Role of Polycomb Repressive Complexes in Neuronal Lineage Differentiation from Human Pluripotent Stem Cells

Abstract

Epigenetic regulators catalyze various histone modifications which is one of newlinethe key mechanisms that regulate neural development in vivo. Polycomb group (PcG) newlineproteins are one of the histone modifiers that regulate gene expression by performing newlinetranscriptional repression by assembling into two broad complexes, viz. - Polycomb newlineRepressive Complex 1 (PRC1) and (PRC2). PRC1 has RING1B as the core catalytic subunit newlinewhich has the RING1 domain that performs monoubiquitination on Histone H2A at Lysine newline119. PRC2 has EZH2 as the core catalytic unit having the SET domain to do di/trimethylation newlineof histone H3 at K27, and these modifications lead to transcriptional silencing. The role of newlinethe PRC1 complex during early neural differentiation and specification in humans is not newlineknown. Hence, my work aimed to understand the role of PRCs in neuronal differentiation newlineusing human pluripotent stem cells. Our study aims to understand the expression of these newlineproteins as well as their localization on neural lineage specific genes. newlineMethodology: We have used human pluripotent stem cell lines (hESCs- human embryonic newlinestem cells: and hiPSCs- human induced pluripotent stem cells) - KIND1 and NIH5 newlinerespectively. We characterized the hPSCs for their pluripotency and then we differentiated newlinehPSCs into neuronal lineage using inhibitors of signaling pathways WNT, TGFand#946;, BMP, newlineACTIVIN, and NODAL, further addition of morphogens such as Retinoic acid and Sonic newlinehedgehog, growth factors like FGF2 and FGF4. These differentiated neuronal cells were newlinecharacterized at transcript and protein levels using immunofluorescence, western blot, and newlineqRT-PCR. Next, we checked the expression profile of PcGs - RING1B, BMI1, and EZH2 newlineduring neuronal differentiation; we also checked the expression of histone modification newlineH2AK119ub1 during neuronal differentiation.