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

Aoki, Aiko; Suetake, Isao; Miyagawa, Junichi; Fujio, Takayuki; Chijiwa, Takahito; Sasaki, Hidetada; Tsuji, Shoji

doi:10.1093/nar/29.17.3506

Cited by 156 publications

(124 citation statements)

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“…Like murine Dnmt3b1 (Okano et al, 1998a), human DNMT3b1 was an active de novo methyltransferase and had equivalent activity on hemimethylated DNA (Figure 2c). Although murine Dnmt3b3 produced in bacteria has been reported to be inactive (Aoki et al, 2001), eucaryotically produced DNMT3b3 was similar to DNMT3b1 in its ability to methylate both substrates. Thus, the predominant DNMT3b splice forms expressed in this model have comparable enzymatic activities, and the significance of alterations in their relative abundance is unclear.…”

Section: Expression Of Dnmts In Transformed Bronchial Epithelial Cellsmentioning

confidence: 97%

DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras

2003

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Transcriptional silencing of tumor suppressor genes in association with DNA methylation contributes to malignant transformation. However, the specific DNA methyltransferases that initiate this process are unknown. Here we show that a de novo DNA methyltransferase, DNMT3b, substantially contributes to the oncogenic phenotype in a lung cancer model. Normal human bronchial epithelial (NHBE) cells expressing telomerase, SV40 large T antigen, and activated Ras were immortal, formed colonies in soft agar, and expressed DNMT3b. Antisense suppression of DNMT3b prevented soft agar growth. Furthermore, mouse embryo fibroblasts expressing T antigen and Ras formed soft agar colonies and large tumors, but fibroblasts from Dnmt3b À/À mice did not grow in soft agar and were much less tumorigenic in vivo. The tumor suppressor genes, FHIT, TSLC1, and RASSF1A were downregulated in transformed NHBE cells, and antisense DNMT3b treatment resulted in reexpression of FHIT and TSLC1. While expression of TSCL1 correlated with methylation of CpG dinucleotides in its promoter region, the expression of FHIT did not, suggesting that DNMT3b may silence genes by several mechanisms including direct DNA methylation or recruitment of proteins that modify chromatin. Regardless of mechanism, our data indicate that DNMT3b plays an important role in transformation.

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Section: Expression Of Dnmts In Transformed Bronchial Epithelial Cellsmentioning

confidence: 97%

DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras

2003

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“…comm.). Non-CpG methylation has been observed for Dnmt3a 9,27,31 . Wild type Dnmt3b2 and Δ218 have some activities on non-CpG-containing oligonucleotides that contain CpA and CpT sites.…”

Section: The Pwwp Domain Of Dnmt3b Binds To Dnamentioning

confidence: 99%

“…Here we present the structure of an amino-proximal PWWP domain of mouse Dnmt3b2 (residues 223-357). Dnmt3b2, an isoform resulting from alternative splicing of the Dnmt3b gene 8,19 , is active in vitro 27 . Although the importance of individual isoforms is unclear, all of the isoforms (3b1-3b5) 21 are the same in the 223-357 rangethis is, all isoforms have the PWWP domain.…”

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The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds

et al. 2002

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The PWWP domain is a weakly conserved sequence motif found in >60 eukaryotic proteins, including the mammalian DNA methyltransferases Dnmt3a and Dnmt3b. These proteins often contain other chromatin-association domains. A 135-residue PWWP domain from mouse Dnmt3b (amino acids 223-357) has been structurally characterized at 1.8 Å resolution. The N-terminal half of this domain resembles a barrel-like five-stranded structure, whereas the C-terminal half contains a five-helix bundle. The two halves are packed against each other to form a single structural module that exhibits a prominent positive electrostatic potential. The PWWP domain alone binds DNA in vitro, probably through its basic surface. We also show that recombinant Dnmt3b2 protein (a splice variant of Dnmt3b) and two N-terminal deletion mutants (Δ218 and Δ369) have approximately equal methyl transfer activity on unmethylated and hemimethylated CpG-containing oligonucleotides. The Δ218 protein, which includes the PWWP domain, binds DNA more strongly than Δ369, which lacks the PWWP domain.Genomic patterns of cytosine methylation at the C5 position play key roles in the organization and control of mammalian chromatin (reviewed in refs 1-3). Mammalian DNA methyltransferases (MTases) all contain a C-terminal catalytic region that is structurally and functionally homologous to bacterial MTases. The eukaryotic DNA MTases have been grouped into three families based on (i) distinctive properties of their N-terminal regions and (ii) intriguing differences in DNA substrate preferences. The first eukaryotic DNA MTase, Dnmt1, contains a large N-terminal regulatory region of ~1,000 amino acids and shows a preference for hemimethylated DNA in vitro [4][5][6] . Dnmt2, a relatively small protein, contains only the MTase domain, and its function is still unclear 7 . Recombinant Dnmt3 acts on unmethylated and hemimethylated DNA at equal rates in vitro 8,9 .The task of dissecting the functional roles of the N-terminal region of the different MTase families is just beginning. These regions vary widely in size and are probably responsible for the diverse biological functions of eukaryotic DNA MTases. These functions include the © 2002 Nature Publishing Group Correspondence should be addressed to X.C. xcheng@emory.edu. Competing interests statementThe authors declare that they have no competing financial interests. HHS Public Access Dnmt3 contains a PWWP domainThe sequences of Dnmt3 orthologs have been determined from human and mouse. They all contain an N-terminal variable region (~280 amino acids in Dnmt3a and ~220 amino acids in Dnmt3b), followed by three stretches of conserved regions: the PWWP motif, six repeats of a CXXC motif and a set of 10 motifs conserved among DNA MTase catalytic regions (Fig. 1a). The PWWP motif 28 was first identified in a gene family related to the hepatomaderived growth factor (HDGF) 29 and WHSC1 genes (Wolf-Hirschhorn Syndrome Candidate) 30 . The corresponding sequence in Dnmt3 is SWWP (Fig. 1b); only the last two positions of the motif...

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“…Dnmt3b1 and Dnmt3b2 are enzymatically active in standard DNA methyltransferase assays, whereas Dnmt3b3, which lacks part of motif IX, appears to be inactive (4,21). Dnmt3b4 and Dnmt3b5 encode truncated proteins that lack motifs IX and X and are presumably inactive (19,20).…”

mentioning

confidence: 99%

“…Dnmt3b4 and Dnmt3b5 encode truncated proteins that lack motifs IX and X and are presumably inactive (19,20). So far, only one form of Dnmt3a has been identified and shown to be capable of methylating DNA both in vitro and in vivo (4,7,9,10,21). DNA methylation may also be regulated through subcellular localization of Dnmt3a and Dnmt3b.…”

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confidence: 99%

A Novel Dnmt3a Isoform Produced from an Alternative Promoter Localizes to Euchromatin and Its Expression Correlates with Activede Novo Methylation

Chen¹,

Ueda²,

Xie

et al. 2002

Journal of Biological Chemistry

321

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Previous studies have shown that the Dnmt3b gene encodes multiple variants via alternative splicing. However, only one form of Dnmt3a has been identified to date. We report here the discovery of a small form of Dnmt3a, denoted Dnmt3a2, from both human and mouse. The transcript encoding Dnmt3a2 is initiated from a downstream intronic promoter. As a result, the Dnmt3a2 protein lacks the N-terminal 223 (human) or 219 (mouse) amino acid residues of the full-length Dnmt3a. Recombinant Dnmt3a2 protein displayed similar cytosine methyltransferase activity as Dnmt3a in vitro. However, Dnmt3a and Dnmt3a2 exhibited strikingly different subcellular localization patterns. Unlike Dnmt3a, which was concentrated on heterochromatin, Dnmt3a2 displayed a localization pattern suggestive of euchromatin association. Dnmt3a2 is the predominant form in embryonic stem cells and embryonal carcinoma cells and can also be detected from testis, ovary, thymus, and spleen, whereas Dnmt3a is expressed at low levels ubiquitously. Comparison of human embryonal carcinoma cell lines with breast/ovarian cancer cell lines indicates that DNMT3A2 expression correlates with high de novo methylation activity. These findings suggest that Dnmt3a and Dnmt3a2 may have distinct DNA targets and different functions in development.

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

Cited by 156 publications

References 29 publications

DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras

DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras

The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds

A Novel Dnmt3a Isoform Produced from an Alternative Promoter Localizes to Euchromatin and Its Expression Correlates with Activede Novo Methylation

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