Large scale preparation of nucleosomes containing site-specifically chemically modified histones lacking the core histone tail domains.
The core histone tail domains are critical regulators of chromatin structure and function and modifications such as acetylation of lysine residues within the tails are central to this regulation. Studies have shown that the removal of core histone tail domains by trypsinization in which one-half to two-thirds of each core histone tail domain are removed in gross aspects mimics the acetylation of core histone tails. In addition, removal of the tails has been useful in understanding general tail function. Thus, removal of native core histone tails by trypsinization is a widely used method. In addition, many in vitro studies now employ core histones site-specifically modified with photo activatable cross-linking probes or fluorescent probes. However, in our experience, standard methods employing trypsinized donor chromatin for reconstitution of nucleosomes containing certain chemically modified histones lacking the core histone tail domains are not uniformly applicable. Here, we describe various methods for preparing nucleosomes containing a core histone modified with a cross-linking agent, APB, and lacking the core histone tail domains.
Yang
Zungyoon
Z
Department of Biochemistry and Biophysics, Box 712 University of Rochester Medical Center, Rochester, NY 14642, USA.
Hayes
Jeffrey J
JJ
D000818:Animals; D002645:Chickens / Q000378:metabolism; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary
D003432:Cross-Linking Reagents; D006657:Histones; D009707:Nucleosomes; D004247:DNA