PRC1 catalytic unit RING1B regulates early neural differentiation of human pluripotent stem cells

Abstract

Background: Polycomb group (PcG) proteins are histone modifiers which control gene expression by assembling into large repressive complexes termed - Polycomb repressive complex (PRC); RING1B, core catalytic subunit of PRC1 that performs H2AK119 monoubiquitination leading to gene repression. The role of PRC1 complex during early neural specification in humans is unclear; we have tried to uncover the role of PRC1 in neuronal differentiation using human pluripotent stem cells as an in vitro model.

Results: We differentiated both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) towards neural progenitor stage evident from the expression of NESTIN, TUJ1, NCAD, and PAX6. When we checked the total expression of RING1B and BMI1, we saw that they were significantly upregulated in differentiated neural progenitors compared to undifferentiated cells. Further, we used Chromatin Immunoprecipitation coupled with qPCR to determine the localization of RING1B, and the repressive histone modification H2AK119ub1 at the promoters of neuronal specific genes. We observed that RING1B localized to and catalyzed H2AK119ub1 modification at promoters of TUJ1, NCAM, and NESTIN during early differentiation and later RING1B was lost from its promoter leading their expression; while functional RING1B persisted significantly on mature neuronal genes such as IRX3, GSX2, SOX1, NEUROD1 and FOXG1 in neural progenitors.

Conclusion: The results of our study show that PRC1 catalytic component RING1B occupies neuronal gene promoters in human pluripotent stem cells and may prevent their precocious expression. However, when neuronal inductive signals are given, RING1B is not only removed from neuronal gene promoters, but the inhibitory H2AK119ub1 modification is also lost.

Keywords: H2AK119 monoubiquitination; Neuronal differentiation; PRC1; RING1B.