Abstract
Primordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs.
Highlights
Germ cells are the only cells capable of giving rise to truly totipotent cells, via differentiation to sperms/eggs and subsequent fertilization
We found that the region near the transcription start site (TSS) was hypomethylated in all cell types tested, but the upstream regulatory region was hypermethylated in the region of epiblast proximal to the adjacent extraembryonic ectoderm at E6.0 before any mil-1 expression was evident (Figure 1, S1, Figure 2A) and in nascent primordial germ cells (PGCs) at E7.5 just as mil-1 expression was evident (Figure 1, S1, Figure 2A)
We demonstrated that regions flanking those genes that contain the Ifitm genes consensus element (ICE) consensus element commonly underwent DNA demethylation that was synchronous with enhancement of co-expression of those PGC genes in differentiating PGCs (Figure 1, 2, 4, S1, S2)
Summary
Germ cells are the only cells capable of giving rise to truly totipotent cells, via differentiation to sperms/eggs and subsequent fertilization. In mouse embryos at around embryonic day (E) 7.25, a small population of primordial germ cells (PGCs) in the extraembryonic mesoderm and derived from the epiblasts is first ‘‘fate-determined’’. Before PGC fate determination, cell type-specific expression of Blimp1/Prdm and Prdm initiates in PGC precursors; these proteins are key transcriptional regulators of PGC development. Many pluripotency-related genes, including Oct, Nanog, and Sox, are expressed in PGCs [2,4]. Oct plays essential roles in PGCs fate determination [5]. Nanos3 [7,8] is initially expressed in PGCs at around PGC fate determination, and it supports survival of migrating PGCs along with Oct4 [9] and Nanog [10,11]
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