Physical and functional interactions between the human DNMT3L protein and members of the de novo methyltransferase family

The de novo methyltransferase-like protein, DNMT3L, is required for methylation of imprinted genes in germ cells. Although enzymatically inactive, human DNMT3L was shown to act as a general stimulatory factor for de novo methylation by murine Dnmt3a. Several isoforms of DNMT3A and DNMT3B with development-stage and tissue-specific expression patterns have been described in mouse and human, thus bringing into question the identity of the physiological partner(s) for stimulation by DNMT3L. Here, we used an episome-based in vivo methyltransferase assay to systematically analyze five isoforms of human DNMT3A and DNMT3B for activity and stimulation by human DNMT3L. Our results show that human DNMT3A, DNMT3A2, DNMT3B1, and DNMT3B2 are catalytically competent, while DNMT3B3 is inactive in our assay. We also report that the activity of all four active isoforms is significantly increased upon co-expression with DNMT3L, albeit to varying extents. This is the first comprehensive description of the in vivo activities of the poorly characterized human DNMT3A and DNMT3B isoforms and of their functional interactions with DNMT3L. To further elucidate the mechanism by which DNMT3L stimulates DNA methylation, we have mapped in detail the domains that mediate interaction of human DNMT3L with human DNMT3A and DNMT3B. Our results show that the C-terminus of DNMT3L is the only region required for interaction with DNMT3A and DNMT3B and that interaction takes place through the C-terminal catalytic domain of DNMT3A and DNMT3B. The implications of these findings for the regulation of de novo methyltransferases and genomic imprinting are discussed. This article contains Supplementary Material available at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/2005/95/chen.html.