Processing mechanism and substrate selectivity of the core NuA4 histone acetyltransferase complex.

Eukaryotic DNA is packaged in the nucleoprotein structure chromatin, the smallest component of which is the nucleosome core particle (NCP). Each core particle consists of 147 bp of DNA wrapped 1.7 times around an octamer of the four canonical histone proteins H4, H3, H2A and H2B (1, 2). Histones can be post-translationally modified on their N-terminal tail domains as well as their globular core domains. Numerous histone modifications have been identified, including methylation, phosphorylation, ubiquitination, sumoylation and acetylation (3). These modifications not only affect structural and intra-molecular interactions (i.e. octamer destabilization, tail-DNA association), but also serve as molecular signals whereby other proteins dynamically associate with chromatin. Mounting evidence suggests that this network of modifications coordinate gene expression via an epigenetic code, in which demarcations are read by enzyme complexes, resulting in altered phenotypes through differential gene activation or repression (4). Histone acetyltransferases (HATs) are a class of enzymes that catalyze the transfer of the acetyl group from acetyl coenzyme-A (AcCoA) to the e-amino group of lysines in histones. HAT-containing complexes are categorized into five distinct families according to the sequence homology of their catalytic domains and their shared substrate specificity. To date, the Gcn5-related N-acetyltransferase (GNAT) and MYST (MOZ, Ybf2/Sas3, Sas2 and Tip60) families have been the most well-characterized. Esa1 (KAT5), a MYST Tip60 homolog, remains the only essential acetyltransferase found in S. cerevisiae, where it is required for cell viability (5, 6), ribosomal gene transcription (7), DNA double strand break repair (8) and cell cycle progression (9). Esa1 can be co-purified in two evolutionarily conserved complexes, 1) a 1.3 MDa nucleosome acetyltransferase of histone H4 (NuA4) complex that contains 13 total subunits, and 2) a small, heterotrimeric complex dubbed piccolo NuA4 (picNuA4) that contains Esa1, Epl1 and Yng2 (8, 10). Recent studies have implicated NuA4 in locus-specific recruitment and transcriptional activation, resulting in targeted H4 and H2A acetylation both in vitro and in vivo (7, (11), while picNuA4 catalyzes non-targeted global H4 acetylation in yeast (12, 13). The molecular determinants dictating substrate specificity for HAT enzyme complexes are still poorly understood. Much of the current information on substrate specificity and recognition originate from crystallographic studies describing Gcn5 catalytic domains bound with H3 tail peptide substrates (14–17). These findings suggested that the tail peptide sequence alone is sufficient for efficient recognition and catalysis by GNAT HAT active sites (18, 19), a supposition supported by the crystal structure of the MYST Esa1 catalytic domain (20). Consistent with this model, Utley et al. has shown that H4 peptides containing S1 phosphorylation can oblate efficient peptide acetylation by NuA4 (21). But recently, Berndsen et al. showed that histone tail peptides alone are surprisingly poor substrates for the MYST picNuA4 complex and that the histone fold domain of H4 is required for efficient nucleosomal recognition (22). Thus, picNuA4 appears to bind substrates in a bipartite manner in which both the histone tails and the histone fold domain are required for efficient acetylation. Unlike Gcn5 acetyltransferases, which preferentially acetylate H3K14 10–100 times more efficiently than other candidate lysines (23), the picNuA4 complex rapidly tetra-acetylates the four sites on the H4 tail at comparable levels of efficiency (22). Here, we have investigated the molecular mechanisms that allow for the efficient tetra-acetylation of H4 by picNuA4. Our results indicate that picNuA4 utilizes a non-processive mechanism of acetylation for nucleosomal substrates, in which picNuA4 dissociates from substrate after each acetylation event. Rapid tetra-acetylation does not require sequential order of acetylation, as picNuA4 randomly acetylates H4 tail lysines (K5, K8, K12 and K16) within the nucleosome. Kinetic analyses of histone mutants suggest a specificity model where site preference is dictated by accessibility on the surface of the nucleosome, the relative proximity from the histone fold domain, and a preference for glycine residues with a minimal (n + 2) spacing between lysines.