A Novel Dnmt3a Isoform Produced from an Alternative Promoter Localizes to Euchromatin and Its Expression Correlates with Activede Novo Methylation
2002; Elsevier BV; Volume: 277; Issue: 41 Linguagem: Inglês
10.1074/jbc.m205312200
ISSN1083-351X
AutoresTaiping Chen, Yoshihide Ueda, Shaoping Xie, En Li,
Tópico(s)Cancer-related gene regulation
ResumoPrevious studies have shown that theDnmt3b 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 Dnmt3ain 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. Previous studies have shown that theDnmt3b 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 Dnmt3ain 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. DNA methyltransferase embryonal carcinoma embryonic stem green fluorescent protein rapid amplification of cDNA ends reverse transcription Four DNA cytosine-5 methyltransferases (Dnmts),1 namely Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b, have been identified in humans and mice (1Bestor T. Laudano A. Mattaliano R. Ingram V. J. Mol. Biol. 1988; 203: 971-983Crossref PubMed Scopus (719) Google Scholar, 2Okano M. Xie S. Li E. Nucleic Acids Res. 1998; 26: 2536-2540Crossref PubMed Scopus (340) Google Scholar, 3Yoder J.A. Bestor T.H. Hum. Mol. Genet. 1998; 7: 279-284Crossref PubMed Scopus (226) Google Scholar, 4Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 5Xie S. Wang Z. Okano M. Nogami M., Li, Y., He, W.W. Okumura K. Li E. Gene. 1999; 236: 87-95Crossref PubMed Scopus (344) Google Scholar). Inactivation of either Dnmt1 or Dnmt3aand Dnmt3b in mice by gene targeting leads to hypomethylation of the genome and embryonic lethality, indicating that DNA methylation is essential for mammalian development (6Li E. Bestor T.H. Jaenisch R. Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3246) Google Scholar, 7Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar). Although both Dnmt1 and the Dnmt3 family of enzymes methylate predominantly CpG dinucleotides in vitro, genetic studies reveal that these two classes of enzymes display different activities and functions in vivo. Dnmt1 is primarily responsible for the maintenance of DNA methylation patterns in proliferating cells, whereas Dnmt3a and Dnmt3b are necessary for de novomethylation and for the establishment of new methylation patterns in mammalian cells and transgenic flies (7Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar, 8Lei H., Oh, S.P. Okano M. Juttermann R. Goss K.A. Jaenisch R. Li E. Development. 1996; 122: 3195-3205Crossref PubMed Google Scholar, 9Lyko F. Ramsahoye B.H. Kashevsky H. Tudor M. Mastrangelo M.A. Orr-Weaver T.L. Jaenisch R. Nat. Genet. 1999; 23: 363-366Crossref PubMed Scopus (163) Google Scholar, 10Hsieh C.L. Mol. Cell. Biol. 1999; 19: 8211-8218Crossref PubMed Scopus (221) Google Scholar). In mice, DNA methylation patterns are established during embryonic development through dynamic regulation of de novomethylation and demethylation. De novo methylation also occurs during gametogenesis in both male and female germ cells and is believed to play a critical role in the establishment of genomic imprinting in the gametes. Genomic imprinting is an epigenetic process that marks alleles according to their parental origin during gametogenesis and results in monoallelic expression of a small set of genes (known as imprinted genes) in the offspring (for reviews, see Refs. 11Jaenisch R. Trends Genet. 1997; 13: 323-329Abstract Full Text PDF PubMed Scopus (320) Google Scholar and 12Reik W. Dean W. Walter J. Science. 2001; 293: 1089-1093Crossref PubMed Scopus (2457) Google Scholar). Recent studies have shown that Dnmt3a is required for the establishment of methylation imprints in the oocytes (13Hata K. Okano M. Lei H. Li E. Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar). De novo methylation has been detected mainly in embryonic stem (ES) cells and embryonal carcinoma (EC) cells, early embryos, and developing germ cells, whereas it is largely suppressed in differentiated somatic cells (8Lei H., Oh, S.P. Okano M. Juttermann R. Goss K.A. Jaenisch R. Li E. Development. 1996; 122: 3195-3205Crossref PubMed Google Scholar, 14Stewart C.L. Stuhlmann H. Jahner D. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4098-4102Crossref PubMed Scopus (199) Google Scholar, 15Kafri T. Ariel M. Brandeis M. Shemer R. Urven L. Mccarrey J. Cedar H. Razin A. Genes Dev. 1992; 6: 705-714Crossref PubMed Scopus (595) Google Scholar, 16Santos F. Hendrich B. Reik W. Dean W. Dev. Biol. 2002; 241: 172-182Crossref PubMed Scopus (1000) Google Scholar). Because aberrant de novo methylation of CpG sites in promoter sequences can lead to abnormal gene silencing and is associated with diseases such as cancers (17Robertson K.D. Wolffe A.P. Nat. Rev. Genet. 2000; 1: 11-19Crossref PubMed Scopus (883) Google Scholar), it is believed thatde novo methylation must be under tight regulation during development. Dnmt1 is expressed ubiquitously, and its function is required for the survival of somatic cells (8Lei H., Oh, S.P. Okano M. Juttermann R. Goss K.A. Jaenisch R. Li E. Development. 1996; 122: 3195-3205Crossref PubMed Google Scholar, 18Tucker K.L. Beard C. Dausmann J. Jackson-Grusby L. Laird P.W. Lei H., Li, E. Jaenisch R. Genes Dev. 1996; 10: 1008-1020Crossref PubMed Scopus (239) Google Scholar). Dnmt3a and Dnmt3b, on the other hand, are regulated during development (4Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 7Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar). Although both Dnmt3a and Dnmt3b transcripts are present at very low levels in somatic cells, they are expressed at high levels in ES cells and germ cells in which active de novo methylation is detected. Several different Dnmt3b transcripts, resulting from alternative splicing of exons 10, 21, and/or 22, have been reported (4Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar,5Xie S. Wang Z. Okano M. Nogami M., Li, Y., He, W.W. Okumura K. Li E. Gene. 1999; 236: 87-95Crossref PubMed Scopus (344) Google Scholar, 19Robertson K.D. Uzvolgyi E. Liang G. Talmadge C. Sumegi J. Gonzales F.A. Jones P.A. Nucleic Acids Res. 1999; 27: 2291-2298Crossref PubMed Scopus (720) Google Scholar, 20Hansen R.S. Wijmenga C. Luo P. Stanek A.M. Canfield T.K. Weemaes C.M. Gartler S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14412-14417Crossref PubMed Scopus (605) Google Scholar). Dnmt3b1 and Dnmt3b2 are enzymatically active in standard DNA methyltransferase assays, whereas Dnmt3b3, which lacks part of motif IX, appears to be inactive (4Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 21Aoki A. Suetake I. Miyagawa J. Fujio T. Chijiwa T. Sasaki H. Tajima S. Nucleic Acids Res. 2001; 29: 3506-3512Crossref PubMed Scopus (149) Google Scholar). Dnmt3b4 andDnmt3b5 encode truncated proteins that lack motifs IX and X and are presumably inactive (19Robertson K.D. Uzvolgyi E. Liang G. Talmadge C. Sumegi J. Gonzales F.A. Jones P.A. Nucleic Acids Res. 1999; 27: 2291-2298Crossref PubMed Scopus (720) Google Scholar, 20Hansen R.S. Wijmenga C. Luo P. Stanek A.M. Canfield T.K. Weemaes C.M. Gartler S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14412-14417Crossref PubMed Scopus (605) Google Scholar). So far, only one form of Dnmt3a has been identified and shown to be capable of methylating DNA bothin vitro and in vivo (4Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 7Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar, 9Lyko F. Ramsahoye B.H. Kashevsky H. Tudor M. Mastrangelo M.A. Orr-Weaver T.L. Jaenisch R. Nat. Genet. 1999; 23: 363-366Crossref PubMed Scopus (163) Google Scholar, 10Hsieh C.L. Mol. Cell. Biol. 1999; 19: 8211-8218Crossref PubMed Scopus (221) Google Scholar, 21Aoki A. Suetake I. Miyagawa J. Fujio T. Chijiwa T. Sasaki H. Tajima S. Nucleic Acids Res. 2001; 29: 3506-3512Crossref PubMed Scopus (149) Google Scholar). DNA methylation may also be regulated through subcellular localization of Dnmt3a and Dnmt3b. Tagged Dnmt3a and Dnmt3b proteins have been shown to localize preferentially to the heterochromatin in transfected cells (22Bachman K.E. Rountree M.R. Baylin S.B. J. Biol. Chem. 2001; 276: 32282-32287Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Dnmt3a and Dnmt3b have been shown to interact with histone deacetylases (HDAC1 and HDAC2) and transcription factors RP58 and PML (22Bachman K.E. Rountree M.R. Baylin S.B. J. Biol. Chem. 2001; 276: 32282-32287Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 23Fuks F. Burgers W.A. Godin N. Kasai M. Kouzarides T. EMBO J. 2001; 20: 2536-2544Crossref PubMed Scopus (470) Google Scholar, 24Di Croce L. Raker V.A. Corsaro M. Fazi F. Fanelli M. Faretta M. Fuks F., Lo Coco F. Kouzarides T. Nervi C. Minucci S. Pelicci P.G. Science. 2002; 295: 1079-1082Crossref PubMed Scopus (694) Google Scholar), which may target Dnmt3a and Dnmt3b to transcriptionally silent heterochromatin. Although the majority of CpG islands, which usually localize in euchromatin regions, are methylation-free, some CpG islands become methylated during development or under pathological conditions such as in cancer cells (25Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5486) Google Scholar). It remains unknown how DNA methylation is differentially regulated in different chromatin regions and what enzymes are involved. In this study, we report the identification of a novel isoform of Dnmt3a, termed Dnmt3a2, which is encoded by transcripts initiated from an intronic promoter. Dnmt3a2 has similar methyltransferase activity to Dnmt3a, but it localizes to euchromatin instead of heterochromatin. Moreover, Dnmt3a2 expression is tightly regulated and correlates with high de novo methylation activity, whereas Dnmt3a is expressed ubiquitously. Therefore, Dnmt3a and Dnmt3a2 may methylate distinct DNA targets and have different functions in development. The GFP-Dnmt3a and GFP-Dnmt3b, the Dnmt3a- and Dnmt3b-pcDNA, and the His6-tagged Dnmt3a constructs were generated by subcloning the corresponding Dnmt3a or Dnmt3b cDNA into pEGFP-C1 (CLONTECH), pcDNA6/V5-HisA (Invitrogen), and pET-28b(+) (Novagen), respectively. The luciferase reporter constructs were generated by inserting a ∼2.0-kb Dnmt3a genomic fragment, which contains a putative promoter (P2), in forward (P2-luc) or reverse (P2R-luc) orientation into pGL3-Basic (Promega). The P2 targeting vector was constructed by sequentially subcloning Dnmt3agenomic fragments (arms), the hCMV-hygTK cassette, and the PGK-DTA cassette into pBluescript II SK. All of the Dnmt3a genomic fragments were generated by PCR using a BAC clone (Genome Systems Inc.) as the template and the following pairs of oligonucleotides as primers: 5′-CTGGGATCCAGGCACCTGGGGTGTTACCT-3′ and 5′-GGTGGATCCCCTCTGCAGTACAGCTC-3′ (for P2-luc and P2R-luc), 5′-CTGGAATTCTCCTACCTT TG-3′ and 5′-CCTGGATCCCAGCCAGTGAGCTGG-3′ (for P2 targeting vector, 5′ arm, 3.7 kb), and 5′-GTTCCGCGGCTGCTCATT-3′ and 5′-CCACCGCGGCCGACTTGCCTCTACTTC-3′ (for P2 targeting vector, 3′ arm, 3.0 kb). (The restriction sites used for cloning are underlined). The identities of the constructs were verified by DNA sequencing. The Dnmt3a and Dnmt3b rabbit polyclonal antibodies, 164 and 157, were generated against mouse Dnmt3a amino acids 15–126 and Dnmt3b amino acids 1–181, respectively. The Dnmt3a monoclonal antibody (mAb) (clone 64B1446) was purchased from Imgenex. Anti-GFP mAb (a mixture of clones 7.1 and 13.1) was obtained from Roche. Anti-tubulin mAb (Ab-1) was obtained from Oncogene Research Products. Anti-DNMT1 (human) polyclonal antibody was purchased from New England Biolabs. Anti-histone H1 (AE-4) and anti-lamin B (M-20) were obtained from Santa Cruz Biotechnology. Transient transfection was carried out in COS-7, NIH 3T3, or ES cells using LipofectAMINE PLUS reagent (Invitrogen). Cell fractionation, immunoprecipitation, immunoblotting, and fluorescence microscopy analyses were performed as previously described (26He D.C. Nickerson J.A. Penman S. J. Cell Biol. 1990; 110: 569-580Crossref PubMed Scopus (369) Google Scholar, 27Chen T. Richard S. Mol. Cell. Biol. 1998; 18: 4863-4871Crossref PubMed Scopus (105) Google Scholar, 28Chen T. Boisvert F.M. Bazett-Jones D.P. Richard S. Mol. Biol. Cell. 1999; 10: 3015-3033Crossref PubMed Scopus (124) Google Scholar). Luciferase reporter constructs as well as pGL-3-Basic (empty vector) were individually co-transfected with pRL-TK (internal control, Promega) into ES cells or NIH 3T3 cells. The cell lysates were analyzed for luciferase activities using the dual-luciferase reporter assay system (Promega). 5′-RACE was carried out on total RNA prepared from ES cells using the 5′-RACE system (Invitrogen) with Dnmt3a-specific primers: 5′-AGCTGCTCGGCTCCGGCC-3′ (for reverse transcription), 5′-TCCCCCACACCAGCTCTCC-3′ (for first round of PCR), and 5′-CTGCAATTACCTTGGCTT-3′ (for second round of PCR). For RT-PCR analysis, total RNA was reverse-transcribed with oligo(dT)12–18, and the resulting cDNAs were amplified by PCR. The PCR products were analyzed either by ethidium bromide staining (for Dnmt3a) or Southern hybridization using cDNA fragments as probes (for Dnmt3b). Dnmt3a-specific primers used are 5′-TCCAGCGGCCCCGGGGAC-3′ (F1), 5′-CCCAACCTGAGGAAGGGA-3′ (F2), 5′-ACCAACATCGAATCCATG-3′ (F3), 5′-TCCCGGGGCCGACTGCGA-3′ (F4), 5′-AGGGGCTGCACCTGGCCTT-3′ (F5), 5′-TCCCCCACACCAGCTCTCC-3′ (R1), and 5′-CCTCTGCAGTACAGCTCA-3′ (R2). Dnmt3b-specific primers used are 5′-TGGGATCGAGGGCCTCAAAC-3′ and 5′-TTCCACAGGACAAACAGCGG-3′ (for exon 10) and 5′-GCGACAACCGTCCATTCTTC-3′ and 5′-CTCTGGGCACTGGCTCTGACC-3′ (for exons 21 and 22). Northern hybridization was performed according to standard protocols. Dnmt3a cDNA fragments used as probes were generated by PCR. The primer pairs used were 5′-GCAGAGCCGCCTGAAGCC-3′ and 5′-CCTTTTCCAACGTGCCAG-3′ (for probe 1) and 5′-GCCAAGGTAATTGCAGTA-3′ and 5′-GATGTTTCTGCACTTCTG-3′ (for probe 2). The P2 targeting vector was electroporated intoDnmt3a +/− ES cells (7Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar), which were subsequently selected in hygromycin-containing medium. Genomic DNA isolated from hygromycin-resistant colonies was digested with ScaI and analyzed by Southern hybridization using a 0.45-kbKpnI-SpeI fragment as a probe. For in vitro DNA methyltransferase activity, His6-tagged Dnmt3a proteins were incubated with double-stranded poly(dI-dC) (Pharmacia) in the presence of S-adenosyl-l-methionine (methyl-3H) (SAM, PerkinElmer Life Sciences), and the incorporation of 3H methyl groups into poly(dI-dC) was measured as previously described (4Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar, 21Aoki A. Suetake I. Miyagawa J. Fujio T. Chijiwa T. Sasaki H. Tajima S. Nucleic Acids Res. 2001; 29: 3506-3512Crossref PubMed Scopus (149) Google Scholar). The reactions were carried out at 37 °C for 1 h in methylation buffer (20 mmTris-HCl, pH 7.4, 40 mm NaCl, 5 mm EDTA, 200 μg/ml bovine serum albumin, 25% glycerol, and 1 mmdithiothreitol) containing the purified enzyme, the substrate DNA, and SAM at concentrations of 0.5 μm, 2 μm, and 2 μm, respectively. For de novo methylation activity, human EC cell lines and breast/ovarian cancer cell lines were infected with Moloney murine leukemia virus, and the methylation status of newly integrated provirus was analyzed as previously described (8Lei H., Oh, S.P. Okano M. Juttermann R. Goss K.A. Jaenisch R. Li E. Development. 1996; 122: 3195-3205Crossref PubMed Google Scholar). The Dnmt3a and Dnmt3b proteins show high sequence homology in the C-terminal catalytic domain, but they share little sequence similarity in the N-terminal regulatory region except for the conserved proline-tryptophan-tryptophan-proline and plant homeodomain domains (Fig. 1 A). To characterize the Dnmt3a and Dnmt3b proteins, we generated rabbit polyclonal antibodies against the N-terminal regions of mouse Dnmt3a (antibody 164) and Dnmt3b (antibody 157) and also obtained a commercial mAb (64B1446), which was raised against the full-length mouse Dnmt3a. The epitope recognized by 64B1446 was mapped to a region (amino acids 705–908) at the C terminus (data not shown). The specificity of these antibodies was examined using GFP fusion proteins expressed in Cos-7 cells (Fig. 1 B). Anti-GFP immunoblotting showed the expression of the GFP fusion proteins (first panel). The polyclonal antibodies, 164 and 157, were specific for Dnmt3a and Dnmt3b, respectively (second andthird panels). The mAb, 64B1446, reacted strongly with Dnmt3a proteins and weakly with Dnmt3b1 and Dnmt3b2, but not Dnmt3b3 (fourth panel), consistent with the epitope-mapping results. Previous studies showed that Dnmt3a and Dnmt3btranscripts were abundant in ES cells (4Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar), but their protein products were not analyzed. To address this question, we analyzed wild-type (J1), Dnmt3a −/− (6aa),Dnmt3b −/− (8bb), and [Dnmt3a −/−,Dnmt3b −/−] (7aabb) mutant ES cells (7Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar) by immunoblotting with the Dnmt3a and Dnmt3b antibodies (Fig. 1,C and D). Two distinct bands, which migrated at ∼120 and ∼110 kDa, were detected by antibody 157 in J1 and 6aa cells, but not in 8bb and 7aabb cells (Fig. 1 C), indicating that these bands represent Dnmt3b proteins. The more abundant 120-kDa band most likely represents Dnmt3b1, and the 110-kDa band represents an isoform smaller than Dnmt3b2 but slightly larger than Dnmt3b3 (Fig.1 C). RT-PCR analysis confirmed the expression of two majorDnmt3b transcripts in ES cells; one corresponds toDnmt3b1 and the other is an alternatively spliced variant that lacks exons 21 and 22 (Fig. 6 and data not shown). We named this new isoform Dnmt3b6 (schematically shown in Fig.1 A). Indeed, the 110-kDa band observed in ES cells co-migrated with protein expressed from Dnmt3b6 cDNA (Fig.1 C, lanes 8 and 9). Dnmt3b6 lacks motif IX and thus may not be enzymatically active, like Dnmt3b3 (21Aoki A. Suetake I. Miyagawa J. Fujio T. Chijiwa T. Sasaki H. Tajima S. Nucleic Acids Res. 2001; 29: 3506-3512Crossref PubMed Scopus (149) Google Scholar). Dnmt3a-specific antibody 164 detected a single band of ∼130 kDa in J1 and 8bb cells, which co-migrated with the control Dnmt3a protein (Fig.1 D, lanes 1, 2, and 5), but not in 6aa and 7aabb cells (lanes 3 and 4). Surprisingly, when the same blot was reprobed with anti-Dnmt3a mAb 64B1446, two more intense bands of ∼120 kDa and ∼100 kDa were detected in addition to the 130-kDa Dnmt3a protein in J1 cells (Fig.1 D, lane 7). The 120-kDa band represents Dnmt3b1, as it was also present in 6aa cells but absent in 8bb cells (lanes 9 and 10). Like the 130-kDa Dnmt3a protein, the 100-kDa band could be detected in 8bb cells (lane 10) but not in 6aa and 7aabb cells (lanes 8and 9), indicating that it is a novel product of theDnmt3a gene. We named this short form Dnmt3a2. Importantly, the immunoblotting result indicates that Dnmt3a2 is the predominantDnmt3a gene product in ES cells (Fig. 1 D). The fact that Dnmt3a2 could not be recognized by antibody 164 suggests that Dnmt3a2 lacks the N-terminal region of Dnmt3a. Inspection of theDnmt3a cDNA sequence revealed that, in addition to the known initiation codon (ATG1), two downstream in-frame ATGs (ATG2 and ATG3), corresponding to Met-159 and Met-220, were found to be within the Kozak consensus sequence. To test the possibility that Dnmt3a2 was produced by translation initiated at one of these ATGs, we expressed in 6aa cells two Dnmt3a proteins with the N-terminal 158 and 219 amino acids truncated and showed that Dnmt3a (220–908) co-migrated with endogenous Dnmt3a2 from J1 cells (Fig. 1 E, comparelanes 3 and 4). This result suggests that ATG3 might be the initiation codon for Dnmt3a2. To further determine whether Dnmt3a2 is produced from the same mRNA transcript as Dnmt3a, we transfected 6aa cells with an expression vector containing the entireDnmt3a coding sequence. Immunoblotting analysis using antibody 64B1446 showed that only Dnmt3a was expressed (Fig.1 F, lane 2). These results suggest that Dnmt3a2 does not derive from Dnmt3a transcript by the use of an alternative ATG or from Dnmt3a protein by proteolytic cleavage or degradation. To determine whether Dnmt3a and Dnmt3a2 are encoded by distinct mRNA transcripts, total RNA from J1, 6aa ES cells, and NIH 3T3 cells (which express only Dnmt3a; see Fig. 7) was analyzed by Northern hybridization with Dnmt3a cDNA probes upstream or downstream of ATG3 (Fig.2 B). The downstream probe (Fig. 2 A, Probe 2) detected two major transcripts of 4.2 kb and 4.0 kb and a weak band of 9.5 kb from J1 cells (Fig.2 B, lane 5), consistent with our previous results (4Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar). All of the transcripts were smaller, and the intensity of 4.2 kb and 4.0 kb bands was substantially reduced in 6aa cells (lane 6), indicating that truncated transcripts were generated. The 9.5-kb transcript was also present at low level in NIH 3T3 cells, but the 4.2-kb and 4.0-kb transcripts were absent (lane 4). Interestingly, the upstream probe (Fig. 2 A, Probe 1) recognized the 9.5-kb transcript in NIH 3T3 and J1 cells and a 7.5-kb truncated form in 6aa cells, but it failed to hybridize to the 4.2-kb and 4.0-kb transcripts in J1 cells (lanes 1–3). Taken together, these observations suggest that Dnmt3a2 is probably encoded by the 4.2-kb and 4.0-kb transcripts. Our previous data indicated that the 4.2-kb and 4.0-kb transcripts differ in their 3′-untranslated region, probably due to alternative 3′ processing (4Okano M. Xie S. Li E. Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1294) Google Scholar). It remains to be determined whether Dnmt3a is encoded by the 9.5-kb transcript or whether the Dnmt3a signal is too weak to be detected.Figure 2Dnmt3a and Dnmt3a2 are encoded by distinct transcripts. A, the structure of mouse and humanDnmt3a gene, mRNAs and proteins. Exons are shown asblack bars. The Dnmt3a2-unique exons are indicated by an asterisk. Dnmt3a and Dnmt3a2 proteins have identical amino acid sequences except that Dnmt3a has 219 (mouse) or 223 (human) extra residues at the N terminus (human DNMT3A amino acid numbering is shown in parentheses). The primers used for RT-PCR are shown under the corresponding exons (F, forward;R, reverse). The probes used for Northern hybridization, shown under the Dnmt3a protein, represent the corresponding cDNA fragments. B, Northern blots of total RNA (20 μg per lane) from NIH 3T3, J1, and 6aa cells were probed with Probe 1 (lanes 1–3) or Probe 2 (lanes 4–6). As a loading control, the 28S rRNA stained with ethidium bromide was shown at thebottom. C, total RNA from J1 cells was reverse transcribed using poly (dT)12–18, and the resulting cDNAs were subjected to PCR amplification with the indicated Dnmt3a primers. Dnmt3a cDNA was used as a positive control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the identity of the Dnmt3a transcripts, we performed 5′-RACE on RNA prepared from J1 ES cells with primers annealing to Dnmt3a sequences downstream of the putative Dnmt3a2 translation start site (ATG3 at M220). Two species ofDnmt3a transcripts were obtained. One of them matched theDnmt3a cDNA sequence, and the other contained a 55-bp sequence at its 5′ end that did not match any known Dnmt3acDNA sequence. Searches of the Celera mouse-genome data base revealed that the 55-bp sequence was part of an exon located in an intron of the Dnmt3a gene. Using the new exon sequence as query, we identified a mouse expressed sequence tag clone,BE855330, which extended the exon to at least 117 bp. Sequencing analysis revealed that the expressed sequence tag clone shared all the downstream exons with Dnmt3a (Fig. 2 A). We conclude that the newly identified transcript encodes Dnmt3a2, because its open reading frame would predict a protein that lacks the N-terminal 219 amino acids of Dnmt3a (Fig. 2 A). As illustrated in Fig. 2 A, the murine Dnmt3a gene consists of 24 exons. Exons 8–24 are shared by both Dnmt3aand Dnmt3a2. Exons 1–6 are present only inDnmt3a, whereas exon 7 (indicated by an asterisk) is unique to Dnmt3a2. By RT-PCR analysis and data base searches, we also identified human DNMT3A2 (Fig.2 A). It is very similar to its murine homologue except that it contains an additional sequence of 68 bp in the 5′-untranslated region, which is encoded by an extra exon located ∼2.5 kb downstream of exon 7 (the newly identified exons are indicated by an asterisk). The predicted mouse Dnmt3a2 and human DNMT3A2 proteins, each consisting of 689 amino acids (Fig. 2 A), show high sequence identity (98.5%). The 5′-RACE results were confirmed by RT-PCR analysis of total RNA from J1 cells using primers annealing to different Dnmt3a exons (Fig. 2 A). Combination of Dnmt3a-specific (F1–F4) or Dnmt3a2-specific (F5) primers with a downstream primer in exon 9 (R1) verified the expression of both Dnmt3aand Dnmt3a2 transcripts in ES cells (Fig. 2 C,lanes 1–4 and 9–16). However, combination of the same Dnmt3a primers (F1–F4) with a primer in the uniqueDnmt3a2 exon (R2) failed to generate any PCR products (lanes 5–8). These results indicate that it is unlikely that the Dnmt3a and Dnmt3a2 transcripts are produced via alternative splicing. The observation that the Dnmt3a2-specific exon is located in a region >80 kb downstream of the putative Dnmt3a promoter suggests that Dnmt3a2 transcription may be driven by a different promoter. Indeed, analysis of the large (∼18 kb) "intron" preceding exon 7 with PROSCAN (bimas.dcrt.nih.gov/molbio/proscan) predicted that a 1.4-kb region immediately upstream of exon 7 has a high probability to function as a promoter. It should also be noted that the uniqueDnmt3a2 exon resides in a GC-rich CpG island, which is a hallmark of the promoter region of genes. We tested the transcriptional activity of the putative promoter using a reporter system (Fig.3). A ∼2.0-kb genomic fragment that includes the putative promoter (P2) was inserted, in both orientations, upstream of the cDNA encoding the firefly luciferase followed by the SV40 late poly(A) signal (Fig. 3 A). Transient-transfection experiments demonstrated that the P2 fragment has high promoter activity in ES cells but much lower activity in NIH 3T3 cells (Fig. 3 B, P2-luc), consistent with the expression levels of Dnmt3a2 in these cell types (Fig.2 B). The transcriptional activity of the P2 fragment is orientation-dependent, as the same fragment showed no promoter activity when subcloned in reverse orientation (Fig.2 B, P2R-luc). As a positive control, SV40 promoter worked equally well in both cell types (data not shown). These data strongly suggest that the region 5′ adjacent to exon 7 functions as a promoter and drives the expression of Dnmt3a2. To confirm that exon 7 and the adjacent promoter are essential for the expression of Dnmt3a2, we deleted the P2 region from the wild-type allele in Dnmt3a +/− ES cells (7Okano M. Bell D.W. Haber D.A. Li E. Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4574) Google Scholar) by gene targeting. An hCMV-hygTK cassette was inserte
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