Investigating the impact of disease-associated mutations in the Polycomb system

About This Grant

Abstract Polycomb group (PcG) complexes are multi-protein, evolutionarily conserved epigenetic machineries that regulate stem cell fate decisions, cell identity and early development. The PcG machinery can be divided into two major complexes: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2). Traditionally, PcG complexes are associated with gene repression mainly via histone-modifying activities. While PRC2 catalyzes methylation on lysine 27 of histone H3 (H3K27me1/2/3) via EZH1/2, PRC1 deposits a ubiquitin group at lysine 119 of histone H2A (H2AK119ub1) via the E3-ligases RING1A/B. Interestingly, several PcG encoding genes are found to be mutated in individuals with developmental disorders. Specifically, de novo missense mutations in the genes encoding for RING1A (RING1), and RING1B (RNF2), have been found in pediatric patients with neurodevelopmental disorders. How mutations at PcG genes impair development in humans is completely unexplored. Additionally, we have discovered novel missense mutations in both genes in children with intellectual disabilities. We conducted predictive analyses using crystal structures to start understanding how these mutations affect PRC1's stability and interaction with nucleosomes. In this proposal, we will focus our efforts in one of the RNF2 mutations, which is associated with intellectual disabilities using novel knock-in ESC lines as well a new mouse model carrying a monoallelic missense mutation on RNF2. Preliminary data reveal that mutant RING1B disrupts Polycomb complex assembly, induces derepression of PRC1 and PRC2 target genes, and impaired differentiation into neurons. By ChIP-seq and mass spectrometry we will investigate chromatin occupancy and recruitment mechanisms and potential rescue strategies. Additionally, this proposal will examine how Rnf2 mutations impact hippocampal structure, and behavioral outcomes in mice. Immunohistochemistry, RNA-seq, and ATAC-seq will determine the cellular diversity and regulatory dynamics in the hippocampus, providing insights into the mutation's molecular and behavioral consequences. Overall, our proposed research aims to define the role of missense mutations in Polycomb genes in neurodevelopment in vitro and in vivo, examining epigenetic mechanisms, behavior, and neuronal architecture. This work will enhance our understanding of how missense mutations influence PRC1 function and their contribution to neurodevelopmental disorders, shedding light on the complex relationship between epigenetics and neurodevelopment. Finally, our findings could pave the way for therapeutic strategies for neurodevelopmental disorders associated with PcG mutations.