Gcn5p, a Transcription-related Histone Acetyltransferase, Acetylates Nucleosomes and Folded Nucleosomal Arrays in the Absence of Other Protein Subunits

Abstract

Gcn5p is the catalytic subunit of several type A histone acetyltransferases (HATs). Previous studies performed under a limited range of solution conditions have found that nucleosome core particles and nucleosomal arrays can be acetylated by Gcn5p only when it is complexed with other proteins, e.g. Gcn5-Ada, HAT-A2, and SAGA. Here we demonstrate that when assayed in buffer containing optimum concentrations of either NaCl or MgCl 2 , purified yeast recombinant Gcn5p (rGcn5p) efficiently acetylates both nucleosome core particles and nucleosomal arrays. Furthermore, under conditions where nucleosomal arrays are extensively folded, rGcn5p acetylates folded arrays ϳ40% faster than nucleosome core particles. Finally, rGcn5p polyacetylates the N termini of free histone H3 but only monoacetylates H3 in nucleosomes and nucleosomal arrays. These results demonstrate both that rGcn5p in and of itself is catalytically active when assayed under optimal solution conditions and that this enzyme prefers folded nucleosomal arrays as a substrate. They further suggest that the structure of the histone H3 N terminus, and concomitantly the accessibility of the H3 acetylation sites, changes upon assembly into nucleosomes and nucleosomal arrays.Histone acetylation is a reversible dynamic process that occurs at specific lysine residues in the N termini of all the core histone proteins and has been correlated with several key biological processes, including nucleosome assembly and modulation of gene expression (1-4). The recent discoveries of the specific histone acetyltransferases (HATs), 1 Hat1p (5, 6) and Gcn5p (7), have directly linked histone acetylation with nucleosome assembly and transcriptional activation, respectively. Hat1p and Gcn5p are specific examples of the two general families of HATs, termed HAT-A and HAT-B. The HAT-A enzymes reside in the nucleus and acetylate the core histone N termini following their assembly into nucleosomes and chromatin. HAT-B type enzymes are primarily cytoplasmic and acetylate only free histones prior to nucleosome assembly (6), although Hat1p has recently been localized in the nucleus as well (8).Hat1p is the catalytic subunit of both the major cytoplasmic HAT-B complex and the nuclear HAT-A3 complex (8). Consistent with their roles in nucleosome assembly, recombinant Hat1p, the HAT-B complex, and the HAT A-3 complex all acetylate free histone H4 in vitro but do not acetylate H4 after its incorporation into nucleosomes (5, 6, 8, 9). However, seemingly at odds with its role as a HAT-A enzyme, Gcn5p has been reported to acetylate free histones H3 and H4 but not nucleosomal H3 or H4 (10 -13). This result has been reconciled by a model in which Gcn5p acetylates nucleosomal substrates only when a component of specific multiprotein complexes and is supported by the recent identification of several Gcn5-Ada complexes that are capable of acetylating histone H3 and to a lesser extent H4 in nucleosomes (12, 14 -16). Nevertheless, because all previous studies of rGcn5p activity were performed using a very...

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Snf1p Regulates Gcn5p Transcriptional Activity by Antagonizing Spt3p

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