SUMMARY DNA methylation and histone modification exert epigenetic control over gene expression. CHG methylation by CHROMOMETHYLASE3 (CMT3) depends on histone H3K9 dimethylation (H3K9me2), but the mechanism underlying this relationship is poorly understood. Here, we report multiple lines of evidence that CMT3 interacts with H3K9me2-containing nucleosomes. CMT3 genome locations nearly perfectly correlated with H3K9me2 and CMT3 stably associated with H3K9me2-containing nucleosomes. Crystal structures of maize CMT3 homologue, ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both BAH- and chromo-domains. The structures reveal an aromatic cage within both BAH- and chromo-domains as interaction interfaces that capture H3K9me2. Mutations that abolish either interaction disrupt CMT3 binding to nucleosomes, and show a complete loss of CMT3 activity in vivo. Our study establishes dual recognition of H3K9me2 marks by BAH- and chromo-domains, and reveals a novel mechanism of interplay between DNA methylation and histone modification.

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.[Keywords: DNMT1; G9a; SUV39H1; methylation; chromatin; replication] Supplemental material is available at http://www.genesdev.org.

Inheritance of epigenetic information encoded by cytosine DNA methylation patterns is crucial for mammalian cell survival, in large part through the activity of the maintenance DNA methyltransferase (DNMT1). Here, we show that SET7, a known histone methyltransferase, is involved in the regulation of protein stability of DNMT1. SET7 colocalizes and directly interacts with DNMT1 and specifically monomethylates Lys-142 of DNMT1. Methylated DNMT1 peaks during the S and G 2 phases of the cell cycle and is prone to proteasome-mediated degradation. Overexpression of SET7 leads to decreased DNMT1 levels, and siRNA-mediated knockdown of SET7 stabilizes DNMT1. These results demonstrate that signaling through SET7 represents a means of DNMT1 enzyme turnover.DNA methyltransferase ͉ methylated lysine ͉ proteasome ͉ protein degradation M ammalian DNA methylation is essential for development and is controlled by a variety of factors including 3 active DNA cytosine methyltransferases (DNMT1, DNMT3A, and DNMT3B) and a methyltransferase-like protein, DNMT3L (1-4). DNMT1 encodes the maintenance DNA methyltransferase (DNMT) responsible for methylating hemimethylated CpG sites shortly after DNA replication, and it is assisted by an accessory factor capable of recognizing hemimethylated DNA called UHRF1 (5, 6). Aberrant DNA methylation of CpG islandcontaining promoters leads to permanent silencing of genes in both physiological and pathological contexts and specifically in cancer cells (7). In cancer cells, disruption of DNMT1 resulted in hemimethylation of a fifth of the CpG sites in the genome and activation of the G 2 /M checkpoint, leading to arrest in the G 2 phase of the cell cycle (8). Apart from DNA methylationmediated gene silencing, DNMT1 also binds to several transcriptional inhibitors and represses gene expression in a DNA methylation-independent manner (9-11). Pharmacological inhibitors of DNMT1 [5-azacytidine (5-aza-CR) and its deoxy analog, 5-aza-2Ј-deoxycytidine (5-aza-CdR)] get incorporated into newly-synthesized DNA (12, 13). Once incorporated into DNA, these compounds form covalent complexes with DNMTs, thereby depleting active enzymes (14, 15) and activating gene expression (16). Recently, 5-aza-CdR-induced depletion of DNMT1 was shown to be mediated by proteasomal pathways in mammalian nuclei (17). However, little is known about other factors regulating DNMT1 levels in cells. Here, we show that DNMT1 stability is regulated by protein methylation coupled to proteasome-mediated protein degradation through the protein methyltransferase activity of SET7. Results DNMT1 Colocalizes and Associates with and Is Methylated by SET7.We used gel filtration and Western blot analysis to analyze DNMT1 from nuclear extracts. Using a highly-specific antibody we observed a major species of DNMT1 at 185 kDa and a higher molecular mass minor species (Fig. 1A). It seemed likely that this minor species of DNMT1 may be posttranslationally modified (18). To test whether DNMT1 might be modified by protein methylation, recombinant DNMT1 wa...

SUMMARY Mediator occupies a central role in RNA polymerase II transcription as a sensor, integrator, and processor of regulatory signals that converge on protein-coding gene promoters. Compared to its role in gene activation, little is known regarding the molecular mechanisms and biological implications of Mediator as a transducer of repressive signals. Here, we describe a protein interaction network required for extra-neuronal gene silencing comprising Mediator, G9a histone methyltransferase, and the RE1 silencing transcription factor (REST; also known as neuron restrictive silencing factor, NRSF). We show that the MED12 interface in Mediator links REST with G9a-dependent histone H3K9 di-methylation to suppress neuronal genes in non-neuronal cells. Notably, missense mutations in MED12 causing the X-linked mental retardation (XLMR) disorders FG syndrome and Lujan syndrome disrupt its REST corepressor function. These findings implicate Mediator in epigenetic restriction of neuronal gene expression to the nervous system and suggest a pathologic basis for MED12-associated XLMR involving impaired REST-dependent neuronal gene regulation.

Lysine-specific murine histone H3 methyltransferase, G9a, was expressed and purified in a baculovirus expression system. The primary structure of the recombinant enzyme is identical to the native enzyme. Enzymatic activity was favorable at alkaline conditions (>pH 8) and low salt concentration and virtually unchanged between 25 and 42°C. Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un-or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9). In vitro methylation of the H3 tail peptide resulted in trimethylation of Lys-9 and the reaction is processive. The turnover number (k cat ) for methylation was 88 and 32 h ؊1 on the wild type and K4AK9 histone H3 tail, respectively. The Michaelis constants for wild type and K4AK9 (K m pep ) were 0.9 and 1.0 M and for S-adenosyl-L-methionine (K m AdoMet ) were 1.8 and 0.6 M, respectively. Comparable kinetic constants were obtained for recombinant histone H3. The conversion of K4AK9 di-to trimethyl-lysine was 7-fold slower than methyl group addition to unmethylated peptide. Preincubation studies showed that G9a-AdoMet and G9a-peptide complexes are catalytically active. Initial velocity data with peptide and S-adenosyl-L-methionine (AdoMet) and product inhibition studies with S-adenosyl-L-homocysteine were performed to assess the kinetic mechanism of the reaction. Double reciprocal plots and preincubation studies revealed S-adenosyl-L-homocysteine as a competitive inhibitor to AdoMet and mixed inhibitor to peptide. Trimethylated peptides acted as a competitive inhibitor to substrate peptide and mixed inhibitor to AdoMet suggesting a random mechanism in a Bi Bi reaction for recombinant G9a where either substrate can bind first to the enzyme, and either product can release first.Histones participate in packaging of eukaryotic DNA. Amino-terminal tails of histones are exposed in packed chromatin and are thus amenable to various post-translational modifications. Histone H3 methylation in mammals is implicated in epigenetic gene regulation (1). Other post-translational modifications namely, acetylation and phosphorylation of histone H3 and H4 N-terminal tails are also documented (2, 3). Histone tail modifications are involved in transcriptional activation or repression of chromatin. Generally, acetylated histones mark transcriptionally active chromatin and hypoacetylated chromatin are silent. Furthermore, histone methylation can be a marker for transcriptionally active or inactive segments of the genome (4). Methylation of histone H3 lysine 9 (H3-K9) is a hallmark of silent chromatin and is globally distributed throughout the heterochromatic regions, such as centromeres and telomeres (5). In the inactivated X chromosome of mammals (6) and transcriptionally silent genes in cancer cells frequent H3-K9 methylation is observed (7).Links between histone methylation and DNA methylation are emerging and have been demonstrated in Neurospora crassa and in plants. Experimental ev...

DNA (cytosine-5)-methyltransferase (DNMT) 1 participates in transcriptional repression of genes by methylation-dependent and -independent mechanisms. Here, DNMT1 is shown to bind p53 and colocalize in the nucleus. DNMT1-mediated methylation is stimulated by p53 in vitro. Upon p53 induction, a reporter construct containing the antiapoptotic gene survivin promoter, which contains a natural p53 binding site, was methylated in WT HCT116 cells but not in DNMT1 null or p53 null cells. Endogenous survivin gene repression involves cooperation between DNMT1 and p53 and is relieved by introduction of DNMT1-or p53-specific small inhibitory RNA. DNMT1 null cells did not exhibit a significant repressive effect for p53 responsive survivin and cdc25C gene expression compared with the parental cells. Normal human fibroblasts also exhibited similar DNMT1-and p53-mediated methylation of the survivin promoter, suggesting cooperation between p53 and DNMT1 in gene silencing.doxorubicin ͉ survivin ͉ small inhibitory RNA

Methylation of lysine residues on histones participates in transcriptional gene regulation. Lysine 9 methylation of histone H3 is a transcriptional repression signal, mediated by a family of SET domain containing AdoMet-dependent enzymes. G9a methyltransferase is a euchromatic histone H3 lysine 9 methyltransferase. Here, G9a is shown to methylate other cellular proteins, apart from histone H3, including automethylation of K239 residue. Automethylation of G9a did not impair or activate the enzymatic activity in vitro. The automethylation motif of G9a flanking target K239 (ARKT) has similarity with histone H3 lysine 9 regions (ARKS), and is identical to amino acids residues in EuHMT (ARKT) and mAM (ARKT). Under steady-state kinetic assay conditions, full-length G9a methylates peptides representing ARKS/T motif of H3, G9a, mAM and EuHMT efficiently. Automethylation of G9a at ARKT motif creates a binding site for HP1 class of protein and mutation of lysine in the motif impairs this binding. In COS-7 cells GFP fusion of the wild-type G9a co-localized with HP1α and HP1γ isoforms whereas the G9a mutant with K239A displayed poor co-localization. Thus, apart from transcriptional repression and regulatory roles of lysine methylation, the non-histone protein methylation may create binding sites for cellular protein–protein interactions.

Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.