Cytosine DNA methyltransferases and CWG islands in Pisum sativum

The aim of this project was to characterise the DNA methyltransferases in plants using Pisum sativum as a model system. In order to purify and assess the target specificity of various forms, I applied the partially purified methyltransferase to a MonoQ column, and fractionated two different forms of the DNA methyltransferase. One, a CG methyltransferase, was 140 kDa in size and appeared to be unstable. This protein only methylates CG and Cl oligonucleotide duplexes. The other enzyme, a 110 kDa protein, methylates 5'-CAG-3' and 5'-CTG-3'trinucleotide targets but not 5'- CCG-3' or 5'-CGG-3' sequences, as revealed by in vitro methylation followed by Maxam-Gilbert chemical sequencing of the methylated strand in the model substrates. I fully purified this second methyltransferase using a CNG hemimethylated DNA affinity chromatography column. The purified protein gave two distinct bands on acrylamide gel, one at 110 and the other 100 kDa. This exciting observation opened up two different avenues in my research project, one to understand more about the targets of CWG methylation and the other to understand more about the enzyme itself. CsCl purified nuclear DNA was digested with several restriction endonuclases, which were sensitive to cytosine methylation such as Pstl, Pvull and EcoRll. After end labelling two to three size range of fragments were produced. I gel-purified and cloned these fragments, which arose from the unmethylated region of the genome. The sequence analysis of fifty such Pstl tiny fragment (PTF) clones shows that these DNAs are rich in unmethylated CWGs and are deficient in CG dinucleotides indicating the occurrence of CWG islands in pea. Occasional unmethylated CGsare found in CWG islands Analysis of the 5' region of 40 published plant gene sequences from the GenEMBL database does not shows any particular trend between CG and CWG content of the plant promoters. It is estimated that 5% of the plant genes are associated with CWG islands, in contrast to most of them being associated with CG islands. To address the biological role of CG vs CWG methylation, I performed transient gene expression studies in collaboration with Dr. Nigel Urwin. Plasmid constructs containing promoters of either CAMV35S or rbcs2 fused to a CAT reporter gene were in vitro methylated and electroporated in to bean protoplasts. Methylation of CWG sequences in the rbcs2 promoter leads to a dramatic inhibition of CAT gene expression suggesting the possible involvement of CNG methyltransferase (MTase) in gene regulation. In order to understand the structure, function and pattern of methyltransferase gene expression during plant development, I have isolated a series of overlapping clones from pea cDNA libraries. The assembled nucleic acid sequence is 5 kb. The deduced amino acid sequence has an open reading frame of 1560 amino acids with a predicted protein of 180 kDa. Like other higher eukaryotic DNA MTase this protein has an N terminal domain fused with a catalytic domain with a short linker which, in case of pea, is RKKKG. The pea enzyme has eight of the ten conserved motifs found in prokaryotic enzymes. The C terminal (catalytic domain) of the protein is homologous to both vertebrate and plant enzymes. However, the N-terminal has poor homology with the vertebrate enzymes. The unique feature of the N terminal region is the presence of several, putative nuclear localisation signals that may also be the sites of action for proteases. The presence of a Zn binding domain on this protein makes it different from the Arabidopsis protein. An uncharacterised acidic domain is also present in the protein. By Southern analysis I concluded that the MTase gene is a single copy gene and Northern analysis shows it to be mostly expressed in the meristematic tissue. Amino acid sequence alignment with the N terminal sequence of the CWG MTase suggests that this protein could be a processed product of the 180 kDa protein

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 HP1alpha and HP1gamma 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

Transcription factor LSF facilitiates lysine methylation of α-tubulin by microtubule-associated SET8

Microtubules are critical for mitosis, cell motility, and protein and organelle transport, and are a validated target for anticancer drugs. However, tubulin regulation and recruitment in these cellular processes is less understood. Post-translational modifications of tubulin are proposed to regulate microtubule functions and dynamics. Although many such modifications have been investigated, tubulin methylations and enzymes responsible for methylation have only recently begun to be described. Here we report that N-lysine methyl transferase KMT5A (SET8/PR-Set7), which methylates histone H4K20, also methylates α-tubulin. Furthermore, the transcription factor LSF binds both tubulin and SET8, and enhances α-tubulin methylation in vitro, countered by FQI1, a specific small molecule inhibitor of LSF. Thus, the three proteins SET8, LSF, and tubulin, all essential for mitotic progression, interact with each other. Overall, these results point to dual functions for both SET8 and LSF not only in chromatin regulation, but also for cytoskeletal modification.First author draf

Transcription factor LSF-DNMT1 complex dissociation by FQI1 leads to aberrant DNA methylation and gene expression

The transcription factor LSF is highly expressed in hepatocellular carcinoma (HCC) and promotes oncogenesis. Factor quinolinone inhibitor 1 (FQI1), inhibits LSF DNA-binding activity and exerts anti-proliferative activity. Here, we show that LSF binds directly to the maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1) and its accessory protein UHRF1 both in vivo and in vitro. Binding of LSF to DNMT1 stimulated DNMT1 activity and FQI1 negated the methyltransferase activation. Addition of FQI1 to the cell culture disrupted LSF bound DNMT1 and UHRF1 complexes, resulting in global aberrant CpG methylation. Differentially methylated regions (DMR) containing at least 3 CpGs, were significantly altered by FQI1 compared to control cells. The DMRs were mostly concentrated in CpG islands, proximal to transcription start sites, and in introns and known genes. These DMRs represented both hypo and hypermethylation, correlating with altered gene expression. FQI1 treatment elicits a cascade of effects promoting altered cell cycle progression. These findings demonstrate a novel mechanism of FQI1 mediated alteration of the epigenome by DNMT1-LSF complex disruption, leading to aberrant DNA methylation and gene expression.We would like to thank Drs. Donald Comb, Rich Roberts, William Jack and Clotilde Carlow at New England Biolabs Inc. for research support and encouragement. The authors thank Dr. Lauren Brown (Boston University Center for Molecular Discovery) for the preparation of FQI1. UH research on this project was supported by Ignition Awards from Boston University and a Johnson & Johnson Clinical Innovator's Award through Boston University. SES research is supported by the NIH (P50 GM067041 & R24 GM111625). Research performed by HGC was partly a requirement for the MCBB graduate program at Boston University and supported by NEB. (Boston University; Johnson & Johnson Clinical Innovator's Award through Boston University; P50 GM067041 - NIH; R24 GM111625 - NIH; NEB)Published versio

5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells

Abstract Background 5-Hydroxymethylcytosine (5hmC) was recently found to be abundantly present in certain cell types, including embryonic stem cells. There is growing evidence that TET proteins, which convert 5-methylcytosine (5mC) to 5hmC, play important biological roles. To further understand the function of 5hmC, an analysis of the genome-wide localization of this mark is required. Results Here, we have generated a genome-wide map of 5hmC in human embryonic stem cells by hmeDIP-seq, in which hydroxymethyl-DNA immunoprecipitation is followed by massively parallel sequencing. We found that 5hmC is enriched in enhancers as well as in gene bodies, suggesting a potential role for 5hmC in gene regulation. Consistent with localization of 5hmC at enhancers, 5hmC was significantly enriched in histone modifications associated with enhancers, such as H3K4me1 and H3K27ac. 5hmC was also enriched in other protein-DNA interaction sites, such as OCT4 and NANOG binding sites. Furthermore, we found that 5hmC regions tend to have an excess of G over C on one strand of DNA. Conclusions Our findings suggest that 5hmC may be targeted to certain genomic regions based both on gene expression and sequence composition

PKC isoforms interact with and phosphorylate DNMT1

<p>Abstract</p> <p>Background</p> <p>DNA methyltransferase 1 (DNMT1) has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC) may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization.</p> <p>Results</p> <p>Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. <it>In vitro </it>phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, <it>in vitro </it>phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity.</p> <p>Conclusions</p> <p>Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.</p

Functional analysis of the N- and C-terminus of mammalian G9a histone H3 methyltransferase

Methylation of lysine 9 (K9) in the N-terminus tail of histone H3 (H3) in chromatin is associated with transcriptionally silenced genes and is mediated by histone methyltransferases. Murine G9a is a 1263 amino acid H3-K9 methyltransferase that possesses characteristic SET domain and ANK repeats. In this paper, we have used a series of green fluorescent protein-tagged deletion constructs to identify two nuclear localization signals (NLS), the first NLS embedded between amino acids 24 and 109 and the second between amino acids 394 and 401 of murine G9a. Our data show that both long and short G9a isoforms were capable of entering the nucleus to methylate chromatin. Full-length or N-terminus-deleted G9a isoforms were also catalytically active enzymes that methylated recombinant H3 or synthetic peptides representing the N-terminus tail of H3. In vitro methylation reactions using N-terminus tail peptides resulted in tri-methylation of K9 that remained processive, even in G9a enzymes that lacked an N-terminus region by deletion. Co-expression of G9a and H3 resulted in di- and tri-methylation of H3-K9, while siRNA-mediated knockdown of G9a in HeLa cells resulted in reduction of global H3-K9 di- and tri-methylation. A recombinant deletion mutant enzyme fused with maltose-binding protein (MBP-G9aΔ634) was used for steady-state kinetic analysis with various substrates and was compared with full-length G9a (G9aFL). Turnover numbers of MBP-G9aΔ634 for various substrates was ∼3-fold less compared with G9aFL, while their Michaelis constants (K(m)) for recombinant H3 were similar. The [Formula: see text] for MBP-G9aΔ634 was ∼2.3–2.65 μM with various substrates. Catalytic efficiencies (k(cat)/K(m)) for both MBP-G9aΔ634 and G9aFL were similar, suggesting that the N-terminus is not essential for catalysis. Furthermore, mutation of conserved amino acids R1097A, W1103A, Y1120A, Y1138A and R1162A, or the metal binding C1168A in the catalytic region, resulted in catalytically impaired enzymes, thereby confirming the involvement of the C-terminus of G9a in catalysis. Thus, distinct domains modulate nuclear targeting and catalytic functions of G9a

Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine

PvuRts1I is a modification-dependent restriction endonuclease that recognizes 5-hydroxymethylcytosine (5hmC) as well as 5-glucosylhydroxymethylcytosine (5ghmC) in double-stranded DNA. Using PvuRts1I as the founding member, we define a family of homologous proteins with similar DNA modification-dependent recognition properties. At the sequence level, these proteins share a few uniquely conserved features. We show that these enzymes introduce a double-stranded cleavage at the 3′-side away from the recognized modified cytosine. The distances between the cleavage sites and the modified cytosine are fixed within a narrow range, with the majority being 11–13 nt away in the top strand and 9–10 nt away in the bottom strand. The recognition sites of these enzymes generally require two cytosines on opposite strand around the cleavage sites, i.e. 5′-CN11–13↓N9–10G-3′/3′-GN9–10↓N11–13C-5′, with at least one cytosine being modified for efficient cleavage. As one potential application for these enzymes is to provide useful tools for selectively mapping 5hmC sites, we have compared the relative selectivity of a few PvuRts1I family members towards different forms of modified cytosines. Our results show that the inherently different relative selectivity towards modified cytosines can have practical implications for their application. By using AbaSDFI, a PvuRts1I homolog with the highest relative selectivity towards 5ghmC, to analyze rat brain DNA, we show it is feasible to map genomic 5hmC sites close to base resolution. Our study offers unique tools for determining more accurate hydroxymethylomes in mammalian cells

The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin

Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein-protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311 Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.R01 GM078240 - NIGMS NIH HHS; R24 GM111625 - NIGMS NIH HHSPublished versio

DNMT1-interacting RNAs block gene specific DNA methylation

Summary DNA methylation was described almost a century ago. However, the rules governing its establishment and maintenance remain elusive. Here, we present data demonstrating that active transcription regulates levels of genomic methylation. We identified a novel RNA arising from the CEBPA gene locus critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extended the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene selective demethylation of therapeutic targets in disease