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1 Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences and Veterinary Medical Sciences, The University of Tokyo, Tokyo 113-8657, Japan
2 Laboratory of Mouse Model, Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
3 Department of Molecular Genetics and Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a–/– embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a–/– cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a–/– cells. These data indicate that G9a site-selectively contributes to DNA methylation.
* Correspondence: E-mail: ashiota{at}mail.ecc.u-tokyo.ac.jp
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