PROJECT SUMMARY Cornelia de Lange Syndrome (CdLS) is a multi-system genetic disorder caused by mutations in the regulatory or structural components of the cohesin complex. Cohesin mediates sister chromatid cohesion during mitosis and is theorized to extrude DNA throughout interphase to form enhancer-promoter chromatin loops and maintain self-interacting clusters of DNA called topologically associating domains (TADs). Chromosome instability is rarely observed in patients. Rather, widespread transcriptional changes have been identified across CdLS patient samples, implicating the structural role of cohesin in the pathogenesis of the disorder. I hypothesize that in the absence of cohesin, chromatin loops that mediate enhancer-promoter interactions are disrupted and instead contacts form between genes and proximal regulatory elements. I further expect that this enhancer reorganization will affect gene expression at the level of transcriptional bursting. To visualize chromatin organization in single cells, I have designed Oligopaints to probe neighboring TADs in the human genome by fluorescence in situ hybridization (FISH). My preliminary data indicate this assay is sensitive to detect changes in local chromosome topology following acute cohesin depletion by both conventional and super resolution microscopy. Therefore, I will apply this and other assays to answer questions regarding the mechanisms by which cohesin folds chromatin to facilitate transcription at the single cell level. In aim 1, I will determine the effect of cohesin loss on promoter topology and transcriptional regulation. To study the role of cohesin in these processes, I will perform these experiments in a cell culture model that facilitates acute cohesin degradation. I will apply a high-throughput RNA FISH technique, seq-FISH, to assess changes in bursting frequencies genome-wide. Then, I will map differences in enhancer-promoter contacts in effort to explain the altered gene expression using a high-resolution Capture-C method. In aim 2, I will detect signatures of chromatin disorganization and transcriptional bursting changes in CdLS. I will probe TAD organization and nascent gene expression in cells derived from CdLS patients. I will then relate phenotypes to the mutation type, clinical severity, and cohesin abundance in each patient. Together, these aims will illuminate the relationship between transcriptional regulation and local genomic architecture while providing new mechanisms to explain how chromosome misfolding may lead to developmental disorders such as CdLS.