Investigating the Role of GSK3A in Cohesin Dynamics and Gene Regulation

Cohesinopathies are multisystemic developmental disorders characterized by limb abnormalities, intellectual disability, and heart defects. They are caused by mutations in the structural or regulatory components of the cohesin complex. Cohesin is essential for interphase genome organization by forming chromatin conformations that position distal genomic regions into spatial proximity. Cohesin-mediated structures are implicated in gene regulation, DNA repair, and somatic recombination. Indeed, cells from individuals with Cornelia de Lange Syndrome (CdLS), the most well-characterized cohesinopathy, exhibit transcriptional dysregulation, suggesting that impaired interphase cohesin function may be responsible for patient phenotypes. The majority of CdLS patients carry a loss-of-function mutation in the cohesin activator NIPBL. Therefore, balancing cohesin activity on chromatin by inhibiting a negative cohesin regulator may provide therapeutic benefits for CdLS patients. However, the development of targeted therapies for CdLS has been hindered by the lack of characterized cohesin regulators during interphase. To address this, our lab performed a high-throughput imaging screen of the human druggable genome, which identified Glycogen synthase kinase-3 alpha (GSK3A) as a negative regulator of cohesin. My preliminary work has identified three phosphorylated residues on the cohesin regulator PDS5A that depend on GSK3 activity, albeit how GSK3A regulates cohesin is still unclear. The goal of this proposal is to elucidate the mechanism by which GSK3A negatively regulates cohesin and the extent to which GSK3A inhibition can rescue expression of cohesin-sensitive genes. In Aim 1, I will determine the role of the GSK3-dependent phosphorylated residues in PDS5A on cohesin regulation. To this end, I will create transgenic cell lines harboring wild-type, phosphomimetic, or phosphodefective PDS5A mutants at the GSK3-dependent sites. Using these cell lines, I will assay how preventing or constitutively mimicking PDS5A phosphorylation affects PDS5A loading onto chromatin and interactions with cohesin subunits. Furthermore, I will determine how the phosphomutants affect cohesin localization and chromatin folding. In Aim 2, I will identify the role of GSK3A in transcriptional regulation of cohesin-sensitive genes. I will first perform PRO-seq to measure changes in nascent expression of cohesin- sensitive genes upon GSK3A inhibition. Then, I will determine the degree to which GSK3A inhibition is able to rescue expression of genes when NIPBL is knocked down. As a functional readout of transcriptional rescue by GSK3A inhibition, I will assess whether GSK3A inhibition is able to restore cell cycle progression, which is impaired in NIPBL-deficient cells. Completion of these aims will provide mechanistic insights into regulation of cohesin by GSK3A and establish the foundations for studying GSK3A or other negative cohesin regulators as therapeutic targets for CdLS. Project Number: 1F31HD118759-01 | Fiscal Year: 2025 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Joanatta Shapiro | Institution: UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, PA | Award Amount: $49,538 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F08-L (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F31HD11875901