Knockdown of histone H2A deubiquitinase BAP1 in human embryonic stem cells restricts their differentiation repertoire but is dispensable for maintaining their undifferentiated state.

Polycomb group proteins (PcGs) such as RING1B catalyze the addition of repressive histone mark (H2AK119ub1), while histone H2A deubiquitinases such as BAP1 remove this repressive mark. Interplay between these two proteins allows for fine control over the gene expression, which is essential during embryonic development. Transgenic mice that lack Bap1 and Ring1b die in embryonic stage. In humans, several cancers such as uveal melanoma, mesothelioma, clear cell carcinoma are caused by BAP1 mutations. Thus, it is clear from literature that Bap1 protein is crucial for murine lineage specification, however whether it controls lineage specification in humans is not reported yet. To understand this, we generated human embryonic stem cells that stably express doxycycline inducible BAP1 shRNAs. In the undifferentiated cells, doxycycline addition induced BAP1 downregulation, without any effect on the undifferentiated state. However, when human embryonic stem cells having BAP1 knocked down were differentiated via the embryoid body formation assay, their differentiation into all three germ lineage was affected. Thus, our results demonstrate that the knockdown of histone H2A deubiquitinase BAP1 restricts the lineage specification ability of human embryonic stem cells to a large extent, while it does not affect their undifferentiated state. Our results have implication in the study of early human development as well as cancer biology.