H3 Lys4 Methylation Research Articles

Id2 gene-targeted crosstalk between Wnt and retinoid signaling regulates proliferation in human keratinocytes

We investigated the effect of all-trans-retinoic acid (atRA) on proliferation in several human skin cell lines and found that antiproliferative potency of atRA correlated with the endogenous activity of canonical Wnt signaling. In HaCaT keratinocytes, we found that atRA significantly suppressed the expression of Id2, a member of the inhibitor of differentiation family of transcription factors that regulate cell growth and differentiation. However, no apparent change in the expression of other Wnt targets, like c-Myc or cyclin D1, was observed. Retinoid-induced Id2 gene suppression was associated with decreased levels of histone H3 and H4 acetylation and histone H3 Lys-4 methylation, and with recruitment of the LSD1 demethylase at the Wnt-response element (WRE) (TCF/LEF-binding site), in the Id2 gene promoter. None of such changes was detected at the WRE of c-Myc and cyclin D1 gene promoters. Inhibition of Id2 by short interfering RNA (siRNA) had a similar effect on the proliferation of HaCaT cells as exposure to atRA, whereas anti-beta-catenin siRNA significantly inhibited its antiproliferative effect. These data suggest that downregulation of Id2 gene expression through transcriptional convergence between Wnt and retinoid signaling pathways underlies the antiproliferative effect of retinoids in keratinocytes, and provide evidence of gene-targeted crosstalk between signaling pathways.

Epigenetic Regulation of Tumor Endothelial Cell Anergy: Silencing of Intercellular Adhesion Molecule-1 by Histone Modifications

Tumors can escape from immunity by repressing leukocyte adhesion molecule expression on tumor endothelial cells and by rendering endothelial cells unresponsive to inflammatory activation. This endothelial cell anergy is induced by angiogenic growth factors and results in reduced leukocyte-vessel wall interactions, thereby attenuating infiltration of leukocytes into the tumor. This report describes a novel mechanism of endothelial cell anergy regulation. We recently reported that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have angiostatic activity. Here, we studied whether epigenetic mechanisms regulate this angiogenesis-mediated escape from immunity. We found that DNMT inhibitors 5-aza-2'-deoxycytidine and zebularine, as well as HDAC inhibitor trichostatin A, reexpressed intercellular adhesion molecule-1 (ICAM-1) on tumor-conditioned endothelial cells in vitro, resulting in restored leukocyte-endothelial cell adhesion. In addition, treatment with DNMT or HDAC inhibitors in vivo also restored ICAM-1 expression on tumor endothelial cells from two different mouse tumor models. Furthermore, leukocyte-vessel wall interactions in mouse tumors were increased by these compounds, as measured by intravital microscopy, resulting in enhanced leukocyte infiltration. We show that ICAM-1 down-regulation in tumor endothelial cells is associated with ICAM-1 promoter histone H3 deacetylation and loss of histone H3 Lys(4) methylation but not with DNA hypermethylation. In conclusion, our data show that ICAM-1 is epigenetically silenced in tumor endothelial cells by promoter histone modifications, which can be overcome by DNMT and HDAC inhibitors, suggesting a new molecular mechanism based on which novel therapeutic approaches for cancer can be pursued.

Regulation of MLL1 H3K4 methyltransferase activity by its core components

Histone H3 Lys4 (H3K4) methylation is a prevalent mark associated with transcription activation. A common feature of several H3K4 methyltransferase complexes is the presence of three structural components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. Here we report the first biochemical reconstitution of a functional four-component mixed-lineage leukemia protein-1 (MLL1) core complex. This reconstitution, combined with in vivo assays, allows direct analysis of the contribution of each component to MLL1 enzymatic activity and their roles in transcriptional regulation. Moreover, taking clues from a crystal structure analysis, we demonstrate that WDR5 mediates interactions of the MLL1 catalytic unit both with the common structural platform and with the histone substrate. Mechanistic insights gained from this study can be generalized to the whole family of SET1-like histone methyltransferases in mammals.

Dynamic and Reversible Changes in Histone H3-Lys4 Methylation and H3 Acetylation Occurring at Submergence-inducible Genes in Rice

Histone modifications such as methylation and acetylation in the chromatin surrounding a gene are thought to regulate transcriptional activity. In this study, to determine whether dynamic changes occur in histone modification on the loci of stress-responsive genes in plants, we chose rice submergence-inducible ADH1 and PDC1 genes. When submerged, the rice ADH1 and PDC1 genes were activated in a biphasic manner: the first and second inductions occurred after approximately 2 and 12 h of submergence, respectively. Their expression was transcriptionally induced as shown by increased binding of RNA polymerase II to the ADH1 and PDC1 loci during submergence. The Lys4 residues of the histone H3 proteins (H3-K4s) at both the 5'- and 3'-coding regions of ADH1 and PDC1 were found to change from a di-methylated state to a tri-methylated state at the first induction period. On the other hand, acetylation of H3 increased throughout ADH1 and PDC1 genes at the later induction period. The methylation and acetylation levels recovered to the initial levels during re-aeration. Treatment of seedlings with a histone deacetylase (HDAC) inhibitor, trichostatin A, increased acetylation of histones H3 and association of RNA polymerase II on the ADH1 and PDC1 loci, thereby increasing transcript levels of ADH1 and PDC1. Together, these results showed dynamic and reversible changes of histone H3-K4 methylation and H3 acetylation in stress-responsive genes in a higher plant in response to the appearance or disappearance of an environmental stress.

Nonadditive Regulation of FRI and FLC Loci Mediates Flowering-Time Variation in Arabidopsis Allopolyploids

Allopolyploidy is formed by combining two or more divergent genomes and occurs throughout the evolutionary history of many plants and some animals. Transcriptome analysis indicates that many genes in various biological pathways, including flowering time, are expressed nonadditively (different from the midparent value). However, the mechanisms for nonadditive gene regulation in a biological pathway are unknown. Natural variation of flowering time is largely controlled by two epistatically acting loci, namely FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FRI upregulates FLC expression that represses flowering in Arabidopsis. Synthetic Arabidopsis allotetraploids contain two sets of FLC and FRI genes originating from Arabidopsis thaliana and A. arenosa, respectively, and flower late. Inhibition of early flowering is caused by upregulation of A. thaliana FLC (AtFLC) that is trans-activated by A. arenosa FRI (AaFRI). Two duplicate FLCs (AaFLC1 and AaFLC2) originating from A. arenosa are expressed in some allotetraploids but silenced in other lines. The expression variation in the allotetraploids is associated with deletions in the promoter regions and first introns of A. arenosa FLCs. The strong AtFLC and AaFLC loci are maintained in natural Arabidopsis allotetraploids, leading to extremely late flowering. Furthermore, FLC expression correlates positively with histone H3-Lys4 methylation and H3-Lys9 acetylation and negatively with H3-Lys9 methylation, epigenetic marks for gene activation and silencing. We provide evidence for interactive roles of regulatory sequence changes, chromatin modification, and trans-acting effects in natural selection of orthologous FLC loci, which determines the fate of duplicate genes and adaptation of allopolyploids during evolution.

Dot1a-AF9 Complex Mediates Histone H3 Lys-79 Hypermethylation and Repression of ENaCα in an Aldosterone-sensitive Manner

Aldosterone is a major regulator of epithelial Na(+) absorption and acts in large part through induction of the epithelial Na(+) channel (ENaC) gene in the renal collecting duct. We previously identified Dot1a as an aldosterone early repressed gene and a repressor of ENaCalpha transcription through mediating histone H3 Lys-79 methylation associated with the ENaCalpha promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaCalpha. AF9 and Dot1a interact in vitro and in vivo as evidenced in multiple assays and colocalize in the nuclei of mIMCD3 renal collecting duct cells. Overexpression of AF9 results in hypermethylation of histone H3 Lys-79 at the endogenous ENaCalpha promoter at most, but not all subregions examined, repression of endogenous ENaCalpha mRNA expression and acts synergistically with Dot1a to inhibit ENaCalpha promoter-luciferase constructs. In contrast, RNA interference-mediated knockdown of AF9 causes the opposite effects. Chromatin immunoprecipitation assays reveal that overexpressed FLAG-AF9, endogenous AF9, and Dot1a are each associated with the ENaCalpha promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaCalpha promoter and represses ENaCalpha transcription in an aldosterone-sensitive manner. This mechanism appears to be more broadly applicable to other aldosterone-regulated genes because overexpression of AF9 alone or in combination with Dot1a inhibited mRNA levels of three other known aldosterone-inducible genes in mIMCD3 cells.

Epigenetic remodeling in colorectal cancer results in coordinate gene suppression across an entire chromosome band

We report a new mechanism in carcinogenesis involving coordinate long-range epigenetic gene silencing. Epigenetic silencing in cancer has always been envisaged as a local event silencing discrete genes. However, in this study of silencing in colorectal cancer, we found common repression of the entire 4-Mb band of chromosome 2q.14.2, associated with global methylation of histone H3 Lys9. DNA hypermethylation within the repressed genomic neighborhood was localized to three separate enriched CpG island 'suburbs', with the largest hypermethylated suburb spanning 1 Mb. These data change our understanding of epigenetic gene silencing in cancer cells: namely, epigenetic silencing can span large regions of the chromosome, and both DNA-methylated and neighboring unmethylated genes can be coordinately suppressed by global changes in histone modification. We propose that loss of gene expression can occur through long-range epigenetic silencing, with similar implications as loss of heterozygosity in cancer.

Effects of KNK437 on heat-induced methylation of histone H3 in human oral squamous cell carcinoma cells

Purpose: Methylation of H3 at lysine 4 (H3 Lys4) may be correlated with active gene trascription, whereas methylation of H3 at lysine 9 (H3 Lys9) may be linked to gene repression in murine cells and Schizosaccharomyces pombe.Methods: Using Western blot analysis, heat-induced changes were studied in two human oral cancer cell lines, HSC4 (thermoresistant cells) and KB (thermosensitive cells). Histone H3 changes were studied; in particular for H3-Lys4 and H3-Lys9 methylation combined with KNK437.Results: Heating of HSC4 cells at 45°C for 20 min and KB cells for 3 min gradually increased H3-Lys4 and H3-Lys9 methylation. Treatment of both cells with 100 µM KNK437 before or after heat-treatment inhibited methylation of H3-Lys4, while methylation of H3 Lys9 remained unaffected. Use of KNK437 either before or after heat treatment inhibited the expression of HSP70.Conclusions: The findings suggest that heat-induced methylation of histone H3 may be correlated with the induction of HSPs by heating.

Role of Histone Modifications in Marking and Activating Genes through Mitosis

The global inhibition of transcription at the mitotic phase of the cell cycle occurs together with the general displacement of transcription factors from the mitotic chromatin. Nevertheless, the DNase- and potassium permanganate-hypersensitive sites are maintained on potentially active promoters during mitosis, helping to mark active genes at this stage of the cell cycle. Our study focuses on the role of histone acetylation and H3 (Lys-4) methylation in the maintenance of the competency of these active genes during mitosis. To this end we have analyzed histone modifications across the promoters and coding regions of constitutively active, inducible, and inactive genes in mitotic arrested cells. Our results show that basal histone modifications are maintained during mitosis at promoters and coding regions of the active and inducible RNA polymerase II-transcribed genes. In addition we have demonstrated that, together with H3 acetylation and H3 (Lys-4) methylation, H4 (Lys-12) acetylation at the coding regions contributes to the formation of a stable mark on active genes at this stage of the cell cycle. Finally, analysis of cyclin B1 gene activation during mitosis revealed that the former occurs with a strong increase of H3 (Lys-4) trimethylation but not H3 or H4 acetylation, suggesting that histone methyltransferases are active during this stage. These data demonstrate a critical role of histone acetylation and H3 (Lys-4) methylation during mitosis in marking and activating genes during the mitotic stage of the cell cycle.

Silencing of the Meiotic Genes SMC1β and STAG3 in Somatic Cells by E2F6

Silencing of the Meiotic Genes SMC1β and STAG3 in Somatic Cells by E2F6

CpG-binding Protein (CXXC Finger Protein 1) Is a Component of the Mammalian Set1 Histone H3-Lys4 Methyltransferase Complex, the Analogue of the Yeast Set1/COMPASS Complex

CpG-binding protein (CXXC finger protein 1 (CFP1)) binds to DNA containing unmethylated CpG motifs and is required for mammalian embryogenesis, normal cytosine methylation, and cellular differentiation. Studies were performed to identify proteins that interact with CFP1 to gain insight into the molecular function of this protein. Immunoprecipitation and mass spectrometry reveal that human CFP1 associates with a approximately 450-kDa complex that contains the mammalian homologues of six of the seven components of the Set1/COMPASS complex, the sole histone H3-Lys4 methyltransferase in yeast. In vitro assays demonstrate that the human Set1/CFP1 complex is a histone methyltransferase that produces mono-, di-, and trimethylated histone H3 at Lys4. Confocal microscopy reveals that CFP1 and Set1 co-localize to nuclear speckles associated with euchromatin. A Set1 complex of reduced mass persists in murine embryonic stem cells lacking CFP1. These cells carry elevated levels of methylated histone H3-Lys4 and reduced levels of methylated histone H3-Lys9. Together with the previous finding of reduced levels of cytosine methylation, these data indicate that cells lacking CFP1 contain reduced levels of heterochromatin. Furthermore, ES cells lacking CFP1 exhibit a 4-fold excess of histone H3-Lys4 methylation following induction of differentiation, indicating that CFP1 restricts the activity of the Set1 histone methyltransferase complex. These results reveal a mammalian counterpart to the yeast Set1/COMPASS complex. The presence of CFP1 in this complex implicates this protein as a critical epigenetic regulator of histone modification in addition to cytosine methylation and reveals one mechanism by which this protein intersects with the epigenetic machinery.

Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27 Kip1 and p18 INK4c

Menin, the product of the Men1 gene mutated in familial multiple endocrine neoplasia type 1 (MEN1), regulates transcription in differentiated cells. Menin associates with and modulates the histone methyltransferase activity of a nuclear protein complex to activate gene expression. However, menin-dependent histone methyltransferase activity in endocrine cells has not been demonstrated, and the mechanism of endocrine tumor suppression by menin remains unclear. Here, we show that menin-dependent histone methylation maintains the in vivo expression of cyclin-dependent kinase (CDK) inhibitors to prevent pancreatic islet tumors. In vivo expression of CDK inhibitors, including p27 and p18, and other cell cycle regulators is disrupted in mouse islet tumors lacking menin. Chromatin immunoprecipitation studies reveal that menin directly associates with regions of the p27 and p18 promoters and increases methylation of lysine 4 (Lys-4) in histone H3 associated with these promoters. Moreover, H3 Lys-4 methylation associated with p27 and p18 is reduced in islet tumors from Men1 mutant mice. Thus, H3 Lys-4 methylation is a crucial function of menin in islet tumor suppression. These studies suggest an epigenetic mechanism of tumor suppression: by promoting histone modifications, menin maintains transcription at multiple loci encoding cell cycle regulators essential for endocrine growth control.

SUMO Modification Is Involved in the Maintenance of Heterochromatin Stability in Fission Yeast

Several studies have suggested that SUMO may participate in the regulation of heterochromatin, but direct evidence is lacking. Here, we present a direct link between sumoylation and heterochromatin stability. SUMO deletion impaired silencing at heterochromatic regions and induced histone H3 Lys4 methylation, a hallmark of active chromatin in fission yeast. Our findings showed that the SUMO-conjugating enzyme Hus5/Ubc9 interacted with the conserved heterochromatin proteins Swi6, Chp2 (a paralog of Swi6), and Clr4 (H3 Lys9 methyltransferase). Moreover, chromatin immunoprecipitation (ChIP) revealed that Hus5 was highly enriched in heterochromatic regions in a heterochromatin-dependent manner, suggesting a direct role of Hus5 in heterochromatin formation. We also found that Swi6, Chp2, and Clr4 themselves can be sumoylated in vivo and defective sumoylation of Swi6 or Chp2 compromised silencing. These results indicate that Hus5 associates with heterochromatin through interactions with heterochromatin proteins and modifies substrates whose sumoylations are required for heterochromatin stability, including heterochromatin proteins themselves.

Methylation of histone H4 by arginine methyltransferase PRMT1 is essential in vivo for many subsequent histone modifications

PRMT1 is a histone methyltransferase that methylates Arg3 on histone H4. When we used siRNA to knock down PRMT1 in an erythroid cell line, it resulted in nearly complete loss of H4 Arg3 methylation across the chicken beta-globin domain, which we use as a model system for studying the relationship of gene activity to histone modification. We observed furthermore a domain-wide loss of histone acetylation on both histones H3 and H4, as well as an increase in H3 Lys9 and Lys27 methylation, both marks associated with inactive chromatin. To determine whether the effect on acetylation was directly related to the loss of H4 Arg3 methylation, we performed an in vitro acetylation reaction on chromatin isolated from PRMT1-depleted cells. We found that nucleosomes purified from these cells, and depleted in methylation at Arg3, are readily acetylated by nuclear extracts from the same cells, if and only if the nucleosomes are incubated with PRMT1 beforehand. Thus, methylation of histones by PRMT1 was sufficient to permit subsequent acetylation. Consistent with earlier reports of experiments in vitro, H4 Arg3 methylation by PRMT1 appears to be essential in vivo for the establishment or maintenance of a wide range of "active" chromatin modifications.

Halogenated Thymidine Analogues Restore the Expression of Silenced Genes without Demethylation

Transcriptional silencing of tumor suppressor genes by aberrant DNA methylation is a characteristic frequently observed in cancer cells. Therefore, reversing this process is a therapeutic target against cancer. In this study, we established a screening system for silencing inhibitors with cell lines transfected by a retroviral vector containing a luciferase gene. More than 100 nucleosides were tested for antisilencing activity with a selected clone in which the silenced expression of luciferase could be recovered by 5-aza-2'-deoxycytidine. A group of halogenated thymidine analogues was found to reactivate transcription of not only the reporter retrovirus vector but also endogenous glutathione-S-transferase 1 gene, without influence to DNA hypermethylation. Gel mobility shift assay showed that 5-bromo-2'-deoxyuridine (BrdUrd) or 5-iodo-2'-deoxyuridine incorporation did not affect the binding of the methyl-CpG binding protein motif to methylated DNA. Finally, in the retroviral promoter, BrdUrd treatment increased the acetylated histone H3 level and decreased methylation of histone H3 Lys9 in accordance with recovered transcription. This study shows that halogenated thymidines have an antisilencing effect without changing DNA methylation status by interfering with step(s) between DNA methylation and histone acetylation.

Ubp10/Dot4p Regulates the Persistence of Ubiquitinated Histone H2B: Distinct Roles in Telomeric Silencing and General Chromatin

We previously discovered that the ubiquitin protease Ubp10/Dot4p is important for telomeric silencing through its interaction with Sir4p. However, the mechanism of Ubp10p action was unknown. We now provide evidence that Ubp10p removes ubiquitin from histone H2B; cells with UBP10 deleted have increased steady-state levels of H2B ubiquitination. As a consequence, ubp10delta cells also have increased steady-state levels of histone H3 Lys4 and Lys79 methylation. Consistent with its role in silencing, Ubp10p is preferentially localized to silent chromatin where its ubiquitin protease activity maintains low levels of H3 Lys4 and Lys79 methylation to allow optimal Sir protein binding to telomeres and global telomeric silencing. The ubiquitin protease Ubp8p has also been shown to remove ubiquitin from H2B, and ubp8delta cells have increased steady-state levels of H2B ubiquitination similar to those in ubp10delta cells. Unlike ubp10delta cells, however, ubp8delta cells do not have increased steady-state levels of H3 Lys4 and Lys79 methylation, nor is telomeric silencing affected. Despite their separate functions in silencing and SAGA-mediated transcription, respectively, deletion of both UBP10 and UBP8 results in a synergistic increase in the steady-state levels of H2B ubiquitination and in the number of genes with altered expression, indicating that Ubp10p and Ubp8p likely overlap in some of their target chromatin regions. We propose that Ubp10p and Ubp8p are the only ubiquitin proteases that normally remove monoubiquitin from histone H2B and, while there are regions of the genome to which each is specifically targeted, both combine to regulate the global balance of H2B ubiquitination.

Small Interfering RNAs That Trigger Posttranscriptional Gene Silencing Are Not Required for the Histone H3 Lys9 Methylation Necessary for Transgenic Tandem Repeat Stabilization in Neurospora crassa

In Neurospora crassa, the introduction of a transgene can lead to small interfering RNA (siRNA)-mediated posttranscriptional gene silencing (PTGS) of homologous genes. siRNAs can also guide locus-specific methylation of Lys9 of histone H3 (Lys9H3) in Schizosaccharomyces pombe. Here we tested the hypothesis that transgenically derived siRNAs may contemporaneously both activate the PTGS mechanism and induce chromatin modifications at the transgene and the homologous endogenous gene. We carried out chromatin immunoprecipitation using a previously characterized albino-1 (al-1) silenced strain but detected no alterations in the pattern of histone modifications at the endogenous al-1 locus, suggesting that siRNAs produced from the transgenic locus do not trigger modifications in trans of those histones tested. Instead, we found that the transgenic locus was hypermethylated at Lys9H3 in our silenced strain and remained hypermethylated in the quelling defective mutants (qde), further demonstrating that the PTGS machinery is dispensable for Lys9H3 methylation. However, we found that a mutant in the histone Lys9H3 methyltransferase dim-5 was unable to maintain PTGS, with transgenic copies being rapidly lost, resulting in reversion of the silenced phenotype. These results indicate that the defect in PTGS of the Deltadim-5 strain is due to the inability to maintain the transgene in tandem, suggesting a role for DIM-5 in stabilizing such repeated sequences. We conclude that in Neurospora, siRNAs produced from the transgenic locus are used in the RNA-induced silencing complex-mediated PTGS pathway and do not communicate with an RNAi-induced initiation of transcriptional gene silencing complex to effect chromatin-based silencing.

Maintenance of Low Histone Ubiquitylation by Ubp10 Correlates with Telomere-Proximal Sir2 Association and Gene Silencing

Low levels of histone covalent modifications are associated with gene silencing at telomeres and other regions in the yeast S. cerevisiae. Although the histone deacetylase Sir2 maintains low acetylation, mechanisms responsible for low H2B ubiquitylation and low H3 methylation are unknown. Here, we show that the ubiquitin protease Ubp10 targets H2B for deubiquitylation, helping to localize Sir2 to the telomere. Ubp10 exhibits reciprocal Sir2-dependent preferential localization proximal to telomeres, where Ubp10 serves to maintain low H2B Lys123 ubiquitylation in this region and, through previously characterized crosstalk, maintains low H3 Lys4 and Lys79 methylation in a slightly broader region. Ubp10 is also localized to the rDNA locus, a second silenced domain, where it similarly maintains low histone methylation. We compare Ubp10 to Ubp8, the SAGA-associated H2B deubiquitylase involved in gene activation, and show that telomeric and gene-silencing functions are specific to Ubp10. Our results suggest that these H2B-deubiquitylating enzymes have distinct genomic functions.

Reversible histone acetylation and deacetylation mediate genome-wide, promoter-dependent and locus-specific changes in gene expression during plant development.

Histone acetylation and deacetylation activate or repress transcription, yet the physiological relevance of reversible changes in chromatin structure and gene expression is poorly understood. We have shown that disrupting the expression of AtHD1 that encodes a putative Arabidopsis thaliana histone deacetylase induces a variety of developmental abnormalities. However, causal effects of the AtHD1 disruption on chromatin structure and gene expression are unknown. Using Arabidopsis spotted oligo-gene microarray analysis, here we report that >7% of the transcriptome was up- or downregulated in A. thaliana plants containing a T-DNA insertion in AtHD1 (athd1-t1), indicating that AtHD1 provides positive and negative control of transcriptional regulation. Remarkably, genes involved in ionic homeostasis and protein synthesis were ectopically expressed, whereas genes in ionic homeostasis, protein transport, and plant hormonal regulation were repressed in athd1-t1 leaves or flowers, suggesting a role of AtHD1 in developmental and environmental regulation of gene expression. Moreover, defective AtHD1 induced site-specific and reversible acetylation changes in H3-Lys9, H4-Lys12, and H4 tetra-lysines (residues 5, 8, 12, and 16) in homozygous recessive and heterozygous plants. Transcriptional activation was locus specific and often associated with specific acetylation sites in the vicinity of promoters, whereas gene repression did not correlate with changes in histone acetylation or correlated directly with H3-Lys9 methylation but not with DNA methylation. The data suggest that histone acetylation and deacetylation are promoter dependent, locus specific, and genetically reversible, which provides a general mechanism for reversible gene regulation responsive to developmental and environmental changes.

Methylation of H3 Lysine 4 at Euchromatin Promotes Sir3p Association with Heterochromatin

Set1p methylates lysine 4 of histone H3 and can activate transcription by recruiting the chromatin-remodeling factor Isw1p. In addition, Lys-4-methylated H3 is required for maintenance of silencing at the telomeres, rDNA, and HML locus in Saccharomyces cerevisiae. The molecular mechanism underlying the role of Set1p in silencing is not known. Here we report that euchromatic methylation of H3 Lys-4 is necessary to maintain silencing at specific heterochromatic sites. Inactivation of Set1p catalytic activity or mutation of H3 Lys-4 leads to decreased binding of the silent information regulator Sir3p at heterochromatic sites. Concomitantly, there is an increase in the amount of Sir3p bound to genes located in subtelomeric regions. Consistent with this result is the finding that in vitro, Sir3p preferentially binds histone H3 tails when methylation is absent at H3 Lys-4, a situation found in heterochromatin. The inability of Sir3p to bind methylated H3 Lys-4 tails suggests a model whereby H3 Lys-4 methylation prevents Sir3p association at euchromatic sites and therefore concentrates Sir3p at unmodified, heterochromatic regions of the genome.