Polycomb Repression via SCMH1 RNA-binding and Nuclear Condensation

Project Summary The goal of this project is to elucidate the biochemical mechanisms of SCMH1, an understudied accessory subunit of Polycomb Repressive Complex 1 (PRC1), during cellular differentiation. Polycomb group (PcG) proteins are critical epigenetic regulators that maintain transcriptional repression during development. Dysregulation of PcG proteins is associated with neurodevelopmental disorders, including Weaver syndrome, and can disrupt normal embryonic development. PRC1 plays a central role in chromatin compaction, long-range chromatin interactions known as Polycomb loops, and the formation of subnuclear clusters called Polycomb bodies. Despite the importance of PRC1, the biochemical role of SCMH1 in Polycomb-mediated repression remains poorly understood. The Drosophila homolog of SCMH1, SCM, is essential for maintaining epigenetic memory during development, but whether SCMH1 fulfills a similar function in mammals is unknown. Preliminary data from our lab reveal that SCMH1 loss leads to ectopic activation of Polycomb target genes, despite unaltered PRC1 occupancy and histone marks. We identified a conserved RNA-binding region (RBR) in SCMH1, suggesting that RNA binding may contribute to its function. Hi-C analysis shows that SCMH1 loss reduces Polycomb loops, implicating it in chromatin architecture. Given its RNA-binding capability and features associated with molecular condensation, such as intrinsically disordered regions and a SAM domain, we hypothesize that SCMH1 contributes to Polycomb body formation and gene regulation through RNA-mediated interactions and phase separation. Aim 1 investigates the role of SCMH1-RNA binding in Polycomb-mediated repression. Using a degron-tagged mouse embryonic stem cell (ESC) line, already in hand, I will degrade and rescue SCMH1 with wild-type or RNA- binding mutants and evaluate transcriptional repression, SCMH1 chromatin occupancy, and differentiation potential. Aim 2 examines the relationship between SCMH1 and Polycomb bodies and Polycomb loops. I will determine the SCMH1 domains required for Polycomb body localization and formation using mutants lacking phase- separation-associated domains. Immunofluorescence and Hi-C will assess how Polycomb body and loop dynamics depend on SCMH1, particularly during ESC differentiation into epiblast-like cells (EpiLCs). Together, these experiments will uncover SCMH1's role in Polycomb repression, nuclear condensation, and chromatin organization, advancing our understanding of epigenetic regulation and the mechanisms underlying Polycomb function during development. My diverse training background, combined with the expertise of my sponsor and the collaborative nature of my research environment, will ensure the success of this project and my development as a researcher.