H2A.Z is a histone H2A variant that is essential for viability in organisms such as Tetrahymena thermophila, Drosophila melanogaster, and mice. In Saccharomyces cerevisiae, loss of H2A.Z is tolerated, but proper regulation of gene expression is affected. Genetics and genome-wide localization studies show that yeast H2A.Z physically localizes to the promoters of genes and functions in part to protect active genes in euchromatin from being silenced by heterochromatin spreading. To date, the function of H2A.Z in mammalian cells is less clear, and evidence so far suggests that it has a role in chromatin compaction and heterochromatin silencing. In this study, we found that the bulk of H2A.Z is excluded from constitutive heterochromatin in differentiated human and mouse cells. Consistent with this observation, analyses of H2A.Z-or H2A-containing mononucleosomes show that the H3 associated with H2A.Z has lower levels of K9 methylation but higher levels of K4 methylation than those associated with H2A. We also found that a fraction of mammalian H2A.Z is monoubiquitylated and that, on the inactive X chromosomes of female cells, the majority of this histone variant is modified by ubiquitin. Finally, ubiquitylation of H2A.Z is mediated by the RING1b E3 ligase of the human polycomb complex, further supporting a silencing role of ubiquitylated H2A.Z. These new findings suggest that mammalian H2A.Z is associated with both euchromatin and facultative heterochromatin and that monoubiquitylation is a specific mark that distinguishes the H2A.Z associated with these different chromatin states.

Epigenetics is the study of heritable changes in gene expression that are not mediated at the DNA sequence level. Molecular mechanisms that mediate epigenetic regulation include DNA methylation and chromatin/histone modifications. With the identification of key histone-modifying enzymes, the biological functions of many histone posttranslational modifications are now beginning to be elucidated. Histone methylation, in particular, plays critical roles in many epigenetic phenomena. In this review, we provide an overview of recent findings that shape the current paradigms regarding the roles of histone methylation and histone variants in heterochromatin assembly and the maintenance of the boundaries between heterochromatin and euchromatin. We also highlight some of the enzymes that mediate histone methylation and discuss the stability and inheritance of this modification.

Histone H3 phosphorylation is a critical step that couples signal transduction pathways to gene regulation. To specifically assess the transcriptional regulatory functions of H3 phosphorylation, we developed an in vivo targeting approach and found that the H3 kinase MSK1 is a direct and potent transcriptional activator. Targeting of this H3 kinase to the endogenous c-fos promoter is sufficient to activate its expression without the need of upstream signaling. Moreover, targeting MSK1 to the α-globin promoter induces H3 S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Importantly, we discovered a mechanism whereby H3 S28 phosphorylation not only displaces binding of the polycomb-repressive complexes, but it also induces a methylacetylation switch of the adjacent K27 residue. Our findings show that signal transduction activation can directly regulate polycomb silencing through a specific histone code-mediated mechanism.chromatin | gene expression | phospho-acetylation | methylation H 3 phosphorylation is a downstream event for a number of signal transduction pathways in mammalian cells (reviewed in refs. 1 and 2). For example, rapid and transient phosphorylation of H3 at S10 (H3S10ph) is detected at the promoters and coding regions of immediate-early (IE) genes upon stimulation of the MAPK or p38 pathways, suggesting that this signalinginduced histone modification functions to activate transcription of IE genes. Thus far, multiple kinases, including RSK2, MSK1/ 2, PIM1, and IKKα, have been shown to directly phosphorylate H3. As a common target of diverse signaling cascades, H3 phosphorylation is thought to be a critical step translating signal transduction information to the chromatin/transcriptional regulatory machinery (3).Currently, how H3 phosphorylation is mechanistically linked to the transcriptional process is still not fully understood. H3 S10 phosphorylation can be physically coupled to acetylation of nearby lysine residues (K9 or K14), suggesting that combinations of these modifications function together to activate transcription (4-6). Specific isoforms of 14-3-3 directly bind H3S10ph, and this interaction is greatly enhanced by additional acetylation of the nearby K14 residue, further illustrating the biological relevance of specific combinations of histone modifications (7,8). Finally, recruitment of 14-3-3 through H3S10ph at the promoters of HDAC1 and several IE genes is thought to facilitate transcription induction of these genes (7, 9, 10). Binding of 14-3-3 to H3S10ph at the FOSL1 enhancer, which is located downstream of the transcription start site, initiates sequential recruitment of the histone acetyltransferase (HAT) males absent on the first (MOF), the bromodomain-containing protein 4 (BRD4), and the positive transcription elongation factor b (P-TEFb) (10). At least for this particular gene, H3 S10 phosphorylation leads to the release of the preinitiated but paused RNA polymerase II (RNAP II) and facilitates transcriptional elongation. In addition to S10, H3 is a...

Histone post-translational modifications (PTMs) often form complex patterns of combinations and cooperate to specify downstream biological processes. In order to systemically analyse combinatorial PTMs and crosstalks among histone PTMs, we have developed a novel nucleosome purification method called Biotinylation-assisted Isolation of CO-modified Nucleosomes (BICON). This technique is based on physical coupling of the enzymatic activity of a histone-modifying enzyme with in vivo biotinylation by the biotin ligase BirA, and using streptavidin to purify the co-modified nucleosomes. Analysing the nucleosomes isolated by BICON allows the identification of PTM combinations that are enriched on the modified nucleosomes and function together within the nucleosome context. We used this new approach to study MSK1-mediated H3 phosphorylation and found that MSK1 not only directly phosphorylated H3, but also induced hyperacetylation of both histone H3 and H4 within the nucleosome. Moreover, we identified a novel crosstalk pathway between H3 phosphorylation and H4 acetylation on K12. Involvement of these acetyl marks in MSK1-mediated transcription was further confirmed by chromatin immunoprecipitation assays, thus validating the biological relevance of the BICON results. These studies serve as proof-of-principle for this new technical approach, and demonstrate that BICON can be further adapted to study PTMs and crosstalks associated with other histone-modifying enzymes.