The Sry (sex determining region on Y chromosome) gene is a master gene for sex determination. We previously reported that the Sry gene has tissue-dependent and differentially methylated regions (T-DMRs) by analyzing the DNA methylation states at CpG sites in the promoter regions. In this study, we found unique non-CpG methylation at the internal cytosine in the 5'-CCTGG-3' pentanucleotide sequence in the Sry T-DMR. This non-CpG methylation was detected in four mouse strains (ICR, BALB/c, DBA2 and C3H), but not in two strains (C57BL/6 and 129S1), suggesting that the CCTGG methylation is tentative and unstable. Interestingly, this CCTGG methylation was associated with demethylation of the CpG sites in the Sry T-DMR in the developmental process. A methylationmediated promoter assay showed that the CCTGG methylation promotes gene expression. Our finding shows that non-CpG methylation has unique characteristic and is still conserved in mammals.
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