Establishment of Cell Lineage-Specific DNA Methylation Profile Follows the Morphological Differentiation of Trophoblast and Embryonic Cell Lineages.

Abstract

There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the mammalian genome. Cell type-specific DNA methylation status of T-DMRs constitutes DNA methylation profile of the cell, which ought to be reconstructed as cells differentiate. The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages resulting in the formation of trophectoderm (TE) and inner cell mass (ICM) in blastocyst. Although lower level of global DNA methylation in TE genome compared to ICM has been suggested, dynamics of DNA methylation profile during the TE/ICM differentiation has not been studied. In this study, we aimed to identify T-DMRs between trophoblast and embryonic cell lineages and analyzed their DNA methylation status in pre- and post-implantation embryos. Oct4 and Nanog are differentially methylated between trophoblast stem (TS) and embryonic stem (ES) cell lines (TS-ES T-DMRs). These two loci are hyper-methylated in TS cells and hypo-methylated in ES cells. Elf5 is another known TS-ES T-DMR but exhibits the opposite methylation pattern. A number of other TS-ES T-DMRs have been found so far in our laboratory. First, we addressed whether these TS-ES T-DMRs are indeed differentially methylated between trophoblast and embryonic cell lineages in vivo. Bisulfite sequencing analysis of 11 TS-ES T-DMRs revealed that the DNA methylation status of them in E6.5 extraembryonic ectoderm and epiblast coincide with those in TS and ES cells, respectively, showing that the TS-ES T-DMRs are surely cell lineage-based T-DMRs in vivo. We then collected TE and ICM cells separately from E3.5 blastocysts and subjected to bisulfite sequencing. Interestingly, genomes of TE and ICM exhibited no difference in the methylation status of all T-DMRs examined. They were in extremely hypomethylated status. Here we identified valuable T-DMRs for delineation of trophoblast/embryonic cell lineages. DNA methylation analysis of these T-DMRs suggested that the DNA methylation profiles specific to each cell lineage are established after the morphological delineation of trophoblast and embryonic cell lineages. DNA methylation may thus contribute to stabilize or fix the cell fate and the differentiation capacity. Supported by Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20062003). (platform)