Nucleosome recognition by the Piccolo NuA4 histone acetyltransferase complex.

In eukaryotic nuclei, DNA is packaged into a complex DNA–protein macromolecule (chromatin) consisting of repeating units of histone protein wrapped by DNA. Alterations in chromatin structure, such as nucleosome sliding, removal of nucleosomes, and disruption of DNA–histone or histone–histone contacts through post-translational modification of the histones, have been linked to transcriptional regulation, DNA damage repair, and replication, among other cellular processes (1–5). Modification of histones occurs primarily in the N-terminal residues or tail region. These modifications include phosphorylation, ubiquitinylation, methylation, sumolyation, and acetylation. A major focus of chromatin research is to understand how the modification state of histones is linked to the resulting phenotype. Acetylation of histones is generally associated with active transcription, constitutes a post-translational mark recognized by specific chromatin factors, and has been shown in vitro to prevent salt-induced folding of nucleosome arrays (6–8). Histone acetyltransferases (HATs)1 modify histones by facilitating the transfer of the acetyl moiety from acetylcoenzyme A (CoA) to the e-amine of lysine residues. The best studied families of HATs are the Gcn5-related N-acetyltransferase (GNAT) family and the MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST) family (9). Members of both HAT families have been shown to acetylate histones as part of large multiprotein complexes. The subunits of HAT complexes have been proposed to target the catalytic subunit to specific loci, regulate HAT activity, and mediate nucleosome recognition (1, 10, 11). Esa1, the essential Sas2-related acetyltransferase in yeast, has been linked to DNA damage repair, riboprotein gene transcription, and global acetylation of H4 (12–16). Orthologues of Esa1 are reported to have similar functional roles (15). In yeast cell extracts, Esa1 co-purifies with a multiprotein complex NuA4 and separately as a trimeric complex, Piccolo NuA4 (picNuA4). The functional roles of all NuA4 subunits are not entirely known, although some are essential for yeast under normal growth conditions (15). The picNuA4 complex consists of the subunits Esa1, Epl1, and Yng2 and displays similar in vitro acetylation patterns to those of the larger NuA4 complex, which also contains the same three subunits (14). The picNuA4 complex is proposed to be responsible for global H4 acetylation in yeast (14). The molecular basis for recognition of nucleosome substrates by HATs is not well-known. Crystal structures of Gcn5 and orthologue p/CAF with bound peptide substrates or bisubstrate inhibitors show numerous contacts with the peptide substrate, suggesting that recognition may be based solely on the sequence of amino acids surrounding the target lysine (11, 17, 18). However, it was noted that some known in vivo substrates of Gcn5 and p/CAF do not conform to this model, suggesting that the other subunits of the GCN5 and p/CAF complexes may alter the specificity of the HAT (11). Because only short peptides were employed in these studies, potential interactions with native nucleosomal substrates are not known. Some models of nucleosome recognition have suggested that post-translational modifications within the histone tail influence the ability of HAT complexes to acetylate chromatin, suggesting that the histone tails themselves play an essential role in nucleosome recognition (19, 20). Unfortunately, the lack of knowledge on the fundamental principles that govern nucleosome recognition make predictions of binding determinants difficult. Here, we have investigated the ability of Esa1 and the picNuA4 complex to acetylate histone substrates of increasing structural complexity, ranging from small peptides to free histones to nucleosome arrays. Most notably, we find that the histone-fold domain (HFD) of histones plays a critical role in the recognition by Esa1 and the picNuA4 complex of both free histones and histones assembled into nucleosomes. While we find that Esa1 can acetylate nucleosomes, the subunits Epl1 and Yng2 dramatically increase the stability of Esa1 and the efficiency of nucleosome recognition, consistent with previous studies that showed that particular regions of Epl1 and Yng2 play critical roles in the ability of picNuA4 to act on its nucleosome substrate (21). Nucleosomes are acetylated at rates approaching the molecular diffusion limit in aqueous solution, indicating that the picNuA4 complex contains all of the components necessary for highly efficient nucleosome recognition and acetylation. The histone tails of H3, H2A, and H2B do not contribute to nucleosome recognition or the efficient acetylation of the preferred H4 tail. We have identified a region within the HFD of H4 that appears to serve as the predominant interaction site for picNuA4. Together, our results provide new insight into the mechanisms by which HAT complexes catalyze specific but processive-like acetylation on nucleosomal lysine residues. The ability of HAT complexes to utilize multiple-binding interactions on the nucleosome (e.g., tails, the HFD, and DNA) may be a general feature of highly specific and efficient catalysis shared by other chromatin-modifying enzyme complexes.