Nucleosome recognition by the Piccolo NuA4 histone acetyltransferase complex

The mechanisms by which multisubunit histone acetyltransferase (HAT) complexes recognize and perform efficient acetylation on nucleosome substrates are largely unknown. Here, we use a variety of biochemical approaches and compare histone-based substrates of increasing complexity to determine the critical components of nucleosome recognition by the MOZ, Ybf2/Sas3, Sas2, Tip60 family HAT complex, Piccolo NuA4 (picNuA4). We find the histone tails to be dispensable for binding to both nucleosomes and free histones and that the H2A, H3, and H2B tails do not influence the ability of picNuA4 to tetra-acetylate the H4 tail within the nucleosome. Most notably, we discovered that the histone-fold domain (HFD) regions of histones, particularly residues 21-52 of H4, are critical for tight binding and efficient tail acetylation. Presented evidence suggests that picNuA4 recognizes the open surface of the nucleosome on which the HFD of H4 is located. This binding mechanism serves to direct substrate access to the tails of H4 and H2A and allows the enzyme to be "tethered", thereby increasing the effective concentration of the histone tail and permitting successive cycles of H4 tail acetylation.     

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

Esa1, an essential MYST histone acetyltransferase found in the yeast piccolo NuA4 complex (picNuA4), is responsible for genome-wide histone H4 and histone H2A acetylation. picNuA4 uniquely catalyzes the rapid tetra-acetylation of nucleosomal H4, though the molecular determinants driving picNuA4 efficiency and specificity have not been defined. Here, we show through rapid substrate trapping experiments that picNuA4 utilizes a nonprocessive mechanism in which picNuA4 dissociates from the substrate after each acetylation event. Quantitative mass spectral analyses indicate that picNuA4 randomly acetylates free and nucleosomal H4, with a small preference for lysines 5, 8, and 12 over lysine 16. Using a series of 24 histone mutants of H4 and H2A, we investigated the parameters affecting catalytic efficiency. Most strikingly, removal of lysine residues did not substantially affect the ability of picNuA4 to acetylate remaining sites, and insertion of an additional lysine into the H4 tail led to rapid quintuple acetylation. Conversion of the native H2A tail to an H4-like sequence resulted in enhanced multisite acetylation. Collectively, the results suggest picNuA4's site selectivity is dictated by accessibility on the nucleosome surface, the relative proximity from the histone fold domain, and a preference for intervening glycine residues with a minimal (n + 2) spacing between lysines. Functionally distinct from other HAT families, the proposed model for picNuA4 represents a unique mechanism of substrate recognition and multisite acetylation.     

The Rtt109-Vps75 histone acetyltransferase complex acetylates non-nucleosomal histone H3

Acetylation of lysine 56 of histone H3 (H3-Lys-56) occurs in S phase and disappears during G(2)/M phase of the cell cycle. However, it is not clear how this modification is regulated during the progression of the cell cycle. We and others have shown that the histone acetyltransferase (HAT) Rtt109 is the primary HAT responsible for acetylating H3-Lys-56 in budding yeast. Here we show that Rtt109 forms a complex with Vps75 and that both recombinant Rtt109-Vps75 complexes and native complexes purified from yeast cells acetylate H3 present in H3/H4/H2A/H2B core histones but not other histones. In addition, both recombinant and native Rtt109-Vps75 HAT complexes exhibited no detectable activity toward nucleosomal H3, suggesting that H3-Lys-56 acetylation is at least in part regulated by the inability of Rtt109-Vps75 complexes to acetylate nucleosomal H3 during G(2)/M phase of the cell cycle. Further, Rtt109 bound mutant H3/H4 tetramers composed of histones lacking their N-terminal tail domains less efficiently than wild-type H3/H4 tetramers, and Rtt109-Vps75 complexes displayed reduced HAT activity toward these mutant H3/H4 tetramers. Thus, the N termini of H3/H4 tetramers are required for efficient acetylation of H3 by the Rtt109-Vps75 complex. Taken together, these studies provide insights into how H3-Lys-56 acetylation is regulated during the cell cycle.     

Methylation of histone H3 lysine 9 occurs during translation

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.     

Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6

The DNA of eukaryotes is wrapped around nucleosomes and packaged into chromatin. Covalent modifications of the histone proteins that comprise the nucleosome alter chromatin structure and have major effects on gene expression. Methylation of lysine 4 of histone H3 by COMPASS is required for silencing of genes located near chromosome telomeres and within the rDNA (Krogan, N. J, Dover, J., Khorrami, S., Greenblatt, J. F., Schneider, J., Johnston, M., and Shilatifard, A. (2002) J. Biol. Chem. 277, 10753-10755; Briggs, S. D., Bryk, M., Strahl, B. D., Cheung, W. L., Davie, J. K., Dent, S. Y., Winston, F., and Allis, C. D. (2001) Genes. Dev. 15, 3286-3295). To learn about the mechanism of histone methylation, we surveyed the genome of the yeast Saccharomyces cerevisiae for genes necessary for this process. By analyzing approximately 4800 mutant strains, each deleted for a different non-essential gene, we discovered that the ubiquitin-conjugating enzyme Rad6 is required for methylation of lysine 4 of histone H3. Ubiquitination of histone H2B on lysine 123 is the signal for the methylation of histone H3, which leads to silencing of genes located near telomeres.     

Thr 3 phosphorylated histone H3 concentrates at centromeric chromatin at metaphase

Thr 3 was one of the newly characterized phosphorylation sites on histone H3. However, the functional significance of histone H3 Thr 3 phosphorylation during mitosis is unclear. In this study, SDS-PAGE and Western blotting analysis showed that histone H3 Thr 3 was phosphorylated specially during mitosis in MCF-10A and ECV-304 cells. Using indirect immunofluorescence labeling and laser confocal microscopy, we demonstrated that histone H3 Thr 3 phosphorylation occurred from prophase to anaphase and dephosphorylated completely in telophase. Remarkably, Thr 3 phosphorylated histone H3 mostly concentrated at centromeric chromatin at metaphase, which was distinct with Ser 10 phosphorylation aggregated at the telomere, but similar to that characteristic of Thr 11 phosphorylated H3 which is largely restricted to the centromeric chromatin. Using chromatin immunoprecipitation (ChIP) assay, we provided direct evidence that the Thr 3 phosphorylated H3 is associated with centromeric DNA at metaphase. These findings suggested that at metaphase Thr 3 phosphorylated histone H3 may also participate in kinetochore assembly to promote faithful chromosome segregation and serve as another recognition code for kinetochore proteins.