Elsevier

Gene

Volume 236, Issue 1, 5 August 1999, Pages 87-95
Gene

Cloning, expression and chromosome locations of the human DNMT3 gene family

https://doi.org/10.1016/S0378-1119(99)00252-8Get rights and content

Abstract

DNA methylation plays an important role in animal development and gene regulation. In mammals, several genes encoding DNA cytosine methyltransferases have been identified. DNMT1 is constitutively expressed and is required for the maintenance of global methylation after DNA replication. In contrast, the murine Dnmt3 family genes appear to be developmentally regulated and behave like de novo DNA methyltransferases in vitro. In this study, we have cloned human DNMT3A and DNMT3B that encode full-length DNMT3A and DNMT3B proteins with 98% and 94% amino acid sequence identity to their murine homologues. The DNMT3A and DNMT3B show high homology in the carboxy terminal catalytic domain and contain a conserved cysteine-rich region, which shares homology with the X-linked ATRX gene of the SNF2/SWI family. We have mapped human DNMT3A and DNMT3B to chromosomes 2p23 and 20q11.2 respectively, and determined the DNMT3B genomic structure. We further show that DNMT3A expression is ubiquitous and can be readily detected in most adult tissues, whereas DNMT3B is expressed at very low levels in most tissues except testis, thyroid and bone marrow. Significantly, both DNMT3A and DNMT3B expression is elevated in several tumor cell lines to levels comparable to DNMT1. The cloning of the human DNMT3 genes will facilitate further biochemical and genetic studies of their functions in establishment of DNA methylation patterns, regulation of gene expression and tumorigenesis.

Introduction

DNA methylation plays an important role in regulation of gene expression (Kass et al., 1997), genomic imprinting (Bartolomei and Tilghman, 1997), and X chromosome inactivation (Jaenisch et al., 1998), and has been shown to be essential for mammalian development (Li et al., 1992). Recent studies have linked hypermethylation of CpG islands in tumor suppressor genes or hypomethylation of bulk genomic DNA to tumorigenesis (Jones and Laird, 1999). However, the mechanism by which DNA methylation is regulated during development and tumorigenesis remains largely unknown. To elucidate the regulatory mechanism, it is crucial to identify all the DNA methyltransferases.

Three distinct families of DNA methyltranferase genes, termed Dnmt1, Dnmt2 and Dnmt3, have been identified in mammalian cells. Dnmt1 is expressed constitutively in proliferating cells and has been shown to be associated with DNA replication foci (Leonhardt et al., 1992, Liu et al., 1998). Inactivation of the Dnmt1 gene results in global demethylation of genomic DNA and embryonic lethality (Lei et al., 1996, Li et al., 1992). These findings are most consistent with Dnmt1 being a maintenance methyltransferase, which restores DNA methylation patterns of the genome shortly after DNA replication.

Dnmt2 contains all the highly conserved methyltransferase motifs and is expressed at low levels in most adult tissues examined. However, methyltransferase activity was not detected with recombinant Dnmt2 (Okano et al., 1998a, Van den Wyngaert et al., 1998, Yoder and Bestor, 1998). Furthermore, inactivation of Dnmt2 in mouse embryonic stem (ES) cells had no effect on either de novo methylation or maintenance methylation (Okano et al., 1998a). Thus, it remains an open question as to whether Dnmt2 encodes a functional methyltransferase.

The Dnmt3 family consists of two related genes, termed Dnmt3a and Dnmt3b (Okano et al., 1998b). Both Dnmt3a and Dnmt3b are highly expressed in ES cells, but their expression is down-regulated upon differentiation of ES cells and remains low in adult somatic tissues. Baculovirus-expressed Dnmt3a and Dnmt3b proteins can methylate cytosine residues in various native and synthetic DNA and show no preference for hemi-methylated oligonucleotide DNA. These results indicate that the Dnmt3 genes probably encode the de novo methyltransferase activities detected in Dnmt1 null mutant ES cells. In this study, we have cloned human DNMT3A and DNMT3B cDNA, mapped their positions on chromosomes and analyzed their expression in normal tissues as well as in tumor cell lines.

Section snippets

Cloning of human DNMT3 genes

A search of the dbEST database was performed with TBLASTN (Altschul et al., 1990) using bacterial cytosine methyltransferases as queries. Four human EST clones (W76111, N88352, f12227, and T66356) were found to match the bacterial cytosine methyltransferase M. Spr. Two of the EST clones (W76111 and T66356) were deposited by the I.M.A.G.E. Consortium (Lawrence Livermore National Laboratory, Livermore, CA) and obtained from American Type Culture Collection (Rockville, MD). Complete sequencing of

Cloning of the human DNMT3A and DNMT3B cDNA

We reported previously that four human EST clones were identified by searching the dbEST database using full-length bacterial type II cytosine-5 methyltransferase sequences as queries (Okano et al., 1998b). To obtain full-length human cDNA, we screened fetal heart and fetal testis cDNA libraries using EST clones as probes. Sequencing analysis of several overlapping DNMT3A cDNA clones indicates that the DNMT3A gene encodes a polypeptide of 912 amino acid residues (Fig. 1). Although DNMT3B cDNA

Acknowledgements

We thank Dr Patricia Donahoe for the human fetal testis cDNA library. This work was supported by grants from NIH (GM52106) and Bristol-Myers/Squibb to E.L. M.O. was a postdoctoral fellow of the Japan Society for the Promotion of Science. S.X. was supported by an NIH postdoctoral training grant.

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    GenBank accession numbers: human DNMT3A, AF067972; human DNMT3B, AF156488; Zebrafish Zmt3, AF135438.

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