Characterizing the functional differences between

Abstract

Stem cells are specialized cells that can self-renew and differentiate into multiple cell types during development and regeneration through gene expression changes. These changes in stem cells are achieved, in part, via chromatin modifications, which include histone methylation, phosphorylation, and acetylation. Polycomb Repressive Complex 2 (PRC2) is a protein complex that methylates lysine 27 in histone H3—a histone modification associated with global silencing of gene expression, especially during stem cell development and differentiation. The PRC2 contains an enzymatic subunit, which can be enhancer of zeste homolog 1 or 2 (EZH1 or EZH2). Previous findings suggest that EZH1 and EZH2 serve different roles in embryonic stem cell (ESC) development and differentiation, despite both of them having EED-dependent (another protein subunit of the PRC2 complex) histone methyltransferase activity. Here, I report my investigation of the distinct roles of EZH1 and EZH2 by generating gene mutations separately in human embryonic stem cells by using the CRISPR/Cas9 genome editing technology. The effect of EZH1 and EZH2 mutations on the expression of pluripotency (e.g. OCT4 and NANOG) and differentiation genes (e.g. NESTIN, Brachyury, and GATA4) was assayed by RT-qPCR. I found that neither EZH1 nor EZH2 mutations affect cell pluripotency; however, EZH2 mutations markedly up-regulate GATA4 expression, while EZH1 mutations up-regulate SOX1 and SOX9 expression.