A native peptide ligation strategy for deciphering nucleosomal histone modifications.
Post-translational modifications of histones influence both chromatin structure and the binding and function of chromatin-associated proteins. A major limitation to understanding these effects has been the inability to construct nucleosomes in vitro that harbor homogeneous and site-specific histone modifications. Here, we describe a native peptide ligation strategy for generating nucleosomal arrays that can harbor a wide range of desired histone modifications. As a first test of this method, we engineered model nucleosomal arrays in which each histone H3 contains a phosphorylated serine at position 10 and performed kinetic analyses of Gcn5-dependent histone acetyltransferase activities. Recombinant Gcn5 shows increased histone acetyltransferase activity on nucleosomal arrays harboring phosphorylated H3 serine 10 and is consistent with peptide studies. However, in contrast to analyses using peptide substrates, we find that the histone acetyltransferase activity of the Gcn5-containing SAGA complex is not stimulated by H3 phosphorylation in the context of nucleosomal arrays. This difference between peptide and array substrates suggests that the ability to generate specifically modified nucleosomal arrays should provide a powerful tool for understanding the effects of post-translational histone modifications.
Shogren-Knaak
Michael A
MA
Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA.
Fry
Christopher J
CJ
Peterson
Craig L
CL
D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D002645:Chickens; D051548:Histone Acetyltransferases; D006657:Histones / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014981:Xenopus
D006657:Histones; D009707:Nucleosomes; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D000123:Acetyltransferases; D051548:Histone Acetyltransferases