RING1B-BMI1 catalyzed dynamic H2AK119ub1 modification in response to sonic hedgehog signalling during pancreatic differentiation of human embryonic stem cells.

Human embryonic stem cells (ESCs) can differentiate into the pancreatic lineage and thus can serve as in vitro models for normal pancreas development. Epigenetic modulators such as RING1B and BMI1, are essential lineage regulators of the pancreatic lineage. Inhibition of the sonic hedgehog (SHH) pathway is critical for development of the pancreatic lineage. In several cancers, SHH signals regulate BMI1 protein. However, it is unclear whether SHH pathway can direct RING1B and BMI1 to the promoters of pancreas-specific genes during human pancreas development to regulate them. We performed chromatin immunoprecipitation coupled with qPCR to assess if RING1B and BMI1 occupy pancreas gene promoters and repress them by adding the repressive H2AK119ub1 mark. We compared the RING1B - BMI1 occupancy at 1000 bp upstream of the pancreas specific gene promoters across cells treated with exogenous sonic hedgehog as well as the pathway inhibitor- SANT1. We observed that the SHH pathway alters the expression of crucial pancreas genes as well as regulates RING1B and BMI1 occupancy at these gene promoters. PcG protein occupancy correlated with expression of HNF4α and HHEX; but surprisingly did not affect the mRNA expression of the master transcription factor, PDX1. Ou results demonstrate that during pancreatic differentiation from human embryonic stem cells, the SHH signalling pathway affects occupancy of PcG proteins - RING1B and BMI1.