Identification of putative targets of DNA (cytosine-5) methylation-mediated transcriptional silencing using a novel conditionally active form of DNA methyltransferase 3a.
Aberrant DNA methylation of gene promoters is a recurrent finding associated with diseases such as cancer and inflammation, and is thought to contribute to disease through its role in transcriptional repression. Indeed, recent evidence suggests that DNA (cytosine-5) methyltransferases (DNMTs) may mediate the activity of factors promoting cell growth. Here, we utilise a novel experimental system for the conditional and reversible activation of a de novo DNMT by constructing a steroid-hormone analogue activated version, Dnmt3a-mERtrade mark. Following treatment with the oestrogen analogue 4-hydroxy tamoxifen of murine embryonic stem cells expressing this protein, we have identified by microarray analysis, several potential targets of Dnmt3a mediated transcriptional repression including the cancer associated genes Ssx2ip, Hmga1 and Wrnip. These results were validated using quantitative reverse transcriptase PCR and we confirm the biological significance of these in vitro observations by demonstrating a reduction in mRNA transcripts of the same genes within the intestinal epithelium of cancer-prone transgenic knock-in mutant mice over-expressing Dnmt3a throughout the intestinal epithelium.
Samuel
Michael S
MS
Ludwig Institute for Cancer Research, P. O. Royal Melbourne Hospital, Parkville, Vic., Australia.
Lundgren-May
Therèse
T
Ernst
Matthias
M
D000818:Animals; D002460:Cell Line; D003596:Cytosine / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D053595:Embryonic Stem Cells / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D008817:Mice, Mutant Strains; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology
C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; D013629:Tamoxifen; D003596:Cytosine; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A