PARENT SESSION
Preimplantation Embryo/Fetal Development
178
DNA METHYLATION STATUS OF OCT-3/4 GENE PROXIMAL ENHANCER IN THE MOUSE ES AND TS CELL LINES.
Sato, Naoko1, Nishino, Koichiro1,2, Hattori, Naka1,2, Ohgane, Jun1, Tanaka, Satoshi1, Shiota, Kunio1, 1 2
ABSTRACT- The first cell differentiation event in mammalian embryogenesis segregates the inner cell mass (ICM) lineage from the trophectoderm (trophoblast lineage) at the blastocyst stage. In the mouse, trophoblast stem (TS) cells appear in the polar trophectoderm. TS cells have pluripotency to differentiate into the various trophoblast subtypes in vitro, and exclusively contribute to the trophoblast lineage in vivo. Oct-3/4, a member of the POU transcription factors, is expressed in the totipotent and pluripotent cells including the oocytes and ICM but not in the TS cells. Repression of Oct-3/4 has been reported to lead ES cells to exhibit TS-like phenotype. DNA methylation pattern is stage- and cell type-specifically formed during development, and methylation of DNA is generally associated with gene silencing. To elucidate possible role of DNA methylation in the first differentiation event of mammalian embryogenesis, we investigated the methylation status of Oct-3/4 proximal enhancer region in the two stem cell lines (mouse ES and TS cell lines) and the adult mouse liver by using bisulfite genomic sequencing method. ES cells, TS cells and the liver were originated from C57BL/6 mouse. Oct-3/4 regulatory region has no TATA sequence and contains putative Sp1, HRE and ETF binding sites. The proximal enhancer (PE) is located between -230 and -1222 (992bp) upstream of Oct-3/4 transcription start site, and contains 14 CpG sites. CpGs in the distal one third of PE (-887 ~ -1222) was methylated both in TS and ES cells. Interestingly, in the proximal two third of PE (-230 ~ -886), the CpGs were hypomethylated in ES cells, while they are hypermethylated in the TS cells. As we assumed, all CpGs are heavily methylated in the liver, in which Oct-3/4 is not expressed. The pattern of the DNA methylation was thus different between the stem cell lines, and there were intimate relations between methylation status of Oct-3/4 gene and its expression. The locus of Oct-3/4 gene is target of methylation in the first cell differentiation.
KEY WORDS: DNA Methylation, Oct-3/4, ES cell, TS cell