Modulators of Cellular and Biochemical PRC2 Activity

• Main Author: Paulk, Joshiawa Lanair James
• Other Authors: Schreiber, Stuart L.
• Language: en_US
• Published: Harvard University 2014
• Subjects: Biochemistry; Chromatin; EZH2; High-throughput screening; Histone crosstalk; PRC2; Small-molecule probes
• Online Access: http://dissertations.umi.com/gsas.harvard.inactive:11829; http://nrs.harvard.edu/urn-3:HUL.InstRepos:13064968

EZH2 is a SET domain-containing methyltransferase and the catalytic component of the multimeric Polycomb- group (PcG) protein complex, PRC2. When in complex with other PRC2 members (EED, SUZ12, AEBP2, and RBBP4), EZH2 catalyzes methylation of H3K27, a histone modification associated with transcriptional repression and developmental regulation. As several PRC2 components are upregulated or mutated in a variety of human cancers, efforts to discover small-molecule modulators of PRC2 and understand its regulation may yield therapeutic insights. Identification of small-molecule probes with distinct chemotypes, MOAs, and selectivity profiles are not only of great value, but necessary in establishing comprehensive probe sets capable of illuminating the various roles of EZH2 in oncogenesis. Here we describe efforts to identify and characterize small-molecule modulators of PRC2 and further understand its regulation. Chapter II outlines the expression and purification of 5-component PRC2 (EZH2-EED-SUZ12-AEBP2-RBBP4) and the establishment of biochemical and cellular HTS assays. These assays were used to screen a diverse set of small molecules (>120,000), identifying biochemical PRC2 inhibitors and activators (described in Chapter III). One biochemical PRC2 inhibitor, BRD1835, appeared to inhibit PRC2 activity through a novel artifactual mechanism involving interaction with peptide substrate, leading to apparent peptide-competitive behavior and putative cellular activity (described in Chapter IV). The characterization of novel biochemical PRC2 activators, BRD3934 and BRD8284, is discussed in Chapter V. Chapter VI describes the use of an HCS assay to identify known bioactive compounds that alter intracellular levels of H3K27me3 through modulating H3K27me3-connected regulatory nodes or by targeting PRC2 directly. These efforts led to the discovery that an antifungal agent, miconazole, is capable of activating PRC2 activity in vitro, while a mucolytic agent, bromhexine, selectively ablates cellular H3K27me3 levels through targeting an activity distinct from PRC2. Finally, Chapter VII discusses novel PRC2-connected crosstalk mechanisms identified through screening libraries of uniquely modified histone peptides for their ability to bind or support methylation by PRC2. These studies enhance our understanding of PRC2 regulation by revealing the effects of H3R26 and H3K23me1 modifications on enzymatic activity, implicating their respective methyltransferases in PRC2 regulation.