DNMT3L Modulates Significant and Distinct Flanking Sequence Preference for DNA Methylation by DNMT3A and DNMT3B In Vivo

The DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the −2 and +2 positions flanking the CpG site for DNMT3A, and at the −1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family.     

Flanking sequence preference modulates de novo DNA methylation in the mouse genome

Mammalian de novo DNA methyltransferases (DNMT) are responsible for the establishment of cell-type-specific DNA methylation in healthy and diseased tissues. Through genome-wide analysis of de novo methylation activity in murine stem cells we uncover that DNMT3A prefers to methylate CpGs followed by cytosines or thymines, while DNMT3B predominantly methylates CpGs followed by guanines or adenines. These signatures are further observed at non-CpG sites, resembling methylation context observed in specialised cell types, including neurons and oocytes. We further show that these preferences result from structural differences in the catalytic domains of the two de novo DNMTs and are not a consequence of differential recruitment to the genome. Molecular dynamics simulations suggest that, in case of human DNMT3A, the preference is due to favourable polar interactions between the flexible Arg836 side chain and the guanine that base-pairs with the cytosine following the CpG. By exchanging arginine to a lysine, the corresponding side chain in DNMT3B, the sequence preference is reversed, confirming the requirement for arginine at this position. This context-dependent enzymatic activity provides additional insights into the complex regulation of DNA methylation patterns.     

Profound flanking sequence preference of Dnmt3a and Dnmt3b mammalian DNA methyltransferases shape the human epigenome

Mammalian DNA methyltransferases methylate cytosine residues within CG dinucleotides. By statistical analysis of published data of the Human Epigenome Project we have determined flanking sequences of up to +/-four base-pairs surrounding the central CG site that are characteristic of high (5'-CTTGCGCAAG-3') and low (5'-TGTTCGGTGG-3') levels of methylation in human genomic DNA. We have investigated the influence of flanking sequence on the catalytic activity of the Dnmt3a and Dnmt3b de novo DNA methyltransferases using a set of synthetic oligonucleotide substrates that covers all possible +/-1 flanks in quantitative terms. Methylation kinetics experiments revealed a >13-fold difference between the preferred (RCGY) and disfavored +/-1 flanking base-pairs (YCGR). In addition, AT-rich flanks are preferred over GC-rich ones. These experimental preferences coincide with the genomic methylation patterns. Therefore, we have expanded our experimental analysis and found a >500-fold difference in the methylation rates of the consensus sequences for high and low levels of methylation in the genome. This result demonstrates a very pronounced flanking sequence preference of Dnmt3a and Dnmt3b. It suggests that the methylation pattern of human DNA is due, in part, to the flanking sequence preferences of the de novo DNA MTases and that flanking sequence preferences could be involved in the origin of CG islands. Furthermore, similar flanking sequence preferences have been found for the stimulation of the immune system by unmethylated CGs, suggesting a co-evolution of DNA MTases and the immune system.     

Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo

While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.     

DNMT1, DNMT3A and DNMT3B proteins are differently expressed in mouse oocytes and early embryos

DNA methylation is one of the epigenetic mechanisms and plays important roles during oogenesis and early embryo development in mammals. DNA methylation is basically known as adding a methyl group to the fifth carbon atom of cytosine residues within cytosine–phosphate–guanine (CpG) and non-CpG dinucleotide sites. This mechanism is composed of two main processes: de novo methylation and maintenance methylation, both of which are catalyzed by specific DNA methyltransferase (DNMT) enzymes. To date, six different DNMTs have been characterized in mammals defined as DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3C, and DNMT3L. While DNMT1 primarily functions in maintenance methylation, both DNMT3A and DNMT3B are essentially responsible for de novo methylation. As is known, either maintenance or de novo methylation processes appears during oocyte and early embryo development terms. The aim of the present study is to investigate spatial and temporal expression levels and subcellular localizations of the DNMT1, DNMT3A, and DNMT3B proteins in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, and early embryos from 1-cell to blastocyst stages. We found that there are remarkable differences in the expressional levels and subcellular localizations of the DNMT1, DNMT3A and DNMT3B proteins in the GV and MII oocytes, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos. The fluctuations in the expression of DNMT proteins in the analyzed oocytes and early embryos are largely compatible with DNA methylation changes and genomic imprintestablishment appearing during oogenesis and early embryo development. To understand precisemolecular biological meaning of differently expressing DNMTs in the early developmental periods, further studies are required.     

DNA甲基化与癌症

DNA甲基化是重要的表观遗传修饰,主要发生在DNA的CpG岛. DNA的甲基化通过DNA甲基转移酶（DNA methyltransferases, DNMTs）完成. DNA甲基化参与了细胞分化、基因组稳定性、X染色体失活、基因印记等多种细胞生物学过程.单基因水平及基因组范围内的DNA甲基化改变在肿瘤发生发展中亦发挥重要作用. 抑癌基因的异常甲基化引起的表达抑制,可导致肿瘤细胞的增殖失控和侵袭转移,并参与肿瘤组织的血管生成过程.在许多肿瘤的研究中都发现了基因组整体DNA低甲基化所导致的染色体不稳定性. 本文从DNA的异常高甲基化和低甲基化两方面论述了DNA甲基化在细胞恶变发生发展过程中的改变及其影响,并阐述了DNA甲基化改变在肿瘤诊断和治疗中的作用.     

Ectopic expression of DNA methyltransferases DNMT3A2 and DNMT3L leads to aberrant hypermethylation and postnatal lethality in mice

DNA methylation is generally known to inactivate gene expression. The DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, catalyze somatic cell lineage‐specific DNA methylation, while DNMT3A and DNMT3L catalyze germ cell lineage‐specific DNA methylation. How such lineage‐ and gene‐specific DNA methylation patterns are created remains to be elucidated. To better understand the regulatory mechanisms underlying DNA methylation, we generated transgenic mice that constitutively expressed DNMT3A and DNMT3L, and analyzed DNA methylation, gene expression, and their subsequent impact on ontogeny. All transgenic mice were born normally but died within 20 weeks accompanied with cardiac hypertrophy. Several genes were repressed in the hearts of transgenic mice compared with those in wild‐type mice. CpG islands of these downregulated genes were highly methylated in the transgenic mice. This abnormal methylation occurred in the perinatal stage. Conversely, monoallelic DNA methylation at imprinted loci was faithfully maintained in all transgenic mice, except H19. Thus, the loci preferred by DNMT3A and DNMT3L differ between somatic and germ cell lineages.     

Preferential Self-interaction of DNA Methyltransferase DNMT3A Subunits Containing the R882H Cancer Mutation Leads to Dominant Changes of Flanking Sequence Preferences

Somatic R882H DNMT3A mutations occur frequently in AML, but their pathogenic mechanism is unclear. As R882H mutations usually are heterozygous, wildtype (WT) and R882H subunits co-exist in affected cells. R882 is located in the RD interface of DNMT3A tetramers, which forms the DNA binding site. R882H causes strong changes in the flanking sequence preferences of DNMT3A. Here, we analyzed flanking sequence preferences for CGNNNN sites showing that most disfavored sites are methylated 4–5 fold slower by R882H than WT, while it methylates most preferred sites 2-fold faster. Overall, R882H was more active than WT at 13% and less active at 52% of all CGNNNN sites. We prepared mixed DNMT3A heterotetramers containing WT and R882H subunits and show that mixed complexes preferentially assemble with an R882H/R882H RD interface. Structural comparisons and MD simulations confirmed the conclusion that the R882H RD interface is more stable than that of WT, in part because H882 forms an inter-subunit contact in the RD interface, while R882 contacts the DNA. As the subunits at the RD interface contribute the two active centers to the DNMT3A tetramer, R882H characteristic flanking sequence preferences of DNMT3A were observed in mixed tetrameric complexes containing WT and R882H subunits, and they are not diluted by the “averaged” effects of mixed or WT interfaces. Hence, R882H has a dominant effect on the flanking sequence preferences and other catalytic properties of DNMT3A in samples containing WT and R882H subunits, which may explain its pathogenic effect in heterozygous state.     

Molecular dynamics simulations of human DNA methyltransferase 3B with selective inhibitor nanaomycin A

DNA methyltransferases (DNMTs) are involved in epigenetic regulation of the genome and are promising targets for therapeutic intervention in cancer and other diseases. Until now, very limited information is available concerning the molecular dynamics of DNMTs. The natural product nanaomycin A is the first selective inhibitor of DNMT3B that induce genomic demethylation. Herein we report long (>100 ns) molecular dynamics simulations for human DNMT3B bound to nanaomycin A with and without the presence of the cofactor S-adenosyl-l-methionine (SAM). We concluded that SAM favors the binding of nanaomycin A to DNMT3B. Key interactions of nanaomycin A with DNMT3B involve long lasting interactions with Arg731, Arg733, Arg832, and the catalytic Cys651. Results further support the previous hypothesis that nanaomycin A has key interactions with amino acid residues involved in the mechanism of methylation. This work represents one of the first molecular dynamics studies of DNMT3B. Results of this work shed light on the structure and binding recognition process of a key epigenetic enzyme with a small molecule inhibitor.     

Down-regulation of DNMT3b in PC3 cells effects locus-specific DNA methylation,and represses cellular growth and migration

Background  

Aberrations in DNA methylation patterns promote changes in gene expression patterns and are invariably associated with neoplasia. DNA methylation is carried out and maintained by several DNA methyltransferases (DNMTs) among which DNMT1 functions as a maintenance methylase while DNMT3a and 3b serve as de novo enzymes. Although DNMT3b has been shown to preferentially target the methylation of DNA sequences residing in pericentric heterochromatin whether it is involved in gene specific methylation remains an open question. To address this issue, we have silenced the expression of DNMT3b in the prostate-derived PC3 cells through RNA interference and subsequently studied the accompanied cellular changes as well as the expression profiles of selected genes.     

DNA Demethylation by DNMT3A and DNMT3B in vitro and of Methylated Episomal DNA in Transiently Transfected Cells

The DNA methylation program in vertebrates is an essential part of the epigenetic regulatory cascade of development, cell differentiation, and progression of diseases including cancer. While the DNA methyltransferases (DNMTs) are responsible for the in vivo conversion of cytosine (C) to methylated cytosine (5mC), demethylation of 5mC on cellular DNA could be accomplished by the combined action of the ten-eleven translocation (TET) enzymes and DNA repair. Surprisingly, the mammalian DNMTs also possess active DNA demethylation activity in vitro in a Ca²⁺- and redox conditions-dependent manner, although little is known about its molecular mechanisms and occurrence in a cellular context. In this study, we have used LC-MS/MS to track down the fate of the methyl group removed from 5mC on DNA by mouse DNMT3B in vitro and found that it becomes covalently linked to the DNA methylation catalytic cysteine of the enzyme. We also show that Ca²⁺ homeostasis-dependent but TET1/TET2/TET3/TDG-independent demethylation of methylated episomal DNA by mouse DNMT3A or DNMT3B can occur in transfected human HEK 293 and mouse embryonic stem (ES) cells. Based on these results, we present a tentative working model of Ca²⁺ and redox conditions-dependent active DNA demethylation by DNMTs. Our study substantiates the potential roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation during Ca²⁺-dependent physiological processes.     

The Dnmt1 Intrinsically Disordered Domain Regulates Genomic Methylation During Development

The DNMT1 cytosine methyltransferase enzyme contains a large ∼300-aa intrinsically disordered domain (IDD) that we previously showed regulated DNA methylation patterns in mouse ES cells. Here we generated seven mouse lines with different mutations in the IDD. Homozygous mutant mice of five lines developed normally, with normal levels of methylation on both imprinted and nonimprinted DNA sequences. The other two lines, however, had alterations in imprinted and/or nonimprinted (global) DNA methylation appearing during embryonic development. Embryos of one line expressing a DNMT1 variant containing a 6-aa rat orthologous sequence in the IDD maintained imprinted methylation, showed very reduced levels of global methylation and occasionally completed fetal development. These in vivo studies demonstrate that at least two DNMT1-dependent methylation processes can be distinguished during fetal development. One process maintains the bulk of genomic methylation on nonimprinted sequences. The other process maintains methylation on a much smaller class of sequences including but not limited to gametic differentially methylated domains (gDMDs) that transmit essential imprinted parent-specific methylation for embryonic development.     

Synthesis of NSC 106084 and NSC 14778 and evaluation of their DNMT inhibitory activity

DNA methylation is an epigenetic modification that is performed by DNA methyltransferases (DNMTs) and that leads to the transfer of a methyl group from S-adenosylmethionine (SAM) to the C5 position of cytosine. This transformation results in hypermethylation and silencing of genes such as tumor suppressor genes. Aberrant DNA methylation has been associated with the development of many diseases, including cancer. Inhibition of DNMTs promotes the demethylation and reactivation of epigenetically silenced genes. NSC 106084 and 14778 have been reported to inhibit DNMTs in the micromolar range. We report herein the synthesis of NSC 106084 and 14778 and the evaluation of their DNMT inhibitory activity. Our results indicate that while commercial NSC 14778 is moderately active against DNMT1, 3A/3L and 3B/3L, resynthesized NSC 14778 is inactive under our assay conditions. Resynthesized 106084 was also found to be inactive.     

An integrated epigenetic and genetic analysis of DNA methyltransferase genes (DNMTs) in tumor resistant and susceptible chicken lines

Both epigenetic alterations and genetic variations play essential roles in tumorigenesis. The epigenetic modification of DNA methylation is catalyzed and maintained by the DNA methyltransferases (DNMT3a, DNMT3b and DNMT1). DNA mutations and DNA methylation profiles of DNMTs themselves and their relationships with chicken neoplastic disease resistance and susceptibility are not yet defined. In the present study, we analyzed the complexity of the DNA methylation variations and DNA mutations in the first exon of three DNMTs genes over generations, tissues, and ages among chickens of two highly inbred White Leghorn lines, Marek's disease-resistant line 6(3) and -susceptible line 7(2), and six recombinant congenic strains (RCSs). Among them, tissue-specific methylation patterns of DNMT3a were disclosed in spleen, liver, and hypothalamus in lines 6(3) and 7(2). The methylation level of DNMT3b on four CpG sites was not significantly different among four tissues of the two lines. However, two line-specific DNA transition mutations, CpG-->TpG (Chr20:10203733 and 10203778), were discovered in line 7(2) compared to the line 6(3) and RCSs. The methylation contents of DNMT1 in blood cell showed significant epimutations in the first CpG site among the two inbred lines and the six RCSs (P<0.05). Age-specific methylation of DNMT1 was detected in comparisons between 15 month-old and 2 month-old chickens in both lines except in spleen samples from line 7(2). No DNA mutations were discovered on the studied regions of DNMT1 and DNMT3a among the two lines and the six RCSs. Moreover, we developed a novel method that can effectively test the significance of DNA methylation patterns consisting of continuous CpG sites. Taken together, these results highlight the potential of epigenetic alterations in DNMT1 and DNMT3a, as well as the DNA mutations in DNMT3b, as epigenetic and genetic factors to neoplastic diseases of chickens.