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1 Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
2 Samuel Runenfeld Research Institute, Mt. Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada
The Nanog and Oct-4 genes are essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that DNA methylation and chromatin remodeling underlie the cell type-specific mechanism of Oct-4 gene expression. In the present study, we found that there is a tissue-dependent and differentially methylated region (T-DMR) in the Nanog up-stream region. The T-DMR is hypomethylated in ES cells, but is heavily methylated in trophoblast stem (TS) cells and NIH/3T3 cells, in which the Nanog gene is repressed. Furthermore, in vitro methylation of T-DMR suppressed Nanog promoter activity in reporter assay. Chromatin immunoprecipitation assay revealed that histone H3 and H4 are highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus in ES cells. Conversely, histone deacetylation and H3-K4 demethylation occurred in TS cells. Importantly, in TS cells, hypermethylation of H3-K9 and -K27 is found only at the Nanog locus, not the Oct-4 locus, indicating that the combination of histone modifications associated with the Nanog gene is distinct from that of the Oct-4 gene. In conclusion, the Nanog gene is regulated by epigenetic mechanisms involving DNA methylation and histone modifications.
aThese authors have contributed equally to this work. * Correspondence: E-mail: ashiota{at}mail.ecc.u-tokyo.ac.jp
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