PUB-NChIP - "in vivo biotinylation" approach to study chromatin in proximity to a protein of interest
- Muhammad Shoaib1,
- Arman Kulyyassov1,
- Chloe Robin1,
- Kinga Winczura1,
- Pavel Tarlykov2,
- Emmanuelle Despras1,
- Patricia Kannouche1,
- Erlan Ramanculov3,
- Marc Lipinski1 and
- Vasily Ogryzko1,4
- 1 Institut Gustave Roussy, CNRS UMR8126, Universite Paris-Sud XI;
- 2 LN Gumilyov Eurasian National University, Munaitpasova street 5, 473021, Astana, Kazakhstan;
- 3 National Center for Biotechnology of the Republic of Kazakhstan
- ↵* Corresponding author; email: vogryzko{at}gmail.com
Abstract
We have developed an approach termed PUB-NChIP (Proximity Utilizing Biotinylation with Native ChIP) to purify and study protein composition of chromatin in the proximity to a nuclear protein of interest. It is based on coexpression of a) a protein of interest, fused with the bacterial biotin ligase BirA together with b) a histone fused to BAP biotin acceptor peptide, which is specifically biotinylated by BirA fusion in the proximity of the protein of interest. Using 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 H4 acetylation pattern starts to resemble acetylation pattern of total H4 after the proximity of chromatin to Rad18 has been lost.
- Received December 9, 2011.
- Accepted October 2, 2012.
- © 2012, Published by Cold Spring Harbor Laboratory Press
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