Histone H2A deubiquitinase BAP1 is required for human neuronal progenitor cell formation

Formation of neuronal progenitor cells, neural crest cells and surface ectoderm cells is a complex process and differential gene expression allows for generation of these three cell types during neurulation process. Histone modifiers such as writers and erasers may play an important role in achieving the differential gene expression during neurulation event, similar to how DNMTs and TET enzymes function in germ cell development. We had previously reported that Polycomb group (PcG) protein RING1B which catalysed repressive H2AK119ub1 mark and prevented precocious activation of neuronal genes in undifferentiated cells, but once they were induced to differentiation, RING1B was dislodged from neuronal promoter sites. However, the role of histone H2A deubiquitinases in neuronal differentiation remains to be investigated. Erasers such as histone H2A deubiquitinases Bap1, are critical for normal development since Bap1 KO mice die in embryonic stage. In humans, the cutaneous and uveal melanomas which are ectodermal in origin typically have a mutated BAP1. Recently, it has been shown that Bap1 regulates the epithelial-mesenchymal transition (EMT) genes in developing mice placenta. We differentiated human pluripotent stem cells into the neuronal lineage and depleted BAP1 via doxycycline inducible shRNA. Our results revealed neuronal progenitor markers viz., PAX6, NESTIN and NCAD were reduced at both protein and transcript level upon BAP1 depletion. We observed that BAP1 regulated EMT genes since, cells with depleted BAP1 were deficient in EMT markers such as of SLUG, TWIST1, VIMENTIN and high levels of E-CADHERIN. At the morphological level, this was evident from distorted neuronal rosettes seen post BAP1 knockdown. Bulk RNA seq analysis showed that neuronal progenitor and neural crest specific transcripts were reduced while surface ectoderm transcripts were higher in BAP1 depleted cells. Thus, our results clearly demonstrate that BAP1 is required for human neuronal progenitor cell formation, since it regulates neuronal and EMT genes.