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Single-copy gene locus chromatin purification in Saccharomyces cerevisiae.
J. Vis. Exp. 2023:16 (2023)
The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Proteins; Capture
ISSN (print) / ISBN
1940-087X
e-ISSN
1940-087X
Quellenangaben
Volume: 2023,
Issue: 201,
Article Number: 16
Publisher
JoVE
Publishing Place
1 Alewife Center, Ste 200, Cambridge, Ma 02140 Usa
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Epigenetics and Stem Cells (IES)
Grants
Helmholtz Gesellschaft
European Research Council (ERC)
DFG
European Research Council (ERC)
DFG