Regulation of PKCi-mediated pluripotency and gene expression by polycomb complex 1 in mouse embryonic stem cells.

Abstract

Objectives: Epigenetic modification of Polycomb repressive complex 1 (PRC1) plays an important role in regulating the pluripotency and self-renewal of embryonic stem cells (ES). Inhibition of protein kinase C (PKCi) can capture and maintain the pluripotency of mouse ES (mES). Here, we characterized the dynamic expression pattern of PRC1 and its mechanism of action in PKCi-derived mES (PKCi-mES).

Methods: The expression of PRC1 components in PKCi-mES, 2iL-derived mES (2iL-mES), and mouse embryonic fibroblasts (MEF) was comparatively analyzed using Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot to identify differentially expressed subunits. Subsequent knockdown of these differential components was performed to examine their effects on cellular differentiation status, along with the expression of pluripotency genes and differentiation genes.

Results: We found that PKCi-mES showed high expression of the PRC1 functional catalytic subunit RING1B, canonical PRC1-specific component CBX7, and non-canonical PRC1 specific component RYBP at both gene and protein levels in comparison with control. Knocking down Ring1b and Cbx7 accelerated the differentiation of PKCi-mES and reduced the expression of core pluripotency genes and naïve marker genes. Genes associated with mesoderm, ectoderm, and endoderm differentiation were broadly upregulated after Ring1b knockdown, while Cbx7 knockdown upregulated the expression of Cbx8 and some ectoderm genes but downregulated the expression of some endoderm and mesoderm genes.

Conclusions: These results indicate that PKCi activates both the canonical and non-canonical PRC1 pathways, finely regulates the expression of mES pluripotent and developmental genes, and helps maintain mES in a poised state between self-renewal and lineage commitment.