Identification and characterization of alternatively spliced variants of DNA methyltransferase 3a in mammalian cells
Introduction
Methylation of cytosine bases in the context of CpG dinucleotides is an epigenetic modification that occurs at specific regions in the DNA as a mechanism intended to silence gene transcription and also to establish heritable markers for transcription-incompetent DNA (Jones and Laird, 1999, Jones and Takai, 2001, Bird, 2002). This process is essential for embryonic development, parental imprinting and X-chromosome inactivation (Jones and Takai, 2001, Robertson, 2001). The distribution of methylated and unmethylated DNA regions creates specific patterns that are established in a de novo fashion and are subsequently preserved by copying the DNA methylation information from the parental strand onto the daughter DNA strand after DNA replication.
Mammalian DNA methylation is performed by at least three active DNA methyltransferases (DNMTs). The preference of DNMT1 for hemimethylated DNA in vitro is suggestive of its role in maintenance methylation (Pradhan et al., 1997, Pradhan et al., 1999, Yoder et al., 1997). DNMT 3a and 3b enzymes are thought to function as de novo DNA methyltransferases (Okano et al., 1998, Xie et al., 1999), and were shown to have equal preference for unmethylated and hemimethylated DNA substrates in vitro (Okano et al., 1998) as well as in vivo (Hsieh, 1999). The murine homologues of Dnmt1, 3a and 3b were shown to be required for embryonic development as these Dnmt −/− knockout mice are lethal (Li et al., 1992, Okano et al., 1999). Unlike Dnmt1, Dnmt3a and 3b are developmentally regulated (Okano et al., 1998), and have recently been shown to be important in establishing maternal imprints in mice (Hata et al., 2002). Mouse ES cells express high levels of Dnmt3a and 3b, which correlate with an increased de novo activity in these cells (Stewart et al., 1982, Lei et al., 1996). The expression levels of Dnmt3a and 3b, as well as de novo activity, are dramatically reduced after differentiation in mouse somatic cells (Okano et al., 1998).
Several alternatively spliced variants of DNMTs have been characterized in human and mouse cells (Mertineit et al., 1998, Okano et al., 1998, Hsu et al., 1999, Robertson et al., 1999). Tissue-specific transcripts of human and mouse DNMT3a (4.0 kb, 4.2 kb (mouse), 4.4 kb (human), and 9.5 kb in length) have been identified by Northern blot analyses (Okano et al., 1998, Robertson et al., 1999, Xie et al., 1999). Recently, the murine 4.0 and 4.2 kb transcripts were shown to be encoded by a Dnmt3a isoform, Dnmt3a2, whose expression was shown to correlate with the de novo methylation in mouse ES cells, and a human DNMT3a2 isoform has also been identified (Chen et al., in press). Mouse and human DNMT3a2 are expressed from separate promoters located in intron 6, and contain additional exonic sequences (exon 7) that are not present in DNMT3a. However, the function of human DNMT3a and DNMT3a2 and their relationship to the 4.0, 4.4 and 9.5 kb mRNA transcripts are not fully understood.
In this work we describe and characterize additional alternatively spliced variants of DNMT3a. Both human and mouse cells expressed novel DNMT3a transcripts that contained an alternate 5′ exon 1 (1β) as well as transcripts that included intron 4. Transcripts containing the intron 4 sequence were less abundant. The exon 1β-containing transcript (Dnmt3a-β) was preferentially expressed in mouse ES cells, whereas the α transcript (Dnmt3a-α) was predominantly expressed in mouse and human somatic cells. These data suggest that both Dnmt3a-β and Dnmt3a2 may be important for establishment of DNA methylation patterns during development.
Section snippets
Cell culture
Wild-type mouse ES cells were provided by Dr. En Li (Massachusetts General Hospital, Boston, MA). Embryoid bodies (EB) were derived from ES cells that were grown in suspension in petri dishes in ES culture media without leukemia inhibitory factor. MCF7, HCT15, T24, and 10T1/2 cells were obtained from the American Type Culture Collection (Rockville, MD). NIH3T3 and MEF cells were provided by Dr. Peter Laird (University of Southern California, Los Angeles, CA). LD419 cells were isolated in our
Identification and characterization of multiple transcripts of human and mouse DNMT3a mRNAs
The DNMT3a gene encodes two known isoforms that are driven by separate promoters, which gives rise to two distinct mRNA species, DNMT3a and DNMT3a2 that differ only in their 5′ regions (Xie et al., 1999, Chen et al., in press). DNMT3a2 does not include exons 1-6, but contains two additional exons (7a-b) in intron 6 (Fig. 1A). We first performed a BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) of the Expressed Sequence Tag (EST) database using the full-length human DNMT3a cDNA sequence and
Acknowledgements
We thank Dr. En Li for the ES cells, Dr. Peter W. Laird for the NIH3T3 and MEF cells, Dr. Louis Dubeau for the MCV50 and HOC7 cells, and Dr. Daiya Takai for assistance with the CpG island analysis. We thank Dr. Peter W. Laird, Dr. Gerhard Coetzee, and Jonathan Cheng for helpful discussions. This work was supported by grants CA 82422-01 and CA 83867-01 (to P.A.J.). M.V. was supported by NIH Training Grant T32 DE07211-11. D.W. was supported by NIH Training Grant in Basic Research in Oncology T32
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