Functional analysis of the core subunits of Polycomb Repressive Complex 2

Abstract

Polycomb Repressive Complex 2 (PRC2) is a highly conserved multisubunit complex that methylates histone 3 on lysine 27 (H3K27). PRC2 function is implicated in diverse biological processes including stem cell regulation, cancer progression and genomic imprinting. This makes it critical to understand the mechanism by which PRC2 mediates gene silencing. In Drosophila, PRC2 consists of 4 core subunits, E(Z) with the SET domain, WD40 repeat proteins ESC and NURF55, and a structurally distinct SU(Z)12. E(Z), the catalytic subunit of PRC2 with SET domain possess the histone methyltransferase activity. A key feature of PRC2 is that E(Z) cannot methylate H3K27 without the help of its partner subunits SU(Z)12 and ESC. There is limited information on the structure and molecular function of SU(Z)12 in PRC2. In Chapter 2 of this thesis, we performed a systemic analysis of Drosophila SU(Z)12 domains to understand its contribution to PRC2. We analyzed the three conserved domains of fly SU(Z)12 and assigned critical functions to these individual domains. Our studies conclude that the N- terminal conserved domain of SU(Z)12 has binding determinants for the subunit E(Z) in PRC2. We show that the conserved, centrally located non-canonical, non-DNA binding C2H2 zinc finger is dispensable for PRC2 enzyme function in vitro. However, its function is critical for PRC2 function in vivo as mutations in the conserved residues of this domain failed to rescue the strong null phenotype of Su(z)123/Su(z)124. We also find that the highly conserved C-terminal VEFS domain is critical for enzyme function as well as assembly of the PRC2 complex. In Chapter 3 of this thesis, we highlight key findings on phosphorylation of EZH2 by Cyclin Dependent Kinases (CDKs), and their modulatory effect on PcG silencing.