Hang Gyeong Chin

Regulation of DNMT1 stability through SET7-mediated lysine methylation in mammalian cells

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 G2 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.

Dual Binding of Chromomethylase Domains to H3K9me2-Containing Nucleosomes Directs DNA Methylation in Plants

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 homolog ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both bromo adjacent homology (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 distinct mechanism of interplay between DNA methylation and histone modification.

Mediator links epigenetic silencing of neuronal gene expression with x-linked mental retardation.

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 extraneuronal gene silencing comprising Mediator, G9a histone methyltransferase, and the RE1 silencing transcription factor (REST; also known as neuron restrictive silencer factor, NRSF). We show that the MED12 interface in Mediator links REST with G9a-dependent histone H3K9 dimethylation to suppress neuronal genes in nonneuronal 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.

Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes

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.

Substrate Specificity and Kinetic Mechanism of Mammalian G9a Histone H3 Methyltransferase

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 (kcat) 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 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((K_{m}^{\mathrm{pep}})\) \end{document}) were 0.9 and 1.0 μm and for S-adenosyl-l-methionine (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{m}^{\mathrm{AdoMet}}\) \end{document}) were 1.8 and 0.6 μm, 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.

Automethylation of G9a and its implication in wider substrate specificity and HP1 binding

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.

Molecular mechanisms of transactivation and doxorubicin-mediated repression of survivin gene in cancer cells.

Human maintenance DNA cytosine methyltransferase (DNMT1) regulates gene expression in a methylation-dependent and -independent manner. Anti-apoptotic survivin gene down-regulation is mediated by p53 recruitment of DNMT1 to its promoter. Survivin inhibits programmed cell death, regulates cell division, and is expressed in cancer cells. The survivin gene promoter is CG-rich containing several Sp1 canonical, Sp1-like, cell cycle-dependent element/cell cycle gene homology region, and p53-binding sites. Here we demonstrate that Sp1 transcription factor(s) play a role in transcriptional activation of the survivin promoter in Drosophila and human cells. Sp1 inhibition in vivo by mithramycin A leads to down-regulation of a luciferase reporter driven by the human survivin promoter in transfected cells. Mithramycin A or Sp1-specific short interfering RNA down-regulated the endogenous survivin gene expression, confirming Sp1 as the primary determinant for transcriptional activation. Furthermore, immobilized DNMT1 ligand bound to seven consensus amino acids corresponding to the N-terminal region of the Sp class of transcription factors in a phage display analysis. In the co-immunoprecipitation assay, the endogenous Sp1 or Sp3 pulled down DNMT1 and methyltransferase activity. Similarly, a glutathione S-transferase pulldown assay between DNMT1 and Sp1 demonstrates a direct interaction between the two proteins. Fluorescent fusions of DNMT1 and Sp1 co-localized in the mammalian nucleus, thus supporting binary complex formation between both the proteins. The kinetics of survivin promoter occupancy via chromatin immunoprecipitation following doxorubicin treatment show the presence of Sp1 and gradual accumulation of transcriptional repressors p53, DNMT1, histone methyltransferase G9a, and HDAC1 onto the promoter along with histone H3K9me2. These data suggest that the Sp1 transcription factor acts as a platform for recruitment of transcriptional repressors.

SET7/9 mediated methylation of non-histone proteins in mammalian cells.

Lysine methylation has emerged as a major posttranslational modification for histones in eukaryotes. Crosstalk between lysine methylation and other posttranslational modifications is crucial for transcriptional gene regulation and epigenetic inheritance. In addition to histones, several other cellular proteins including transcription factors, tumor suppressor and membrane-associated receptors are subject to lysine methylation. SET7/9 plays a prominent role in lysine methylation of histone and non-histone proteins. Recent reports have suggested a new mechanism of epigenetic gene regulation via SET7/9 modulated DNMT1 methylation. In this mechanism, SET7/9 may methylate DNMT1 leading to proteasome mediated protein degradation, and antagonist lysine specific demethylase (LSD), may prevent this degradation by removing the methyl mark. Thus a fine-tuning and balance between cellular SET7/9 and LSD interaction with DNMT1 may be means for epigenetic gene regulation.

A unique family of Mrr-like modification-dependent restriction endonucleases

Mrr superfamily of homologous genes in microbial genomes restricts modified DNA in vivo. However, their biochemical properties in vitro have remained obscure. Here, we report the experimental characterization of MspJI, a remote homolog of Escherichia coli’s Mrr and show it is a DNA modification-dependent restriction endonuclease. Our results suggest MspJI recognizes mCNNR (R = G/A) sites and cleaves DNA at fixed distances (N12/N16) away from the modified cytosine at the 3′ side (or N9/N13 from R). Besides 5-methylcytosine, MspJI also recognizes 5-hydroxymethylcytosine but is blocked by 5-glucosylhydroxymethylcytosine. Several other close homologs of MspJI show similar modification-dependent endonuclease activity and display substrate preferences different from MspJI. A unique feature of these modification-dependent enzymes is that they are able to extract small DNA fragments containing modified sites on genomic DNA, for example ∼32 bp around symmetrically methylated CG sites and ∼31 bp around methylated CNG sites. The digested fragments can be directly selected for high-throughput sequencing to map the location of the modification on the genomic DNA. The MspJI enzyme family, with their different recognition specificities and cleavage properties, provides a basis on which many future methods can build to decode the epigenomes of different organisms.

CXXC domain of human DNMT1 is essential for enzymatic activity.

DNA cytosine methylation is one of the major epigenetic gene silencing marks in the human genome facilitated by DNA methyltransferases. DNA cytosine-5 methyltransferase 1 (DNMT1) performs maintenance methylation in somatic cells. In cancer cells, DNMT1 is responsible for the aberrant hypermethylation of CpG islands and the silencing of tumor suppressor genes. Here we show that the catalytically active recombinant DNMT1, lacking 580 amino acids from the amino terminus, binds to unmethylated DNA with higher affinity than hemimethylated or methylated DNA. To further understand the binding domain of enzyme, we have used gel shift assay. We have demonstrated that the CXXC region (C is cysteine; X is any amino acid) of DNMT1 bound specifically to unmethylated CpG dinucleotides. Furthermore, mutation of the conserved cysteines abolished CXXC mediated DNA binding. In transfected COS-7 cells, CXXC deleted DNMT1 (DNMT1 (DeltaCXXC)) localized on replication foci. Both point mutant and DNMT1 (DeltaCXXC) enzyme displayed significant reduction in catalytic activity, confirming that this domain is crucial for enzymatic activity. A permanent cell line with DNMT1 (DeltaCXXC) displayed partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme. Thus, the CXXC domain encompassing the amino terminus region of DNMT1 cooperates with the catalytic domain for DNA methyltransferase activity.