PUB-NChIP—“in vivo biotinylation” approach to study chromatin in proximity to a protein of interest
- Muhammad Shoaib1,
- Arman Kulyyassov1,2,
- Chloé Robin1,
- Kinga Winczura1,
- Pavel Tarlykov1,3,
- Emmanuelle Despas4,
- Patricia Kannouche4,
- Erlan Ramanculov2,
- Marc Lipinski1 and
- Vasily Ogryzko1,5
- 1UMR8126, Université Paris-Sud 11, CNRS, Institut de Cancérologie Gustave Roussy, 94805 Villejuif, France;
- 2National Center of Biotechnology, 01000 Astana, Kazakhstan;
- 3LN Gumilyov Eurasian National University, 010008 Astana, Kazakhstan;
- 4UMR8200, Université Paris-Sud 11, CNRS, Institut de Cancérologie Gustave Roussy, 94805 Villejuif, France
Abstract
We have developed an approach termed PUB-NChIP (proximity utilizing biotinylation with native ChIP) to purify and study the protein composition of chromatin in proximity to a nuclear protein of interest. It is based on coexpression of (1) a protein of interest, fused with the bacterial biotin ligase BirA, together with (2) a histone fused to a biotin acceptor peptide (BAP), which is specifically biotinylated by BirA-fusion in the proximity of the protein of interest. Using the RAD18 protein as a model, we demonstrate that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2AZ has a different pattern of H4 acetylation. Finally, biotin pulse-chase experiments show that the H4 acetylation pattern starts to resemble the acetylation pattern of total H4 after the proximity of chromatin to RAD18 has been lost.
Footnotes
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↵5 Corresponding author
E-mail vogryzko{at}gmail.com
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.134874.111.
- Received December 9, 2011.
- Accepted October 2, 2012.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/.
