Structure and Mechanism of Epigenetic Gene Silencing - Project Summary The human body has ~37 trillion cells and over 200 different cell types. Polycomb Repressive Complex (PRC1, PRC2) and DNA methyltransferase 1 (DNMT1) are evolutionarily conserved chromatin modifier enzymes that are essential for establishing and maintaining cellular identity by silencing lineage-inappropriate genes during embryonic development and cellular differentiation. PRC1, PRC2, and DNMT1 are also key enzymes in cardiac development, and defective activity of these enzymes is a hallmark of cardiac diseases and loss of cell identity in various cancers and tumors. Polycomb-mediated gene silencing involves the coordinated activity of PRC1 and PRC2 through histone post-translational modifications. PRC1 is a histone E3 ligase that catalyzes the mono- ubiquitination of H2A at lysine 119 (H2AK119ub1) on nucleosomes, which aids in the recruitment and activation of PRC2 for the trimethylation of histone H3 (H3K27me3) leading to gene silencing. Similar crosstalk between trimethylation of histone H3 at lysine 9 (H3K9me3) and mono-ubiquitination of histone H3 at lysine 18 and 23 (H3K18K23ub2) is thought to be responsible for the recruitment and activation of DNMT1 at replication forks for the maintenance of DNA methylation. The major research goal in the lab over the next five years is to uncover the mechanistic basis for the recruitment and activation of PRC1 and DNMT1 on chromatin. We will utilize an integrated approach combining cryo-electron microscopy, chemical biology, mass spectrometry, and biochemical assays to (a) uncover the mechanistic basis for how DNA and histone post- translational modifications activate PRC1 and DNMT1 on chromatin and (b) design and develop new inhibitor and activator candidates that can target specific regions of PRC2 that are important for chromatin interactions and enzymatic activity. We will use biochemical mutational and activity assays to validate our results from the structural studies. We will also biochemically characterize how known disease-causing mutations lead to defects in the enzymatic activity of PRC1 and DNMT1 on chromatin. The vision of our research program is to provide novel structural and mechanistic insights into the role played by chromatin in activating gene-silencing enzymes such as PRC1, PRC2, and DNMT1. We envisage that such insights will aid in developing small molecule effectors targeting these enzymes in malignancies such as solid tumors, which have so far lacked targeted treatments. One example of this structural biology approach is our proposed work on developing effector molecules targeting specific regions of PRC2 in this proposal.
