Activation of a Yeast Pseudo DNA Methyltransferase by Deletion of a Single Amino Acid

Pınarbaşı, Ergün; Elliott, John; Hornby, D.

doi:10.1006/jmbi.1996.0203

Cited by 67 publications

(51 citation statements)

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“…Our data are consistent with several previous studies implicating that, except for yeast pmt1 (7,12), the Dnmt2 protein family members might be active DNA MTases. First, the functional motifs conserved among the well known DNA MTases such as Dnmt1 are also conserved in dDnmt2 and mammalian Dnmt2 (8 -11).…”

Section: Figsupporting

confidence: 93%

“…The eukaryotic Dnmt2 protein family consists of the yeast pmt1 (7), the mammalian Dnmt2 including mouse mDnmt2 and human hDnmt2 (8,9), and Drosophila dDnmt2 (10,11). Of the Dnmt2 proteins known, pmt1 has been demonstrated to be enzymatically inactive due to amino acid change at a potential catalytic site (12). Sequence analysis showed that mDnmt2, hDnmt2, and dDnmt2 all contain the conserved DNA MTase motifs (8 -11).…”

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confidence: 99%

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The Eukaryotic DNMT2 Genes Encode a New Class of Cytosine-5 DNA Methyltransferases

Tang¹,

Reddy²,

Rasheva

et al. 2003

Journal of Biological Chemistry

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Methylation of the vertebrate genomes at cytosines is known to be accomplished by the combined actions of proteins encoded by different cytosine-5 DNA methyltransferases or DNA MTases. These proteins include the de novo DNA MTases Dnmt3a and Dnmt3b, the maintenance DNA MTase Dnmt1, and their isomers (1-4). There are also other DNA MTase-like proteins expressed in the eukaryotic cells, but they are without well documented DNA methylation activities. These include the Dnmt3L (5) and Dnmt2 proteins.The Dnmt2 proteins are relatively shorter than Dnmt3a, Dnmt3b, or Dnmt1, and structurally they are similar to the bacteria dcm enzyme (6). The eukaryotic Dnmt2 protein family consists of the yeast pmt1 (7), the mammalian Dnmt2 including mouse mDnmt2 and human hDnmt2 (8, 9), and Drosophila dDnmt2 (10, 11). Of the Dnmt2 proteins known, pmt1 has been demonstrated to be enzymatically inactive due to amino acid change at a potential catalytic site (12). Sequence analysis showed that mDnmt2, hDnmt2, and dDnmt2 all contain the conserved DNA MTase motifs (8 -11). DNA methylation analysis of ES cells with homozygous knock-out of the mouse mD-NMT2 genes suggested that mDnmt2 protein might also be an inactive DNA MTase (8). Finally, there has been no report on the DNA methylation activity of the Drosophila dDnmt2 until very recently. By overexpression of dDnmt2 in Drosophila S2 cells and subsequent analysis of the S2 cell genome with the sodium bisulfite sequencing approach, it was shown that specific regions were anomalously methylated in comparison to S2 cells without overexpression of the dDnmt2 protein (13).To avoid potential side effects resulting from use of the long term-selected S2 cell culture in the above study, we have now examined the genome of transgenic Drosophila flies stably overexpressing dDnmt2. Interestingly, specific genomic regions of the transgenic flies were also found to be anomalously hypermethylated. To complement the fly analysis, we further carried out DNA transfection experiments to transiently express fly dDnmt2 or mouse mDnmt2 in S2 cells. As shown below, dDnmt2 as well as mDnmt2 are capable of methylating cytosines of a cotransfected plasmid. The conservation of the enzymatically active Dnmt2 proteins from mammals to the flies suggests that this DNA MTase subfamily likely carries out important and to-be-identified function(s). EXPERIMENTAL PROCEDURESPlasmid Constructs-For transgenic fly work, the dDNMT2 cDNA was released from pGEM(1)-dDNMT2 with PacI-NcoI and blunt ended. This dDNMT2 fragment, which contains bp 6964 -7024 linked to 7074 -8051 of the dDNMT2 gene, was cloned at the EcoRI site of the polylinker of pUAST (14), and the resulting plasmid, pUAST-dDNMT2, was used for germ line transformation. For transient transfection, pAC5.1/V5-HisA vector (Invitrogen) containing Drosophila actin promoter was digested with KpnI, blunt ended, and redigested with EcoRI. pSG424 plasmid (15) was cut with BglII, blunt ended, and the GAL4 DNA binding domain-containing fragment was released by EcoRI digestion. Th...

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Section: Figsupporting

confidence: 93%

mentioning

confidence: 99%

The Eukaryotic DNMT2 Genes Encode a New Class of Cytosine-5 DNA Methyltransferases

Tang¹,

Reddy²,

Rasheva

et al. 2003

Journal of Biological Chemistry

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“…Therefore, the other MTases might compensate for the function of Dnmt2, which also could explain the weak knock-out phenotype. The consensus sequence rules out that the enzyme recognizes CCWGG sites as suggested by the fact that this sequence specificity has been reported for a mutant of the S. pombe Dnmt2 homolog pmt1 (37) and by the observation that CCWGG methylation has been detected in mammals (cf. Ref.…”

Section: Discussionmentioning

confidence: 88%

The Human Dnmt2 Has Residual DNA-(Cytosine-C5) Methyltransferase Activity

Hermann

Schmitt

Jeltsch

2003

Journal of Biological Chemistry

198

132

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The human Dnmt2 protein is one member of a protein family conserved from Schizosaccharomyces pombe and Drosophila melanogaster to Mus musculus and Homo sapiens. It contains all of the amino acid motifs characteristic for DNA-(Cytosine-C5) methyltransferases, and its structure is very similar to prokaryotic DNA methyltransferases. Nevertheless, so far all attempts to detect catalytic activity of this protein have failed. We show here by two independent assay systems that the purified Dnmt2 protein has weak DNA methyltransferase activity. Methylation was observed at CG sites in a loose ttnCGga(g/a) consensus sequence, suggesting that Dnmt2 has a more specialized role than other mammalian DNA methyltransferases.In mammals, DNA methylation is the only known physiological modification of DNA. It primarily occurs at CG sites, which are methylated to 70 -80% and encode epigenetic information on the DNA. DNA methylation is implicated in the regulation of gene expression, control of development, X chromosome inactivation, parental imprinting, and the protection of the genome against parasitic genetic elements such as transposons, retrotransposons, and viruses (for review see Refs. 1, 2-4). Aberrant methylation is among the most important causes of the inactivation of tumor suppressor genes in cancer (5). DNA-(Cytosine-C5) methyltransferases (MTases) 1 are characterized by a set of highly conserved amino acid motifs (6) that easily allows the identification of putative enzymes based on genomic sequences. In mammals, four candidate enzymes have been identified (1, 7). Dnmt1 is known to have a high preference for hemimethylated CG sites and has an important role in maintenance of methylation (8, 9). Dnmt3a and Dnmt3b do not recognize preexisting patterns of methylation and are de novo MTases (10 -12). However, it is also clear that in vivo the functions of maintenance and de novo methylation overlap, because accurate maintenance of the methylation at certain sequences also requires the presence of Dnmt3a and 3b and de novo methylation also relies on support by Dnmt1 (13-16). Dnmt1, Dnmt3A, and Dnmt3B are large proteins comprising an N-terminal part implicated in protein targeting and regulation and a C-terminal part that contains all 10 motifs characteristic for DNA-(Cytosine-C5) MTases. All three MTases are essential in mammals. Knock-out mice die during embryogenesis or shortly after birth (8, 11). The fourth putative DNA MTase in the genome of mammals, Dnmt2, has been discovered in 1998 (17, 18). Expression of Dnmt2 has been found in many human and mouse tissues albeit at low levels (10, 17, 18). Lacking a large N-terminal part, the human protein comprises 391 amino acid residues, which show clear homology to prokaryotic DNA-(Cytosine-C5) MTases. Members of the Dnmt2 family are found in many species including man and mice, which also contain other DNA MTases, but also in D. melanogaster and S. pombe where these enzymes are the only obvious DNA MTase candidate genes. The strict conservation of all of the motifs characte...

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“…This finding predicts that an enzyme with bacteria-like dcm methyltransferase activity is present in mammalian cells. A potential candidate would be Dnmt2, a mammalian structural homologue of the Schizosaccharomyces pombe pmt1, which can specifically bind C m C(A͞ T)GG sequences in vitro (37). However, methyltransferase activity for Dnmt2 has not been demonstrated (38).…”

Section: Pel and Rpmi-8226 Myeloma Cells Must Have Expressed B29mentioning

confidence: 99%

C ^m C(A/T)GG DNA methylation in mature B cell lymphoma gene silencing

Malone¹,

Miner²,

Doerr³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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P rimary effusion lymphoma (PEL) is an infrequent neoplasia of severe immunodeficiency and is usually observed in those with longstanding AIDS (1, 2). PEL exhibit features both in common with and distinct from other B cell neoplasms. Like myeloma, PEL arise from postgerminal center, developmentally mature B lymphocytes (1-3). Unlike myeloma, PEL grow in body cavities as liquid effusions, are almost always infected with human herpesvirus-8 (HHV-8) and lack expression of B-lineagespecific genes (reviewed in ref.2). The loss of B lineage gene expression in PEL could be caused by DNA methylation and epigenetic silencing. In accord with this idea, ␥-herpesvirus genomes such as HHV-8, herpesvirus saimiri, and Epstein-Barr virus, are commonly methylated (4, 5). The same mechanism(s) responsible for viral DNA methylation may also be involved in methylation and silencing of B lineage-specific genes in PEL.DNA methylation in mammalian cells largely occurs on cytosines in symmetric CpG dinucleotides and is associated with repressed gene transcription (6 -10). Methyl-binding domain proteins engage methylated CpG ( m CpG) and recruit histone deacetylase (HDAC) and transcriptional repressors to form stable repression complexes that induce local chromatin remodeling and gene silencing (11-16). Early mammalian embryos and germ cells, like plants and fungi, also methylate non-CpG cytosines (17-21). A recent report indicates that peripheral blood leukocytes may methylate the internal cytosine of symmetric CCTGG but not CCAGG motifs (22). However, little is known about the gene specificity, frequency, and functional significance of this latter type of symmetric non-CpG methylation.The B29 (Ig␤͞CD79b) component of the B cell surface receptor is encoded by the B29 gene and is absent in all PEL lines and some myelomas (1, 23). The B cell-specific B29 promoter is well characterized and provides an ideal model to analyze DNA methylation in B lineage gene silencing in PEL and myeloma. Early B cell factor (EBF), in concert with Octamer, Ets, Sp1, and Ikaros transcription factors, regulates B29 promoter activity in early B cell development, whereas non-EBF factors control B29 gene expression at later stages of B cell differentiation (24). The human B29 promoter contains 20 CpG dinucleotides and 6 CCAGG or CCTGG motifs within a 450-bp span (25). CpG dinucleotides are present in single Sp1 and EBF consensusbinding sites, whereas the CCAGG and CCTGG motifs are concentrated in a central promoter control region containing essential EBF sites.Here we report that the B29 promoter in PEL and nonexpressing myeloma cells is methylated at CpG and CC(A͞T)GG sites. Because the methylation pattern observed at these B29 promoter sites is similar to that reported in epigenetic retroviral silencing, we propose that B cell gene extinction occurs through a similar mechanism. We also find that CC(A͞T)GG methylation repressed B29 promoter activity and replaced transcription factors with new protein complexes. Materials and MethodsDNA Demethylation and HDAC In...

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Activation of a Yeast Pseudo DNA Methyltransferase by Deletion of a Single Amino Acid

Cited by 67 publications

References 0 publications

The Eukaryotic DNMT2 Genes Encode a New Class of Cytosine-5 DNA Methyltransferases

The Eukaryotic DNMT2 Genes Encode a New Class of Cytosine-5 DNA Methyltransferases

The Human Dnmt2 Has Residual DNA-(Cytosine-C5) Methyltransferase Activity

C ^m C(A/T)GG DNA methylation in mature B cell lymphoma gene silencing

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Activation of a Yeast Pseudo DNA Methyltransferase by Deletion of a Single Amino Acid

Cited by 67 publications

References 0 publications

The Eukaryotic DNMT2 Genes Encode a New Class of Cytosine-5 DNA Methyltransferases

The Eukaryotic DNMT2 Genes Encode a New Class of Cytosine-5 DNA Methyltransferases

The Human Dnmt2 Has Residual DNA-(Cytosine-C5) Methyltransferase Activity

C m C(A/T)GG DNA methylation in mature B cell lymphoma gene silencing

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C ^m C(A/T)GG DNA methylation in mature B cell lymphoma gene silencing