PARENT SESSION
Preimplantation Embryo/Fetal Development

A CPG ISLAND DE NOVO METHYLATED IN THE DIFFERENTIATION OF MOUSE ES CELLS BUT NOT IN TS CELLS.

Ohgane, Jun¹, Takei, Yuki¹, Hattori, Naka^1,2, Tanaka, Satoshi¹, Shiota, Kunio¹, ^{1 2}

ABSTRACT- Cytosine methylation occurs mainly in CpG dinucleotides in the mammalian genome. The methylation is heritable event and affects gene activity in the genomic DNA through the transcription or replication. CpG sequences that remain are distributed very unevenly in the mammalian genome forming so-called CpG islands, which are generally found around promoter regions and replication origins. We previously found that there were differentially methylated CpG islands in the placenta, although CpG islands had been generally recognized as unmethylated area. Our previous genome-wide analysis revealed that 3% of the total CpG islands we detected were methylated differentially between the rat placenta and kidney. In the present study, we analyzed changes in methylation status between undifferentiated and differentiated embryonic stem (ES) cells and trophoblast stem (TS) cells by methylation sensitive arbitrarily primed PCR (MS-AP-PCR). We identified a differentially methylated locus (DML) during the first differentiation process of embryonic development. The DML is hypomethylated in the ES cells and gradually de novo methylated during the in vitro differentiation. TS cell lines have been established from trophectoderm of the mouse blastocyst and can differentiate into all subtypes of trophoblast cells both in vivo and in vitro. Interestingly, the DML is hypomethylated in the TS cells regardless of differentiation status. Southern blot analysis indicated that the locus is completely methylated in the various tissues in fetal, neonatal and adult tissues. The ES- and TS-specifically hypomethylated locus is embedded in the small CpG island (340 bp) which was characterized as high CG content and CpG frequency. The CpG island was located at chromosome 11 and close to the gene cluster including PMP22. Thus, we have identified a CpG island with differentially methylated region. These results clearly indicate that changes in the methylation status are involved in the earliest embryonic development.

KEY WORDS: CpG methylation, ES cells, TS cells, differentiation