Elucidating the Dynamics and Function of Long-Range Polycomb Interactions - Project Summary/Abstract Precise spatiotemporal regulation of gene expression is fundamental to biological function. Polycomb- group proteins (PcGs) are critical regulators that establish and maintain repression of developmental genes. Polycomb proteins are conserved across multicellular eukaryotes and are essential in mammals, in which their deletion causes embryonic lethality or developmental defects. However, how the Polycomb system drives repression of target genes and how cell type-specific Polycomb genes are selectively derepressed during development remain unknown. PcGs form two distinct complexes, Polycomb repressive complex 1 and 2 (PRC1 and PRC2), which modify chromatin and mediate 3D interactions between Polycomb-bound regions. PcG binding and histone modifications alone are insufficient to explain Polycomb repression, suggesting a functional role for 3D chromatin interactions. In particular, long-range looping interactions between distal Polycomb loci appear to be functionally important, as they are observed in both flies and mammals and disappear during differentiation. Yet, the dynamics of these interactions, including whether they are frequent enough to drive gene silencing, and how they contribute to changes in gene expression during development remain unclear. The proposed research will examine the dynamics and function of long-range Polycomb interactions in gene repression by combining cutting-edge super-resolution live-cell imaging, genomics, polymer simulations, and perturbations. Existing methods relying on cellular fixation cannot capture the inherently dynamic nature of these processes. I will therefore directly visualize Polycomb loops, target genes, and nascent transcription. Aim 1 will address whether the interactions are stable and frequent enough to confer function, determine chromatin organizing principles that drive loop formation, and directly test the impact of abolishing Polycomb looping on gene expression. In Aim 2, I will determine whether changes in long-range Polycomb looping dynamics correspond to gene activation by simultaneous imaging of Polycomb target genes and nascent transcription. These studies will be complemented with genomics approaches to address genome-wide features that correlate with selective derepression of developmental genes. Together, the proposed research will define the functional relationship between distal Polycomb interactions and transcriptional regulation, revealing whether long-range 3D chromatin interactions represent a key mechanism of Polycomb-mediated gene repression. These studies will inform our understanding of how cells are able to precisely control gene expression during development and provide a basis for future studies to correct disrupted Polycomb regulation in disease. The proposed research is designed to provide the necessary conceptual and technical training to achieve my goal of becoming an independent PI at a research institution. Research training will be conducted in parallel with career development training to develop necessary skills to establish and lead a research lab, including training in the responsible conduct of research, scientific communication, leadership, and lab management.
