The Set2 methyltransferase associates with Ssn6 yet Tup1-Ssn6 repression is independent of histone methylation

The Tup1-Ssn6 corepressor regulates the expression of diverse classes of genes in Saccharomyces cerevisiae. Chromatin is an important component of Tup1-Ssn6-mediated repression. Tup1 binds to underacetylated tails of histones H3 and H4, and requires multiple histone deacetylases for the repression. Here we examine if histone methylation, in addition to histone deacetylation, plays a role in Tup1-Ssn6 repression. We found that like other genes, Tup1-Ssn6 target genes exhibit increased levels of histone H3 lysine 4 trimethylation upon activation. However, deletion of individual or multiple histone methyltransferases and other SET-domain containing genes has no apparent effect on Tup1-Ssn6-mediated repression of a number of well-defined targets. Interestingly, we discovered that Ssn6 interacts with Set2. Although deletion of SET2 does not affect Tup1-Ssn6 repression of a number of target genes, Ssn6 may utilize Set2 in specific contexts to regulate gene repression.     

Reduction of Hox gene expression by histone H1 depletion

The evolutionarily conserved homeotic (Hox) genes are organized in clusters and expressed collinearly to specify body patterning during embryonic development. Chromatin reorganization and decompaction are intimately connected with Hox gene activation. Linker histone H1 plays a key role in facilitating folding of higher order chromatin structure. Previous studies have shown that deletion of three somatic H1 subtypes together leads to embryonic lethality and that H1c/H1d/H1e triple knockout (TKO) embryonic stem cells (ESCs) display bulk chromatin decompaction. To investigate the potential role of H1 and higher order chromatin folding in the regulation of Hox gene expression, we systematically analyzed the expression of all 39 Hox genes in triple H1 null mouse embryos and ESCs by quantitative RT-PCR. Surprisingly, we find that H1 depletion causes significant reduction in the expression of a broad range of Hox genes in embryos and ESCs. To examine if any of the three H1 subtypes (H1c, H1d and H1e) is responsible for decreased expression of Hox gene in triple-H1 null ESCs, we derived and characterized H1c(-/-), H1d(-/-), and H1e(-/-) single-H1 null ESCs. We show that deletion of individual H1 subtypes results in down-regulation of specific Hox genes in ESCs. Finally we demonstrate that, in triple-H1- and single-H1-null ESCs, the levels of H3K4 trimethylation (H3K4me3) and H3K27 trimethylation (H3K27me3) were affected at specific Hox genes with decreased expression. Our data demonstrate that marked reduction in total H1 levels causes significant reduction in both expression and the level of active histone mark H3K4me3 at many Hox genes and that individual H1 subtypes may also contribute to the regulation of specific Hox gene expression. We suggest possible mechanisms for such an unexpected role of histone H1 in Hox gene regulation.     

The cap binding complex influences H2B ubiquitination by facilitating splicing of the SUS1 pre-mRNA

Pre-messenger RNA splicing is carried out by a large ribonucleoprotein complex called the spliceosome. Despite the striking evolutionary conservation of the spliceosomal components and their functions, controversy persists about the relative importance of splicing in Saccharomyces cerevisiae—particularly given the paucity of intron-containing genes in yeast. Here we show that splicing of one pre-messenger RNA, SUS1, a component of the histone H2B ubiquitin protease machinery, is essential for establishing the proper modification state of chromatin. One protein complex that is intimately involved in pre-mRNA splicing, the yeast cap-binding complex, appears to be particularly important, as evidenced by its extensive and unique genetic interactions with enzymes that catalyze histone H2B ubiquitination. Microarray studies show that cap binding complex (CBC) deletion has a global effect on gene expression, and for ∼20% of these genes, this effect is suppressed when ubiquitination of histone H2B is eliminated. Consistent with this finding of histone H2B dependent effects on gene expression, deletion of the yeast cap binding complex leads to overubiquitination of histone H2B. A key component of the ubiquitin-protease module of the SAGA complex, Sus1, is encoded by a gene that contains two introns and is misspliced when the CBC is deleted, leading to destabilization of the ubiquitin protease complex and defective modulation of cellular H2B levels. These data demonstrate that pre-mRNA splicing plays a critical role in histone H2B ubiquitination and that the CBC in particular helps to establish the proper state of chromatin and proper expression of genes that are regulated at the level of histone H2B ubiquitination.     

Genome-wide studies of histone demethylation catalysed by the fission yeast homologues of mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1(+) gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1Delta strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression.     

Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression

The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing. Purified COMPASS lacking Cps60 can mono- and dimethylate but is not capable of trimethylating H3(K4). Chromatin immunoprecipitation (ChIP) studies indicate that the loss subunits of COMPASS required for histone trimethylation do not affect the localization of Set1 to chromatin for the genes tested. Collectively, our results suggest a molecular requirement for several components of COMPASS for proper histone H3 trimethylation and regulation of telomere-associated gene expression, indicating multiple roles for different forms of histone methylation by COMPASS.     

Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation

Linker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.     

Histone H3 lysine 4 monomethylation (H3K4me1) and H3 lysine 9 monomethylation (H3K9me1): Distribution and their association in regulating gene expression under hyperglycaemic/hyperinsulinemic conditions in 3T3 cells

Hyperglycemia/hyperinsulinemia are leading cause for the induction type 2 diabetes and the role of post-translational histone modifications in dysregulating the expression of genes has emerged as potential important contributor in the progression of disease. The paradoxical nature of histone H3-Lysine 4 and Lysine 9 mono-methylation (H3K4me1 and H3K9me1) in both gene activation and repression motivated us to elucidate the functional relationship of these histone modifications in regulating expression of genes under hyperglycaemic/hyperinsulinemic condition. Chromatin immunoprecipitation–microarray analysis (ChIP-chip) was performed with H3 acetylation, H3K4me1 and H3K9me1 antibody. CLUSTER analysis of ChIP-chip (Chromatin immunoprecipitation–microarray analysis) data showed that mRNA expression and H3 acetylation/H3K4me1 levels on genes were inversely correlated with H3K9me1 levels on the transcribed regions, after 30 min of insulin stimulation under hyperglycaemic condition. Interestingly, we provide first evidence regarding regulation of histone de/acetylases and de/methylases; Myst4, Jmjd2b, Aof1 and Set by H3Ac, H3K4me1 and H3K9me1 under hyperinsulinemic/hyperglycaemic condition. ChIP–qPCR analysis shows association of increased H3Ac/H3K4me1 and decreased levels of H3K9me1 in up regulation of Myst4, Jmjd2, Set and Aof1 genes. We further analyse promoter occupancy of histone modifications by ChIP walking and observed increased occupancy of H3Ac/H3K4me1 on promoter region (−1000 to −1) of active genes and H3K9me1 on inactive genes under hyperglycemic/hyperinsulinemic condition. To best of our knowledge this is the first report that shows regulation of chromatin remodelling genes by alteration in the occupancy of histone H3Ac/H3K4/K9me on both promoter and transcribed regions.     

Hir Proteins Are Required for Position-Dependent Gene Silencing in Saccharomyces cerevisiae in the Absence of Chromatin Assembly Factor I

Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans. Yeast cells lacking CAF-I (cacΔ mutants) have defects in heterochromatic gene silencing. In this study, we showed that deletion of HIR genes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci in cacΔ mutants, although hirΔ mutants had no silencing defects when CAF-I was intact. Therefore, Hir proteins are required for an alternative silencing pathway that becomes important in the absence of CAF-I. Because Hir proteins regulate expression of histone genes, we tested the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and H4 levels or in core histone stoichiometry reduced silencing in cacΔ mutants but not in wild-type cells. We therefore propose that Hir proteins contribute to silencing indirectly via regulation of histone synthesis. However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects of chromosome function may be affected by the loss of members of both gene families.     

Crosstalk of prolyl isomerases, Pin1/Ess1, and cyclophilin A.

Previous studies have indicated that Ess1/Pin1, a gene in the parvulin family of peptidyl-prolyl isomerases (PPIases), plays an important role in regulating the G(2)/M transition of the cell cycle by binding cell-cycle-regulating proteins in eukaryotic cells. Although the ess1 gene has been considered to be essential in yeast, we have isolated viable ess1 deletion mutants and demonstrated, via analysis of yeast gene expression profiles using microarray techniques, a novel regulatory role for ESS1 in the G(1) phase. Although the overall expression profiles in the tested strains (C110-1, W303, S288c, and RAY-3AD) were similar, marked changes were detected for a number of genes involved in the molecular action of ESS1. Among these, the expression levels of a cyclophilin A gene, also a member of the PPIase family, increased in the ess1 null mutant derived from C110-1. Subsequent treatment with cyclosporin A significantly retarded growth, which suggests that ESS1 and cyclophilin A are functionally linked in yeast cells and play important roles at the G(1) phase of the cell cycle.     

PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression

Histone methylation occurs on both lysine and arginine residues, and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD finger (PHD(UHRF1)), an important regulator of DNA CpG methylation, as a histone H3 unmodified arginine 2 (H3R2) recognition modality. This conclusion is based on binding studies and cocrystal structures of PHD(UHRF1) bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1's ability to repress its direct target gene expression is dependent on PHD(UHRF1) binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.     

Trimethylation of histone H3K4 is associated with the induction of fructose-inducible genes in rat jejunum

We previously reported that fructose force-feeding rapidly induces jejunal Slc2a5 gene expression in rats. In this study, we conducted microarray analyses using total RNA to identify genes upregulated in rat jejunum by fructose force-feeding. Rats were force-fed fructose, glucose or distilled water for 6h. Genes such as Slc2a5, Cdkn1c, Cabp2, Ranbp3, Vwce and Gcgr were induced by force-feeding with fructose compared with glucose or distilled water. Chromatin immunoprecipitation assays revealed that trimethylation of histone H3K4, and acetylation of histones H3 and H4, on the transcribed region of these fructose-inducible genes were enhanced by force-feeding of fructose, but not glucose or distilled water. These results suggest that the induction of genes in the rat jejunum by fructose force-feeding is coordinately regulated by histone modifications, particularly trimethylation of histone H3K4.