Methylation of histone H3 mediates the association of the NuA3 histone acetyltransferase with chromatin

The SAS3-dependent NuA3 histone acetyltransferase complex was originally identified on the basis of its ability to acetylate histone H3 in vitro. Whether NuA3 is capable of acetylating histones in vivo, or how the complex is targeted to the nucleosomes that it modifies, was unknown. To address this question, we asked whether NuA3 is associated with chromatin in vivo and how this association is regulated. With a chromatin pulldown assay, we found that NuA3 interacts with the histone H3 amino-terminal tail, and loss of the H3 tail recapitulates phenotypes associated with loss of SAS3. Moreover, mutation of histone H3 lysine 14, the preferred site of acetylation by NuA3 in vitro, phenocopies a unique sas3Delta phenotype, suggesting that modification of this residue is important for NuA3 function. The interaction of NuA3 with chromatin is dependent on the Set1p and Set2p histone methyltransferases, as well as their substrates, histone H3 lysines 4 and 36, respectively. These results confirm that NuA3 is functioning as a histone acetyltransferase in vivo and that histone H3 methylation provides a mark for the recruitment of NuA3 to nucleosomes.     

Dynamic Acetylation of All Lysine 4–Methylated Histone H3 in the Mouse Nucleus: Analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.     

The Dnmt3a PWWP Domain Reads Histone 3 Lysine 36 Trimethylation and Guides DNA Methylation

The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2′-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.     

Histone H3 lysine 4 mono-methylation does not require ubiquitination of histone H2B

The yeast Set1-complex catalyzes histone H3 lysine 4 (H3K4) methylation. Using N-terminal Edman sequencing, we determined that 50% of H3K4 is methylated and consists of roughly equal amounts of mono, di and tri-methylated H3K4. We further show that loss of either Paf1 of the Paf1 elongation complex, or ubiquitination of histone H2B, has only a modest effect on bulk histone mono-methylation at H3K4. Despite the fact that Set1 recruitment decreases in paf1delta cells, loss of Paf1 results in an increase of H3K4 mono-methylation at the 5' coding region of active genes, suggesting a Paf1-independent targeting of Set1. In contrast to Paf1 inactivation, deleting RTF1 affects H3K4 mono-methylation at the 3' coding region of active genes and results in a decrease of global H3K4 mono-methylation. Our results indicate that the requirements for mono-methylation are distinct from those for H3K4 di and tri-methylation, and point to differences among members of the Paf1 complex in the regulation of H3K4 methylation.     

Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.     

Overlapping Functions between SWR1 Deletion and H3K56 Acetylation in Candida albicans

Nucleosome destabilization by histone variants and modifications has been implicated in the epigenetic regulation of gene expression, with the histone variant H2A.Z and acetylation of H3K56 (H3K56ac) being two examples. Here we find that deletion of SWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin in exchange for H2A, promotes epigenetic white-opaque switching in Candida albicans. We demonstrate through nucleosome mapping that SWR1 is required for proper nucleosome positioning on the promoter of WOR1, the master regulator of switching, and that its effects differ in white and opaque cells. Furthermore, we find that H2A.Z is enriched adjacent to nucleosome-free regions at the WOR1 promoter in white cells, suggesting a role in the stabilization of a repressive chromatin state. Deletion of YNG2, a subunit of the NuA4 H4 histone acetyltransferase (HAT) that targets SWR1 activity through histone acetylation, produces a switching phenotype similar to that of swr1, and both may act downstream of the GlcNAc signaling pathway. We further uncovered a genetic interaction between swr1 and elevated H3K56ac with the discovery that the swr1 deletion mutant is highly sensitive to nicotinamide. Our results suggest that the interaction of H2A.Z and H3K56ac regulates epigenetic switching at the nucleosome level, as well as having global effects.     

Combined Interactions of Plant Homeodomain and Chromodomain Regulate NuA4 Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1Δ set2Δ mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site.     

Methylations of histone H3 lysine 9 and lysine 36 are functionally linked to DNA replication checkpoint control in fission yeast

Recently, histone H4 lysine 20 and H3 lysine 79 methylations were functionally linked to DNA damage checkpoint. The crosstalk between histone methylation and the S-M checkpoint, however, has remained unclear. Here, we show that H3 lysine 9 (K9) and lysine 36 (K36) methylations catalyzed by two histone methyltransferases Clr4 and Set2 are involved in hydroxyurea (HU)-induced replication checkpoint. The clr4-set2 double mutants besides histone H3-K9 and K36 double mutants exhibited HU-sensitivity, a defective HU-induced S-M checkpoint, and a significant reduction of HU-induced phosphorylation of Cdc2. Intriguingly, the clr4-set2 double mutations impaired the HU-induced accumulation of a mitotic inhibitor Mik1. Double mutants in Alp13 and Swi6, which can specifically bind to H3-K36 and K9 methylations, exhibited phenotypes similar to those of the clr4-set2 mutants. Together, these findings suggest that methylations of histone H3-K9 and K36 by Clr4 and Set2 are functionally linked to DNA replication checkpoint via accumulation of Mik1.     

Effects of histone acetylation by Piccolo NuA4 on the structure of a nucleosome and the interactions between two nucleosomes

We characterized the effect of histone acetylation on the structure of a nucleosome and the interactions between two nucleosomes. In this study, nucleosomes reconstituted with the Selex "Widom 601" sequence were acetylated with the Piccolo NuA4 complex, which acetylates mainly H4 N-terminal tail lysine residues and some H2A/H3 N-terminal tail lysine residues. Upon the acetylation, we observed directional unwrapping of nucleosomal DNA that accompanies topology change of the DNA. Interactions between two nucleosomes in solution were also monitored to discover multiple transient dinucleosomal states that can be categorized to short-lived and long-lived (～1 s) states. The formation of dinucleosomes is strongly Mg(2+)-dependent, and unacetylated nucleosomes favor the formation of long-lived dinucleosomes 4-fold as much as the acetylated ones. These results suggest that the acetylation of histones by Piccolo NuA4 disturbs not only the structure of a nucleosome but also the interactions between two nucleosomes. Lastly, we suggest a structural model for a stable dinucleosomal state where the two nucleosomes are separated by ～2 nm face-to-face and rotated by 34° with respect to each other.     

Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo

Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin-lysine linkage and ubiquitination cycles are not critical for trans-histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation.     

Both combinatorial K4me0-K36me3 marks on sister histone H3s of a nucleosome are required for Dnmt3a-Dnmt3L mediated de novo DNA methylation

A nucleosome contains two copies of each histone H2A,H2B,H3 and H4.Histone H3 K4me0 and K36me3are two key chromatin marks for de novo DNA methylation catalyzed by DNA methyltransferases in mammals.However,it remains unclear whether K4me0 and K36me3 marks on both sister histone H3s regulate de novo DNA methylation independently or cooperatively.Here,taking advantage of the bivalent histone H3 system in yeast,we examined the contributions of K4 and K36 on sister histone H3s to genomic DNA methylation catalyzed by ectopically co-expressed murine Dnmt3a and Dnmt3L.The results show that lack of both K4me0 and K36me3 on one sister H3 tail,or lack of K4me0 and K36me3 on respective sister H3s results in a dramatic reduction of 5mC,revealing a synergy of two sister H3s in DNA methylation regulation.Accordingly,the Dnmt3a or Dnmt3L mutation that disrupts the interaction of Dnmt3aADD domain-H3K4me0,Dnmt3LADD domain-H3K4me0,orDnmt3aPWWP domain-H3K36me3 causes a significant reduction of DNA methylation.These results support the model that each heterodimeric Dnmt3a-Dnmt3L reads both K4me0 and K36me3 marks on one tail of sister H3s,and the dimer of heterodimeric Dnmt3a-Dnmt3L recognizes two tails of sister histone H3s to efficiently execute de novo DNA methylation.