Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin

Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.     

Mapping global histone methylation patterns in the coding regions of human genes

Histone methylation patterns in the human genome, especially in euchromatin regions, have not been systematically characterized. In this study, we examined the profile of histone H3 methylation (Me) patterns at different lysines (Ks) in the coding regions of human genes by genome-wide location analyses by using chromatin immunoprecipitation linked to cDNA arrays. Specifically, we compared H3-KMe marks known to be associated with active gene expression, namely, H3-K4Me, H3-K36Me, and H3-K79Me, as well as those associated with gene repression, namely, H3-K9Me, H3-K27Me, and H4-K20Me. We further compared these to histone lysine acetylation (H3-K9/14Ac). Our results demonstrated that: first, close correlations are present between active histone marks except between H3-K36Me2 and H3-K4Me2. Notably, histone H3-K79Me2 is closely associated with H3-K4Me2 and H3-K36Me2 in the coding regions. Second, close correlations are present between histone marks associated with gene silencing such as H3-K9Me3, H3-K27Me2, and H4-K20Me2. Third, a poor correlation is observed between euchromatin marks (H3-K9/K14Ac, H3-K4Me2, H3-K36Me2, and H3-K79Me2) and heterochromatin marks (H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2). Fourth, H3-K9Me2 is neither associated with active nor repressive histone methylations. Finally, histone H3-K4Me2, H3-K4Me3, H3-K36Me2, and H3-K79Me2 are associated with hyperacetylation and active genes, whereas H3-K9Me2, H3-K9Me3, H3-K27Me2, and H4-K20Me2 are associated with hypoacetylation. These data provide novel new information regarding histone KMe distribution patterns in the coding regions of human genes.     

DSIF and RNA polymerase II CTD phosphorylation coordinate the recruitment of Rpd3S to actively transcribed genes

Histone deacetylase Rpd3 is part of two distinct complexes: the large (Rpd3L) and small (Rpd3S) complexes. While Rpd3L targets specific promoters for gene repression, Rpd3S is recruited to ORFs to deacetylate histones in the wake of RNA polymerase II, to prevent cryptic initiation within genes. Methylation of histone H3 at lysine 36 by the Set2 methyltransferase is thought to mediate the recruitment of Rpd3S. Here, we confirm by ChIP-Chip that Rpd3S binds active ORFs. Surprisingly, however, Rpd3S is not recruited to all active genes, and its recruitment is Set2-independent. However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive. Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes. Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain.     

G9a histone methyltransferase contributes to imprinting in the mouse placenta

Whereas DNA methylation is essential for genomic imprinting, the importance of histone methylation in the allelic expression of imprinted genes is unclear. Imprinting control regions (ICRs), however, are marked by histone H3-K9 methylation on their DNA-methylated allele. In the placenta, the paternal silencing along the Kcnq1 domain on distal chromosome 7 also correlates with the presence of H3-K9 methylation, but imprinted repression at these genes is maintained independently of DNA methylation. To explore which histone methyltransferase (HMT) could mediate the allelic H3-K9 methylation on distal chromosome 7, and at ICRs, we generated mouse conceptuses deficient for the SET domain protein G9a. We found that in the embryo and placenta, the differential DNA methylation at ICRs and imprinted genes is maintained in the absence of G9a. Accordingly, in embryos, imprinted gene expression was unchanged at the domains analyzed, in spite of a global loss of H3-K9 dimethylation (H3K9me2). In contrast, the placenta-specific imprinting of genes on distal chromosome 7 is impaired in the absence of G9a, and this correlates with reduced levels of H3K9me2 and H3K9me3. These findings provide the first evidence for the involvement of an HMT and suggest that histone methylation contributes to imprinted gene repression in the trophoblast.     

Human SirT1 interacts with histone H1 and promotes formation of facultative heterochromatin

We characterized human SirT1, one of the human homologs of the budding yeast Sir2p, an NAD+-dependent histone deacetylase involved in establishing repressive chromatin and increased life span. SirT1 deacetylates histone polypeptides with a preference for histone H4 lysine 16 (H4-K16Ac) and H3 lysine 9 (H3-K9Ac) in vitro. RNAi-mediated decreased expression of SirT1 in human cells causes hyperacetylation of H4-K16 and H3-K9 in vivo. SirT1 interacts with and deacetylates histone H1 at lysine 26. Using an inducible system directing expression of SirT1 fused to the Gal4-DNA binding domain and a Gal4-reporter integrated in euchromatin, Gal4-SirT1 expression resulted in the deacetylation of H4-K16 and H3-K9, recruitment of H1 within the promoter vicinity, drastically reduced reporter expression, and loss of H3-K79 methylation, a mark restricting silenced chromatin. We propose a model for SirT1-mediated heterochromatin formation that includes deacetylation of histone tails, recruitment and deacetylation of histone H1, and spreading of hypomethylated H3-K79 with resultant silencing.     

Molecular basis of the interaction of Saccharomyces cerevisiae Eaf3 chromo domain with methylated H3K36

Eaf3 is a component of both NuA4 histone acetyltransferase and Rpd3S histone deacetylase complexes in Saccharomyces cerevisiae. It is involved in the regulation of the global pattern of histone acetylation that distinguishes promoters from coding regions. Eaf3 contains a chromo domain at the N terminus that can bind to methylated Lys-36 of histone H3 (H3K36). We report here the crystal structures of the Eaf3 chromo domain in two truncation forms. Unlike the typical HP1 and Polycomb chromo domains, which contain a large groove to bind the modified histone tail, the Eaf3 chromo domain assumes an autoinhibited chromo barrel domain similar to the human MRG15 chromo domain. Compared with other chromo domains, the Eaf3 chromo domain contains a unique 38-residue insertion that folds into two short beta-strands and a long flexible loop to flank the beta-barrel core. Both isothermal titration calorimetry and surface plasmon resonance studies indicate that the interaction between the Eaf3 chromo domain and the trimethylated H3K36 peptide is relatively weak, with a K(D) of approximately 10(-4) m. NMR titration studies demonstrate that the methylated H3K36 peptide is bound to the cleft formed by the C-terminal alpha-helix and the beta-barrel core. Site-directed mutagenesis study and in vitro binding assay results show that the conserved aromatic residues Tyr-23, Tyr-81, Trp-84, and Trp-88, which form a hydrophobic pocket at one end of the beta-barrel, are essential for the binding of the methylated H3K36. These results reveal the molecular mechanism of the recognition and binding of the methylated H3K36 by Eaf3 and provide new insights into the functional roles of the Eaf3 chromo domain.     

Homodimeric PHD Domain-containing Rco1 Subunit Constitutes a Critical Interaction Hub within the Rpd3S Histone Deacetylase Complex

Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way.