PROJECT SUMMARY Cornelia de Lange Syndrome (CdLS) is multi-system, dominant genetic disorder caused by mutations in the structural or regulatory subunits of the cohesin complex. Cohesin regulates the 3D organization of chromatin, especially at the level of chromatin looping and topologically associated domains (TAD). We recently showed that cohesin depletion disrupts TAD boundaries and that differentially expressed genes in CdLS patient cells are enriched at these boundaries, suggesting that 3D chromatin organization is key to disease pathogenesis. We have additional preliminary evidence that co-depletion of WAPL, a negative regulator of cohesin, is able to rescue the effects of cohesin loss of genome organization and gene expression. No drugs are known to target cohesin or WAPL, however, and discovering novel cohesin regulators remains difficult with current approaches. To discover genes that modulate the role of cohesin on TAD boundaries, I developed TAD high-throughput fluorescence in situ hybridization (TAD Hi-FISH). TAD Hi-FISH uses Oligopaints and high-content imaging to quantitatively identify genes that regulate TAD boundaries in a cohesin- or WAPL-like manner. To identify potential drug targets for CdLS, I applied TAD Hi-FISH to the human “druggable genome,” i.e. 3,083 genes targeted by known drugs. My primary screen uncovered 70 cohesin-like and 57 WAPL-like hits. GSK3A, a multifunctional kinase, has been preliminarily validated as a lead WAPL-like hit that can suppress cohesin loss. I propose to apply TAD Hi-FISH and other assays to further characterize the effects of GSK3A and all of my hits on TAD boundaries and cohesin regulation. In aim 1, I will curate a set of druggable regulators of cohesin’s chromatin architectural function. I will first use secondary TAD Hi-FISH screens to determine which hits regulate TAD boundares in both a region non-specific and cohesin-dependent manner. Of these top secondary hits, I will evaluate which affect cohesin chromatin binding by performing chromatin fractionation and western blot experiments. Finally, I will evaluate the genome-wide effects of the strongest cohesin regulators using ChIP-seq for cohesin. In aim 2, I will investigate the relationship of GSK3A to genome organization, cohesin, and transcription. To study both GSK3A protein and its catalytic function, I will develop an inducible degron cell line for GSK3A, and also optimize conditions in this cell line for a recently developed GSK3A chemical inhibitor. I will then perform Hi-C after GSK3A depletion and inhibition to investigate the genome-wide effects of GSK3A on chromatin architecture. Next, I will apply nascent transcriptomics using PRO-seq to determine whether GSK3A co-depletion or inhibition can rescue the gene expression changes of cohesin loss. Together, these aims will curate a novel set of cohesin regulators, including GSK3A, that will advance our understanding of 3D genome organization and potential CdLS therapeutic developm...