PRC1 coordinates timing of sexual differentiation of female primordial germ cells

Abstract

In mammalian somatic cells, PRC1 and PRC2 proteins are transcriptional repressors that function in large multi-protein complexes and that modify chromatin by monoubiquitinating histone H2A at lysine 119 (H2AK119u1) and tri-methylating H3 at lysine 27 (H3K27me3), respectively 9,12 . At day 12.5 of embryonic development (E12.5), we observed in PGCs marked by Cdh1 (E-cadherin) staining 13 nuclear localization of PRC1 components Rnf2/Ring1b, Mel18/Pcgf2 and Rybp ( Fig. 1a; Supplementary Fig. 1) as well as a robust H2AK119u1 signal suggesting the presence of catalytically active PRC1 complexes (Fig. 1a). To address the function of PRC1 in PGC development ( Supplementary Fig. 2), we conditionally deleted Rnf2 in PGCs from E9.5 onwards by Fig. 3, 6a). This strategy resulted in ~90% deletion efficiency at E11.5 ( Supplementary Fig. 4). At E12.5, Rnf2, Mel18, Rybp and H2AK119u1 were lost in PGCs of Rnf2 cko but not Rnf2 F/Δ embryos indicating that complex stability and catalytic activity of PRC1 is regulated by Rnf2 in PGCs at E12.5 ( Fig. 1a; Supplementary Fig. 4). In contrast, Ezh2 and H3K27me3 levels were similar in Rnf2 cko versus control PGCs suggesting globally unaltered PRC2 function in Rnf2 cko PGCs ( Supplementary Fig. 5).To study the fate of Rnf2-deficient PGCs, we analyzed expression of the pluripotency and germ cell marker Oct4 in whole gonads of E10.5 to E13.5 embryos ( Fig. 1b; Supplementary Fig. 2). We observed a strong reduction of Oct4-positive PGCs specifically in female Rnf2 cko embryos but not in male Rnf2 cko or control embryos, starting around E12.5 of gestation (Fig. 1b, c). In contrast, double deficiency of Ring1 and Rnf2 caused a strong reduction of Oct4-positive PGCs already at E11.5 in both sexes ( Supplementary Fig. 6b, c), indicating an essential role for PRC1 in PGCs after their migration into the embryonic gonad ( Supplementary Fig. 2) 11.To further dissect the role of Rnf2 in regulating Oct4 expression versus PGC development, we assessed in Rnf2 cko embryos co-expression of Oct4 and Rnf2 at E12.5 in Cdh1-positive PGCs (Fig. 1d). The number of PGCs lacking detectable Rnf2 protein was strongly reduced in female but not male gonads, despite that gonads of opposite sexes harbored comparable numbers of Rnf2-deficient PGCs at E12.0 ( Supplementary Fig. 4a, data not shown). We further observed a pronounced downregulation of Oct4 protein in female and some male Rnf2-deficient PGCs (Fig. 1d).These data indicate that Rnf2 contributes to maintaining Oct4 expression particularly in female PGCs beginning between E12.0 and E12.5. At E11.5, gonads still possess the potential to develop either into ovaries or testes while they are committed to a sex-specific differentiation process two days later.To relate aberrant expression in Rnf2 ∆ PGCs to changes occurring during normal PGC differentiation, we profiled expression in Rnf2 + PGCs isolated between E11.5 and E13.5. In this developmental period, 6-fold more genes were up-regulated in female (810) than male (132) Rnf2 + PGCs, with an additional 196 ge...

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