Abstract
Polycomb group (PcG) proteins are essential for maintenance of lineage fidelity by coordinating developmental gene expression programs. Polycomb group ring finger 6 (PCGF6) has been previously reported to repress expression of lineage-specific genes, especially germ cell-related genes in mouse embryonic stem cells (ESCs) via the noncanonical polycomb repressive complex PRC1.6. However, the molecular mechanism of this repression remains largely unknown. Here, using RNA-Seq, real-time RT-PCR, immunohistochemistry, immunoprecipitation, and ChIP analyses, we demonstrate that PCGF6 plays an essential role in embryonic development, indicated by the partially penetrant embryonic lethality in homozygous PCGF6 (Pcgf6-/-)-deficient mice. We also found that surviving Pcgf6-deficient mice exhibit reduced fertility. Using the Pcgf6-deficient mice, we observed that ablation of Pcgf6 in somatic tissues robustly derepresses germ cell-related genes. We further provide evidence that these genes are direct targets of PCGF6 in ESCs and that endogenous PCGF6 co-localizes with the histone-modifying proteins G9A histone methyltransferase (G9A)/G9a-like protein (GLP) and histone deacetylase 1/2 (HDAC1/2) on the promoters of the germ cell-related genes. Moreover, the binding of these proteins to their target genes correlated with methylation of Lys-9 of histone 3 and with the status of histone acetylation at these genes. Moreover, the recruitment of G9A/GLP and HDAC1/2 to target promoters depended on the binding of PCGF6. Our findings indicate that PCGF6 has a critical role in safeguarding lineage decisions and in preventing aberrant expression of germ cell-related genes.
Highlights
Polycomb group (PcG) proteins act as evolutionarily conserved epigenetic mediators of transcription that have integral roles in stem cell identity and development by repressing key developmental genes [1,2,3]
We found that Pcgf6 selectively recognized Ring1B and did not exhibit detectable binding to G9a/Glp or Hdac1/2, likely suggesting that Pcgf6 interacts with G9a/Glp and Hdac1/2 through other polycomb repressive complex 1 (PRC1).6 subunits (Fig. S6)
Taking advantage of Pcgf6-deficient mice, we showed that Pcgf6 targets were enriched for meiosis– and germ cell– related genes in liver and brain and that such genes were dramatically derepressed in all Pcgf6 null tissues we examined
Summary
To uncover the biological role of Pcgf in vivo, we generated mice with targeted disruption of the Pcgf locus (Fig. 1A). The presence of centrally enriched E-box and E2f6/Dp1 motifs in these germ cell promoters suggests that Pcgf represses germ cell gene expression in the context of a PRC1.6 complex To build on this observation, we chose to focus our efforts on the set of germline-specific genes showing aberrant expression in adult somatic tissues derived from Pcgf null mice. We found that the Pcgf was enriched at the promoters of selected germ cell–related genes—including Mael, Tcam, Slc25a31, Syce, Tdrkh, and Ddx4—whereas enrichment of Pcgf was not observed at the promoter of Hprt, whose expression in ESCs was not changed by absence of Pcgf (Fig. 6B) This was accompanied by considerable enrichment of Ring1B, G9a/Glp, and Hdac1/2, suggesting that Pcgf is involved in the recruitment of the PRC1.6 complex (Fig. 6, B–D). These results suggest that Pcgf coordinates epigenetic repression of germ cell–related genes by recruitment of G9a/ Glp and Hdac1/2 (Fig. 6E)
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