The Roles of E2F6 and DNA Methylation in the Regulation of Gene Expression During Germ Cell Development.

Abstract

Mammalian primordial germ cells (PGCs), the embryonic progenitors of mature gametes, are specified in the extraembryonic mesoderm. These cells actively migrate to the developing genital ridge where they subsequently differentiate into gonocytes. Colonization of the gonad coincides with a widespread DNA demethylation event in the germ cell genome, leading to the upregulation of a subset of gonocyte-specific genes. DNA methylation contributes to silencing these gonocyte genes in migratory PGCs and in somatic tissues. Additionally, recent studies suggest a role for the transcription factor E2F6 in silencing germ cell-specific genes in somatic tissues. A subset of germ cell-specific genes, including Tuba3, Stag3, and Smc1β, is ectopically expressed in somatic tissues lacking E2f6. Furthermore, DNA hypomethylation is found at the promoter region of at least one of these genes, Tuba3, in E2f6-deficient tissues. These observations have led us to investigate a potential relationship between E2F6 and DNA methylation in the temporal regulation of gonocyte-specific gene expression in the developing germ line. We have focused our initial studies on the Tuba3 locus to gain further knowledge about the regulation of germ cell-specific, E2F6 regulated genes. Here we provide evidence that: 1) Members of the E2f family, including E2f6, are expressed in PGCs, 2) The Tuba3 promoter contains two CpG islands within 1kb of exon 1 and the downstream island is highly methylated in PGCs. Remarkably, unlike other known genes, this region of methylation appears to be stable through major stages of early embryogenesis and germ cell development, 3) Similar to several other gonocyte-specific genes, Tuba3 expression is activated as gonocytes differentiate and undergo genome-wide DNA demethylation. This activation coincides with demethylation of the upstream CpG island at the Tuba3 locus, however, the downstream island retains its methylation, 4) E2f6-deficient somatic tissue displays hypomethylation at both CpG islands on the Tuba3 promoter. From these data, we conclude that E2f6 is required for establishing or maintaining DNA methylation at the Tuba3 promoter in certain tissues. Current efforts are focused on creating an E2f6 gene trap mouse line to precisely determine the expression pattern of E2f6 in early development and its contribution to stability of DNA methylation in the germ line.