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PubMed2006

Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair.

While methylcytosines serve as the fifth base encoding epigenetic information, they are also a dangerous endogenous mutagen due to their intrinsic instability. Methylcytosine undergoes spontaneous deamination, at a rate much higher than cytosine, to generate thymine. In mammals, two repair enzymes, thymine DNA glycosylase (TDG) and methyl-CpG binding domain 4 (MBD4), have evolved to counteract the mutagenic effect of methylcytosines. Both recognize G/T mismatches arising from methylcytosine deamination and initiate base-excision repair that corrects them to G/C pairs. However, the mechanism by which the methylation status of the repaired cytosines is restored has remained unknown. We show here that the DNA methyltransferase Dnmt3a interacts with TDG. Both the PWWP domain and the catalytic domain of Dnmt3a are able to mediate the interaction with TDG at its N-terminus. The interaction affects the enzymatic activity of both proteins: Dnmt3a positively regulates the glycosylase activity of TDG, while TDG inhibits the methylation activity of Dnmt3a in vitro. These data suggest a mechanistic link between DNA repair and remethylation at sites affected by methylcytosine deamination.

Animals↗
PubMed2006

Aberrant expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3A, and DNMT3B in women with endometriosis.

OBJECTIVE: Since endometriosis is a persistent disease with substantial gene dysregulation, there must be cellular memory of some sort that constitutes a unique cell identity for endometriotic cells. Epigenetic regulation, especially through DNA methylation, is a flexible, yet stable, mechanism for maintaining such a cellular memory. The aim of this study was to determine gene expression levels of DNMT1, DNMT3A, and DNMT3B, the three genes coding for DNA methyltransferases that are responsible for methylation. DESIGN: Cross-sectional measurements of gene expression levels of DNMT1, DNMT3A, and DNMT3B on endometriotic tissue. SETTING: Academic. PATIENT(S): Seventeen patients with laparoscopically confirmed endometriosis and 8 healthy women who underwent tubal sterilization who were free of endometriosis were recruited for the study. INTERVENTION(S): Epithelial cells were harvested from tissue samples by laser capture microdissection and messenger RNA abundance was measured by quantitative real-time reverse transcription-polymerase chain reaction. MAIN OUTCOME MEASURE(S): The expression levels of these genes in epithelial cells from 13 ectopic endometrial tissue samples, 10 eutopic endometrial tissue samples taken from women with endometriosis, and 8 normal endometrial tissue samples from women without endometriosis. RESULT(S): The genes DNMT1, DNMT3A, and DNMT3B were over-expressed in the ectopic endometrium as compared with normal control subjects or the eutopic endometrium of women with endometriosis, and their expression levels were correlated positively with each other. CONCLUSION(S): The aberrant expression of these genes suggests that aberrant methylation may be rampant in endometriosis. This also provides a strong piece of evidence that endometriosis ultimately may be an epigenetic disease.

Adult↗
PubMed2006

Mouse follicular and marginal zone B cells show differential expression of Dnmt3a and sensitivity to 5'-azacytidine.

Mature B cells in the spleen of mouse and human comprise of two main subsets, the follicular (Fo) B cells and the marginal zone (MZ) B cells. In this study, we report that Fo and MZ B cells express different levels of DNA methyltransferase Dnmt3a. By using RT-PCR and immunohistochemistry, we found that Fo B cells expressed high level of Dnmt3a while MZ B cells expressed little. Treatment of mice by in vivo administration of 5'-azacytidine, an inhibitor of DNA methyltransferases, induced B cell loss in both the bone marrow and the spleen. We noticed that this treatment resulted in a much faster and more severe disappearance of Fo B cells than MZ B cells in the spleen. Further analysis showed that MZ progenitors increased significantly in mice treated with 5'-azacytidine. These results suggest that epigenetic mechanisms involving Dnmt3a might participate in the development of B cells including the differentiation of Fo B cells and MZ B cells in the periphery.

Animals↗
PubMed2006

Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase.

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.

3' Flanking Region↗
PubMed1999

DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease.

Animals↗
PubMed2004

Dimethyl sulfoxide stimulates the catalytic activity of de novo DNA methyltransferase 3a (Dnmt3a) in vitro.

Mammalian DNA methyltransferase Dnmt3a is required for de novo methylation of CpG dinucleotides in genomic DNA. While DNA methyltransferase inhibitors have been extensively utilized both in vitro and in vivo, no stimulator of catalytic activity has been identified thus far. Here we show that the methyltransfer activity of Dnmt3a is stimulated by the addition of dimethyl sulfoxide (DMSO) to the reaction solution in vitro. Enzymatic analysis of initial reaction velocity suggests that the DMSO stimulation effect depends on the interaction between DMSO and the reaction substrates (DNA and AdoMet), but not the enzyme itself.

Animals↗
PubMed2005

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.

Base Sequence↗
PubMed2006

Recruitment of the de novo DNA methyltransferase Dnmt3a by Kaposi's sarcoma-associated herpesvirus LANA.

The Kaposi's sarcoma-associated herpesvirus LANA protein is expressed in all Kaposi's sarcoma-associated herpesvirus-infected cells, including the tumor cells of endemic and AIDS-associated Kaposi sarcoma, primary effusion lymphoma, and Castleman disease. LANA modulates cell gene expression, but the mechanisms of LANA-mediated transcriptional reprogramming are poorly understood. LANA-repressed cell genes were identified by using retroviral-transduced telomerase-immortalized microvascular endothelial cells. Transciptional repression of targeted genes was relieved by treatment with the methyltransferase inhibitor 5-aza-2'-deoxycytidine, suggesting a role for DNA methylation in repression. We found that LANA coprecipitated with DNA methyltransferases (Dnmts) and recruited endogenous DNA methyltransferase activity from the cell extract. LANA preferentially relocalized Dnmt3a from the nuclear matrix into the chromatin fraction. Further, LANA associated with repressed cellular promoters, recruited Dnmt3a to DNA, and facilitated de novo promoter methylation of a down-regulated gene, cadherin 13 (H-cadherin). The data provide an example of promoter-specific epigenetic DNA modification through viral protein recruitment of de novo Dnmt activity.

Antigens, Viral↗
PubMed2006

Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1(-/-) or Dnmt3a(-/-)Dnmt3b(-/-) ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

Amino Acid Sequence↗
PubMed2005

Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells.

BACKGROUND: Acquired drug resistance is a major obstacle to the successful treatment of neuroblastoma by chemotherapy. Recent studies from our laboratory have demonstrated that drug-induced alterations in DNA methylation play an important role in this process. METHODS: The reversal of resistance to cisplatin in murine neuroblastoma (MNB) was induced by inhibition of DNA methyltransferase activity. MNB cells overexpressing DNA methyltransferase activity (Dnmt3a or Dnmt3b) were established by stable co-transfection of wild type MNB cells with plasmids containing Dnmt3a or Dnmt3b cDNA. Cytotoxic response (IC50), total DNA methyltransferase activity and expression of Dnmt3a or Dnmt3b methyltransferase were determined in Dnmt3a or Dnmt3b transfected MNB cells, respectively. RESULTS: These data demonstrated that total DNA methyltransferase activity was increased to 3-fold above controls (P<0.001) in cisplatin resistant MNB cells, 3-fold in Dnmt3a and 4-fold in Dnmt3b transfected MNB cells. Western blot and RT-PCR data confirmed a corresponding increase in Dnmt3a and 3b expression in cisplatin resistant and transfected cells when compared with control MNB cells (P<0.001). MNB clones overexpressing Dnmt3a or Dnmt3b proteins were resistant to cisplatin treatment (10(-6) M). Incubation of cisplatin resistant, Dnmt3a or Dnmt3b overexpressing MNB cells with 5'-azacytidine (5'-azaC), a methylation inhibitor (2.5 microM) significantly decreased DNA methyltransferase activity, expression of Dnmt3a and Dnmt3b proteins and mRNA levels of cisplatin resistant, Dnmt3a and Dnmt3b transfected MNB cells. Cytotoxicity studies using the MTT assay demonstrated that the sensitivity of cisplatin resistant, Dnmt3a and Dnmt3b overexpressing MNB cells to cisplatin was increased 10-fold (P<0.001) following 5'-azaC treatment. CONCLUSIONS: These findings suggest that the overexpression of DNA methyltransferase is associated with a cisplatin resistant phenotype in MNB cells that may or may not be true in animal studies or in the clinical setup. Thus, DNA methylation plays a central role in onset of drug resistance in cisplatin resistant neuroblastoma cells in vitro.

Animals↗
PubMed2005

Physical and functional interactions between the human DNMT3L protein and members of the de novo methyltransferase family.

The de novo methyltransferase-like protein, DNMT3L, is required for methylation of imprinted genes in germ cells. Although enzymatically inactive, human DNMT3L was shown to act as a general stimulatory factor for de novo methylation by murine Dnmt3a. Several isoforms of DNMT3A and DNMT3B with development-stage and tissue-specific expression patterns have been described in mouse and human, thus bringing into question the identity of the physiological partner(s) for stimulation by DNMT3L. Here, we used an episome-based in vivo methyltransferase assay to systematically analyze five isoforms of human DNMT3A and DNMT3B for activity and stimulation by human DNMT3L. Our results show that human DNMT3A, DNMT3A2, DNMT3B1, and DNMT3B2 are catalytically competent, while DNMT3B3 is inactive in our assay. We also report that the activity of all four active isoforms is significantly increased upon co-expression with DNMT3L, albeit to varying extents. This is the first comprehensive description of the in vivo activities of the poorly characterized human DNMT3A and DNMT3B isoforms and of their functional interactions with DNMT3L. To further elucidate the mechanism by which DNMT3L stimulates DNA methylation, we have mapped in detail the domains that mediate interaction of human DNMT3L with human DNMT3A and DNMT3B. Our results show that the C-terminus of DNMT3L is the only region required for interaction with DNMT3A and DNMT3B and that interaction takes place through the C-terminal catalytic domain of DNMT3A and DNMT3B. The implications of these findings for the regulation of de novo methyltransferases and genomic imprinting are discussed. This article contains Supplementary Material available at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/2005/95/chen.html.

Cells, Cultured↗
PubMed2005

Physical and functional interaction of DNA methyltransferase 3A with Mbd3 and Brg1 in mouse lymphosarcoma cells.

Dnmt3a and Dnmt3b are de novo DNA methyltransferases that also act as transcriptional repressors independent of methyltransferase activity. To elucidate the underlying mechanism of transcriptional repression, Dnmt3a was purified from mouse lymphosarcoma cells (P1798) by extensive fractionation on five different chromatographic matrices followed by glycerol density gradient centrifugation. Liquid chromatography electrospray tandem mass spectrometry analysis of Dnmt3a-associated polypeptides identified the methyl CpG binding protein Mbd3, histone deacetylase 1(Hdac1), and components of Brg1 complex (Brg1, Baf155, and Baf57) in the purified preparation. Association of Dnmt3a with Mbd3 and Brg1 was confirmed by coimmunoprecipitation and coimmunolocalization studies. Glutathione S-transferase pulldown assay showed that the NH2-terminal ATRX homology domain of Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and with both bromo and ATPase domains of Brg1. Chromatin immunoprecipitation assay revealed that all three proteins are associated with transcriptionally silent methylated metallothionein (MT-I) promoter in the mouse lymphosarcoma cells. To understand the functional significance of their association with the promoter, their role on the MT-I promoter activity was analyzed by transient transfection assay. The results showed that Mbd3 and Dnmt3a specifically inhibited the methylated promoter, and the catalytic activity of Dnmt3a was dispensable for the suppression. In contrast, the wild-type but not the ATPase-inactive mutant of Brg1 suppressed MT-I promoter irrespective of its methylation status, implicating involvement of ATP-dependent chromatin remodeling in the process. Coexpression of two of the three interacting proteins at a time augmented their repressor function. This study shows physical and functional interaction of Dnmt3a with components of nucleosome remodeling machinery.

Animals↗
PubMed2026

Clonal Hematopoiesis and Incident Heart Failure.

IMPORTANCE: Clonal hematopoiesis of indeterminate potential (CHIP), the age-related clonal expansion of hematopoietic cells with acquired preleukemic variants, has been associated with cardiometabolic diseases, including heart failure (HF). However, prior studies have lacked power to examine less common CHIP driver variants and have not investigated potential mediators of the CHIP-HF association. OBJECTIVE: To test whether specific CHIP subtypes are associated with incident HF and determine the extent to which CHIP-associated comorbidities mediate this association. DESIGN, SETTING, AND PARTICIPANTS: This was a UK Biobank prospective population-based cohort study of community-dwelling adults in the UK, with enrollment from 2006 to 2010 and follow-up through 2020. Included were participants with whole-exome sequencing (WES) and without prevalent HF, hematologic malignancy, or other CHIP-associated comorbidities (coronary artery disease [CAD], atrial fibrillation [AF], type 2 diabetes [T2D], or chronic kidney disease [CKD]) at baseline. Study data were analyzed from April through October 2025. EXPOSURES: Presence of CHIP and gene-specific CHIP subtypes (DNMT3A, non-DNMT3A, TET2, ASXL1, JAK2, DNA damage repair genes, and spliceosome genes). Mediation analyses examined CHIP-associated comorbidities (CAD, AF, T2D, and CKD). MAIN OUTCOMES AND MEASURES: The primary outcome was incident HF. Cox regression tested associations of CHIP and CHIP subtypes with incident HF, adjusted for age, sex, race, and cardiovascular risk factors. RESULTS: Among 417&#x202f;616 participants (mean [SD] age, 56.1 [8.1] years; 234&#x202f;868 female [56.2%]), 7183 (1.7%) developed incident HF over a median (IQR) of 11.1 (10.4-11.8) years of follow-up. CHIP was associated with HF risk (adjusted hazard ratio [aHR], 1.27; 95% CI, 1.15-1.40; P&#x2009;<&#x2009;.001), driven by non-DNMT3A subtypes (aHR, 1.52; 95% CI, 1.33-1.75; P&#x2009;<&#x2009;.001), including associations with TET2, ASXL1, JAK2, and spliceosome CHIP. DNMT3A CHIP was more modestly associated with HF (aHR, 1.15; 95% CI, 1.00-1.31; P&#x2009;=&#x2009;.04). In mediation analyses, development of CAD, AF, T2D, and/or CKD collectively accounted for 28.2% of the association (95% CI, 11.6%-45.4%; P&#x2009;=&#x2009;.001) between non-DNMT3A CHIP and HF. CONCLUSIONS AND RELEVANCE: Results of this cohort study suggest that CHIP, especially non-DNMT3A CHIP, was associated with incident HF. Other CHIP-associated comorbidities explained only a minority of the association between non-DNMT3A CHIP and HF. These findings suggest that CHIP is an HF risk factor and potential therapeutic target.

Adult↗
PubMed2005

Transcriptional regulation of the human DNA methyltransferase 3A and 3B genes by Sp3 and Sp1 zinc finger proteins.

The DNMT3A (DNA methyltransferase 3A) and DNMT3B genes encode putative de novo methyltransferases and show complex transcriptional regulation in the presence of three and two different promoters respectively. All promoters of DNMT3A and DNMT3B lack typical TATA sequences adjacent to their transcription start sites and contain several Sp1-binding sites. The importance of these Sp1-binding sites was demonstrated by using a GC-rich DNA-binding protein inhibitor, mithramycin A, i.e. on the basis of decrease in the promoter activities and mRNA expression levels of DNMT3A and DNMT3B. Overexpression of Sp1 and Sp3 up-regulated the promoter activities of these two genes. The physical binding of Sp1 and Sp3 to DNMT3A and DNMT3B promoters was confirmed by a gel shift assay. Interestingly, Sp3 overexpression in HEK-293T cells (human embryonic kidney 293T cells) resulted in 3.3- and 4.0-fold increase in DNMT3A and DNMT3B mRNA expression levels respectively by quantitative reverse transcriptase-PCR, whereas Sp1 overexpression did not. Furthermore, an antisense oligonucleotide to Sp3 significantly decreased the mRNA levels of DNMT3A and DNMT3B. These results indicate the functional importance of Sp proteins, particularly Sp3, in the regulation of DNMT3A and DNMT3B gene expression.

Adenoviridae↗
PubMed2001

Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases.

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K(m)(AdoMet), for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 microM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K(m)(DNA) values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 microM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT >/= CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.

Binding Sites↗
PubMed2026

Mammalian DNA methyltransferases in DNA methylation and imprinted gene expression in extraembryonic ectoderm of post-implantation embryos.

DNA methylation in mammals is mainly catalyzed by three DNA methyltransferases (DNMTs). Conventionally, DNMT1 is considered the primary DNMT protein for maintenance DNA methylation, whereas DNMT3A and DNMT3B function in de novo DNA methylation. In two previous studies, we demonstrated that DNMT3A and DNMT3B maintain genome-wide DNA methylation in embryonic stem (ES) cells and in the epiblast of post-implantation embryos. Interestingly, DNMT3A and DNMT3B also sustain genome-wide DNA methylation in the extraembryonic ectoderm (EXE) of post-implantation embryos, including repeats, genic and intergenic regions. Although DNMT1 plays a major role in maintaining DNA methylation at the imprinting control regions (ICRs) in the imprinted regions, DNMT3A and DNMT3B are required for preserving DNA methylation at the ICRs of a subset of imprinted regions in EXE, similar to the observations in ES cells and epiblast. Surprisingly, de novo DNA methylation mediated by DNMT3A and DNMT3B leads to increased DNA methylation at a large subset of imprinted regions. These results are consistent with what we previously elucidated in the epiblast of post-implantation embryos. Importantly, loss of DNA methylation at the ICR of an imprinted region, resulting from the absence of DNMT1 or two DNMT3 proteins, causes allelic expression switch of the corresponding imprinted genes in that imprinted region. This study provides further evidence that DNMT3A and DNMT3B exert both maintenance and de novo DNA methylation functions across the genome in post-implantation embryos. It also validates some previous findings for DNA methylation-dependent allelic expression switch of imprinted genes.

DNA methylation↗
PubMed2003

Biochemical fractionation reveals association of DNA methyltransferase (Dnmt) 3b with Dnmt1 and that of Dnmt 3a with a histone H3 methyltransferase and Hdac1.

De novo DNA methyltransferases, Dnmt3a and 3b, were purified by fractionation of S-100 extract from mouse lymphosarcoma cells through several chromatographic matrices followed by glycerol density gradient centrifugation. Dnmt3a was separated from Dnmt3b and Dnmt1 in the first column, Q-Sepharose whereas Dnmt3b co-purified with Dnmt1 after further fractionation through Mono-S and Mono-Q columns and glycerol density gradient centrifugation. Following purification, the majority of de novo DNA methyltransfearse activity was associated with Dnmt3b/Dnmt1 fractions. By contrast, the fractions containing Dnmt3a alone exhibited markedly reduced activity, which correlated with diminished expression of this isoform in these cells. Histone deacetylase 1(Hdac1) cofractionated with Dnmt3a throughout purification whereas Hdac1 was separated from Dnmt3b/Dnmt1 following chromatography on Mono-Q column. Dnmt3a purified through glycerol gradient centrifugation was also associated with a histone H3 methyltransferase (HMTase) activity whereas purified Dnmt3b/Dnmt1 was devoid of any HMTase activity. The activity of this HMTase was abolished when lysine 9 of N-terminal histone H3 peptide was replaced by leucine whereas mutation of lysine 4 to leucine inhibited this activity only partially. This is the first report on the identification of a few key co-repressors associated with endogenous Dnmt3a and of a complex containing Dnmt3b and a minor form of Dnmt1 following extensive biochemical fractionation.

Animals↗