A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast
A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast
Jeffrey A. Yoder, Timothy H. Bestor*
Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, 701 West 168 Street, New York, NY 10032, USA
Received September 24, 1997;Revised and Accepted November 2, 1997
Trace levels of 5-methylcytosine persist in the DNA of mouse embryonic stem cells that are homozygous for null mutations in Dnmt1, the gene for the one previously recognized metazoan DNA methyltransferase. This residual 5-methylcytosine may be the product of a candidate second DNA methyltransferase, Dnmt2, that has now been identified in human and mouse. Dnmt2 contains all the sequence motifs diagnostic of DNA (cytosine-5)-methyltransferases but appears to lack the large N-terminal regulatory domain common to other eukaryotic methyltransferases. Dnmt2 is more similar to a putative DNA methyltransferase of the fission yeast Schizosaccharomyces pombe than to Dnmt1. Dnmt2 produces multiple mRNA species that are present at low levels in all tissues of human and mouse and is not restricted to those cell types known to be active in de novo methylation. The human DNMT2 gene was mapped to chromosome 10p12-10p14 in a panel of radiation hybrids. Dnmt2 is a candidate for the activity that methylates newly integrated retroviral DNA and maintains trace levels of 5-methylcytosine in the DNA of embryonic stem cells homozygous for null mutations in Dnmt1.
About 1% of the bases in the mammalian genome are 5-methylcytosine (m5C), nearly all of which are in parasitic sequence elements and other repetitive sequences, imprinted genes and genes silenced by X inactivation (reviewed in 1). The primary function of cytosine methylation appears to be suppression of parasitic sequence elements (endogenous retroviruses and transposable elements), with additional crucial roles in genomic imprinting and X inactivation (1,2). While genomic methylation patterns are in part transmitted from parent to offspring, most genomic m5C is created in a wave of de novo methylation that occurs shortly after implantation of the mammalian embryo (1).
The DNA methyltransferases previously isolated from vertebrates (3-7), echinoderms (8) and flowering plants (9) comprise a C-terminal domain that is closely related to bacterial cytosine-5 restriction methyltransferases and a large N-terminal domain that has multiple regulatory functions (10). The human gene that encodes this ubiquitous enzyme is called DNMT1; the mouse homolog is Dnmt1. Homozygous targeted disruptions of Dnmt1 are lethal at embryonic day 8.5 and the embryos show a loss of allele-specific gene expression at endogenous imprinted loci (11) and ectopic expression of Xist and inactivation of all X chromosomes in some cells (12,13). The first Dnmt1 disruptions were partial loss-of-function alleles (14) which allowed homozygous mutant embryonic stem cells to retain [sim]30% of the wild-type level of m5C. A null mutation that deleted the enzymatic active site from Dnmt1 removed all but trace levels of m5C in homozygous embryonic stem cells (15), but the mutant cells retained some capacity for de novo methylation of newly integrated retroviral DNA. These findings require that there be at least one additional DNA methyltransferase in mammals. Such enzymes have been highly refractory to identification by biochemical methods (16).
A set of six well-conserved sequence motifs of invariant order is diagnostic of all DNA (cytosine-5)-methyltransferases (17). We report the identification of a new candidate methyltransferase in humans and mice that contains all six diagnostic motifs as well as other features characteristic of DNA methyltransferases. This new protein, Dnmt2, is less closely related to the product of the Dnmt1 gene than to a putative DNA methyltransferase of unknown function from the fission yeast Schizosaccharomyces pombe (18). Dnmt2 may be responsible for de novo methylation of newly integrated retroviral DNA in mammalian DNA.
The search algorithm TBLASTN (19) was used to screen the dbEST database of expressed sequence tags (ESTs) for sequences that contain DNA methyltransferase diagnostic motifs. The sequences of the C-terminal catalytic domain of Dnmt1 and the bacterial restriction methyltransferase M.XorI were used as query sequences. The Dnmt1 sequence did not give a significant match to sequences other than Dnmt1 itself in the BLAST search, but M.XorI identified a human EST that appeared to have three of the diagnostic DNA methyltransferase motifs but which was highly diverged from Dnmt1. The EST clone (ID 198982) was obtained from the IMAGE Consortium (20) and sequenced in its entirety. It was found to contain all the diagnostic DNA methyltransferase motifs (Fig. 1B) and to display a hydropathy profile that is very similar to a bacterial DNA methyltransferase (Fig. 1A). The sequence comparisons and secondary structure predictions strongly indicate that the sequence is that of a DNA methyltransferase and it was therefore termed DNMT2. The homologous mouse sequence (Dnmt2) was obtained by screening mouse cDNA libraries with a DNMT2 cDNA probe.
Figure 1 Sequence analysis of the candidate DNA methyltransferase Dnmt2. (A) Hydropathy plot and motif location in Dnmt2 compared with a bacterial restriction methyltransferase. Note that the motifs occupy corresponding environments in both enzymes. The six conserved motifs are colored to correspond to the coloring of (B) and (C). (B) Comparison of Dnmt2 sequence with the consensus sequence of DNA (cytosine-5)-methyltransferase motifs. The functions of the motifs were assigned from the crystal structure of a covalent DNA-DNA methyltransferase transition state intermediate (21). Dashes indicate that there is no strong consensus at that position. Red underlining indicates the prolylcysteinyl dipeptide at the catalytic center. This sequence is serylcysteinyl in pmt1. (C) Sequence alignment of mouse Dnmt2, human DNMT2 and S.pombe pmt1. Solid boxes indicate amino acid identity, gray boxes indicate similarity.
TBLASTN searches of GenBank with the Dnmt2/DNMT2 sequence identified pmt1 of S.pombe as the most closely related sequence. pmt1 (for pombe methyltransferase 1) was cloned fortuitously and recognized to bear similarities to cytosine methyltransferases (18). An alignment of the Dnmt2, DNMT2 and pmt1 sequences is shown in Figure 1C and the locations and functions of conserved diagnostic motifs (21) are also indicated. The conserved prolylcysteinyl (PC) active site is present in motif IV of Dnmt2 and DNMT2 but is interrupted by a seryl residue in pmt1 (Fig. 1B and C). Restoration of the canonical PC in pmt1 has been reported to confer the ability to methylate the sequence CCWGG (22).
ClustalW analysis (23) was used to evaluate the relationship of DNMT2 to other cytosine methyltransferases. As shown in Figure 2A, this analysis revealed that the known DNA cytosine methyltransferases sort into three families: the Dnmt1 family (which includes DNA methyltransferases from several vertebrate species, a sea urchin and the flowering plant Arabidopsis thaliana), the bacterial C5 restriction methyltransferase family and the Dnmt2 family. The adenine-specific methyltransferase M.EcoRI, which is not recognizably related to C5 methyltransferases and which utilizes a catalytic mechanism very different from that of the cytosine-specific enzymes, was used as an outgroup. As shown in Figure 1, the most pronounced similarities are confined to the conserved motifs and this sequence analysis allows Dnmt2 to be categorized as a DNA (cytosine-5)-methyltransferase. It is distinguished from the Dnmt1 family in that the predominant form lacks the large N-terminal regulatory domain (Fig. 2B).
Figure 2Comparison of relatedness of the Dnmt2 family to the Dnmt1 family and to the bacterial DNA (cytosine-5)-methyltransferase family. (A) Evaluation of relatedness of cytosine methyltransferases by ClustalW analysis. M.EcoRI is an adenosine methyltransferase and was included as an outgroup to root the tree. (B) Schematic view of the organization of the Dnmt1, Dnmt2 and bacterial C5 restriction methyltransferases. The large N-terminal regulatory domain is unique to the Dnmt1 family; the Dnmt2 and bacterial restriction methyltransferase families are of similar gross organization.
Most de novo methylation occurs in germ cells and early post-implantation embryos (1). Cultured embryonic stem cells have also been reported to be active in de novo methylation of retroviral DNA, even when the Dnmt1 gene has been inactivated by deletion of an exon that encodes the key active site residues (15). RNase protection and RNA blot hybridization were used to determine whether Dnmt2 is expressed only in those cell types and tissues known to be active in de novo methylation.
As shown in Figure 3, Dnmt2 mRNA is expressed in all cell types. There are multiple RNA species, presumably the product of alternative splicing, whose relative amounts differ only slightly in different tissues, with highest expression in testes, ovary and thymus (Fig. 3A, C and D). The predominant Dnmt2 mRNA in all mouse tissues has an apparent length of 1.6 kb (Fig. 3C), which corresponds to the length of the recovered cDNA [1691 nt without the poly(A) tail]. The longest mouse cDNA therefore represents all or nearly all of the predominant Dnmt2 mRNA. Human DNMT2 has a very different distribution of mRNA sizes; the predominant form has a length of 3.7 kb (Fig. 3A) and the largest species is 7.2 kb. There is little evidence that the observed alternative splicing is tissue specific for DNMT2 or Dnmt2 mRNA. It is concluded that Dnmt2 is present at low but variable levels in all tissues and is not restricted to those cell types that have been shown to be active in de novo methylation.
Figure 3 Analysis of Dnmt2, DNMT2 and DNMT1 mRNA. (A) RNA blot analysis of DNMT2 in RNA from human tissues. (B) The blot shown in (A) was stripped and sequentially hybridized with cDNA probes for DNMT1 and [beta]-actin. DNMT1 and DNMT2 mRNA are expressed at highest levels in testis and thymus. (C) Blot hybridization analysis and (D) RNase protection analysis of Dnmt2 mRNA in mouse tissues and cultured cells. As in the case of DNMT1, Dnmt2 mRNA is expressed at highest levels in testis and thymus. sm. int., small intestine; leuk., peripheral blood leukocytes; e10.5, day 10.5 mouse embryo; e8.5, day 8.5 mouse embryo.
Low stringency hybridization of a Dnmt2 cDNA probe for motifs VIII-IX to DNA blots did not identify additional bands indicative of a gene family (data not shown). This suggests that closely related genes do not exist and that the larger transcripts seen in Figure 3 are due to alternative splicing of Dnmt2. Exhaustive screening of cDNA libraries has failed to identify longer transcripts, apparently due to obstruction of reverse transcriptase by structural features in the mRNA. Some alternative splicing within the region shown in Figure 1C was observed; all truncated the reading frame upstream of conserved methyltransferase motifs (data not shown).
DNMT1 mRNA in human tissues is visualized as a single band that varies in amount among tissue types. DNMT2 is present at lower levels than is DNMT1, as shown in Figure 3A and B. The filter probed for DNMT2 required a 5 day exposure to X-ray film, while the DNMT1 blot required only 0.5 days and dbEST contains only four DNMT2 ESTs but 25 DNMT1 ESTs. These data suggest that Dnmt1 mRNA is 5- to 10-fold more abundant than Dnmt2 mRNA.
DNMT2 was expressed in Escherichia coli and tested for transmethylase activity in biochemical assays as described (16). Clear evidence of activity was not obtained (data not shown), but this may be due to the absence of accessory factors, inappropriate post-translational modification in bacteria or a requirement for alternative secondary structures in the DNA substrate that were not present in the duplex oligonucleotides used in the assays.
DNMT2-specific PCR primers as described in Materials and Methods were used to map the DNMT2 gene from the Genebridge 4 radiation hybrid panel (24). The gene was found to map near the center of chromosome arm 10p (Fig. 4), [sim]21.57 cR (5.83 Mb) centromeric of the framework marker NIB1436 and telomeric of GATA70E11. This region shows strong conserved gene order with a segment of mouse chromosome 2 (25). Human DNMT1 has been shown to map to chromosome 19p (5) and mouse Dnmt1 to proximal chromosome 9.
Figure 4 Genomic location of DNMT2 on human chromosome 10p. DNMT2 was mapped in the Genebridge 4 radiation hybrid panel framework (24). An ideogram of chromosome 10 is shown at left and markers flanking DNMT2 are shown at right. The location of flanking markers places DNMT2 in the region 10p12-10p14.
The human DNMT2 and murine Dnmt2 cDNA sequences are available from GenBank as accession nos AF012128 and AF012129 respectively.
It has been thought that genomic methylation patterns might be established by developmentally regulated de novo methyltransferases and maintained by a different family of maintenance methyltransferases; these latter enzymes would act only on hemimethylated DNA to preserve existing methylation patterns. However, there has been virtually no evidence in support of this traditional view. The first DNA methyltransferase (now termed Dnmt1) to be isolated from mammals was shown to possess both de novo and maintenance activities (16,26), with an [sim]10-fold preference for hemimethylated over unmethylated substrates. The establishment and maintenance of genomic methylation patterns could in principle be mediated by one enzyme (27). However, embryonic stem cells bearing homozygous deletions of the portion of the Dnmt1 gene that encodes the catalytic center of the enzyme retain trace levels of m5C and are capable of methylating newly integrated retroviral DNA with reduced efficiency (15). This requires that at least one additional DNA methyltransferase must exist in the mouse.
The large and increasing number of expressed sequence tags (ESTs) in public domain databases has greatly aided searches for genes that contain diagnostic sequence motifs and searches of such databases resulted in recovery of human cDNA clones with strong sequence similarities to DNA methyltransferases; this new candidate enzyme has been named DNMT2. A very similar sequence was recovered from mouse cDNA libraries by hybridization screening. Dnmt2 contains the six highly conserved DNA methyltransferase signature motifs but has greatly diverged from Dnmt1; Dnmt1 and Dnmt2 are much more distant from each other than Dnmt1 is from MET1, the Dnmt1 homolog of the flowering plant A.thaliana (Fig. 2A). This implies that Dnmt1 and Dnmt2 diverged at a very early time (when the common ancestor of plants and animals was unicellular and perhaps prior to the eukaryote-prokaryote separation) or that Dnmt2 had a separate origin and came to resemble Dnmt1 by convergent evolution.
Convergent evolution seems unlikely, as the product of the pmt1 gene of the fission yeast S.pombe is closely related to Dnmt2 (Fig. 2A). This suggests a common origin rather than convergent evolution. pmt1 was cloned by chance and null mutants have no apparent phenotype under laboratory conditions. The DNA of S.pombe has not been reported to contain large amounts of m5C, although other fungi are known to contain this base (28). Schizosaccaromyces pombe shows imprinting of mating type switching and pmt1 mutants have not been investigated for defects in switching (A.Klar and P.Nurse, personal communication). Small amounts of m5C in S.pombe DNA would have escaped detection. It is significant that the genome of Saccharomyces cerevisiae does not have an open reading frame that contains DNA methyltransferase signature motifs and S.cerevisiae is not known to possess methylated DNA or to display imprinted gene expression. Furthermore, it has been reported that a point mutation which restores the consensus sequence at the catalytic center endows pmt1 with DNA methyltransferase activity. DNMT2 and Dnmt2 already bear the DNA methyltransferase consensus at the catalytic center and would be expected to be active, although activity may require cell type-specific post-transcriptional regulation or modification, the participation of accessory factors or specific alternative secondary structures in the DNA substrate.
It has been held that programmed methylation and demethylation of regulatory sequences is involved in gene control during development. The importance of direct sequence recognition in transcriptional regulation provoked predictions of sequence-specific DNA methyltransferases that would act during gametogenesis and early development to establish methylation patterns around developmentally regulated genes. However, it has not been proven that any cellular gene is regulated by reversible methylation and it has been argued that de novo methylation is not regulated by direct sequence recognition (27); in keeping with the hypothetical role as a host defense system that suppresses parasitic sequence elements, de novo methylation might be contingent on interaction of the DNA methylating system with repeated sequences or structural features characteristic of integration intermediates (1,27). As Dnmt1 null embryonic stem cells retain the capacity to methylate newly integrated retroviral DNA, it is tempting to speculate that this special reaction is mediated by Dnmt2 and that cells doubly homozygous for null mutations of Dnmt1 and Dnmt2 will be unable to methylate incoming retroviral DNA. In this context it can be noted that Lengauer et al. (29) recently reported that a subset of colorectal carcinoma cell lines are proficient in mismatch repair but unable to silence and methylate newly integrated retroviral DNA. The destabilization of the genome observed in these methylation-deficient cells was suggested to result from demethylation of DNA (1,29). These cells may suffer from alterations in a second methylation pathway that involves Dnmt2 and other factors.
BLASTN (19) was used to search NCBI dbEST through the Baylor College of Medicine Search Launcher via the World Wide Web (http://dot.imgen.bcm.tmc.edu:9331/). Multiple sequence alignments of the entire regions encoding motifs I-VIII of the DNA methyltransferase proteins were performed by ClustalW (23) at the same site. The alignments were downloaded and displayed as trees with the PHYLIP software package v.3.5c (30) run locally on a desktop computer.
An 8.5 days post-coitum mouse embryo cDNA library in vector [lambda]gt10 (courtesy of Brigid Hogan) was screened by hybridization with a DNMT2 cDNA probe (encoding nt 24-1155). Overlapping clones were identified and sequenced. The combined Dnmt2 sequences lacked the 5[prime]-ATG present in DNMT2. PCR between primers complementary to 5[prime] cDNA sequences and flanking vector sequence was used to amplify 5[prime] sequences from normalized mouse cDNA libraries (courtesy of Bento Soares).
Total mouse RNA was purified from cells and tissues as described (31). RNA was fractionated by electrophoresis on a 1.5% agarose, 2.2 M formaldehyde gel, transferred to Nytran Plus membranes (Schleicher & Schuell) and cross-linked by UV irradiation (32). The blot was hybridized (33) with a Dnmt2 cDNA probe encoding nt 41-1166. An end-labeled oligonucleotide probe for 18S rRNA was used as a RNA loading control. A human RNA blot (Clontech) was hybridized with a DNMT2 probe (encoding nt 24-1155). The blot was then stripped and hybridized with a probe for DNMT1 (encoding nt 4468-5037) and with a probe for [beta]-actin (Clontech).
Riboprobes for RNase protection assays were generated by ligating an EcoRI-XbaI fragment of a Dnmt2 cDNA clone (encoding motifs I-IV) into pZero (Invitrogen), linearizing the plasmid with BamHI and transcribing with T7 polymerase as described by the vendor (Ambion). RNase protection assays were performed with the HybSpeed RPA kit from Ambion.
Restriction digests of human or C57Bl/6 mouse DNA were fractionated by electrophoresis on a 1% agarose gel, blotted to Nytran Plus (Schleicher & Schuell) and cross-linked by UV irradiation (32). Blots were hybridized to a Dnmt2 probe (encoding nt 479-1054) at 55°C in 6× SSC, 5× Denhardt's solution and 0.5% SDS and washed twice at 55°C in 2× SSC and 0.5% SDS (low stringency conditions) or 65°C in 0.5× SSC and 0.5% SDS (high stringency conditions) prior to autoradiography.
Primers HPMT4 (5[prime]-CAGCATACAGTGTTCTGG-3[prime]) and HPMT6 (5[prime]-CAGCAATGACTTTGGTGG-3[prime]), both specific for human DNMT2, were used in PCR screening of the Genebridge 4 radiation hybrid panel (24). Data from each radiation hybrid cell line were submitted to the Whitehead/MIT server via the World Wide Web (http://www-genome.wi.mit.edu), where it was tested against the Genebridge 4 panel framework. These results were then compared with the human transcript map (34) (http://www.ncbi.nlm.nih.gov/SCIENCE96/).
We thank B.Hogan, B.Soares, J.Gearhardt and J.Dean for cDNA libraries, Linda Lieberman for assistance with screening libraries and A.Klar and P.Nurse for sharing unpublished data. Supported by NIH grants CA60610 and GM00616.
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