Histone Methylation

The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by examining histone methylation in mouse embryonic stem (ES) cells across 56 large HCNE-rich loci. We identified a specific modification pattern, termed ''bivalent domains,'' consisting of large regions of H3 lysine 27 methylation harboring smaller regions of H3 lysine 4 methylation. Bivalent domains tend to coincide with TF genes expressed at low levels. We propose that bivalent domains silence developmental genes in ES cells while keeping them poised for activation. We also found striking correspondences between genome sequence and histone methylation in ES cells, which become notably weaker in differentiated cells. These results highlight the importance of DNA sequence in defining the initial epigenetic landscape and suggest a novel chromatin-based mechanism for maintaining pluripotency.

Gene regulation in eukaryotes requires the coordinate interaction of chromatin-modulating proteins with specific transcription factors such as the androgen receptor 1 . Gene activation and repression is specifically regulated by histone methylation status at distinct lysine residues 2 . Here we show that lysine-specific demethylase 1 (LSD1; also known as BHC110) 3 co-localizes with the androgen receptor in normal human prostate and prostate tumour. LSD1 interacts with androgen receptor in vitro and in vivo, and stimulates androgen-receptor-dependent transcription. Conversely, knockdown of LSD1 protein levels abrogates androgen-induced transcriptional activation and cell proliferation. Chromatin immunoprecipitation analyses demonstrate that androgen receptor and LSD1 form chromatin-associated complexes in a ligand-dependent manner. LSD1 relieves repressive histone marks by demethylation of histone H3 at lysine 9 (H3-K9), thereby leading to de-repression of androgen receptor target genes. Furthermore, we identify pargyline as an inhibitor of LSD1. Pargyline blocks demethylation of H3-K9 by LSD1 and consequently androgen-receptor-dependent transcription. Thus, modulation of LSD1 activity offers a new strategy to regulate androgen receptor functions. Here, we link demethylation of a repressive histone mark with androgen-receptor-dependent gene activation, thus providing a mechanism by which demethylases control specific gene expression.

search Centre, Vancouver, British Columbia V6H 1Z3, Canada. in mouse embryonic fibroblasts (MEFs) that are either 4 Present address: University Medical Center, Department of Obstetwild-type (wt) or double null (dn) for both Suv39h1 and rics and Gynaecology, Catholic University Nijmegen, 6500 HC Nij-Suv39h2. Immunoblots of nuclear extracts revealed that megen, The Netherlands. 5 These authors contributed equally to this work.

DNA methylation plays an important role in mammalian development and correlates with chromatinassociated gene silencing. The recruitment of MeCP2 to methylated CpG dinucleotides represents a major mechanism by which DNA methylation can repress transcription. MeCP2 silences gene expression partly by recruiting histone deacetylase (HDAC) activity, resulting in chromatin remodeling. Here, we show that MeCP2 associates with histone methyltransferase activity in vivo and that this activity is directed against Lys 9 of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys 9 H3 methylation to the H19 gene, whose activity it represses. We show that the presence of MeCP2 on nucleosomes within the repressor region of the H19 gene (the differentially methylated domain) coincides with an increase in H3 Lys 9 methylation. Our data provide evidence that MeCP2 reinforces a repressive chromatin state by acting as a bridge between two global epigenetic modifications, DNA methylation and histone methylation.

Histone methylation regulates chromatin structure, transcription, and epigenetic state of the cell. Histone methylation is dynamically regulated by histone methylases and demethylases such as LSD1 and JHDM1, which mediate demethylation of di-and monomethylated histones. It has been unclear whether demethylases exist that reverse lysine trimethylation. We show the JmjC domain-containing protein JMJD2A reversed trimethylated H3-K9/K36 to di-but not mono-or unmethylated products. Overexpression of JMJD2A but not a catalytically inactive mutant reduced H3-K9/K36 trimethylation levels in cultured cells. In contrast, RNAi depletion of the C. elegans JMJD2A homolog resulted in an increase in general H3-K9Me3 and localized H3-K36Me3 levels on meiotic chromosomes and triggered p53-dependent germline apoptosis. Additionally, other human JMJD2 subfamily members also functioned as trimethylation-specific demethylases, converting H3-K9Me3 to H3-K9Me2 and H3-K9Me1, respectively. Our finding that this family of demethylases generates different methylated states at the same lysine residue provides a mechanism for fine-tuning histone methylation.

SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show that mice lacking Suz12, like Ezh2 and Eed mutant mice, are not viable and die during early postimplantation stages displaying severe developmental and proliferative defects. Consistent with this, we demonstrate that SUZ12 is required for proliferation of cells in tissue culture. Furthermore, we demonstrate that SUZ12 is essential for the activity and stability of the PRC2/3 complexes in mouse embryos, in tissue culture cells and in vitro. Strikingly, Suz12-deficient embryos show a specific loss of di-and trimethylated H3K27, demonstrating that Suz12 is indeed essential for EZH2 activity in vivo. In conclusion, our data demonstrate an essential role of SUZ12 in regulating the activity of the PRC2/3 complexes, which are required for regulating proliferation and embryogenesis.

activity. Thus, SET domain is believed to be a signature motif for histone lysine methyltransferase [3]. So far, all the known methylation sites are located at the N terminus of histones H3 and H4 [2, 3]. Due to the Chapel Hill, North Carolina 27599 2 Department of Biological Chemistry and limitations of detection methods, other potential modifications either at low abundance or located in the histone Molecular Pharmacology Harvard Medical School globular domain might be missed. We therefore employed mass spectrometry to identify potential novel Boston, Massachusetts 02115 3 Molecular Biology Program modification sites. Histone H3 purified from HeLa cells was subjected to tryptic digestion. Analysis of the re-Memorial Sloan Kettering Cancer Center New York, New York 10021 sulting peptides revealed that most of the major ions matched the calculated monoisotopic masses of predicted peptides (Figure 1Aa). However, one ion (m/z ϭ 1349.72) is 14.03 Da heavier than the predicted mass Summary of an H3 tryptic peptide "EIAQDFKTDLR" corresponding to amino acids 73-83. Such a mass difference could The N-terminal tails of core histones are subjected to be a result of methylation (DeltaMass database: http:// multiple covalent modifications, including acetylation, www.abrf.org/index.cfm/dm.home). To explore this methylation, and phosphorylation [1]. Similar to acetpossibility, the two peptides with masses of 1335.69 ylation, histone methylation has emerged as an imporand 1349.72 were subjected to tandem (MS/MS) mass tant player in regulating chromatin dynamics and gene spectrometric sequencing. Results shown in Figures activity [2-4]. Histone methylation occurs on arginine 1Ab and 1Ac unequivocally demonstrate that both pepand lysine residues and is catalyzed by two families of tides correspond to EIAQDFKTDLR and that the heavier proteins, the protein arginine methyltransferase family peptide is monomethylated on K79 (Figures 1Ab and and the SET-domain-containing methyltransferase 1Ac). family [3]. Here, we report that lysine 79 (K79) of H3, Based on the published nucleosome structure [15], located in the globular domain, can be methylated.

Mutations in isocitrate dehydrogenases (IDHs) have a gain-of-function effect leading to R(−)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R- and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC50) values for the R-form of 2HG varied from approximately 25 μM for the histone Nepsilon-lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

The DNA methyltransferases, Dnmts, are the enzymes responsible for methylating DNA in mammals, which leads to gene silencing. Repression by DNA methylation is mediated partly by recruitment of the methyl-CpG-binding protein MeCP2. Recently, MeCP2 was shown to associate and facilitate histone methylation at Lys9 of H3, which is a key epigenetic modi®cation involved in gene silencing. Here, we show that endogenous Dnmt3a associates primarily with histone H3-K9 methyltransferase activity as well as, to a lesser extent, with H3-K4 enzymatic activity. The association with enzymatic activity is mediated by the conserved PHD-like motif of Dnmt3a. The H3-K9 histone methyltransferase that binds Dnmt3a is likely the H3-K9 speci®c SUV39H1 enzyme since we ®nd that it interacts both in vitro and in vivo with Dnmt3a, using its PHD-like motif. We ®nd that SUV39H1 also binds to Dnmt1 and, consistent with these interactions, SUV39H1 can purify DNA methyltransferase activity from nuclear extracts. In addition, we show that HP1b, a SUV39H1-interacting partner, binds directly to Dnmt1 and Dnmt3a and that native HP1b associates with DNA methyltransferase activity. Our data show a direct connection between the enzymes responsible for DNA methylation and histone methylation. These results further substantiate the notion of a self-reinforcing repressive chromatin state through the interplay between these two global epigenetic modi®cations.

The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. Here we show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development.

Post-translational modification of chromatin has profound effects on many biological processes including transcriptional regulation, heterochromatin organization, and X-chromosome inactivation 1,2 . Recent studies indicate that methylation on specific histone lysine (K) residues participates in many of these processes 3 . Lysine methylation occurs in three distinct states, having either one (me1), two (me2) or three (me3) methyl groups attached to the amine group of the lysine side chain. These differences in modification state have an important role in defining how methylated chromatin is recognized and interpreted 4-6 . Until recently, histone lysine methylation was considered a stable modification 7,8 , but the identification of histone demethylase enzymes has demonstrated the reversibility of this epigenetic mark 9-11 . So far, all characterized histone demethylases show enzymatic activity towards lysine residues modified in the me1 or me2 state , leaving open the possibility that me3 constitutes an irreversible modification. Here we demonstrate that JHDM3A (jumonji C (JmjC)-domaincontaining histone demethylase 3A; also known as JMJD2A) is capable of removing the me3 group from modified H3 lysine 9 (H3K9) and H3 lysine 36 (H3K36). Overexpression of JHDM3A abrogates recruitment of HP1 (heterochromatin protein 1) to heterochromatin, indicating a role for JHDM3A in antagonizing methylated H3K9 nucleated events. siRNA-mediated knockdown of JHDM3A leads to increased levels of H3K9 methylation and upregulation of a JHDM3A target gene, ASCL2, indicating that JHDM3A may function in euchromatin to remove histone methylation marks that are associated with active transcription 12 .

Mutations in MECP2 are associated with Rett syndrome, an X-linked neurodevelopmental disorder. To identify genes targeted by Mecp2, we sequenced 100 in vivo Mecp2-binding sites in mouse brain. Several sequences mapped to an imprinted gene cluster on chromosome 6, including Dlx5 and Dlx6, whose transcription was roughly two times greater in brains of Mecp2-null mice compared with those of wild-type mice. The maternally expressed gene DLX5 showed a loss of imprinting in lymphoblastoid cells from individuals with Rett syndrome. Because Dlx5 regulates production of enzymes that synthesize c-aminobutyric acid (GABA), loss of imprinting of Dlx5 may alter GABAergic neuron activity in individuals with Rett syndrome. In mouse brain, Dlx5 imprinting was relaxed, yet Mecp2-mediated silent-chromatin structure existed at the Dlx5-Dlx6 locus in brains of wild-type, but not Mecp2-null, mice. Mecp2 targeted histone deacetylase 1 to a sharply defined, B1-kb region at the Dlx5-Dlx6 locus and promoted repressive histone methylation at Lys9 at this site. Chromatin immunoprecipitation-combined loop assays showed that Mecp2 mediated the silent chromatin-derived 11-kb chromatin loop at the Dlx5-Dlx6 locus. This loop was absent in chromatin of brains of Mecp2-null mice, and Dlx5-Dlx6 interacted with far distant sequences, forming distinct active chromatin-associated loops. These results show that formation of a silent-chromatin loop is a new mechanism underlying gene regulation by Mecp2.

Epigenetic modifications are crucial for proper lineage specification and embryo development. To explore the chromatin modification landscapes in human ES cells, we profiled two histone modifications, H3K4me3 and H3K27me3, by ChIP coupled with the paired-end ditags sequencing strategy. H3K4me3 was found to be a prevalent mark and occurred in close proximity to the promoters of two-thirds of total human genes. Among the H3K27me3 loci identified, 56% are associated with promoters and the vast majority of them are comodified by H3K4me3. By deep-transcript digital counting, 80% of H3K4me3 and 36% of comodified promoters were found to be transcribed. Remarkably, we observed that different combinations of histone methylations are associated with genes from distinct functional categories. These global histone methylation maps provide an epigenetic framework that enables the discovery of novel transcriptional networks and delineation of different genetic compartments of the pluripotent cell genome.

Cocaine-induced alterations in gene expression cause changes in neuronal morphology and behavior that may underlie cocaine addiction. We identified an essential role for histone 3 lysine 9 (H3K9) dimethylation and the lysine dimethyltransferase G9a in cocaine-induced structural and behavioral plasticity. Repeated cocaine administration reduced global levels of H3K9 dimethylation in the nucleus accumbens. This reduction in histone methylation was mediated through the repression of G9a in this brain region, which was regulated by the cocaine-induced transcription factor ΔFosB. Using conditional mutagenesis and viral-mediated gene transfer, we found that G9a downregulation increased dendritic spine plasticity of nucleus accumbens neurons and enhanced preference for cocaine, thereby establishing a crucial role for histone methylation in the long-term actions of cocaine.

In higher eukaryotes, histone methylation is involved in maintaining cellular identity during somatic development. As most nucleosomes are replaced by protamines during spermatogenesis, it is unclear whether histone modifications function in paternal transmission of epigenetic information. Here we show that two modifications important for Trithorax- and Polycombmediated gene regulation have methylation-specific distributions at regulatory regions in human spermatozoa. Histone H3 Lys4 dimethylation (H3K4me2) marks genes that are relevant in spermatogenesis and cellular homeostasis. In contrast, histone H3 Lys27 trimethylation (H3K27me3) marks developmental regulators in sperm, as in somatic cells. However, nucleosomes are only moderately retained at regulatory regions in human sperm. Nonetheless, genes with extensive H3K27me3 coverage around transcriptional start sites in particular tend not to be expressed during male and female gametogenesis or in preimplantation embryos. Promoters of orthologous genes are similarly modified in mouse spermatozoa. These data are compatible with a role for Polycomb in repressing somatic determinants across generations, potentially in a variegating manner.

The amino-terminal histone tails are subject to covalent post-translational modifications such as acetylation, methylation, and phosphorylation. In the histone code hypothesis, these exposed and unstructured histone tails are accessible to a repertoire of regulatory factors that specifically recognize the various modified histones, thereby generating altered chromatin structures that mediate specific biological responses. Here, we report that lysine (Lys) 79 of histone H3, which resides in the globular domain, is methylated in eukaryotic organisms. In the yeast Saccharomyces cerevisiae, Lys 79 of histone H3 is methylated by Dot1, a protein shown previously to play a role in telomeric silencing.

and against the reversible nature of methylation and consider ways in which this modification may be dis-University of Cambridge Tennis Court Road placed from the histones if methylation were to be dynamically controlled. Cambridge CB2 1QR United Kingdom Methylation of Histones The major methylation sites within histone tails are the Methylation of histones mediates transcriptional sibasic amino acid side chains of lysine and arginine resilencing at heterochromatin sites and affects regulated dues (Kouzarides, 2002; Zhang and Reinberg, 2001). The transcription at euchromatic loci. So is the methyl positions of many of these methylated residues within group a permanent mark on histones, or can it be the histone N-terminal tails have now been mapped (Figremoved by an active process necessary for regulated ure 1A), and the histone methyltransferases (HMTs) pergene expression?

Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27.

Imprinting Genome-wide reprogramming of DNA methylation oc-Developmental Genetics Programme curs in mammalian embryos. In the mouse there are two Babraham Institute phases of reprogramming, one during germline develop-Cambridge CB2 4AT ment, when DNA methylation imprints are erased [13, United Kingdom 14], and the second during preimplantation develop-2 Institute of Molecular Animal Breeding ment [15-18]. The second phase is initiated upon fertil-Gene Centre ization and includes chromatin remodeling of the male Ludwig-Maximilian University pronucleus and selective demethylation of its DNA. The Munich remaining methylation is largely lost during cleavage Germany divisions by passive demethylation. By the blastocyst 3 Research Institute of Molecular Pathology stage, de novo methylation has begun in a lineage-spe-University of Vienna cific manner such that the inner cell mass (ICM) is highly Vienna methylated while the trophectoderm is hypomethylated Austria [18]. A number of studies show that epigenetic reprogramming is severely deficient in cloned embryos, where aberrant patterns of DNA methylation [7, 8, 10, 12] and gene expression occur [11, 19, 20]. These observations Summary beg the question as to whether other epigenetic marking systems are reprogrammed during early development During differentiation, somatic nuclei acquire highly and whether they could be similarly deregulated during specialized DNA and chromatin modifications, which cloning [2-6].

Chromatin methylation is necessary for stable repression of gene expression during mammalian development. During cell division, DNMT1 maintains the DNA methylation pattern of the newly synthesized daughter strand, while G9a methylates H3K9. Here, DNMT1 is shown to directly bind G9a both in vivo and in vitro and to colocalize in the nucleus during DNA replication. The complex of DNMT1 and G9a colocalizes with dimethylated H3K9 (H3K9me2) at replication foci. Similarly, another H3K9 histone methyltransferase, SUV39H1, colocalizes with DNMT1 on heterochromatic regions of the nucleoli exclusively before cell division. Both DNMT1 and G9a are loaded onto the chromatin simultaneously in a ternary complex with loading factor PCNA during chromatin replication. Small interfering RNA (siRNA) knockdown of DNMT1 impairs DNA methylation, G9a loading, and H3K9 methylation on chromatin and rDNA repeats, confirming DNMT1 as the primary loading factor. Additionally, the complex of DNMT1 and G9a led to enhanced DNA and histone methylation of in vitro assembled chromatin substrates. Thus, direct cooperation between DNMT1 and G9a provides a mechanism of coordinated DNA and H3K9 methylation during cell division.

It has been established that regulation of chromatin structure through post-translational modification of histone proteins, primarily histone H3 phosphorylation and acetylation, is an important early step in the induction of synaptic plasticity and formation of long-term memory. In this study, we investigated the contribution of another histone modification, histone methylation, to memory formation in the adult hippocampus. We found that tri-methylation of histone H3 at lysine 4 (H3K4), an active mark for transcription, is upregulated in hippocampus one hour following contextual fear conditioning. In addition, we found that di-methylation of histone H3 at lysine 9 (H3K9), a molecular mark associated with transcriptional silencing, is increased one hour after fear conditioning and decreased twenty-four hours after context exposure alone and contextual fear conditioning. Tri-methylated H3K4 levels returned to baseline levels at twentyfour hours. We also found that mice deficient in the H3K4-specific histone methyltransferase, Mll, displayed deficits in contextual fear conditioning relative to wildtype animals. This suggests that histone methylation is required for proper long-term consolidation of contextual fear memories. Interestingly, inhibition of histone deacetylases (HDACs) with sodium butyrate (NaB) resulted in increased H3K4 tri-methylation and decreased H3K9 di-methylation in hippocampus following contextual fear conditioning. Correspondingly, we found that fear learning triggered increases in H3K4 tri-methylation at specific gene promoter regions (Zif268 and bdnf) with altered DNA methylation and MeCP2 DNA binding. Zif268 DNA methylation levels returned to baseline at twenty-four hours. Together, these data demonstrate that histone methylation is actively regulated in the hippocampus and facilitates long-term memory formation.

Histones package DNA, and post-translational modifications of histones can regulate access to DNA. Until recently, histone methylation-unlike all other histone modifications-was considered a permanent mark. The discovery of enzymes that reverse the methylation of lysines and arginines challenges our current thinking on the unique nature of histone methylation, and substantially increases the complexity of histone modification pathways.

Nuclear processes such as transcription, DNA replication, and recombination are dynamically regulated by chromatin structure. Transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent posttranslational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here, we show that RAG2 -an essential component of the RAG1/2 V(D)J recombinase, that mediates antigen receptor gene assembly 1 -contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together our results identify a novel function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence suggesting that disrupting the read-out of histone modifications can cause an inherited human disease.

The tumor suppressor proteins p16 INK4A and p14 ARF , encoded by the INK4A-ARF locus, are key regulators of cellular senescence. The locus is epigenetically silenced by the repressive H3K27me3 mark in normally growing cells, but becomes activated in response to oncogenic stress. Here, we show that expression of the histone H3 Lys 27 (H3K27) demethylase JMJD3 is induced upon activation of the RAS-RAF signaling pathway. JMJD3 is recruited to the INK4A-ARF locus and contributes to the transcriptional activation of p16 INK4A in human diploid fibroblasts. Additionally, inhibition of Jmjd3 expression in mouse embryonic fibroblasts results in suppression of p16 Ink4a and p19 Arf expression and in their immortalization.

On fertilisation, gametes undergo epigenetic reorganisation and re-establish totipotency. Here, we investigate links between chromatin remodelling and asymmetric maintenance of DNA methylation in the early mouse embryo. Using antibodies for lysine specific H3 methylation reveals that the male pronucleus is negative for di-and trimethyl H3-K9 yet the female is positive for these residues. However, the male is positive for monomethyl H3-K9 and H3-K27 and these signals increase during pronuclear maturation. Non-histone chromatin proteins of the Polycomb group are found in the paternal compartment as early as sperm decondensation. However, trimethyl H3-K27 is not observed in the male until the completion of DNA replication. Heterochromatin protein 1 beta (HP1h) is abundant in the male pronucleus, despite the absence of di-and trimethyl H3-K9, and co-localises with monomethyl H3-K9. Recent evidence identifies monomethyl H3-K9 as the preferred substrate of Suvar39h, the histone methyl transferase (HMT) responsible for heterochromatic H3-K9 trimethylation. The association of HPIh with monomethyl H3-K9 may assist in preventing further modification of H3-K9. Association of dimethylation but not trimethylation of H3-K9 with DNA methylation, in the female pronucleus, suggests a mechanistically significant link. These differences begin to provide a chromatin based explanation for paternal-specific active DNA demethylation and maternal specific protection in the mouse. D

Monoubiquitination of histone H2B, catalyzed by Rad6-Bre1, is required for methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively. The Paf1 protein complex, which associates with RNA polymerase II, is known to be required for these histone H3 methylation events. During the early elongation stage of transcription, the Paf1 complex is required for association of COMPASS with RNA polymerase II, but the role the Paf1 complex plays at the promoter has not been clear. We present evidence that the Paf1 complex is required for monoubiquitination of histone H2B at promoters. Strains deleted for several components of the Paf1 complex are defective in monoubiquitination of histone H2B, which results in the loss of methylation of lysines 4 and 79 of histone H3. We also show that Paf1 complex is required for the interaction of Rad6 and COMPASS with RNA polymerase II. Finally, we show that the Paf1 complex is required for Rad6-Bre1 catalytic activity but not for the recruitment of Rad6-Bre1 to promoters. Thus, in addition to its role during the elongation phase of transcription, the Paf1 complex appears to activate the function but not the placement of the Rad6-Bre1 ubiquitinprotein ligase at the promoters of active genes.

H3K27me3 methylation is not sufficient for silencing of targets. We suggest that the spread of H3K27me3 contributes to the mitotic heritability of Pc-G silencing, and that the loss of silencing caused by transposon insertions at plant Pc-G targets reflects impaired spreading.

The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12DCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12DCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12DCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane.

tion of histone H3 lysine 9 staining was observed between wild-type and Eed null/null TS and ES cells (data 2 Curriculum in Genetics and Molecular Biology 3 Carolina Center for Genome Sciences not shown), suggesting that the defect is specific to H3K27 methylation.

Adaptation to hypoxia is mediated through a coordinated transcriptional response driven largely by hypoxia-inducible factor 1 (HIF-1). We used ChIP-chip and gene expression profiling to identify direct targets of HIF-1 transactivation on a genome-wide scale. Several hundred direct HIF-1 targets were identified and, as expected, were highly enriched for proteins that facilitate metabolic adaptation to hypoxia. Surprisingly, there was also striking enrichment for the family of 2-oxoglutarate dioxygenases, including the jumonji-domain histone demethylases. We demonstrate that these histone demethylases are direct HIF targets, and their up-regulation helps maintain epigenetic homeostasis under hypoxic conditions. These results suggest that the coordinated increase in expression of several oxygendependent enzymes by HIF may help compensate for decreased levels of oxygen under conditions of cellular hypoxia.

Histone methylation has emerged as an important mechanism for regulating the transcriptional accessibility of chromatin. Several methyltransferases have been shown to target histone aminoterminal tails and mark nucleosomes associated with either euchromatic or heterochromatic states. However, the biochemical machinery responsible for regulating histone methylation and integrating it with other cellular events has not been well characterized. We report here the purification, molecular identification, and genetic and biochemical characterization of the Set1 protein complex that is necessary for methylation of histone H3 at lysine residue 4 in Saccharomyces cerevisiae. The seven-member 363-kDa complex contains homologs of Drosophila melanogaster proteins Ash2 and Trithorax and Caenorhabditis elegans protein DPY-30, which are implicated in the maintenance of Hox gene expression and regulation of X chromosome dosage compensation, respectively. Mutations of Set1 protein comparable to those that disrupt developmental function of its Drosophila homolog Trithorax abrogate histone methylation in yeast. These studies suggest that epigenetic regulation of developmental and sex-specific gene expression are species-specific readouts for a common chromatin remodeling machinery associated mechanistically with histone methylation.

Distinct histone lysine methylation marks are involved in transcriptional repression linked to the formation and maintenance of facultative heterochromatin, although the underlying mechanisms remain unclear. We demonstrate that the malignant-brain-tumor (MBT) protein L3MBTL1 is in a complex with core histones, histone H1b, HP1g, and Rb. The MBT domain is structurally related to protein domains that directly bind methylated histone residues. Consistent with this, we found that the L3MBTL1 MBT domains compact nucleosomal arrays dependent on mono-and dimethylation of histone H4 lysine 20 and of histone H1b lysine 26. The MBT domains bind at least two nucleosomes simultaneously, linking repression of transcription to recognition of different histone marks by L3MBTL1. Consistently, L3MBTL1 was found to negatively regulate the expression of a subset of genes regulated by E2F, a factor that interacts with Rb.

In mammals and plants, formation of heterochromatin is associated with hypermethylation of DNA at CpG sites and histone H3 methylation at lysine 9. Previous studies have revealed that maintenance of DNA methylation in Neurospora and Arabidopsis requires histone H3 methylation. A feedback loop from DNA methylation to histone methylation, however, is less understood. Its recent examination in Arabidopsis with a partial loss of function in DNA methyltransferase 1 (responsible for maintenance of CpG methylation) yielded conflicting results. Here we report that complete removal of CpG methylation in an Arabidopsis mutant null for DNA maintenance methyltransferase results in a clear loss of histone H3 methylation at lysine 9 in heterochromatin and also at heterochromatic loci that remain transcriptionally silent. Surprisingly, these dramatic alterations are not reflected in heterochromatin relaxation.

Alterations in GABAergic mRNA expression play a key role for prefrontal dysfunction in schizophrenia and other neurodevelopmental disease. Here, we show that histone H3-lysine 4 methylation, a chromatin mark associated with the transcriptional process, progressively increased at GAD1 and other GABAergic gene promoters (GAD2, NPY, SST) in human prefrontal cortex (PFC) from prenatal to peripubertal ages and throughout adulthood. Alterations in schizophrenia included decreased GAD1 expression and H3K4-trimethylation, predominantly in females and in conjunction with a risk haplotype at the 5Ј end of GAD1. Heterozygosity for a truncated, lacZ knock-in allele of mixed-lineage leukemia 1 (Mll1), a histone methyltransferase expressed in GABAergic and other cortical neurons, resulted in decreased H3K4 methylation at GABAergic gene promoters. In contrast, Gad1 H3K4 (tri)methylation and Mll1 occupancy was increased in cerebral cortex of mice after treatment with the atypical antipsychotic, clozapine. These effects were not mimicked by haloperidol or genetic ablation of dopamine D 2 and D 3 receptors, suggesting that blockade of D 2-like signaling is not sufficient for clozapine-induced histone methylation. Therefore, chromatin remodeling mechanisms at GABAergic gene promoters, including MLL1-mediated histone methylation, operate throughout an extended period of normal human PFC development and play a role in the neurobiology of schizophrenia.

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

In Arabidopsis thaliana, the promotion of flowering by cold temperatures, vernalization, is regulated via a floral-repressive MADS box transcription factor, FLOWERING LOCUS C (FLC). Vernalization leads to the epigenetic repression of FLC expression, a process that requires the polycomb group (PcG) protein VERNALIZATION 2 (VRN2) and the plant homeodomain protein VERNALIZATION IN-SENSITIVE 3 (VIN3). We demonstrate that the repression of FLC by vernalization requires homologues of other Polycomb Repressive Complex 2 proteins and VRN2. We show in planta that VRN2 and VIN3 are part of a large protein complex that can include the PcG proteins FERTILIZATION INDEPENDENT ENDOSPERM, CURLY LEAF, and SWINGER. These findings suggest a single protein complex is responsible for histone deacetylation at FLC and histone methylation at FLC in vernalized plants. The abundance of the complex increases during vernalization and declines after plants are returned to higher temperatures, consistent with the complex having a role in establishing FLC repression. epigenetics ͉ polycomb group proteins

Introductory paragraph DNA-double strand break (DSB) repair involves complex interactions between chromatin and repair proteins, including the Tip60 tumor suppressor1. Tip60 is an acetyltransferase which acetylates both histones2-5 and the ATM kinase6, 7. Inactivation of Tip60 leads to defective DNA repair2-4 and increased cancer risk8-11. However, how DNA damage activates Tip60's acetyltransferase activity is not known. Here, we show that direct interaction between Tip60's chromodomain and histone H3 trimethylated on lysine 9 (H3K9me3) at DSBs activates Tip60's acetyltransferase activity. Depletion of intracellular H3K9me3 blocks activation of Tip60's acetyltransferase activity, resulting in defective ATM activation and widespread defects in DSB repair. In addition, the ability of Tip60 to access H3K9me3 is dependent on the DNA damage induced displacement of HP1β from H3K9me3. Finally, we demonstrate that the mre11-rad50-nbs1 complex targets Tip60 to H3K9me3, and is required to activate Tip60's acetyltransferase activity. These results reveal a new function for H3K9me3 in coordinating activation of Tip60-dependent DNA repair pathways, and imply that aberrant patterns of histone methylation may contribute to cancer by altering the efficiency of DSB repair. The Tip60 acetyltransferase is a tumor suppressor gene whose expression is altered in both breast and prostate tumors8-11. Tip60 acetylates key proteins involved in the DNA damage response2-7, 12 and plays a critical role in the detection and repair DNA double strand breaks (DSBs)2-5, 7. Currently, the mechanism by which Tip60's acetyltransferase activity is increased by DNA damage is not known. Here, we examined if interaction between Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Polycomb group (PcG) proteins convey epigenetic inheritance of repressed transcriptional states. Although the mechanism of the action of PcG is not completely understood, methylation of histone H3 lysine 27 (H3K27) is important in establishing PcG-mediated transcriptional repression. We show that the plant PcG target gene PHERES1 is regulated by histone trimethylation on H3K27 residues mediated by at least two different