Cloning of a mammalian DNA methyltransferase

Bestor, TH

doi:10.1016/0378-1119(88)90238-7

Cited by 126 publications

(75 citation statements)

References 15 publications

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“…The existence of a two-component DNA methylation system consisting of an enzyme activity that methylates unmethylated DNA (de novo) and one that methylates hemimethylated sites (''maintenance'') has long been proposed (12)(13)(14). Genetic disruption of mouse DNMT1 (15) leads to extensive demethylation of several classes of genomic DNA sequences and embryonic lethality during midgestation (15,16).…”

Section: Introductionmentioning

confidence: 99%

De novo CpG Island Methylation in Human Cancer Cells

et al. 2006

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A major obstacle toward understanding how patterns of abnormal mammalian cytosine DNA methylation are established is the difficulty in quantitating the de novo methylation activities of DNA methyltransferases (DNMT) thought to catalyze these reactions. Here, we describe a novel method, using native human CpG island substrates from genes that frequently become hypermethylated in cancer, which generates robust activity for measuring de novo CpG methylation. We then survey colon cancer cells with genetically engineered deficiencies in different DNMTs and find that the major activity against these substrates in extracts of these cells is DNMT1, with minor contribution from DNMT 3b and none from DNMT3a, the only known bona fide de novo methyltransferases. The activity of DNMT1 against unmethylated CpG rich DNA was further tested by introducing CpG island substrates and DNMT1 into Drosophila melanogaster cells. The exogenous DNMT1 methylates the integrated mammalian CpG islands but not the Drosophila DNA. Additionally, in human cancer cells lacking DNMT1 and DNMT3b and having nearly absent genomic methylation, gene-specific de novo methylation can be initiated by reintroduction of DNMT1. Our studies provide a new assay for de novo activity of DNMTs and data suggesting a potential role for DNMT1 in the initiation of promoter CpG island hypermethylation in human cancer cells. (Cancer Res 2006; 66(2): 682-92)

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Section: Introductionmentioning

confidence: 99%

De novo CpG Island Methylation in Human Cancer Cells

et al. 2006

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“…1 The DNA MTase gene has been cloned and characterised but it still is unclear whether there is only a single enzyme responsible for both de novo and maintenance methylation activities in the cell. 2 In vitro studies have shown that the intact DNA MTase enzyme favours maintenance methylation activities because of its strong substrate preference for hemimethylated DNA. 3 However, in vitro cleavage of the N-terminal domain greatly increases the de novo activity of DNA MTase.…”

Section: Introductionmentioning

confidence: 99%

Increased DNA methyltransferase expression in leukaemia

et al. 1998

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Aberrant DNA methylation has been observed consistently in many human tumours, in particular in the CpG islands of tumour suppressor genes, but the underlying mechanism of these changes remains unclear. To determine whether DNA methyltransferase expression is increased in leukaemia, we developed a standarised competitive RT-PCR assay to measure the level of DNA methyltransferase transcripts. Using this assay on bone marrow RNA samples from 12 patients with acute leukaemia, we observed a 4.4-fold mean increase in the level of DNA methyltransferase mRNA compared with normal bone marrow. These results support but do not prove the hypothesis that an increase in DNA methyltransferase activity is associated with malignant haematological diseases and may constitute a key step in carcinogenesis.

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“…The mammalian DNMT1 is a protein postulated to be composed, based on its similarity to other cytosine DNA methyltransferases, of at least three structural components (21)(22)(23)(24)(25)(26). These domains are as follows: a catalytic domain at the C terminus, an N-terminal domain that is responsible for localization of the protein to the nucleus and replication foci, and another poorly characterized central domain.…”

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The DNMT1 Target Recognition Domain Resides in the N Terminus

Araujo¹,

Croteau

Slack

et al. 2001

Journal of Biological Chemistry

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DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. Vertebrate genomes are modified by methylation of ϳ60 -80% of the cytosines residing at the CG dinucleotide sequence (1). The distribution of methylated cytosines is not random, resulting in gene-and tissue-specific patterns of methylation (2). A large body of evidence supports the hypothesis that both methylation patterns and activity of DNA methyltransferases (DNMTs) 1 play critical roles in development and in controlling genome functions such as differential gene expression, chromosome imprinting, and X-chromosome inactivation (3-5). It has also been suggested that DNMT1 is a downstream effector of many oncogenic pathways and a potential target for anticancer therapy (6 -11). We have previously demonstrated that inhibition of DNMT1 leads to an inhibition of DNA replication (12). Recently, it has been shown that DNMT1 is able to form a complex with Rb, E2F, and HDAC1 and repress E2F-responsive expression (13,14). Furthermore, it has been shown that DNMT1 can establish a transcriptional repressive complex with HDAC2 and DMAP1 at replication foci (15). These data suggest that DNMT1 has multiple functions in the cell. However, because the DNMT1 target recognition domain is unknown it is not possible to determine how these multiple functions and protein-protein interactions relate to its target specificity.If DNA methylation patterns contain significant information, there must be a mechanism that ensures its proper inheritance in cell lineages. Razin and Riggs (16) have proposed that patterns of methylation are inherited, because DNMT1 is more proficient in methylating hemimethylated DNA than nonmethylated DNA. This hypothesis has been verified by a number of experiments (17, 18). Another level of specificity is the ability of DNMT1 to recognize CG sequences almost exclusively (19,20). Thus, DNMT1 exhibits both substrate and sequence specificity.The mammalian DNMT1 is a protein postulated to be composed, based on its similarity to other cytosine DNA methyltransferases, of at least three structural components (21-26). These domains are as follows: a catalytic domain at the C terminus, an N-terminal domain that is responsible for localization of the protein to the nucleus and replication foci, and another poorly characterized central domain. It is unclear as yet which segment is responsible for determining its specificity for hemimethylated CG sequences. Previous reports have shown that when the N-terminal domain is cleaved by proteolysis, the enzyme loses its ability to discriminate between hemimethylated and un...

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Cloning of a mammalian DNA methyltransferase

Cited by 126 publications

References 15 publications

De novo CpG Island Methylation in Human Cancer Cells

De novo CpG Island Methylation in Human Cancer Cells

Increased DNA methyltransferase expression in leukaemia

The DNMT1 Target Recognition Domain Resides in the N Terminus

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