisms, and to explore them interactively using the Flash-based Cytoscape Web software. AU - Luo, Q.* AU - Pagel, P. AU - Vilne, B.* AU - Frishman, D. C1 - 6614 C2 - 28978 SP - D724-D729 TI - DIMA 3.0: Domain Interaction Map. JO - Nucleic Acids Res. VL - 39 IS - SUPPL. 1 PB - Oxford Univ. Press PY - 2011 SN - 0305-1048 ER - TY - JOUR AB - The International Knockout Mouse Consortium (IKMC) aims to mutate all protein-coding genes in the mouse using a combination of gene targeting and gene trapping in mouse embryonic stem (ES) cells and to make the generated resources readily available to the research community. The IKMC database and web portal (www.knockoutmouse.org) serves as the central public web site for IKMC data and facilitates the coordination and prioritization of work within the consortium. Researchers can access up-to-date information on IKMC knockout vectors, ES cells and mice for specific genes, and follow links to the respective repositories from which corresponding IKMC products can be ordered. Researchers can also use the web site to nominate genes for targeting, or to indicate that targeting of a gene should receive high priority. The IKMC database provides data to, and features extensive interconnections with, other community databases. AU - Ringwald, M.* AU - Iyer, V.* AU - Mason, J.C.* AU - Stone, K.R.* AU - Tadepally, H.D.* AU - Kadin, J.A.* AU - Bult, C.J.* AU - Eppig, J.T.* AU - Oakley, D.J.* AU - Briois, S.* AU - Stupka, E.* AU - Maselli, V.* AU - Smedley, D.* AU - Liu, S.Y.* AU - Hansen, J. AU - Baldock, R.* AU - Hicks, G.G.* AU - Skarnes, W.C.* C1 - 2596 C2 - 28380 SP - D849-D855 TI - The IKMC web portal: A central point of entry to data and resources from the International Knockout Mouse Consortium. JO - Nucleic Acids Res. VL - 39 IS - SUPPL. 1 PB - Oxford Univ Press PY - 2011 SN - 0305-1048 ER - TY - JOUR AB - The MIPS Fusarium graminearum Genome Database (FGDB) was established as a comprehensive genome database on one of the most devastating fungal plant pathogens of wheat, barley and maize. The current version of FGDB v3.1 provides information on the full manually revised gene set based on the Broad Institute assembly FG3 genome sequence. The results of gene prediction tools were integrated with the help of comparative data on related species to result in a set of 13.718 annotated protein coding genes. This rigorous approach involved adding or modifying gene models and represents a coding sequence gold standard for the genus Fusarium. The gene loci improvements results in 2461 genes which either are new or have different structures compared to the Broad Institute assembly 3 gene set. Moreover the database serves as a convenient entry point to explore expression data results and to obtain information on the Affymetrix GeneChip probe sets. The resource is accessible on http://mips.gsf.de/genre/proj/FGDB/. AU - Wong, P. AU - Walter, M. AU - Lee, W. AU - Mannhaupt, G. AU - Münsterkötter, M. AU - Mewes, H.-W. AU - Adam, G.* AU - Güldener, U. C1 - 4731 C2 - 28215 SP - D637-D639 TI - FGDB: Revisiting the genome annotation of the plant pathogen Fusarium graminearum. JO - Nucleic Acids Res. VL - 39 IS - SUPPL. 1 PB - Oxford Univ. Press PY - 2011 SN - 0305-1048 ER - TY - JOUR AB - R spider is a web-based tool for the analysis of a gene list using the systematic knowledge of core pathways and reactions in human biology accumulated in the Reactome and KEGG databases. R spider implements a network-based statistical framework, which provides a global understanding of gene relations in the supplied gene list, and fully exploits the Reactome and KEGG knowledge bases. R spider provides a user-friendly dialog-driven web interface for several model organisms and supports most available gene identifiers. R spider is freely available at http://mips.helmholtz-muenchen.de/proj/rspider. AU - Antonov, A.V. AU - Schmidt, E.E.* AU - Dietmann, S. AU - Krestyaninova, M.* AU - Hermjakob, H.* C1 - 6137 C2 - 28142 SP - W78-W83 TI - R spider: A network-based analysis of gene lists by combining signaling and metabolic pathways from Reactome and KEGG databases. JO - Nucleic Acids Res. VL - 38 IS - SUPPL. 2 PB - Oxford Univ. Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - CCancer is an automatically collected database of gene lists, which were reported mostly by experimental studies in various biological and clinical contexts. At the moment, the database covers 3369 gene lists extracted from 2644 papers published in approximately 80 peer-reviewed journals. As input, CCancer accepts a gene list. An enrichment analyses is implemented to generate, as output, a highly informative survey over recently published studies that report gene lists, which significantly intersect with the query gene list. A report on gene pairs from the input list which were frequently reported together by other biological studies is also provided. CCancer is freely available at http://mips.helmholtz-muenchen.de/proj/ccancer. AU - Dietmann, S.* AU - Lee, W. AU - Wong, P. AU - Rodchenkov, I.* AU - Antonov, A.V. C1 - 1799 C2 - 28139 SP - W118-W123 TI - CCancer: A bird's eye view on gene lists reported in cancer-related studies. JO - Nucleic Acids Res. VL - 38 IS - SUPPL. 2 PB - Oxford Univ. Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - In contrast to lower eukaryotes, most vertebrate cells are characterized by a moderate efficiency of homologous recombination (HR) and limited feasibility of targeted genetic modifications. As a notable exception, the chicken DT40 B cell line is distinguished by efficient homology-mediated repair of DNA lesions during Ig gene conversion, and also shows exceptionally high gene-targeting efficiencies. The molecular basis of these phenomena is elusive. Here we show that the activity levels of Ubc13, the E2 enzyme responsible for non-canonical K63-linked polyubiquitination, are critical for high efficiency of Ig gene conversion and gene targeting in DT40. Ubc13(+/-) cells show substantially lower homology-mediated repair, yet do not display changes in somatic hypermutation, overall DNA repair or cell proliferation. Our results suggest that modulation of the activity of K63-linked polyubiquitination may be used to customize HR efficiencies in vertebrate cells. AU - Ertongur, I. AU - Tomi, N.S. AU - Kutzera, A. AU - Fischer-Burkart, S. AU - Jungnickel, B. C1 - 4992 C2 - 27440 SP - 4701-4707 TI - Ubc13 dosage is critical for immunoglobulin gene conversion and gene targeting in vertebrate cells. JO - Nucleic Acids Res. VL - 38 IS - 14 PB - Oxford Univ. Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - The Munich Information Center for Protein Sequences (MIPS at the Helmholtz Center for Environmental Health, Neuherberg, Germany) has many years of experience in providing annotated collections of biological data. Selected data sets of high relevance, such as model genomes, are subjected to careful manual curation, while the bulk of high-throughput data is annotated by automatic means. High-quality reference resources developed in the past and still actively maintained include Saccharomyces cerevisiae, Neurospora crassa and Arabidopsis thaliana genome databases as well as several protein interaction data sets (MPACT, MPPI and CORUM). More recent projects are PhenomiR, the database on microRNA-related phenotypes, and MIPS PlantsDB for integrative and comparative plant genome research. The interlinked resources SIMAP and PEDANT provide homology relationships as well as up-to-date and consistent annotation for 38,000,000 protein sequences. PPLIPS and CCancer are versatile tools for proteomics and functional genomics interfacing to a database of compilations from gene lists extracted from literature. A novel literature-mining tool, EXCERBT, gives access to structured information on classified relations between genes, proteins, phenotypes and diseases extracted from Medline abstracts by semantic analysis. All databases described here, as well as the detailed descriptions of our projects can be accessed through the MIPS WWW server (http://mips.helmholtz-muenchen.de). AU - Mewes, H.-W. AU - Ruepp, A. AU - Theis, F.J. AU - Rattei, T.* AU - Walter, M.C. AU - Frishman, D. AU - Suhre, K. AU - Spannagl, M. AU - Mayer, K.F.X. AU - Stuempflen, V. AU - Antonov, A. C1 - 4181 C2 - 28154 SP - D220-D224 TI - MIPS: Curated databases and comprehensive secondary data resources in 2010. JO - Nucleic Acids Res. VL - 39 IS - SUPPL. 1 PB - Oxfort Univ. Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - The broad aim of biomedical science in the postgenomic era is to link genomic and phenotype information to allow deeper understanding of the processes leading from genomic changes to altered phenotype and disease. The EuroPhenome project (http://www.EuroPhenome.org) is a comprehensive resource for raw and annotated high-throughput phenotyping data arising from projects such as EUMODIC. EUMODIC is gathering data from the EMPReSSslim pipeline (http://www.empress.har.mrc.ac.uk/) which is performed on inbred mouse strains and knock-out lines arising from the EUCOMM project. The EuroPhenome interface allows the user to access the data via the phenotype or genotype. It also allows the user to access the data in a variety of ways, including graphical display, statistical analysis and access to the raw data via web services. The raw phenotyping data captured in EuroPhenome is annotated by an annotation pipeline which automatically identifies statistically different mutants from the appropriate baseline and assigns ontology terms for that specific test. Mutant phenotypes can be quickly identified using two EuroPhenome tools: PhenoMap, a graphical representation of statistically relevant phenotypes, and mining for a mutant using ontology terms. To assist with data definition and cross-database comparisons, phenotype data is annotated using combinations of terms from biological ontologies. AU - Morgan, H.* AU - Beck, T.* AU - Blake, A.* AU - Gates, H.* AU - Adams, N.* AU - Debouzy, G.* AU - Leblanc, S.* AU - Lengger, C. AU - Maier, H. AU - Melvin, D.* AU - Meziane, H.* AU - Richardson, D.* AU - Wells, S.* AU - White, J.* AU - Wood, J.* AU - EUMODIC Consortium (Hrabě de Angelis, M. AU - Gailus-Durner, V. AU - Fuchs, H. AU - Adler, T. AU - Aguilar-Pimentel, J.A. AU - Becker, L. AU - Beckeredjian, R AU - Busch, D.H. AU - Calzada-Wack, J. AU - Da Silva-Buttkus, P. AU - Esposito, I. AU - Favor, J. AU - Fleischmann, W. AU - Garrett, L. AU - Glasl, L. AU - Götz, A. AU - Graw, J. AU - Hans, W, AU - Hölter, S.M. AU - Ivandic, B. AU - Katus, H.A. AU - Klingenspor, M. AU - Klopstock, T. AU - Lengger, C. AU - Ludwig, T. AU - Maier, H. AU - Micklich, K. AU - Minxuan, S. AU - Naton, B. AU - Neff, F. AU - Ollert, M. AU - Puk, O. AU - Quintanilla-Fend, L. AU - Rácz, I. AU - Rathkolb, B. AU - Rozman, J. AU - Schäble, K.-H. AU - Schiller, E. AU - Schrewe, A. AU - Steinkamp, R. AU - Stoeger, C. AU - Stöger, T. AU - Schulz, S. AU - Tost, M. AU - Treise, I. AU - Vogt Weisenhorn, D.M. AU - Willershäuser, M. AU - Wolf, E. AU - Zimprich, A. AU - Wurst, W. AU - Zeh, R.M. AU - Zimmer, A.) AU - Hrabě de Angelis, M. AU - Brown, S.* AU - Hancock, J.M.* AU - Mallon, A.M.* C1 - 973 C2 - 27056 SP - D577-D585 TI - EuroPhenome: A repository for high-throughput mouse phenotyping data. JO - Nucleic Acids Res. VL - 38 IS - Database Issue PB - Oxford Univ. Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - The prediction of protein function as well as the reconstruction of evolutionary genesis employing sequence comparison at large is still the most powerful tool in sequence analysis. Due to the exponential growth of the number of known protein sequences and the subsequent quadratic growth of the similarity matrix, the computation of the Similarity Matrix of Proteins (SIMAP) becomes a computational intensive task. The SIMAP database provides a comprehensive and up-to-date pre-calculation of the protein sequence similarity matrix, sequence-based features and sequence clusters. As of September 2009, SIMAP covers 48 million proteins and more than 23 million non-redundant sequences. Novel features of SIMAP include the expansion of the sequence space by including databases such as ENSEMBL as well as the integration of metagenomes based on their consistent processing and annotation. Furthermore, protein function predictions by Blast2GO are pre-calculated for all sequences in SIMAP and the data access and query functions have been improved. SIMAP assists biologists to query the up-to-date sequence space systematically and facilitates large-scale downstream projects in computational biology. Access to SIMAP is freely provided through the web portal for individuals (http://mips.gsf.de/simap/) and for programmatic access through DAS (http://webclu.bio.wzw.tum.de/das/) and Web-Service (http://mips.gsf.de/webservices/services/SimapService2.0?wsdl). AU - Rattei, T.* AU - Tischler, P.* AU - Götz, S.* AU - Jehl, M.A.* AU - Hoser, J.D.S.* AU - Arnold, R.* AU - Conesa, A.* AU - Mewes, H.-W. C1 - 1422 C2 - 27072 SP - D223-D226 TI - SIMAP - a comprehensive database of pre-calculated protein sequence similarities, domains, annotations and clusters. JO - Nucleic Acids Res. VL - 38 IS - Database Issue PB - Oxford Univ. Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - CORUM is a database that provides a manually curated repository of experimentally characterized protein complexes from mammalian organisms, mainly human (64%), mouse (16%) and rat (12%). Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. The new CORUM 2.0 release encompasses 2837 protein complexes offering the largest and most comprehensive publicly available dataset of mammalian protein complexes. The CORUM dataset is built from 3198 different genes, representing approximately 16% of the protein coding genes in humans. Each protein complex is described by a protein complex name, subunit composition, function as well as the literature reference that characterizes the respective protein complex. Recent developments include mapping of functional annotation to Gene Ontology terms as well as cross-references to Entrez Gene identifiers. In addition, a 'Phylogenetic Conservation' analysis tool was implemented that analyses the potential occurrence of orthologous protein complex subunits in mammals and other selected groups of organisms. This allows one to predict the occurrence of protein complexes in different phylogenetic groups. CORUM is freely accessible at (http://mips.helmholtz-muenchen.de/genre/proj/corum/index.html). AU - Ruepp, A. AU - Wägele, B. AU - Lechner, M. AU - Brauner, B. AU - Dunger-Kaltenbach, I. AU - Fobo, G. AU - Frishman, G. AU - Montrone, C. AU - Mewes, H.-W. C1 - 1800 C2 - 28101 SP - D497-D501 TI - CORUM: The comprehensive resource of mammalian protein complexes - 2009. JO - Nucleic Acids Res. VL - 38 IS - SUPPL.1 PB - Oxford Univ. Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - Recombinase-mediated cassette exchange (RMCE) exploits the possibility to unidirectionally exchange any genetic material flanked by heterotypic recombinase recognition sites (RRS) with target sites in the genome. Due to a limited number of available pre-fabricated target sites, RMCE in mouse embryonic stem (ES) cells has not been tapped to its full potential tRecombinase-mediated cassette exchange (RMCE) exploits the possibility to unidirectionally exchange any genetic material flanked by heterotypic recombinase recognition sites (RRS) with target sites in the genome. Due to a limited number of available pre-fabricated target sites, RMCE in mouse embryonic stem (ES) cells has not been tapped to its full potential to date. Here, we introduce a universal system, which allows the targeted insertion of any given transcriptional unit into 85 742 previously annotated retroviral conditional gene trap insertions, representing 7013 independent genes in mouse ES cells, by RMCE. This system can be used to express any given cDNA under the control of endogenous trapped promoters in vivo, as well as for the generation of transposon 'launch pads' for chromosomal region-specific 'Sleeping Beauty' insertional mutagenesis. Moreover, transcription of the gene-of-interest is only activated upon Cre-recombinase activity, a feature that adds conditionality to this expression system, which is demonstrated in vivo. The use of the RMCE system presented in this work requires one single-cloning step followed by one overnight gateway clonase reaction and subsequent cassette exchange in ES cells with efficiencies of 40% in average.o date. Here, we introduce a universal system, which allows the targeted insertion of any given transcriptional unit into 85 742 previously annotated retroviral conditional gene trap insertions, representing 7013 independent genes in mouse ES cells, by RMCE. This system can be used to express any given cDNA under the control of endogenous trapped promoters in vivo, as well as for the generation of transposon 'launch pads' for chromosomal region-specific 'Sleeping Beauty' insertional mutagenesis. Moreover, transcription of the gene-of-interest is only activated upon Cre-recombinase activity, a feature that adds conditionality to this expression system, which is demonstrated in vivo. The use of the RMCE system presented in this work requires one single-cloning step followed by one overnight gateway clonase reaction and subsequent cassette exchange in ES cells with efficiencies of 40% in average. AU - Schebelle, L. AU - Wolf, C. AU - Stribl, C.B. AU - Javaheri, T. AU - Schnütgen, F.* AU - Ettinger, A. AU - Ivics, Z.* AU - Hansen, J. AU - Ruiz, P.* AU - von Melchner, H.* AU - Wurst, W. AU - Floß, T. C1 - 1622 C2 - 27215 SP - e106-e106 TI - Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps. JO - Nucleic Acids Res. VL - 38 IS - 9 PB - Oxford Univ. Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - The Negatome is a collection of protein and domain pairs that are unlikely to be engaged in direct physical interactions. The database currently contains experimentally supported non-interacting protein pairs derived from two distinct sources: by manual curation of literature and by analyzing protein complexes with known 3D structure. More stringent lists of non-interacting pairs were derived from these two datasets by excluding interactions detected by high-throughput approaches. Additionally, non-interacting protein domains have been derived from the stringent manual and structural data, respectively. The Negatome is much less biased toward functionally dissimilar proteins than the negative data derived by randomly selecting proteins from different cellular locations. It can be used to evaluate protein and domain interactions from new experiments and improve the training of interaction prediction algorithms. The Negatome database is available at http://mips.helmholtz-muenchen.de/proj/ppi/negatome. AU - Smialowski, P. AU - Pagel, P. AU - Wong, P. AU - Brauner, B. AU - Dunger, I. AU - Fobo, G. AU - Frishman, G. AU - Montrone, C. AU - Rattei, T.* AU - Frishman, D. AU - Ruepp, A. C1 - 2417 C2 - 27048 SP - D540-D544 TI - The Negatome database: A reference set of non-interacting protein pairs. JO - Nucleic Acids Res. VL - 38 IS - Database Issue PB - Oxford Univ. Presss PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - The Mre11/Rad50/Xrs2 (MRX) complex in Saccharomyces cerevisiae has well-characterized functions in DNA double-strand break processing, checkpoint activation, telomere length maintenance and meiosis. In this study, we demonstrate an involvement of the complex in the base excision repair (BER) pathway. We studied the repair of methyl-methanesulfonate-induced heat-labile sites in chromosomal DNA in vivo and the in vitro BER capacity for the repair of uracil- and 8-oxoG-containing oligonucleotides in MRX-deficient cells. Both approaches show a clear BER deficiency for the xrs2 mutant as compared to wildtype cells. The in vitro analyses revealed that both subpathways, long-patch and short-patch BER, are affected and that all components of the MRX complex are similarly important for the new function in BER. The investigation of the epistatic relationship of XRS2 to other BER genes suggests a role of the MRX complex downstream of the AP-lyases Ntg1 and Ntg2. Analysis of individual steps in BER showed that base recognition and strand incision are not affected by the MRX complex. Reduced gap-filling activity and the missing effect of aphidicoline treatment, an inhibitor for polymerases, on the BER efficiency indicate an involvement of the MRX complex in providing efficient polymerase activity. AU - Steininger, S. AU - Ahne, F. AU - Winkler, K. AU - Kleinschmidt, A. AU - Eckardt-Schupp, F. AU - Mörtl, S. C1 - 4543 C2 - 27950 SP - 1853-1865 TI - A novel function for the Mre11-Rad50-Xrs2 complex in base excision repair. JO - Nucleic Acids Res. VL - 38 IS - 6 PB - Oxford Univ Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - The laboratory mouse is the premier animal model for studying human disease and thousands of mutants have been identified or produced, most recently through gene-specific mutagenesis approaches. High throughput strategies by the International Knockout Mouse Consortium (IKMC) are producing mutants for all protein coding genes. Generating a knock-out line involves huge monetary and time costs so capture of both the data describing each mutant alongside archiving of the line for distribution to future researchers is critical. The European Mouse Mutant Archive (EMMA) is a leading international network infrastructure for archiving and worldwide provision of mouse mutant strains. It operates in collaboration with the other members of the Federation of International Mouse Resources (FIMRe), EMMA being the European component. Additionally EMMA is one of four repositories involved in the IKMC, and therefore the current figure of 1700 archived lines will rise markedly. The EMMA database gathers and curates extensive data on each line and presents it through a user-friendly website. A BioMart interface allows advanced searching including integrated querying with other resources e.g. Ensembl. Other resources are able to display EMMA data by accessing our Distributed Annotation System server. EMMA database access is publicly available at http://www.emmanet.org. AU - Wilkinson, P.* AU - Sengerova, J.* AU - Matteoni, R.* AU - Chen, C-K.* AU - Soulat, G.* AU - Ureta-Vidal, A.* AU - Fessele, S. AU - Hagn, M. AU - Massimi, M.* AU - Pickford, K.* AU - Butler, R.H.* AU - Marschall, S. AU - Mallon, A.-M.* AU - Pickard, A.* AU - Raspa, M.* AU - Scavizzi, F.* AU - Fray, M.* AU - Larrigaldie, V.* AU - Leyritz, J.* AU - Birney, E.* AU - Tocchini-Valentini, G.P.* AU - Brown, S.* AU - Herault, Y.* AU - Montoliu, L.* AU - Hrabě de Angelis, M. AU - Smedley, D.* C1 - 33 C2 - 27089 SP - D570-D576 TI - EMMA - mouse mutant resources for the international scientific community. JO - Nucleic Acids Res. VL - 38 IS - Database Issue PB - Oxford Univ. Press PY - 2010 SN - 0305-1048 ER - TY - JOUR AB - GeneSet2miRNA is the first web-based tool which is able to identify whether or not a gene list has a signature of miRNA-regulatory activity. As input, GeneSet2miRNA accepts a list of genes. As output, a list of miRNA-regulatory models is provided. A miRNA-regulatory model is a group of miRNAs (single, pair, triplet or quadruplet) that is predicted to regulate a significant subset of genes from the submitted list. GeneSet2miRNA provides a user friendly dialog-driven web page submission available for several model organisms. AU - Antonov, A.V. AU - Dietmann, S. AU - Wong, P. AU - Lutter, D. AU - Mewes, H.-W. C1 - 495 C2 - 26285 SP - W323-W328 TI - GeneSet2miRNA: Finding the signature of cooperative miRNA activities in the gene lists. JO - Nucleic Acids Res. VL - 37 IS - SUPPL. 2 PB - Oxford Univ Press PY - 2009 SN - 0305-1048 ER - TY - JOUR AB - Next-generation sequencing allows now the sequencing of small RNA molecules and the estimation of their expression levels. Consequently, there will be a high demand of bioinformatics tools to cope with the several gigabytes of sequence data generated in each single deep-sequencing experiment. Given this scene, we developed miRanalyzer, a web server tool for the analysis of deep-sequencing experiments for small RNAs. The web server tool requires a simple input file containing a list of unique reads and its copy numbers (expression levels). Using these data, miRanalyzer (i) detects all known microRNA sequences annotated in miRBase, (ii) finds all perfect matches against other libraries of transcribed sequences and (iii) predicts new microRNAs. The prediction of new microRNAs is an especially important point as there are many species with very few known microRNAs. Therefore, we implemented a highly accurate machine learning algorithm for the prediction of new microRNAs that reaches AUC values of 97.9% and recall values of up to 75% on unseen data. The web tool summarizes all the described steps in a single output page, which provides a comprehensive overview of the analysis, adding links to more detailed output pages for each analysis module. miRanalyzer is available at http://web.bioinformatics.cicbiogune.es/microRNA/. AU - Hackenberg, M.* AU - Sturm, M. AU - Langenberger, D.* AU - Falcón-Pérez, J.M.* AU - Aransay, A.M.* C1 - 1746 C2 - 26411 SP - W68-W76 TI - miRanalyzer: A microRNA detection and analysis tool for next-generation sequencing experiments. JO - Nucleic Acids Res. VL - 37 IS - Web Server issue PB - Oxford Univ Press PY - 2009 SN - 0305-1048 ER - TY - JOUR AB - The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during S-and M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites. These phospho-specific RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recognized mitotically phosphorylated RPA2 with high specificity. In addition, the RPA2-(P)-S23 antibody recognized the S-phase-specific phosphorylation of RPA2, suggesting that during S-phase only S23 is phosphorylated, whereas during M-phase both CDK sites, S23 and S29, are phosphorylated. Immunofluorescence microscopy revealed that the mitotic phosphorylation of RPA2 starts at the onset of mitosis, and dephosphorylation occurs during late cytokinesis. In mitotic cells treated with ionizing radiation (IR), we observed a rapid hyperphosphorylation of RPA2 in addition to its mitotic phosphorylation at S23 and S29, associated with a significant change in the subcellular localization of RPA. Our data also indicate that the RPA2 hyperphosphorylation in response to IR is facilitated by the activity of both ATM and DNAPK, and is associated with activation of the Chk2 pathway. AU - Stephan, H.* AU - Concannon, C.* AU - Kremmer, E. AU - Carty, M.P.* AU - Nasheuer, H.P.* C1 - 1973 C2 - 26522 SP - 6028-6041 TI - Ionizing radiation-dependent and independent phosphorylation of the 32-kDa subunit of replication protein A during mitosis. JO - Nucleic Acids Res. VL - 37 IS - 18 PB - Oxford Univ Press PY - 2009 SN - 0305-1048 ER - TY - JOUR AB - ProfCom is a web-based tool for the functional interpretation of a gene list that was identified to be related by experiments. A trait which makes ProfCom a unique tool is an ability to profile enrichments of not only available Gene Ontology (GO) terms but also of 'complex functions'. A 'Complex function' is constructed as Boolean combination of available GO terms. The complex functions inferred by ProfCom are more specific in comparison to single terms and describe more accurately the functional role of genes. ProfCom provides a user friendly dialog-driven web page submission available for several model organisms and supports most available gene identifiers. In addition, the web service interface allows the submission of any kind of annotation data. ProfCom is freely available at http://webclu.bio.wzw.tum.de/profcom/. AU - Antonov, A.V. AU - Schmidt, T.* AU - Wang, Y. AU - Mewes, H.-W. C1 - 5724 C2 - 25524 SP - W347-W351 TI - ProfCom: A web tool for profiling the complex functionality of gene groups identified from high-throughput data. JO - Nucleic Acids Res. VL - 36 IS - S PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - Genome-wide mutations and selection within a population are the basis of natural evolution. A similar process occurs during antibody affinity maturation when immunoglobulin genes are hypermutated and only those B cells which express antibodies of improved antigen-binding specificity are expanded. Protein evolution might be simulated in cell culture, if transgene-specific hypermutation can be combined with the selection of cells carrying beneficial mutations. Here, we describe the optimization of a GFP transgene in the B cell line DT40 by hypermutation and iterative fluorescence activated cell sorting. Artificial evolution in DT40 offers unique advantages and may be easily adapted to other transgenes, if the selection for desirable mutations is feasible. AU - Arakawa, H. AU - Kudo, H. AU - Batrak, V. AU - Caldwell, R.B. AU - Rieger, M. AU - Ellwart, J.W. AU - Buerstedde, J.-M. C1 - 2561 C2 - 24996 TI - Protein evolution by hypermutation and selection in the B cell line DT40. JO - Nucleic Acids Res. VL - 36 IS - 1 PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - MicroRNAs (miRNAs) have been implicated in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr virus (EBV) encodes 23 miRNAs of unknown function. Here we show that the EBV-encoded miRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. MiR-BART2 guides cleavage within the 3'-untranslated region (3'UTR) of BALF5 by virtue of its complete complementarity to its target. Induction of the lytic viral replication cycle results in a reduction of the level of miR-BART2 with a strong concomitant decrease of cleavage of the BALF5 3'UTR. Expression of miR-BART2 down-regulates the activity of a luciferase reporter gene containing the BALF5 3'UTR. Forced expression of miR-BART2 during lytic replication resulted in a 40-50% reduction of the level of BALF5 protein and a 20% reduction of the amount of virus released from EBV-infected cells. Our results are compatible with the notion that EBV-miR-BART2 inhibits transition from latent to lytic viral replication. AU - Barth, S.* AU - Pfuhl, T.* AU - Mamiani, A.* AU - Ehses, C.* AU - Roemer, K.* AU - Kremmer, E. AU - Jäker, C.* AU - Höck, J.* AU - Meister, G.* AU - Grässer, F.A.* C1 - 2514 C2 - 25689 SP - 666-675 TI - Epstein-Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. JO - Nucleic Acids Res. VL - 36 IS - 2 PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - All RNA sequences that fold into hairpins possess the intrinsic potential to form intermolecular duplexes because of their high self-complementarity. The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations. We developed and applied a novel approach to unambiguously distinguish RNA hairpin and duplex conformations for the structural analysis of a Xist RNA A-repeat. Using a combination of a quantitative HNN-COSY experiment and an optimized double isotope-filtered NOESY experiment we could define the conformation of the 26-mer A-repeat RNA. In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat. The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter-and intra-molecular base-pairing patterns. AU - Duszczyk, M.M. AU - Zanier, K.* AU - Sattler, M. C1 - 2565 C2 - 25935 SP - 7068-7077 TI - A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat. JO - Nucleic Acids Res. VL - 36 IS - 22 PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - The flowering plant genus Oenothera is uniquely suited for studying molecular mechanisms of speciation. It assembles an intriguing combination of genetic features, including permanent translocation heterozygosity, biparental transmission of plastids, and a general interfertility of well-defined species. This allows an exchange of plastids and nuclei between species often resulting in plastome-genome incompatibility. For evaluation of its molecular determinants we present the complete nucleotide sequences of the five basic, genetically distinguishable plastid chromosomes of subsection Oenothera (=Euoenothera) of the genus, which are associated in distinct combinations with six basic genomes. Sizes of the chromosomes range from 163 365 bp (plastome IV) to 165 728 bp (plastome I), display between 96.3% and 98.6% sequence similarity and encode a total of 113 unique genes. Plastome diversification is caused by an abundance of nucleotide substitutions, small insertions, deletions and repetitions. The five plastomes deviate from the general ancestral design of plastid chromosomes of vascular plants by a subsection-specific 56 kb inversion within the large single-copy segment. This inversion disrupted operon structures and predates the divergence of the subsection presumably 1 My ago. Phylogenetic relationships suggest plastomes I-III in one clade, while plastome IV appears to be closest to the common ancestor. AU - Greiner, S.* AU - Wang, X. AU - Rauwolf, U.* AU - Silber, M.V.* AU - Mayer, K.F.X. AU - Meurer, J.* AU - Haberer, G. AU - Herrmann, R.G.* C1 - 1201 C2 - 25507 SP - 2366-2378 TI - The complete nucleotide sequences of the five genetically distinct plastid genomes of Oenothera, subsection Oenothera: I. Sequence evaluation and plastome evolution. JO - Nucleic Acids Res. VL - 36 IS - 7 PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster leads to male-specific lethality due to a failure of X chromosome dosage compensation. MLE is presumably involved in incorporating the non-coding roX RNA into the dosage compensation complex (DCC), which is an essential but poorly understood requirement for faithful targeting of the complex to the X chromosome. Sequence comparison predicts several RNA-binding domains in MLE but their properties have not been experimentally verified. We evaluated the RNA-binding characteristics of these conserved motifs and their contributions to RNA-stimulated ATPase activity, to helicase activity, as well as to the targeting of MLE to the nucleus and to the X chromosome territory. We find that RB2 is the dominant, conditional RNA-binding module, which is indispensable for ATPase and helicase activity whereas the N-terminal RB1 motif does not bind RNA, but is involved in targeting MLE to the X chromosome. The C-terminal domain containing a glycine-rich heptad repeat adds potential dimerization and RNA-binding surfaces which are not required for helicase activity. AU - Izzo, A.* AU - Regnard, C.* AU - Morales, V.* AU - Kremmer, E. AU - Becker, P.B.* C1 - 2171 C2 - 25694 SP - 950-962 TI - Structure-function analysis of the RNA helicase maleless. JO - Nucleic Acids Res. VL - 36 IS - 3 PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - The Munich Information Center for Protein Sequences (MIPS-GSF, Neuherberg, Germany) combines automatic processing of large amounts of sequences with manual annotation of selected model genomes. Due to the massive growth of the available data, the depth of annotation varies widely between independent databases. Also, the criteria for the transfer of information from known to orthologous sequences are diverse. To cope with the task of global in-depth genome annotation has become unfeasible. Therefore, our efforts are dedicated to three levels of annotation: (i) the curation of selected genomes, in particular from fungal and plant taxa (e.g. CYGD, MNCDB, MatDB), (ii) the comprehensive, consistent, automatic annotation employing exhaustive methods for the computation of sequence similarities and sequence-related attributes as well as the classification of individual sequences (SIMAP, PEDANT and FunCat) and (iii) the compilation of manually curated databases for protein interactions based on scrutinized information from the literature to serve as an accepted set of reliable annotated interaction data (MPACT, MPPI, CORUM). All databases and tools described as well as the detailed descriptions of our projects can be accessed through the MIPS web server (http://mips.gsf.de). AU - Mewes, H.-W. AU - Dietmann, S. AU - Frishman, D.* AU - Gregory, R. AU - Mannhaupt, G. AU - Mayer, K.F.X. AU - Münsterkötter, M. AU - Ruepp, A. AU - Spannagl, M. AU - Stuempflen, V. AU - Rattei, T.* C1 - 5721 C2 - 25516 SP - D196-D201 TI - MIPS: Analysis and annotation of genome information in 2007. JO - Nucleic Acids Res. VL - 36 IS - SI PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR. AU - Mierau, M. AU - Drexler, G.A. AU - Kutzera, A. AU - Braunschmidt, K. AU - Ellwart, J.W. AU - Eckardt-Schupp, F. AU - Fritz, E.* AU - Bachl, J.* AU - Jungnickel, B. C1 - 2980 C2 - 25744 SP - 5591-5601 TI - Non-conservative homologous recombination in human B lymphocytes is promoted by activation-induced cytidine deaminase and transcription. JO - Nucleic Acids Res. VL - 36 IS - 17 PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - DIMA-the domain interaction map has evolved from a simple web server for domain phylogenetic profiling into an integrative prediction resource combining both experimental data on domain-domain interactions and predictions from two different algorithms. With this update, DIMA obtains greatly improved coverage at the level of genomes and domains as well as with respect to available prediction approaches. The domain phylogenetic profiling method now uses SIMAP as its backend for exhaustive domain hit coverage: 7038 Pfam domains were profiled over 460 completely sequenced genomes.Domain pair exclusion predictions were produced from 83 969 distinct protein-protein interactions obtained from IntAct resulting in 21 513 domain pairs with significant domain pair exclusion algorithm scores. Additional predictions applying the same algorithm to predicted protein interactions from STRING yielded 2378 high-confidence pairs. Experimental data comes from iPfam (3074) and 3did (3034 pairs), two databases identifying domain contacts in solved protein structures. Taken together, these two resources yielded 3653 distinct interacting domain pairs. DIMA is available at http://mips.gsf.de/genre/proj/dima. AU - Pagel, P. AU - Oesterheld, M. AU - Tovstukhina, O.* AU - Strack, N.* AU - Stuempflen, V. AU - Frishman, D. C1 - 3195 C2 - 25392 SP - D651-655 TI - DIMA 2.0 - predicted and known domain interactions. JO - Nucleic Acids Res. VL - 36 IS - SI PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - Non-integrating gene vectors, which are stably and extrachromosomally maintained in transduced cells would be perfect tools to support long-term expression of therapeutic genes but preserve the genomic integrity of the cellular host. Small extrachromosomal plasmids share some of these ideal characteristics but are primarily based on virus blueprints. These plasmids are dependent on viral trans-acting factors but they can replicate their DNA molecules in synchrony with the chromosome of the cellular host and segregate to daughter cells in an autonomous fashion. On the basis of the concept of the latent origin of DNA replication of Epstein-Barr virus, oriP, we devised novel derivatives, which exclusively rely on an artificial replication factor for both nuclear retention and replication of plasmid DNA. In addition, an allosteric switch regulates the fate of the plasmid molecules, which are rapidly lost upon addition of doxycycline. Conditional maintenance of these novel plasmid vectors allows the reversible transfer of genetic information into target cells for the first time. AU - Pich, D. AU - Hummer, S. AU - Spindler, M.-P. AU - Schepers, A. AU - Hammerschmidt, W. C1 - 4894 C2 - 25416 TI - Conditional gene vectors regulated in cis. JO - Nucleic Acids Res. VL - 36 IS - 13 PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - Protein sequences are the most important source of evolutionary and functional information for new proteins. In order to facilitate the computationally intensive tasks of sequence analysis, the Similarity Matrix of Proteins (SIMAP) database aims to provide a comprehensive and up-to-date dataset of the pre-calculated sequence similarity matrix and sequence-based features like InterPro domains for all proteins contained in the major public sequence databases. As of September 2007, SIMAP covers approximately 17 million proteins and more than 6 million non-redundant sequences and provides a complete annotation based on InterPro 16. Novel features of SIMAP include a new, portlet-based web portal providing multiple, structured views on retrieved proteins and integration of protein clusters and a unique search method for similar domain architectures. Access to SIMAP is freely provided for academic use through the web portal for individuals at http://mips.gsf.de/simap/and through Web Services for programmatic access at http://mips.gsf.de/webservices/services/SimapService2.0?wsdl. AU - Rattei, T.* AU - Tischler, P.* AU - Arnold, R.* AU - Hamberger, F.* AU - Krebs, J.* AU - Krumsiek, J.* AU - Wachinger, B.* AU - Stuempflen, V. AU - Mewes, H.-W. C1 - 5722 C2 - 25520 SP - D289-D292 TI - SIMAP­-structuring the network of protein similarities. JO - Nucleic Acids Res. VL - 36 IS - SI PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. The CORUM (http://mips.gsf.de/genre/proj/corum/index.html) database is a collection of experimentally verified mammalian protein complexes. Information is manually derived by critical reading of the scientific literature from expert annotators. Information about protein complexes includes protein complex names, subunits, literature references as well as the function of the complexes. For functional annotation, we use the FunCat catalogue that enables to organize the protein complex space into biologically meaningful subsets. The database contains more than 1750 protein complexes that are built from 2400 different genes, thus representing 12% of the protein-coding genes in human. A web-based system is available to query, view and download the data. CORUM provides a comprehensive dataset of protein complexes for discoveries in systems biology, analyses of protein networks and protein complex-associated diseases. Comparable to the MIPS reference dataset of protein complexes from yeast, CORUM intends to serve as a reference for mammalian protein complexes. AU - Ruepp, A. AU - Brauner, B. AU - Dunger-Kaltenbach, I. AU - Frishman, G. AU - Montrone, C. AU - Stransky, M. AU - Wägele, B. AU - Schmidt, T.* AU - Doudieu, O.N. AU - Stuempflen, V. AU - Mewes, H.-W. C1 - 5719 C2 - 25511 SP - D646-D650 TI - CORUM: The comprehensive resource of mammalian protein complexes. JO - Nucleic Acids Res. VL - 36 IS - SI PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes. AU - Schnütgen, F.* AU - Hansen, J. AU - De-Zolt, S.* AU - Horn, C.* AU - Lutz, M.* AU - Floß, T. AU - Wurst, W. AU - Ruiz Noppinger, P.* AU - von Melchner, H.* C1 - 703 C2 - 25895 TI - Enhanced gene trapping in mouse embryonic stem cells. JO - Nucleic Acids Res. VL - 36 IS - 20 PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - Recent technical advances in mass spectrometry (MS) have brought the field of metabolomics to a point where large numbers of metabolites from numerous prokaryotic and eukaryotic organisms can now be easily and precisely detected. The challenge today lies in the correct annotation of these metabolites on the basis of their accurate measured masses. Assignment of bulk chemical formula is generally possible, but without consideration of the biological and genomic context, concrete metabolite annotations remain difficult and uncertain. MassTRIX responds to this challenge by providing a hypothesis-driven approach to high precision MS data annotation. It presents the identified chemical compounds in their genomic context as differentially colored objects on KEGG pathway maps. Information on gene transcription or differences in the gene complement (e.g. samples from different bacterial strains) can be easily added. The user can thus interpret the metabolic state of the organism in the context of its potential and, in the case of submitted transcriptomics data, real enzymatic capacities. The MassTRIX web server is freely accessible at http://masstrix.org. AU - Suhre, K. AU - Schmitt-Kopplin, P. C1 - 5720 C2 - 25513 SP - W481-W484 TI - MassTRIX: Mass translator into pathways. JO - Nucleic Acids Res. VL - 36 IS - S PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - The Rice Annotation Project Database (RAP-DB) was created to provide the genome sequence assembly of the International Rice Genome Sequencing Project (IRGSP), manually curated annotation of the sequence, and other genomics information that could be useful for comprehensive understanding of the rice biology. Since the last publication of the RAP-DB, the IRGSP genome has been revised and reassembled. In addition, a large number of rice-expressed sequence tags have been released, and functional genomics resources have been produced worldwide. Thus, we have thoroughly updated our genome annotation by manual curation of all the functional descriptions of rice genes. The latest version of the RAP-DB contains a variety of annotation data as follows: clone positions, structures and functions of 31 439 genes validated by cDNAs, RNA genes detected by massively parallel signature sequencing (MPSS) technology and sequence similarity, flanking sequences of mutant lines, transposable elements, etc. Other annotation data such as Gnomon can be displayed along with those of RAP for comparison. We have also developed a new keyword search system to allow the user to access useful information. The RAP-DB is available at: http://rapdb.dna.affrc.go.jp/ and http://rapdb.lab.nig.ac.jp/. AU - Tanaka, T.* AU - Antonio, B.A.* AU - Kikuchi, S.* AU - Matsumoto, T.* AU - Nagamura, Y.* AU - Numa, H.* AU - Sakai, H.* AU - Wu, J.* AU - Itoh, T.* AU - Sasaki, T.* AU - Aono, R.* AU - Fujii, Y.* AU - Habara, T.* AU - Harada, E.* AU - Kanno, M.* AU - Kawahara, Y.* AU - Kawashima, H.* AU - Kubooka, H.* AU - Matsuya, A.* AU - Nakaoka, H.* AU - Saichi, N.* AU - Sanbonmatsu, R.* AU - Sato, Y.* AU - Shinso, Y.* AU - Suzuki, M.* AU - Takeda, J.* AU - Tanino, M.* AU - Todokoro, F.* AU - Yamaguchi, K.* AU - Yamamoto, N.* AU - Yamasaki, C.* AU - Imanishi, T.* AU - Okido, T.* AU - Tada, M.* AU - Ikeo, K.* AU - Tateno, Y.* AU - Gojobori, T.* AU - Lin, YC.* AU - Wei, F.J.* AU - Hsing, Y.I.* AU - Zhao, Q.* AU - Han, B.* AU - Kramer, M.R.* AU - McCombie, R.W.* AU - Lonsdale, D.* AU - O'Donovan, C.C.* AU - Whitfield, E.J.* AU - Apweiler, R.* AU - Koyanagi, K.O.* AU - Khurana, J.P.* AU - Raghuvanshi, S.* AU - Singh, N.K.* AU - Tyagi, A.K.* AU - Haberer, G. AU - Fujisawa, M.* AU - Hosokawa, S.* AU - Ito, Y.* AU - Ikawa, H.* AU - Shibata, M.* AU - Yamamoto, M.* AU - Bruskiewich, R.M.* AU - Hoen, D.R.* AU - Bureau, T.E.* AU - Namiki, N.* AU - Ohyanagi, H.* AU - Sakai, Y.* AU - Nobushima, S.* AU - Sakata, K.* AU - Barrero, R.A.* AU - Souvorov, A.* AU - Smith-White, B.* AU - Tatusova, T.* AU - An, S.* AU - An, G.* AU - Oota, S.* AU - Fuks, G.* AU - Messing, J.* AU - Christie, K.R.* AU - Lieberherr, D.* AU - Kim, H.* AU - Zuccolo, A.* AU - Wing, R.A.* AU - Nobuta, K.* AU - Green, P.J.* AU - Lu, C.* AU - Meyers, B.C.* AU - Chaparro, C.* AU - Piegu, B.* AU - Panaud, O.* AU - Echeverria, M.* AU - Rice Annotation Project (*) C1 - 5718 C2 - 25508 SP - D1028-D1033 TI - The Rice Annotation Project Database (RAP-DB): 2008 update. JO - Nucleic Acids Res. VL - 36 IS - SI PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - The generation of expressed sequence tag (EST) libraries offers an affordable approach to investigate organisms, if no genome sequence is available. OREST (http://mips.gsf.de/genre/proj/orest/index.html) is a server-based EST analysis pipeline, which allows the rapid analysis of large amounts of ESTs or cDNAs from mammalia and fungi. In order to assign the ESTs to genes or proteins OREST maps DNA sequences to reference datasets of gene products and in a second step to complete genome sequences. Mapping against genome sequences recovers additional 13% of EST data, which otherwise would escape further analysis. To enable functional analysis of the datasets, ESTs are functionally annotated using the hierarchical FunCat annotation scheme as well as GO annotation terms. OREST also allows to predict the association of gene products and diseases by Morbid Map (OMIM) classification. A statistical analysis of the results of the dataset is possible with the included PROMPT software, which provides information about enrichment and depletion of functional and disease annotation terms. OREST was successfully applied for the identification and functional characterization of more than 3000 EST sequences of the common marmoset monkey (Callithrix jacchus) as part of an international collaboration. AU - Wägele, B. AU - Schmidt, T.* AU - Mewes, H.-W. AU - Ruepp, A. C1 - 5723 C2 - 25521 SP - W140-W144 TI - OREST: The online resource for EST analysis. JO - Nucleic Acids Res. VL - 36 IS - S PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - The PEDANT genome database provides exhaustive annotation of nearly 3000 publicly available eukaryotic, eubacterial, archaeal and viral genomes with more than 4.5 million proteins by a broad set of bioinformatics algorithms. In particular, all completely sequenced genomes from the NCBI's Reference Sequence collection (RefSeq) are covered. The PEDANT processing pipeline has been sped up by an order of magnitude through the utilization of pre-calculated similarity information stored in the similarity matrix of proteins (SIMAP) database, making it possible to process newly sequenced genomes immediately as they become available. PEDANT is freely accessible to academic users at http://pedant.gsf.de. For programmatic access Web Services are available at http://pedant.gsf.de/webservices.jsp. AU - Walter, M.C. AU - Rattei, T.* AU - Arnold, R.* AU - Güldener, U. AU - Münsterkötter, M. AU - Nenova, K. AU - Kastenmüller, G. AU - Tischler, P.* AU - Wölling, A.* AU - Volz, A.* AU - Pongratz, N.* AU - Jost, R.* AU - Mewes, H.-W. AU - Frishman, D. C1 - 658 C2 - 25945 SP - D408-D411 TI - Pedant covers all complete RefSeq genomes. JO - Nucleic Acids Res. VL - 37 IS - SI PB - Oxford Univ. Press PY - 2008 SN - 0305-1048 ER - TY - JOUR AB - Heterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatal diagnosis would benefit from the ability to characterize individual single cells. As the amount of DNA from single cells is so small, several PCR protocols have been developed in an attempt to achieve unbiased amplification. Many of these approaches are suitable for subsequent cytogenetic analyses using conventional methodologies such as comparative genomic hybridization (CGH) to metaphase spreads. However, attempts to harness array-CGH for single-cell analysis to provide improved resolution have been disappointing. Here we describe a strategy that combines single-cell amplification using GenomePlex library technology (GenomePlex) Single Cell Whole Genome Amplification Kit, Sigma-Aldrich, UK) and detailed analysis of genomic copy number changes by high-resolution array-CGH. We show that single copy changes as small as 8.3 Mb in single cells are detected reliably with single cells derived from various tumor cell lines as well as patients presenting with trisomy 21 and Prader-Willi syndrome. Our results demonstrate the potential of this technology for studies of tumor biology and for clinical diagnostics. AU - Fiegler, H.* AU - Geigl, J.B.* AU - Langer, S.* AU - Rigler, D.* AU - Porter, K.* AU - Unger, K. AU - Carter, N.P.* AU - Speicher, M.R.* C1 - 1980 C2 - 24955 TI - High resolution array-CGH analysis of single cells. JO - Nucleic Acids Res. VL - 35 IS - 3 PB - Oxford Univ. Press PY - 2007 SN - 0305-1048 ER - TY - JOUR AB - In the last years, RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of this technique in developing or adult mice, short hairpin (sh)RNA vectors expressed stably from the genome are a faster alternative to conventional knockout approaches. Here we describe an advanced strategy for conditional gene knockdown in mice, where we used the Cre/loxP system to activate RNAi in a time and tissue dependent manner in the adult mouse brain. By placing conditional RNAi constructs into the defined genomic Rosa26 locus and by using recombinase mediated cassette exchange (RMCE) instead of laborious homologous recombination, we developed a fast, easy and reproducible approach to assess gene function in adult mice. We applied this technique to three genes of the MAPK signaling pathway-Braf, Mek1 and Mek2-and demonstrate here the potential of this new tool in mouse mutagenesis. AU - Hitz, C. AU - Wurst, W. AU - Kühn, R. C1 - 1380 C2 - 24847 TI - Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference. JO - Nucleic Acids Res. VL - 35 IS - 12 PB - Oxford Univ. Press PY - 2007 SN - 0305-1048 ER - TY - JOUR AB - RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down–knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein. AU - Hölzel, M. AU - Rohrmoser, M. AU - Orban, M. AU - Hömig, C. AU - Harasim, T. AU - Malamoussi, A. AU - Gruber-Eber, A. AU - Heissmeyer, V. AU - Bornkamm, G.W. AU - Eick, D. C1 - 2520 C2 - 24342 TI - Rapid conditional knock-down–knock-in system for mammalian cells. JO - Nucleic Acids Res. VL - 35 IS - 3 PB - Oxford Univ. Press PY - 2007 SN - 0305-1048 ER - TY - JOUR AB - The nucleolar protein Pes1 interacts with Bop1 and WDR12 in a stable complex (PeBoW-complex) and its expression is tightly associated with cell proliferation. The yeast homologue Nop7p (Yph1p) functions in both, rRNA processing and cell cycle progression. The presence of a BRCT-domain (BRCA1 C-terminal) within Pes1 is quite unique for an rRNA processing factor, as this domain is normally found in factors involved in DNA-damage or repair pathways. Thus, the function of the BRCT-domain in Pes1 remains elusive. We established a conditional siRNA-based knock-down-knock-in system and analysed a panel of Pes1 truncation mutants for their functionality in ribosome synthesis in the absence of endogenous Pes1. Deletion of the BRCT-domain or single point mutations of highly conserved residues caused diffuse nucleoplasmic distribution and failure to replace endogenous Pes1 in rRNA processing. Further, the BRCT-mutants of Pes1 were less stable and not incorporated into the PeBoW-complex. Hence, the integrity of the BRCT-domain of Pes1 is crucial for nucleolar localization and its function in rRNA processing. AU - Hölzel, M. AU - Grimm, T. AU - Rohrmoser, M. AU - Malamoussi, A. AU - Harasim, T. AU - Gruber-Eber, A. AU - Kremmer, E. AU - Eick, D. C1 - 3935 C2 - 24467 SP - 789-800 TI - The BRCT domain of mammalian Pes1 is crucial for nucleolar localization and rRNA processing. JO - Nucleic Acids Res. VL - 35 IS - 3 PB - Oxford Univ. Press PY - 2007 SN - 0305-1048 ER - TY - JOUR AB - The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response. AU - Messaoudi, L.* AU - Yang, Y.G.* AU - Kinomura, A.* AU - Stavreva, D.A.* AU - Yan, G.* AU - Bortolin-Cavaillé, ML.* AU - Arakawa, H. AU - Buerstedde, J.M. AU - Hainaut, P.* AU - Cavaillé, J.* AU - Takata, M.* AU - van Dyck, E.* C1 - 2215 C2 - 24993 SP - 6571-6587 TI - Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress. JO - Nucleic Acids Res. VL - 35 IS - 19 PB - Oxford Univ. Press PY - 2007 SN - 0305-1048 ER - TY - JOUR AB - The PEDANT genome database provides exhaustive annotation of 468 genomes by a broad set of bioinformatics algorithms. We describe recent developments of the PEDANT Web server. The all-new Graphical User Interface (GUI) implemented in JavaTM allows for more efficient navigation of the genome data, extended search capabilities, user customization and export facilities. The DNA and Protein viewers have been made highly dynamic and customizable. We also provide Web Services to access the entire body of PEDANT data programmatically. Finally, we report on the application of association rule mining for automatic detection of potential annotation errors. PEDANT is freely accessible to academic users at http://pedant.gsf.de. AU - Riley, M.L. AU - Schmidt, T.* AU - Artamonova, I.I. AU - Wagner, C.* AU - Volz, A.* AU - Heumann, K.* AU - Mewes, H.-W. AU - Frishman, D. C1 - 5818 C2 - 24575 SP - D354-D357 TI - PEDANT genome database: 10 years online. JO - Nucleic Acids Res. VL - 35 IS - SI PB - Oxford Univ. Press PY - 2007 SN - 0305-1048 ER - TY - JOUR AB - Genome-oriented plant research delivers rapidly increasing amount of plant genome data. Comprehensive and structured information resources are required to structure and communicate genome and associated analytical data for model organisms as well as for crops. The increase in available plant genomic data enables powerful comparative analysis and integrative approaches. PlantsDB aims to provide data and information resources for individual plant species and in addition to build a platform for integrative and comparative plant genome research. PlantsDB is constituted from genome databases for Arabidopsis, Medicago, Lotus, rice, maize and tomato. Complementary data resources for cis elements, repetive elements and extensive cross-species comparisons are implemented. The PlantsDB portal can be reached at http://mips.gsf.de/projects/plants. AU - Spannagl, M. AU - Noubibou, O. AU - Haase, D. AU - Yang, L. AU - Gundlach, H. AU - Hindemitt, T. AU - Klee, K. AU - Haberer, G. AU - Schoof, H. AU - Mayer, K.F.X. C1 - 5819 C2 - 24576 SP - 834-840 TI - MIPSPlantsDB - plant database resource for integrative and comparative plant genome research. JO - Nucleic Acids Res. VL - 35 IS - SI PB - Oxford Univ. Press PY - 2007 SN - 0305-1048 ER - TY - JOUR AB - The development of high-throughput technologies has generated the need for bioinformatics approaches to assess the biological relevance of gene networks. Although several tools have been proposed for analysing the enrichment of functional categories in a set of genes, none of them is suitable for evaluating the biological relevance of the gene network. We propose a procedure and develop a web-based resource (BIOREL) to estimate the functional bias (biological relevance) of any given genetic network by integrating different sources of biological information. The weights of the edges in the network may be either binary or continuous. These essential features make our web tool unique among many similar services. BIOREL provides standardized estimations of the network biases extracted from independent data. By the analyses of real data we demonstrate that the potential application of BIOREL ranges from various benchmarking purposes to systematic analysis of the network biology. AU - Antonov, A.V. AU - Tetko, I.V. AU - Mewes, H.-W. C1 - 4914 C2 - 24030 SP - e6 TI - A systematic approach to infer biological relevance and biases of gene network structures. JO - Nucleic Acids Res. VL - 34 IS - 1 PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community. AU - De-Zolt, S.* AU - Schnütgen, F.* AU - Seisenberger, C. AU - Hansen, J. AU - Hollatz, M. AU - Floß, T. AU - Ruiz, P.* AU - Wurst, W. AU - von Melchner, H.* C1 - 4535 C2 - 23647 TI - High-throughput trapping of secretory pathway genes in mouse embryonic stem cells. JO - Nucleic Acids Res. VL - 34 IS - 3 PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - The nucleolar PeBoW-complex, consisting of Pes1, Bop1 and WDR12, is essential for cell proliferation and processing of ribosomal RNA in mammalian cells. Here we have analysed the physical and functional interactions of Pes1 deletion mutants with the PeBoW-complex. Pes1 mutants M1 and M5, with N- and C-terminal truncations, respectively, displayed a dominant-negative phenotype. Both mutants showed nucleolar localization, blocked processing of the 36S/32S precursors to mature 28S rRNA, inhibited cell proliferation, and induced high p53 levels in proliferating, but not in resting cells. Mutant M1 and M5 proteins associated with large pre-ribosomal complexes and co-immunoprecipitated Bop1 and WDR12 proteins indicating their proper incorporation into the PeBoW-complex. We conclude that the dominant-negative effect of the M1 and M5 mutants is mediated by the impaired function of the PeBoW-complex. AU - Grimm, T. AU - Hölzel, M. AU - Rohrmoser, M. AU - Harasim, T. AU - Malamoussi, A. AU - Gruber-Eber, A. AU - Kremmer, E. AU - Eick, D. C1 - 4931 C2 - 23820 SP - 3030-3043 TI - Dominant-negative Pes1 mutants inhibit ribosomal RNA processing and cell proliferation via incorporation into the PeBoW-complex. JO - Nucleic Acids Res. VL - 34 IS - 10 PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - In recent years, the Munich Information Center for Protein Sequences (MIPS) yeast protein-protein interaction (PPI) dataset has been used in numerous analyses of protein networks and has been called a gold standard because of its quality and comprehensiveness [H. Yu, N. M. Luscombe, H. X. Lu, X. Zhu, Y. Xia, J. D. Han, N. Bertin, S. Chung, M. Vidal and M. Gerstein (2004) Genome Res., 14, 1107-1118]. MPact and the yeast protein localization catalog provide information related to the proximity of proteins in yeast. Beside the integration of high-throughput data, information about experimental evidence for PPIs in the literature was compiled by experts adding up to 4300 distinct PPIs connecting 1500 proteins in yeast. As the interaction data is a complementary part of CYGD, interactive mapping of data on other integrated data types such as the functional classification catalog [A. Ruepp, A. Zollner, D. Maier, K. Albermann, J. Hani, M. Mokrejs, I. Tetko, U. Güldener, G. Mannhaupt, M. Münsterkötter and H. W. Mewes (2004) Nucleic Acids Res., 32, 5539-5545] is possible. A survey of signaling proteins and comparison with pathway data from KEGG demonstrates that based on these manually annotated data only an extensive overview of the complexity of this functional network can be obtained in yeast. The implementation of a web-based PPI-analysis tool allows analysis and visualization of protein interaction networks and facilitates integration of our curated data with high-throughput datasets. The complete dataset as well as user-defined sub-networks can be retrieved easily in the standardized PSI-MI format. The resource can be accessed through http://mips.gsf.de/genre/proj/mpact. AU - Güldener, U. AU - Münsterkötter, M. AU - Oesterheld, M. AU - Pagel, P. AU - Ruepp, A. AU - Mewes, H.-W. AU - Stuempflen, V. C1 - 3844 C2 - 24108 SP - 436-441 TI - MPact: The MIPS protein interaction resource on yeast. JO - Nucleic Acids Res. VL - 34 IS - SI PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - The MIPS Fusarium graminearum Genome Database (FGDB) is a comprehensive genome database on one of the most devastating fungal plant pathogens of wheat and barley. FGDB provides information on two gene sets independently derived by automated annotation of the F.graminearum genome sequence. A complete manually revised gene set will be completed within the near future. The initial results of systematic manual correction of gene calls are already part of the current gene set. The database can be accessed to retrieve information from bioinformatics analyses and functional classifications of the proteins. The data are also organized in the well established MIPS catalogs and novel query techniques are available to search the data. The comprehensive set of gene calls was also used for the design of an Affymetrix GeneChip. The resource is accessible on http://mips.gsf.de/genre/proj/fusarium/. AU - Güldener, U. AU - Mannhaupt, G.* AU - Münsterkötter, M. AU - Haase, M. AU - Oesterheld, M. AU - Stuempflen, V. AU - Mewes, H.-W. AU - Adam, G.* C1 - 3846 C2 - 24110 SP - D456-D458 TI - FGDB: A comprehensive fungal genome resource on the plant pathogen Fusarium graminearum. JO - Nucleic Acids Res. VL - 34 IS - SI PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - The Munich Information Center for Protein Sequences (MIPS at the GSF), Neuherberg, Germany, provides resources related to genome information. Manually curated databases for several reference organisms are maintained. Several of these databases are described elsewhere in this and other recent NAR database issues. In a complementary effort, a comprehensive set of >400 genomes automatically annotated with the PEDANT system are maintained. The main goal of our current work on creating and maintaining genome databases is to extend gene centered information to information on interactions within a generic comprehensive framework. We have concentrated our efforts along three lines (i) the development of suitable comprehensive data structures and database technology, communication and query tools to include a wide range of different types of information enabling the representation of complex information such as functional modules or networks Genome Research Environment System, (ii) the development of databases covering computable information such as the basic evolutionary relations among all genes, namely SIMAP, the sequence similarity matrix and the CABiNet network analysis framework and (iii) the compilation and manual annotation of information related to interactions such as protein-protein interactions or other types of relations (e.g. MPCDB, MPPI, CYGD). All databases described and the detailed descriptions of our projects can be accessed through the MIPS WWW server (http://mips.gsf.de). AU - Mewes, H.-W. AU - Frishman, D.* AU - Mayer, K.F.X. AU - Münsterkötter, M. AU - Noubibou, O. AU - Pagel, P. AU - Rattei, T.* AU - Oesterheld, M. AU - Ruepp, A. AU - Stuempflen, V. C1 - 5346 C2 - 24105 SP - D169-D172 TI - MIPS: Analysis and annotation of proteins from whole genomes in 2005. JO - Nucleic Acids Res. VL - 34 IS - SI PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - Gene trapping is a method of generating murine embryonic stem (ES) cell lines containing insertional mutations in known and novel genes. A number of international groups have used this approach to create sizeable public cell line repositories available to the scientific community for the generation of mutant mouse strains. The major gene trapping groups worldwide have recently joined together to centralize access to all publicly available gene trap lines by developing a user-oriented Website for the International Gene Trap Consortium (IGTC). This collaboration provides an impressive public informatics resource comprising approximately 45 000 well-characterized ES cell lines which currently represent approximately 40% of known mouse genes, all freely available for the creation of knockout mice on a non-collaborative basis. To standardize annotation and provide high confidence data for gene trap lines, a rigorous identification and annotation pipeline has been developed combining genomic localization and transcript alignment of gene trap sequence tags to identify trapped loci. This information is stored in a new bioinformatics database accessible through the IGTC Website interface. The IGTC Website (www.genetrap.org) allows users to browse and search the database for trapped genes, BLAST sequences against gene trap sequence tags, and view trapped genes within biological pathways. In addition, IGTC data have been integrated into major genome browsers and bioinformatics sites to provide users with outside portals for viewing this data. The development of the IGTC Website marks a major advance by providing the research community with the data and tools necessary to effectively use public gene trap resources for the large-scale characterization of mammalian gene function. AU - Nord, A.S.* AU - Chang, P.J.* AU - Conklin, B.R.* AU - Cox, A.V.* AU - Harper, C.A.* AU - Hicks, G.G.* AU - Huang, C.C.* AU - Johns, S.J.* AU - Kawamoto, M.* AU - Liu, S.* AU - Meng, E.C.* AU - Morris, J.H.* AU - Rossant, J.* AU - Ruiz, P.* AU - Skarnes, W.C.* AU - Soriano, P.* AU - Stanford, W.L.* AU - Stryke, D.* AU - von, Melchner, H.* AU - Wurst, W. AU - Yamamura, K.* AU - Young, S.G.* AU - Babbitt, P.C.* AU - Ferrin, T.E. C1 - 378 C2 - 23485 SP - D642-D648 TI - The International Gene Trap Consortium website: A portal to all publicly available gene trap cell lines in mouse. JO - Nucleic Acids Res. VL - 34 IS - SI PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - The MitoP2 database (http://www.mitop.de) integrates information on mitochondrial proteins, their molecular functions and associated diseases. The central database features are manually annotated reference proteins localized or functionally associated with mitochondria supplied for yeast, human and mouse. MitoP2 enables (i) the identification of putative orthologous proteins between these species to study evolutionarily conserved functions and pathways; (ii) the integration of data from systematic genome-wide studies such as proteomics and deletion phenotype screening; (iii) the prediction of novel mitochondrial proteins using data integration and the assignment of evidence scores; and (iv) systematic searches that aim to find the genes that underlie common and rare mitochondrial diseases. The data and analysis files are referenced to data sources in PubMed and other online databases and can be easily downloaded. MitoP2 users can explore the relationship between mitochondrial dysfunctions and disease and utilize this information to conduct systems biology approaches on mitochondria. AU - Prokisch, H. AU - Andreoli, C.* AU - Ahting, U.* AU - Heiss, K.* AU - Ruepp, A. AU - Scharfe, C.* AU - Meitinger, T. C1 - 1912 C2 - 23451 SP - D705-D711 TI - MitoP2: The mitochondrial proteome database-now including mouse data. JO - Nucleic Acids Res. VL - 34 IS - SI PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - Similarity Matrix of Proteins (SIMAP) (http://mips.gsf.de/simap) provides a database based on a pre-computed similarity matrix covering the similarity space formed by >4 million amino acid sequences from public databases and completely sequenced genomes. The database is capable of handling very large datasets and is updated incrementally. For sequence similarity searches and pairwise alignments, we implemented a grid-enabled software system, which is based on FASTA heuristics and the Smith-Waterman algorithm. Our ProtInfo system allows querying by protein sequences covered by the SIMAP dataset as well as by fragments of these sequences, highly similar sequences and title words. Each sequence in the database is supplemented with pre-calculated features generated by detailed sequence analyses. By providing WWW interfaces as well as web-services, we offer the SIMAP resource as an efficient and comprehensive tool for sequence similarity searches. AU - Rattei, T.* AU - Arnold, R. AU - Tischler, P. AU - Lindner, D.* AU - Stuempflen, V. AU - Mewes, H.-W. C1 - 4969 C2 - 24113 SP - 252-256 TI - SIMAP: The similarity matrix of proteins. JO - Nucleic Acids Res. VL - 34 IS - SI PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - MfunGD (http://mips.gsf.de/genre/proj/mfungd/) provides a resource for annotated mouse proteins and their occurrence in protein networks. Manual annotation concentrates on proteins which are found to interact physically with other proteins. Accordingly, manually curated information from a protein-protein interaction database (MPPI) and a database of mammalian protein complexes is interconnected with MfunGD. Protein function annotation is performed using the Functional Catalogue (FunCat) annotation scheme which is widely used for the analysis of protein networks. The dataset is also supplemented with information about the literature that was used in the annotation process as well as links to the SIMAP Fasta database, the Pedant protein analysis system and cross-references to external resources. Proteins that so far were not manually inspected are annotated automatically by a graphical probabilistic model and/or superparamagnetic clustering. The database is continuously expanding to include the rapidly growing amount of functional information about gene products from mouse. MfunGD is implemented in GenRE, a J2EE-based component-oriented multi-tier architecture following the separation of concern principle. AU - Ruepp, A. AU - Doudieu, O.N. AU - van den Oever, J. AU - Brauner, B. AU - Dunger-Kaltenbach, I. AU - Fobo, G. AU - Frishman, G. AU - Montrone, C. AU - Skornia, C. AU - Wanka, S. AU - Rattei, T.* AU - Pagel, P. AU - Riley, M.L. AU - Frishman, D.* AU - Surmeli, D. AU - Tetko, I.V. AU - Oesterheld, M. AU - Stuempflen, V. AU - Mewes, H.-W. C1 - 5483 C2 - 24114 SP - D568–D571 TI - The mouse functional genome database (MfunGD) :Functional annotation of proteins in the light of their cellular context. JO - Nucleic Acids Res. VL - 34 IS - SI PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - A total of 10 B-lymphocyte-specific DNase I hypersensitive sites located in the chicken Ig-beta locus were divided into four regions and combinations of deletions of these regions were carried out. A decrease in transcription of the Ig-beta gene to <3% was demonstrated in cells with deletions in all four regions. The Ig-beta chromatin was resistant to DNase I digestion in these cells. Thus, the collaboration is shown to convert the Ig-beta chromatin from the condensed state to a relaxed state. H3 and H4 acetylation decreased to <8% but H3K4 hypermethylation was observed at the Ig-beta promoter and exon 3. The collaboration of four regions had virtually no effect on CG hypomethylation in the region upstream the transcriptional start site. Accordingly, neither the DNase I general sensitive state in the Ig-beta chromatin nor hyperacetylation of H3 and H4 histones in the promoter proximal region causes H3K4 di-methylation or CG hypomethylation in the promoter. From these analyses, a chromatin situation was found in which both an active state, such as enhanced H3K4 methylation, or CG hypomethylation, and an inactive state, such as DNase I resistance in the Ig-beta chromatin or hypoacetylation of H3 and H4 histones in the Ig-beta locus, coexist. AU - Shimada, N.* AU - Matsudo, H.* AU - Osano, K.* AU - Arakawa, H. AU - Buerstedde, J.-M. AU - Matsumoto, Y.* AU - Chayahara, K.* AU - Torihata, A.* AU - Ono, M.* C1 - 893 C2 - 24149 SP - 3794-3802 TI - Activation of the chicken Ig-ß locus by the collaboration of scattered regulatory regions through changes in chromatin structure. JO - Nucleic Acids Res. VL - 34 IS - 13 PB - Oxford Univ. Press PY - 2006 SN - 0305-1048 ER - TY - JOUR AB - Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1. AU - Bornkamm, G.W. AU - Berens, Ch.* AU - Kuklik-Roos, C. AU - Bechet, J.-M.* AU - Laux, G. AU - Bachl, J. AU - Korndörfer, M. AU - Schlee, M. AU - Hölzel, M. AU - Malamoussi, A. AU - Chapman, R. C1 - 4644 C2 - 23195 SP - 1-11 TI - Stringent doxycycline-dependent control of gene activities using an episomal one-vector system. JO - Nucleic Acids Res. VL - 33 IS - 16 PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - Tolerance to replication-blocking DNA lesions is achieved by means of ubiquitylation of PCNA, the processivity clamp for replicative DNA polymerases, by components of the RAD6 pathway. In the yeast Saccharomyces cerevisiae the ubiquitin ligase (E3) responsible for polyubiquitylation of the clamp is the RING finger protein Rad5p. Interestingly, the RING finger, responsible for the protein's E3 activity, is embedded in a conserved DNA-dependent ATPase domain common to helicases and chromatin remodeling factors of the SWI/SNF family. Here, we demonstrate that the Rad5p ATPase domain provides the basis for a function of the protein in DNA double-strand break repair via a RAD52- and Ku-independent pathway mediated by the Mre11/Rad50/Xrs2 protein complex. This activity is distinct and separable from the contribution of the RING domain to ubiquitin conjugation to PCNA. Moreover, we show that the Rad5 protein physically associates with the single-stranded DNA regions at a processed double-strand break in vivo. Our observations suggest that Rad5p is a multifunctional protein that--by means of independent enzymatic activities inherent in its RING and ATPase domains--plays a modulating role in the coordination of repair events and replication fork progression in response to various different types of DNA lesions. AU - Chen, S.* AU - Davies, A.A.* AU - Sagan, D. AU - Ulrich, H.D.* C1 - 5000 C2 - 23242 SP - 5878-5886 TI - The RING finger ATPase Rad5p of Saccharomyces cerevisiae contributes to DNA double-strand break repair in a ubiquitin-independent manner. JO - Nucleic Acids Res. VL - 33 IS - 18 PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - Pathway- or disease-associated genes may participate in more than one transcriptional co-regulation network. Such gene groups can be readily obtained by literature analysis or by high-throughput techniques such as microarrays or protein-interaction mapping. We developed a strategy that defines regulatory networks by in silico promoter analysis, finding potentially co-regulated subgroups without a priori knowledge. Pairs of transcription factor binding sites conserved in orthologous genes (vertically) as well as in promoter sequences of co-regulated genes (horizontally) were used as seeds for the development of promoter models representing potential co-regulation. This approach was applied to a Maturity Onset Diabetes of the Young (MODY)-associated gene list, which yielded two models connecting functionally interacting genes within MODY-related insulin/glucose signaling pathways. Additional genes functionally connected to our initial gene list were identified by database searches with these promoter models. Thus, data-driven in silico promoter analysis allowed integrating molecular mechanisms with biological functions of the cell. AU - Döhr, S. AU - Klingenhoff, A.* AU - Maier, H. AU - Hrabě de Angelis, M. AU - Werner, T.* AU - Schneider, R. C1 - 4635 C2 - 22495 SP - 864-872 TI - Linking disease-associated genes to regulatory networks via promoter organization. JO - Nucleic Acids Res. VL - 33 IS - 3 PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - The Comprehensive Yeast Genome Database (CYGD) compiles a comprehensive data resource for information on the cellular functions of the yeast Saccharomyces cerevisiae and related species, chosen as the best understood model organism for eukaryotes. The database serves as a common resource generated by a European consortium, going beyond the provision of sequence information and functional annotations on individual genes and proteins. In addition, it provides information on the physical and functional interactions among proteins as well as other genetic elements. These cellular networks include metabolic and regulatory pathways, signal transduction and transport processes as well as co-regulated gene clusters. As more yeast genomes are published, their annotation becomes greatly facilitated using S.cerevisiae as a reference. CYGD provides a way of exploring related genomes with the aid of the S.cerevisiae genome as a backbone and SIMAP, the Similarity Matrix of Proteins. The comprehensive resource is available under http://mips.gsf.de/genre/proj/yeast/. AU - Güldener, U. AU - Münsterkötter, M. AU - Kastenmüller, G. AU - Strack, N.* AU - van Helden, J.* AU - Lemer, C.* AU - Richelles, J.* AU - Wodak, S.J.* AU - García-Martínez, J.* AU - Pérez-Ortín, J.E.* AU - Michael, H.* AU - Kaps, A.* AU - Talla, E.* AU - Dujon, B.* AU - André, B.* AU - Souciet, J.L.* AU - de Montigny, J.* AU - Bon, E.* AU - Gaillardin, C.* AU - Mewes, H.-W. C1 - 2922 C2 - 23418 SP - D364-368 TI - CYGD: The Comprehensive Yeast Genome Database. JO - Nucleic Acids Res. VL - 33 IS - DATABASE ISS. PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - To test the inferences of spotted microarray technology against a biochemically well-studied process, we performed transcriptional profiling of conidial germination in the filamentous fungus, Neurospora crassa. We first constructed a 70 base oligomer microarray that assays 3366 predicted genes. To estimate the relative gene expression levels and changes in gene expression during conidial germination, we analyzed a circuit design of competitive hybridizations throughout a time course using a Bayesian analysis of gene expression level. Remarkable consistency of mRNA profiles with previously published northern data was observed. Genes were hierarchically clustered into groups with respect to their expression profiles over the time course of conidial germination. A functional classification database was employed to characterize the global picture of gene expression. Consensus motif searches identified a putative regulatory component associated with genes involved in ribosomal biogenesis. Our transcriptional profiling data correlate well with biochemical and physiological processes associated with conidial germination and will facilitate functional predictions of novel genes in N.crassa and other filamentous ascomycete species. Furthermore, our dataset on conidial germination allowed comparisons to transcriptional mechanisms associated with germination processes of diverse propagules, such as teliospores of the phytopathogenic fungus Ustilago maydis and spores of the social amoeba Dictyostelium discoideum. AU - Kasuga, T.* AU - Townsend, J.P.* AU - Tian, C.* AU - Gilbert, L.B.* AU - Mannhaupt, G. AU - Taylor, J.W.* AU - Glass, N.L.* C1 - 466 C2 - 23347 SP - 6469-6485 TI - Long-oligomer microarray profiling in Neurospora crassa reveals the transcriptional program underlying biochemical and physiological events of conidial germination. JO - Nucleic Acids Res. VL - 33 IS - 20 PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - Genome-wide mapping in the identification of novel candidate genes has always been the standard method in genetics and genomics to correlate a clinically interesting phenotypic trait with a genotype. However, the performance of a mapping experiment using classical microsatellite approaches can be very time consuming. The high-throughput analysis of single-nucleotide polymorphisms (SNPs) has the potential of being the successor of microsatellite analysis routinely used for these mapping approaches, where one of the major obstacles is the design of the appropriate SNP marker set itself. Here we report on ARTS, an advanced retrieval tool for SNPs, which allows researchers to comb freely the public mouse dbSNP database for multiple reference and test strains. Several filters can be applied in order to improve the sensitivity and the specificity of the search results. By employing the panel generator function of this program, it is possible to abbreviate the extraction of reliable sequence data for a large marker panel including several different mouse strains from days to minutes. The concept of ARTS is easily adaptable to other species for which SNP databases are available, making it a versatile tool for the use of SNPs as markers for genotyping. The web interface is accessible at http://andromeda.gsf.de/arts. AU - Klaften, M. AU - Hrabě de Angelis, M. C1 - 5124 C2 - 22722 SP - W496-W500 TI - ARTS: A web-based tool for the set-up of high-throughput genome-wide mapping panels for the SNP genotyping of mouse mutants. JO - Nucleic Acids Res. VL - 33 IS - SUPPL. 2 PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - The C-terminal domain (CTD) of mammalian RNA polymerase II consists of 52 repeats of the consensus hepta-peptide YSPTSPS, and links transcription to the processing of pre-mRNA. Although Pol II with a CTD shortened to five repeats (Pol II Delta5) is transcriptionally inactive on chromatin templates, it is not clear whether CTD is required for promoter recognition in vivo. Here, we demonstrate that in the context of chromatin, Pol II Delta5 can bind to the c-myc promoter with the same efficiency as wild type Pol II. However, Pol II Delta5 does not form a stable initiation complex, and does not transcribe promoter proximal sequences. Fluorescence recovery after photobleaching (FRAP) experiments with cells expressing enhanced green fluorescent protein (EGFP)-tagged Delta5 or wildtype Pol II revealed a single, highly mobile Pol II Delta5 fraction whereas wildtype Pol II yielded less mobile fractions. These data suggest that CTD is not required for promoter recognition, but rather for subsequent formation of a stable initiation complex and isomerization to an elongation competent complex. AU - Lux, Ch. AU - Albiez, H.* AU - Chapman, R.D. AU - Heidinger, M. AU - Meininghaus, M. AU - Brack-Werner, R. AU - Lang, A. AU - Ziegler, M. AU - Cremer, T. AU - Eick, D. C1 - 3978 C2 - 23045 SP - 5139-5144 TI - Transition from initiation to promoter proximal pausing requires the CTD of RNA polymerase II. JO - Nucleic Acids Res. VL - 33 IS - 16 PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - The LitMiner software is a literature data-mining tool that facilitates the identification of major gene regulation key players related to a user-defined field of interest in PubMed abstracts. The prediction of gene-regulatory relationships is based on co-occurrence analysis of key terms within the abstracts. LitMiner predicts relationships between key terms from the biomedical domain in four categories (genes, chemical compounds, diseases and tissues). Owing to the limitations (no direction, unverified automatic prediction) of the co-occurrence approach, the primary data in the LitMiner database represent postulated basic gene-gene relationships. The usefulness of the LitMiner system has been demonstrated recently in a study that reconstructed disease-related regulatory networks by promoter modelling that was initiated by a LitMiner generated primary gene list. To overcome the limitations and to verify and improve the data, we developed WikiGene, a Wiki-based curation tool that allows revision of the data by expert users over the Internet. LitMiner (http://andromeda.gsf.de/litminer) and WikiGene (http://andromeda.gsf.de/wiki) can be used unrestricted with any Internet browser. AU - Maier, H. AU - Döhr, S. AU - Grote, K. AU - O'Keeffe, S. AU - Werner, T. AU - Hrabě de Angelis, M. AU - Schneider, R. C1 - 2327 C2 - 22735 SP - W779-W782 TI - LitMiner and WikiGene: Identifying problem-related key players of gene regulation using publication abstracts. JO - Nucleic Acids Res. VL - 33 IS - SUPPL. 2 PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - The PEDANT genome database (http://pedant.gsf.de) contains pre-computed bioinformatics analyses of publicly available genomes. Its main mission is to provide robust automatic annotation of the vast majority of amino acid sequences, which have not been subjected to in-depth manual curation by human experts in high-quality protein sequence databases. By design PEDANT annotation is genome-oriented, making it possible to explore genomic context of gene products, and evaluate functional and structural content of genomes using a category-based query mechanism. At present, the PEDANT database contains exhaustive annotation of over 1,240,000 proteins from 270 eubacterial, 23 archeal and 41 eukaryotic genomes. AU - Riley, M.L. AU - Schmidt, T.* AU - Wagner, C.* AU - Mewes, H.-W. AU - Frishman, D. C1 - 1360 C2 - 23417 SP - D308-D310 TI - The PEDANT genome database in 2005. JO - Nucleic Acids Res. VL - 33 IS - DATABASE ISS. PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - Molecular markers are required in a broad spectrum of gene screening approaches, ranging from gene-mapping within traditional 'forward'-genetics approaches through QTL identification studies to genotyping and haplotyping studies. As we enter the post-genomics era, the need for genetic markers does not diminish, even in the species with fully sequenced genomes. PlantMarkers is a genetic marker database that contains a comprehensive pool of predicted molecular markers. We have adopted contemporary techniques to identify putative single nucleotide polymorphism (SNP), simple sequence repeat (SSR) and conserved orthologue set markers. A systematic approach to identify as broad a range of putative markers has been undertaken by screening the available openSputnik unigene consensus sequences from over 50 plant species. A web presence at http://markers.btk.fi provides functionality so that a user may search for species-specific markers on the basis of many specific criteria not limited to non-synonymous SNPs segregating between different varieties or measured polymorphic SSRs. Feedback forms are provided with all sequence entries to enable inclusion of, for example, map location for markers validated by the research community. AU - Rudd, S.* AU - Schoof, H. AU - Mayer, K.F.X. C1 - 4420 C2 - 23345 SP - 628-632 TI - PlantMarkers - a database of predicted molecular markers from plants. JO - Nucleic Acids Res. VL - 33 IS - DATABASE ISS. PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - The identification of the genes that participate at the biological interface of two species remains critical to our understanding of the mechanisms of disease resistance, disease susceptibility and symbiosis. The sequencing of complementary DNA (cDNA) libraries prepared from the biological interface between two organisms provides an inexpensive way to identify the novel genes that may be expressed as a cause or consequence of compatible or incompatible interactions. Sequence classification and annotation of species origin typically use an orthology-based approach and require access to large portions of either genome, or a close relative. Novel species- or clade-specific sequences may have no counterpart within existing databases and remain ambiguous features. Here we present a web-service, Eclair, which utilizes support vector machines for the classification of the origin of expressed sequence tags stemming from mixed host cDNA libraries. In addition to providing an interface for the classification of sequences, users are presented with the opportunity to train a model to suit their preferred species pair. Eclair is freely available at http://eclair.btk.fi. AU - Rudd, S.* AU - Tetko, I.V. C1 - 4848 C2 - 23416 SP - W724-W727 TI - Éclair - a web service for unravelling species origin of sequences sampled from mixed host interfaces. JO - Nucleic Acids Res. VL - 33 IS - SUPPL. 2 PB - Oxford Univ. Press PY - 2005 SN - 0305-1048 ER - TY - JOUR AB - The aim of the MitoP2 database (http://ihg.gsf.de/mitop2) is to provide a comprehensive list of mitochondrial proteins of yeast and man. Based on the current literature we created an annotated reference set of yeast and human proteins. In addition, data sets relevant to the study of the mitochondrial proteome are integrated and accessible via search tools and links. They include computational predictions of signalling sequences, and summarize results from proteome mapping, mutant screening, expression profiling, protein-protein interaction and cellular sublocalization studies. For each individual approach, specificity and sensitivity for allocating mitochondrial proteins was calculated. By providing the evidence for mitochondrial candidate proteins the MitoP2 database lends itself to the genetic characterization of human mitochondriopathies. AU - Andreoli, C. AU - Prokisch, H. AU - Hörtnagel, K. AU - Mueller, J.C. AU - Münsterkötter, M. AU - Scharfe, C.* AU - Meitinger, T. C1 - 2865 C2 - 21813 SP - 459-462 TI - MitoP2, an integrated databases on mitochondrial proteins in yeast and man. JO - Nucleic Acids Res. VL - 32 PB - Oxford Univ. Press PY - 2004 SN - 0305-1048 ER - TY - JOUR AB - The phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD) has been shown to affect the initiation, and transition to elongation of the Pol II complex. The differential phosphorylation of serines within this domain coincides with the recruitment of factors important for pre-mRNA processing and transcriptional elongation. A role for tyrosine and threonine phosphorylation has yet to be described. The discovery of kinases that express a preference for specific residues within this sequence suggests a mechanism for the controlled recruitment and displacement of CTD-interacting partners during the transcription cycle. The last CTD repeat (CTD52) contains unique interaction sites for the only known CTD tyrosine kinases, Abl1/c-Abl and Abl2/Arg, and the serine/threonine kinase casein kinase II (CKII). Here, we show that removal or severe disruption of the last CTD repeat, but not point mutation of its CKII sites, results in its proteolytic degradation to the Pol IIb form in vivo, but does not appear to affect the specific transcription of genes. These results suggest a possible mechanism of transcription control through the proteolytic removal of the Pol II CTD. AU - Chapman, R.D. AU - Palancade, B.* AU - Lang, A. AU - Bensaude, O.* AU - Eick, D. C1 - 2540 C2 - 21678 SP - 35-44 TI - The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability. JO - Nucleic Acids Res. VL - 32 IS - 1 PB - Oxford Univ. Press PY - 2004 SN - 0305-1048 ER - TY - JOUR AB - The Munich Information Center for Protein Sequences (MIPS-GSF), Neuherberg, Germany, provides protein sequence-related information based on whole-genome analysis. The main focus of the work is directed toward the systematic organization of sequence-related attributes as gathered by a variety of algorithms, primary information from experimental data together with information compiled from the scientific literature. MIPS maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the database of complete cDNAs (German Human Genome Project, NGFN), the database of mammalian protein-protein interactions (MPPI), the database of FASTA homologies (SIMAP), and the interface for the fast retrieval of protein-associated information (QUIPOS). The Arabidopsis thaliana database, the rice database, the plant EST databases (MATDB, MOsDB, SPUTNIK), as well as the databases for the comprehensive set of genomes (PEDANT genomes) are described elsewhere in the 2003 and 2004 NAR database issues, respectively. All databases described, and the detailed descriptions of our projects can be accessed through the MIPS web server (http://mips.gsf.de). AU - Mewes, H.-W. AU - Amid, C. AU - Arnold, R. AU - Frishman, D.* AU - Güldener, U. AU - Mannhaupt, G.* AU - Münsterkötter, M. AU - Pagel, P. AU - Strack, N.* AU - Stuempflen, V. AU - Warfsmann, J. AU - Ruepp, A. C1 - 9967 C2 - 21438 SP - D41-D44 TI - MIPS : analysis and annotation of proteins from whole genomes. JO - Nucleic Acids Res. VL - 32 IS - SI PB - Oxford Univ. Press PY - 2004 SN - 0305-1048 ER - TY - JOUR AB - In this paper, we present the Functional Catalogue (FunCat), a hierarchically structured, organism-independent, flexible and scalable controlled classification system enabling the functional description of proteins from any organism. FunCat has been applied for the manual annotation of prokaryotes, fungi, plants and animals. We describe how FunCat is implemented as a highly efficient and robust tool for the manual and automatic annotation of genomic sequences. Owing to its hierarchical architecture, FunCat has also proved to be useful for many subsequent downstream bioinformatic applications. This is illustrated by the analysis of large-scale experiments from various investigations in transcriptomics and proteomics, where FunCat was used to project experimental data into functional units, as 'gold standard' for functional classification methods, and also served to compare the significance of different experimental methods. Over the last decade, the FunCat has been established as a robust and stable annotation scheme that offers both, meaningful and manageable functional classification as well as ease of perception. AU - Ruepp, A. AU - Zollner, A.* AU - Maier, D.* AU - Albermann, K.* AU - Hani, J.* AU - Mokrejs, M. AU - Tetko, I.V. AU - Güldener, U. AU - Mannhaupt, G. AU - Münsterkötter, M. AU - Mewes, H.-W. C1 - 2755 C2 - 22390 SP - 5539-5545 TI - The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes. JO - Nucleic Acids Res. VL - 32 IS - 18 PB - Oxford Univ. Press PY - 2004 SN - 0305-1048 ER - TY - JOUR AB - Pathbase is a database that stores images of the abnormal histology associated with spontaneous and induced mutations of both embryonic and adult mice including those produced by transgenesis, targeted mutagenesis and chemical mutagenesis. Images of normal mouse histology and strain-dependent background lesions are also available. The database and the images are publicly accessible (http://www.pathbase.net) and linked by anatomical site, gene and other identifiers to relevant databases; there are also facilities for public comment and record annotation. The database is structured around a novel ontology of mouse disorders (MPATH) and provides high-resolution downloadable images of normal and diseased tissues that are searchable through orthogonal ontologies for pathology, developmental stage, anatomy and gene attributes (GO terms), together with controlled vocabularies for type of genetic manipulation or mutation, genotype and free text annotation for mouse strain and additional attributes. The database is actively curated and data records assessed by pathologists in the Pathbase Consortium before publication. The database interface is designed to have optimal browser and platform compatibility and to interact directly with other web-based mouse genetic resources. AU - Schofield, P.N.* AU - Bard, J.B.L.* AU - Booth, C.* AU - Boniver, J.* AU - Covelli, V.* AU - Delvenne, P.* AU - Ellender, M.* AU - Engstrom, W.* AU - Goessner, W. AU - Gruenberger, M.* AU - Höfler, H. C1 - 2697 C2 - 22236 SP - D512-D515 TI - Pathbase: A database of mutant mouse pathology. JO - Nucleic Acids Res. VL - 32 PB - Oxford Univ. Press PY - 2004 SN - 0305-1048 ER - TY - JOUR AB - Arabidopsis thaliana is the most widely studied model plant. Functional genomics is intensively underway in many laboratories worldwide. Beyond the basic annotation of the primary sequence data, the annotated genetic elements of Arabidopsis must be linked to diverse biological data and higher order information such as metabolic or regulatory pathways. The MIPS Arabidopsis thaliana database MAtDB aims to provide a comprehensive resource for Arabidopsis as a genome model that serves as a primary reference for research in plants and is suitable for transfer of knowledge to other plants, especially crops. The genome sequence as a common backbone serves as a scaffold for the integration of data, while, in a complementary effort, these data are enhanced through the application of state-of-the-art bioinformatics tools. This information is visualized on a genome-wide and a gene-by-gene basis with access both for web users and applications. This report updates the information given in a previous report and provides an outlook on further developments. The MAtDB web interface can be accessed at http://mips.gsf.de/proj/thal/db. AU - Schoof, H. AU - Ernst, R. AU - Nazarov, V. AU - Pfeifer, L. AU - Mewes, H.-W. AU - Mayer, K.F.X. C1 - 2185 C2 - 22216 SP - 373-376 TI - MIPS Arabidopsis thaliana database (MAtDB) An integrated biological knowledge resource for plant genomics. JO - Nucleic Acids Res. VL - 32 PB - Oxford Univ. Press PY - 2004 SN - 0305-1048 ER - TY - JOUR AB - As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5' end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns' features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts. AU - Bon, E.* AU - Casaregola, S.* AU - Blandin, G.* AU - Llorente, B.* AU - Neuvéglise, C.* AU - Münsterkötter, M. AU - Güldener, U. AU - Mewes, H.-W. AU - van Helden, J.* AU - Dujon, B.* AU - Gaillardin, C.* C1 - 22355 C2 - 21233 SP - 1121-1135 TI - Molecular evolution of eukaryotic genomes: Hemiascomycetous yeast spliceosomal introns. JO - Nucleic Acids Res. VL - 31 IS - 4 PB - Oxford Univ. Press PY - 2003 SN - 0305-1048 ER - TY - JOUR AB - The cDNA-chip technology is a highly versatile tool for the comprehensive analysis of gene expression at the transcript level. Although it has been applied successfully in expression profiling projects, there is an ongoing dispute concerning the quality of such expression data. The latter critically depends on the specificity of hybridisation. SAFE (specificity assessment from fractionation experiments) is a novel method to discriminate between nonspecific cross-hybridisation and specific signals. We applied in situ fractionation of hybridised target on DNA-chips by means of repeated washes with increasing stringencies. Different fractions of hybridised target are washed off at defined stringencies and the collected fluorescence intensity data at each step comprise the fractionation curve. Based on characteristic features of the fractionation curve, unreliable data can be filtered and eliminated from subsequent analyses. The approach described here provides a novel experimental tool to identify probes that produce specific hybridisation signals in DNA-chip expression profiling approaches. The iterative use of the SAFE procedure will result in increasingly reliable sets of probes for microarray experiments and significantly improve the overall efficiency and reliability of RNA expression profiling data from DNA-chip experiments. AU - Drobyshev, A.L. AU - Machka, C. AU - Horsch, M. AU - Seltmann, M. AU - Liebscher, V. AU - Hrabě de Angelis, M. AU - Beckers, J. C1 - 9965 C2 - 21084 SP - 2-10 TI - Specificity assessment from fractionation experiments (SAFE) : a novel method to evaluate microarray probe specificity based on hybridisation strinencies. JO - Nucleic Acids Res. VL - 31 IS - 2 PB - OXFORD UNIV PRESS PY - 2003 SN - 0305-1048 ER - TY - JOUR AB - The MIPS Rice (Oryza sativa) database (MOsDB; http://mips.gsf.de/proj/rice) provides a comprehensive data collection dedicated to the genome information of rice. Rice (O. sativa L.) is one of the most important food crops for over half the world's population and serves as a major model system in cereal genome research. MOsDB integrates data from two publicly available rice genomic sequences, O. sativa L. ssp. indica and O. sativa L. ssp. japonica. Besides regularly updated rice genome sequence information, MOsDB provides an integrated resource for associated analysis data, e. g. internal and external annotation information as well as a complex characterization of all annotated rice genes. The MOsDB web interface supports various search options and allows browsing the database content. MOsDB is continuously expanding to include an increasing range of data type and the growing amount of information on the rice genome. AU - Karlowski, W.M. AU - Schoof, H. AU - Janakiraman, V. AU - Stuempflen, V. AU - Mayer, K.F.X. C1 - 9966 C2 - 21447 SP - 190-192 TI - MOsDB : an integrated information resource for rice genomics. JO - Nucleic Acids Res. VL - 31 IS - 1 PB - OXFORD UNIV PRESS PY - 2003 SN - 0305-1048 ER - TY - JOUR AB - The proto-oncogene c-myc encodes a basic helix-loop-helix leucine zipper transcription factor (c-Myc) that has a profound role in growth control and cell cycle progression. Previous microarray studies identified various classes of c-Myc target genes, including genes involved in ribosome biogenesis. By screening the human B-cell line P493-6 and rat fibroblasts conditionally expressing c-Myc, we could substantially extend the list of c-Myc target genes, particularly those required for ribosome biogenesis. The identification of 38 new c-Myc target genes with nucleolar function, prompted us to investigate processing of ribosomal RNA (rRNA). Using pulse-chase labelling experiments we show that c-Myc regulates the efficiency of rRNA maturation. In serum-stimulated P493-6 cells, only the processing of the 47S rRNA precursor to mature 18S and 28S rRNA, but not the synthesis of the 47S transcript, was dependent on the presence of c-Myc. As processing of rRNA is sensitive to inhibition of cyclin-dependent kinase (cdk) activity by roscovitine, we conclude that c-Myc regulates cell growth and proliferation by the coordinated induction of cdk activity and rRNA processing. AU - Schlosser, I. AU - Hölzel, M. AU - Mürnseer, M. AU - Burtscher, H.* AU - Weidle, U.H.* AU - Eick, D. C1 - 22386 C2 - 21305 SP - 6148-6156 TI - A role for c-Myc in the regulation of ribosomal RNA processing. JO - Nucleic Acids Res. VL - 31 IS - 21 PB - Oxford Univ. Press PY - 2003 SN - 0305-1048 ER - TY - JOUR AB - Regulation of gene expression involves sequence elements in nucleic acids. In promoters, multiple sequence elements cooperate as functional modules, which in combination determine overall promoter activity. We previously developed computational tools based on this hierarchical structure for in silico promoter analysis. Here we address the functional organization of post-transcriptional control elements, using the HIV-1 genome as a model. Numerous mutagenesis studies demonstrate that expression of HIV structural proteins is restricted by inhibitory sequences (INS) in HIV mRNAs in the absence of the HIV-1 Rev protein. However, previous attempts to detect conserved sequence patterns of HIV-1 INS have failed. We defined four distinct sequence patterns for inhibitory motifs (weight matrices), which identified 22 out of the 25 known INS as well as several new candidate INS regions contained in numerous HIV-1 strains. The conservation of INS motifs within the HIV genome was not due to overall sequence conservation. The functionality of two candidate INS regions was analyzed with a new assay that measures the effect of non-coding mRNA sequences on production of red fluorescent reporter protein. Both new INS regions showed inhibitory activity in sense but not in antisense orientation. Inhibitory activity increased by combining both INS regions in the same mRNA. Inhibitory activity of known and new INS regions was overcome by co-expression of the HIV-1 Rev protein. AU - Wolff, H. AU - Brack-Werner, R. AU - Neumann, M. AU - Werner, T. AU - Schneider, R. C1 - 9964 C2 - 21080 SP - 2839-2851 TI - Integrated functional and bioinformatics approach for the identification and experimental verification of RNA signals : Application to HIV-1 INS. JO - Nucleic Acids Res. VL - 31 IS - 11 PB - OXFORD UNIV PRESS PY - 2003 SN - 0305-1048 ER - TY - JOUR AB - Non-homologous insertion (NHI) of DNA fragments into genomic DNA is a method widely used in insertional mutagenesis screens. In the yeast Saccharomyces cerevisiae, the efficiency of NHI is very low. Here we report that its efficiency can be increased by gamma-irradiation of recipient cells at the time of transformation. Radiation-assisted NHI depends on YKU70, but its efficiency is not improved by inactivation of RAD5 or RAD52. In a pilot study, we generated 102 transformant clones expressing a lacZ reporter gene under standard conditions (30degreesC, rich medium). The site of insertion was determined in a subset of eight clones in which lacZ expression was altered by UV-irradiation. A comparison with published data revealed that three of the eight genes identified in our screen have not been targeted by large-scale transposon-based insertion screens. This suggests that radiation-assisted NHI offers a more homogeneous coverage of the genome than methods relying on transposons or retroviral elements. AU - Kiechle, M.* AU - Manivasakam, P.* AU - Eckardt-Schupp, F. AU - Schiestl, R.H.* AU - Friedl, A.A.* C1 - 9968 C2 - 20432 TI - Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion. JO - Nucleic Acids Res. VL - 30 IS - 24 PB - Oxford Univ. Press PY - 2002 SN - 0305-1048 ER - TY - JOUR AB - The Munich Information Center for Protein Sequences (MIPS-GSF, Neuherberg, Germany) continues to provide genome-related information in a systematic way. MIPS supports both national and European sequencing and functional analysis projects, develops and maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences, and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the databases for the comprehensive set of genomes (PEDANT genomes), the database of annotated human EST clusters (HIB), the database of complete cDNAs from the DHGP (German Human Genome Project), as well as the project specific databases for the GABI (Genome Analysis in Plants) and HNB (Helmholtz-Netzwerk Bioinformatik) networks. The Arabidospsis thaliana database (MATDB), the database of mitochondrial proteins (MITOP) and our contribution to the PIR International Protein Sequence Database have been described elsewhere [Schoof et al. (2002) Nucleic Acids Res., 30, 91-93; Scharfe et al. (2000) Nucleic Acids Res., 28, 155-158; Barker et al. (2001) Nucleic Acids Res., 29, 29-32]. All databases described, the protein analysis tools provided and the detailed descriptions of our projects can be accessed through the MIPS World Wide Web server (http://mips.gsf.de). AU - Mewes, H.-W. AU - Frishman, D. AU - Güldener, U. AU - Mannhaupt, G. AU - Mayer, K.F.X. AU - Mokrejs, M. AU - Morgenstern, B. AU - Münsterkötter, M. AU - Rudd, S. AU - Weil, B. C1 - 22361 C2 - 21239 SP - 31-34 TI - MIPS: A database for genomes and protein sequences. JO - Nucleic Acids Res. VL - 30 IS - 1 PB - Oxford Univ. Press PY - 2002 SN - 0305-1048 ER - TY - JOUR AB - Arabidopsis thaliana is the first plant for which the complete genome has been sequenced and published. Annotation of complex eukaryotic genomes requires more than the assignment of genetic elements to the sequence. Besides completing the list of genes, we need to discover their cellular roles, their regulation and their interactions in order to understand the workings of the whole plant. The MIPS Arabidopsis thaliana Database (MAtDB; http://mips.gsf.de/proj/thal/db) started out as a repository for genome sequence data in the European Scientists Sequencing Arabidopsis (ESSA) project and the Arabidopsis Genome Initiative. Our aim is to transform MAtDB into an integrated biological knowledge resource by integrating diverse data, tools, query and visualization capabilities and by creating a comprehensive resource for Arabidopsis as a reference model for other species, including crop plants. AU - Schoof, H. AU - Zaccaria, P. AU - Gundlach, H. AU - Lemcke, K. AU - Rudd, S. AU - Kolesov, G. AU - Arnold, R. AU - Mewes, H.-W. AU - Mayer, K.F.X. C1 - 22359 C2 - 21237 SP - 91-93 TI - MIPS Arabidopsis thaliana Database (MAtDB): An integrated biological konwledge resource based on the first complete plant genome. JO - Nucleic Acids Res. VL - 30 IS - 1 PB - Oxford Univ. Press PY - 2002 SN - 0305-1048 ER - TY - JOUR AB - Gamma-crystallin genes are specifically expressed in the eye lens. Their promoters constitute excellent models to analyse tissue-specific gene expression. We investigated murine CRYGE/f promoters of different length in lens epithelial cell lines. The most active fragment extends from position -219 to +37. Computer analysis predicts homeodomain and paired-domain binding sites for all rodent CRYGD/e/f core promoters. As examples, we analysed the effects of Prox1 and Six3, which are considered important transcription factors involved in lens development. Because of endogenous Prox1 expression in N/N1003A cells, a weak stimulation of CRYGE/f promoter activity was found for PROX1. In contrast, PROX1 stimulated the CRYGF promoter 10-fold in CD5A cells without endogenous PROX1. In both cell lines Six3 repressed the CRYGF promoter to 10% of its basal activity. Our cell transfection experiments indicated that CRYG expression increases as Six3 expression decreases. Prox1 and Six3 act antagonistically on regulation of the CRYGD/e/f promoters. Functional assays using randomly mutated gammaF-crystallin promoter fragments define a Six3-responsive element between -101 and -123 and a Prox1-responsive element between -151 and -174. Since Prox1 and Six3 are present at the beginning of lens development, expression of CRYGD/e/f is predicted to remain low at this time. It increases as Six3 expression decreases during ongoing lens development. AU - Lengler, J. AU - Krausz, E. AU - Tomarev, S.* AU - Prescott, A.* AU - Quinlan, R.A.* AU - Graw, J. C1 - 9962 C2 - 19670 SP - 515-526 TI - Antagonistic action of Six3 and Prox1 at the ϒ-crystallin promoter. JO - Nucleic Acids Res. VL - 29 IS - 2 PB - Oxford Univ. Press PY - 2001 SN - 0305-1048 ER - TY - JOUR AB - MITOP (http://www.mips.biochem.mpg.de/proj/medgen/mitop/) is a comprehensive database for genetic and functional information on both nuclear- and mitochondrial-encoded proteins and their genes. The five species files--Saccharomyces cerevisiae, Mus musculus, Caenorhabditis elegans, Neurospora crassa and Homo sapiens--include annotated data derived from a variety of online resources and the literature. A wide spectrum of search facilities is given in the overlapping sections 'Gene catalogues', 'Protein catalogues', 'Homologies', 'Pathways and metabolism' and 'Human disease catalogue' including extensive references and hyperlinks to other databases. Central features are the results of various homology searches, which should facilitate the investigations into interspecies relationships. Precomputed FASTA searches using all the MITOP yeast protein entries and a list of the best human EST hits with graphical cluster alignments related to the yeast reference sequence are presented. The orthologue tables with cross-listings to all the protein entries for each species in MITOP have been expanded by adding the genomes of Rickettsia prowazeckii and Escherichia coli. To find new mitochondrial proteins the complete yeast genome has been analyzed using the MITOPROT program which identifies mitochondrial targeting sequences. The 'Human disease catalogue' contains tables with a total of 110 human diseases related to mitochondrial protein abnormalities, sorted by clinical criteria and age of onset. MITOP should contribute to the systematic genetic characterization of the mitochondrial proteome in relation to human disease. AU - Scharfe, C.* AU - Zaccaria, P. AU - Hoertnagel, K.* AU - Jaksch, M.* AU - Klopstock, T.* AU - Dembowski, M.* AU - Lill, R.* AU - Prokisch, H.* AU - Gerbitz, K.D.* AU - Neupert, W.* AU - Mewes, H.-W. AU - Meitinger, T.* C1 - 23794 C2 - 31406 SP - 155-158 TI - MITOP, the mitochondrial proteome database: 2000 update. JO - Nucleic Acids Res. VL - 28 IS - 1 PB - Oxford Univ. Press PY - 2000 SN - 0305-1048 ER - TY - JOUR AB - eregulated expression of the proto-oncogene c-myc in Burkitt lymphoma (BL) cells carrying a t(2;8) translocation is mediated by a synergistic interaction of the translocated immunoglobulin (Ig) kappa gene intron (kappa Ei) and 3' (kappa E3') enhancers and characterized by a strong activation of the promoter P1, We have investigated the functional role of distinct kappa enhancer sequence motifs in P1 activation on both minichromosomes and reporter gene constructs, Stable and transient transfections of BL cells revealed critical roles of the kappa Ei and kappa E3' elements kappa B and PU, respectively, Joint mutation of kappa B and PU completely abolished P1 activity, implying that an interaction of kappa B- and PU-binding factors is essential for the enhancer synergism, Mutation of the E box 1 and E box 2 motifs markedly decreased P1 activity in transient but not in stable transfection experiments. Co-expression of the NF-kappa B subunit p65(RelA) and Sp1, an essential factor for P1 transcription, in Drosophila melanogaster SL2 cells synergistically enhanced promoter activity. Our results support a model which proposes cross-talk between promoter and enhancer binding factors as the basic mechanism for kappa enhancer-mediated c-myc activation in BL cells AU - Wittekindt, N.E. AU - Hörtnagel, K. AU - Geltinger, C. C1 - 9969 C2 - 19350 SP - 800-808 TI - Activation of c-myc promoter P1 by immunoglobulin κ gene enhancers in Burkitt lymphoma: functional characterization of the intron enhancer motifs kB, E box 1 and E box 2 and of the 3' enhancer motif PU. JO - Nucleic Acids Res. VL - 28 IS - 3 PB - Oxford Univ. Press PY - 2000 SN - 0305-1048 ER - TY - JOUR AU - Immervoll, T. AU - Wjst, M. C1 - 20931 C2 - 18977 SP - 213-214 TI - Current status of the asthma and allergy database. JO - Nucleic Acids Res. VL - 27 PY - 1999 SN - 0305-1048 ER - TY - JOUR AB - Many cell lines are sensitive to growth at low cell density and undergo apoptosis induced by oxidative stress if the cell density is decreased below a critical threshold. In stable transfection experiments this cell density-dependent growth may be the limiting factor, since during drug selection the cell density falls below the critical threshold, precluding outgrowth of transfected clones. We describe here a simple protocol for the establishment of stably transfected human B cell lines making use of the protective action of antioxidants. The protocol includes: (i) seeding the cells in medium supplemented with sodium pyruvate, alpha-thioglycerol and bathocuproine disulfonate; (ii) delaying the onset of dominant marker selection to improve recovery of the cells after electroporation. Stably transfected clones have thus been obtained from Burkitt's lymphoma lines, which have been regarded as untransfectable. Using this protocol the stable transfection efficiency with episomal plasmids approaches the transient transfection efficiency, indicating that virtually every transfected cell can be established as a stably transfected clone. This protocol should also prove useful for other cell lines, e.g. neuronal cells, having similar sensitivities to oxidative stress. AU - Brielmeier, M. AU - Bechet, J.-M.* AU - Falk, M.H.* AU - Pawlita, M.* AU - Polack, A. AU - Bornkamm, G.W. C1 - 9963 C2 - 22868 SP - 2082-2085 TI - Improving stable transfection efficiency : antioxidants dramatically improve the outgrowth of clones under dominant marker selection. JO - Nucleic Acids Res. VL - 26 IS - 9 PB - OXFORD UNIV PRESS PY - 1998 SN - 0305-1048 ER - TY - JOUR AB - We present a method to determine the location and extent of protein binding regions in nucleic acids by computer-assisted analysis of sequence data. The program Consindex establishes a library of consensus descriptions based on sequence sets containing known regulatory elements. These defined consensus descriptions are used by the program Consinspector to predict binding sites in new sequences. We show the programs to correctly determine the significant regions involved in transcriptional control of seven sequence elements. The internal profile of relative variability of individual nucleotide positions within these regions paralleled experimental profiles of biological significance. Consensus descriptions are determined by employing an anchored alignment scheme, the results of which are then evaluated by a novel method which is superior to cluster algorithms. The alignment procedure is able to include several closely related sequences without biasing the consensus description. Moreover, the algorithm detects additional elements on the basis of a moderate distance correlation and is capable of discriminating between real binding sites and false positive matches. The software is well suited to cope with the frequent phenomenon of optional elements present in a subset of functionally similar sequences, while taking maximal advantage of the existing sequence data base. Since it requires only a minimum of seven sequences for a single element, it is applicable to a wide range of binding sites. AU - Frech, K. AU - Herrmann, G. AU - Werner, T. C1 - 40327 C2 - 38005 SP - 1655-1664 TI - Computer-assisted prediction, classification, and delimitation of protein binding sites in nucleic acids. JO - Nucleic Acids Res. VL - 21 IS - 7 PY - 1993 SN - 0305-1048 ER - TY - JOUR AU - Möller, E.M. AU - Bahnweg, G. AU - Sandermann, H. AU - Geiger, H.H. C1 - 20261 C2 - 13447 SP - 6115-6116 TI - A Simple and Efficient Protocol for Isolation of High Molecular Weight DNA from Filamentous Fungi, Fruit Bodies, and infected Plant Tissues. JO - Nucleic Acids Res. VL - 20 PY - 1992 SN - 0305-1048 ER - TY - JOUR AU - Werner, T. C1 - 40840 C2 - 38918 SP - 1711 TI - A HyperCard-shell for the Human Genome Mapping database (HGM) including a stand-alone reference database. JO - Nucleic Acids Res. VL - 19 IS - 7 PY - 1991 SN - 0305-1048 ER - TY - JOUR AU - Eick, D. C1 - 18208 C2 - 11419 SP - 1199-1205 TI - Elongation and Maturation of c-myc RNA is Inhibited by Differentiation Inducing Agents in HL60 Cells. JO - Nucleic Acids Res. VL - 18 PY - 1990 SN - 0305-1048 ER - TY - JOUR AU - Rump, A. C1 - 18228 C2 - 11441 TI - Purification of DNA from agarose by polyethylene glycol induced precipitation. JO - Nucleic Acids Res. PY - 1990 SN - 0305-1048 ER - TY - JOUR AU - Werner, T. C1 - 18380 C2 - 11576 TI - A HyperCard-shell for the Human Genome Mapping Database (HGM) Including a Stand-alone Reference Database. JO - Nucleic Acids Res. PY - 1990 SN - 0305-1048 ER - TY - JOUR AU - Brack-Werner, R. AU - Werner, T. AU - Leib-Mösch, C. AU - Hehlmann, R. AU - Erfle, V.F. C1 - 42563 C2 - 40156 SP - 5382 TI - Primary structure of the SSAV tether-RNase H endonuclease (pol) region deleted in SSV. JO - Nucleic Acids Res. VL - 17 IS - 13 PY - 1989 SN - 0305-1048 ER - TY - JOUR AU - Leib-Mösch, C. AU - Barton, D.E. AU - Geigl, E.M. AU - Brack-Werner, R. AU - Erfle, V.F. AU - Hehlmann, R. AU - Francke., U. C1 - 41917 C2 - 36498 SP - 2367 TI - Two RFLPs associated with the human endogenous retroviral element S71 on chromosome 18q21. JO - Nucleic Acids Res. VL - 17 IS - 6 PY - 1989 SN - 0305-1048 ER -