The Yeast Histone Acetyltransferase A2 Complex, but Not Free Gcn5p, Binds Stably to Nucleosomal Arrays

Abstract

We have investigated the structural basis for the differential catalytic function of the yeast Gcn5p-containing histone acetyltransferase (HAT) A2 complex and free recombinant yeast Gcn5p (rGcn5p). HAT A2 is shown to be a unique complex that contains Gcn5p, Ada2p, and Ada3p, but not proteins specific to other related HAT A complexes, e.g. ADA, SAGA. Nevertheless, HAT A2 produces the same unique polyacetylation pattern of nucleosomal substrates reported previously for ADA and SAGA, demonstrating that proteins specific to the ADA and SAGA complexes do not influence the enzymatic activity of Gcn5p within the HAT A2 complex. To investigate the role of substrate interactions in the differential behavior of free and complexed Gcn5p, sucrose density gradient centrifugation was used to characterize the binding of HAT A2 and free rGcn5p to intact and trypsinized nucleosomal arrays, H3/H4 tetramer arrays, and nucleosome core particles. We find that HAT A2 forms stable complexes with all nucleosomal substrates tested. In distinct contrast, rGcn5p does not interact stably with nucleosomal arrays, despite being able to specifically monoacetylate the H3 N terminus of nucleosomal substrates. Our data suggest that the ability of the HAT A2 complex to bind stably to nucleosomal arrays is functionally related to both local and global acetylation by the complexed and free forms of Gcn5p.Histone acetylation is a dynamic process in vivo involving multiple acetyltransferase and deacetylase enzymes (1-3). Acetylation occurs at specific lysine residues of each of the core histone N termini, and influences chromatin structure and function at all levels from the folded chromatin fiber to the nucleosome (4 -6). Because of the key role of acetylation in transcription and replication, a great deal of attention has been focused on the properties and functions of recently identified histone acetyltransferase enzymes (HATs).1 HAT A enzymes (e.g. Gcn5p, Esa1p) primarily are involved in transcriptional regulation, whereas HAT B enzymes (e.g. Hat1p) are thought to participate in replication coupled-chromatin assembly. The Tetrahymena equivalent of yeast Gcn5p (Gcn5p) was the first specific HAT A enzyme to be cloned and overexpressed (7), and the Gcn5 family of HATs remains the focus of intense investigation (see Refs. 5,8,and 9, and references therein).The catalytic function of yeast Gcn5p in vitro is strongly dependent on whether the enzyme is free or is a component of large multiprotein complexes such as HAT A2, ADA, and SAGA (8, 9). Purified ADA and SAGA complexes both have been shown to contain the non-DNA-binding, transcriptional adaptor proteins Ada2p and Ada3p (8 -10). Consistent with these in vitro results, genetic experiments in yeast have documented that mutations in Ada2p or Ada3p yield phenotypes identical to loss of Gcn5p in terms of both transcriptional regulation at specific gene loci (11, 12) and cell growth (11, 13). In addition, several specific proteins have recently been identified as unique components of ADA or SAGA. For exam...

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