Histone H2A Ubiquitination Inhibits the Enzymatic Activity of H3 Lysine 36 Methyltransferases

Histone H3 lysine 27 (H3K27) methylation and H2A monoubiquitination (ubH2A) are two closely related histone modifications that regulate Polycomb silencing. Previous studies reported that H3K27 trimethylation (H3K27me3) rarely coexists with H3K36 di- or tri-methylation (H3K36me2/3) on the same histone H3 tails, which is partially controlled by the direct inhibition of the enzymatic activity of H3K27-specific methyltransferase PRC2. By contrast, H3K27 methylation does not affect the catalytic activity of H3K36-specific methyltransferases, suggesting other Polycomb mechanism(s) may negatively regulate the H3K36-specific methyltransferase(s). In this study, we established a simple protocol to purify milligram quantities of ubH2A from mammalian cells, which were used to reconstitute nucleosome substrates with fully ubiquitinated H2A. A number of histone methyltransferases were then tested on these nucleosome substrates. Notably, all of the H3K36-specific methyltransferases, including ASH1L, HYPB, NSD1, and NSD2 were inhibited by ubH2A, whereas the other histone methyltransferases, including PRC2, G9a, and Pr-Set7 were not affected by ubH2A. Together with previous reports, these findings collectively explain the mutual repulsion of H3K36me2/3 and Polycomb modifications.     

Histone monoubiquitylation position determines specificity and direction of enzymatic cross-talk with histone methyltransferases Dot1L and PRC2

It is well established that chromatin is a destination for signal transduction, affecting many DNA-templated processes. Histone proteins in particular are extensively post-translationally modified. We are interested in how the complex repertoire of histone modifications is coordinately regulated to generate meaningful combinations of "marks" at physiologically relevant genomic locations. One important mechanism is "cross-talk" between pre-existing histone post-translational modifications and enzymes that subsequently add or remove modifications on chromatin. Here, we use chemically defined "designer" nucleosomes to investigate novel enzymatic cross-talk relationships between the most abundant histone ubiquitylation sites, H2AK119ub and H2BK120ub, and two important histone methyltransferases, Dot1L and PRC2. Although the presence of H2Bub in nucleosomes greatly stimulated Dot1L methylation of H3K79, we found that H2Aub did not influence Dot1L activity. In contrast, we show that H2Aub inhibited PRC2 methylation of H3K27, but H2Bub did not influence PRC2 activity. Taken together, these results highlight how the position of nucleosome monoubiquitylation affects the specificity and direction of cross-talk with enzymatic activities on chromatin.     

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.