The Regulatory Mechanism Of Non-canonical PRC1-RYBP Complex In Neural Differentiation Of Embryonic Stem Cells

The PcG complexs play important roles in the maintenance of pluripotency and differentiation of mouse embryonic stem cells.Polycomb group repressive complexes(PRCs),which are divided into two major complexes,called PRC1 and PRC2.PRC1 and PRC2 catalyze monoubiquitination of histone H2 A at lysine-119(H2AK119ub1)and trimethylation of histone H3 lysine 27(H3K27me3),respectively.RING1 and YY1 Binding Protein(RYBP)is a subunit of the non-classical PRC1 complex,which interacts with RING1 B,a core subunit of PRC1 complex and enhances the enzymatic activity of RING1 B.In this study,we found that the expression of RYBP gradually decreases during the differentiation of ESCs towards neural progenitior cells,but the role of RYBP in the neural differentiation of ESCs is still unclear.To study the biological function of Rybp during neural differentiation of m ESCs,we have generated Rybp homozygous knockout mouse ESC line using CRISPR/Cas9 genome editing technology.The exons 4 and 5 of Rybp gene containing 115 amino acids were replaced with PGK-puro by homologous recombination.Genomic DNA was isolated,PCR and Sanger sequencing were utilized to verify the knockout efficiency.The expression of Rybp was detected though real-time PCR and Western blot.This cell line was verified to be pluripotent since Rybp-/-has no effect on the expression of pluripotency-associated markers,NANOG and OCT3/4 and has no effect on the expression of pluripotency-associated surface marker SSEA1 via flow cytometric analysis.Rybp-/-also had no significant effect on cell cycle and apoptosis of m ESCs.By inducing differentiation of m ESCs to embryoid bodies(EB)and the expression of the triploblastic layer marker genes was detected by RT-q PCR on day 9,our results indicated that Rybp knockout disrupted the differentiation of endoderm and mesoderm,significantly inhibited ectoderm differentiation.What’s more,Rybp-/-ESCs were further induced differentiation to neural fate using N2B27 culture,and the expression of neural stem cell marker genes was detected by immunofluorescence and RT-q PCR.We found that the differentiation of m ESCs into neural progenitior cells was inhibited after Rybp knockout.Meanwhile,RNA sequencing data indicated that the expression of neural-related genes were inhibited after Rybp knockout.Transcriptome analysis revealed that Wnt signal pathwayrelated genes were abnormally activated during neural differentiation of m ESCs after Rybp knockout.Taken together,this study suggests that knockout of inhibits neural differentiation of m ESCs by activating Wnt signal pathway.