Abstract Cornelia de Lange Syndrome (CdLS) is a rare developmental disorder affecting a multitude of organs including the central nervous system, inducing a variable neurodevelopmental delay. CdLS is primarily caused by mutations in regulatory or structural components of the cohesin complex, which are essential Structural Maintenance of Chromosomes (SMC) protein-containing complexes that interact with chromatin and modulate genome folding. It is hypothesized that CdLS symptoms are a consequence of transcriptional dysregulation due to changes in genome architecture upon cohesin disruption. To date, there are no therapies aimed at correcting cohesin dysfunction or chromatin misfolding directly. These issues are due, in part, to the paucity of factors known to regulate cohesin-mediated chromatin folding. We performed a high-throughput fluorescent in situ hybridization (Hi-FISH)-based screen in human cells, which uses Oligopaint probe technology to detect and measure chromatin interactions in a large number of samples. We targeted ~8,000 human genes, which represent the “druggable genome” and isolated five candidate genes whose depletion can offset cellular phenotypes associated with cohesin dysfunction. The goal of this proposal is to characterize the role of these novel ‘anti-cohesin factors’ in nuclear architecture and cohesin function and test the ability of their inhibition to correct gene misexpression in CdLS models. Through understanding the molecular factors involved in cohesin biology and chromatin folding, we can begin to consider targeted approaches to CdLS therapeutic development.

Project Narrative The proposed work tests new ideas regarding cohesin and related transcriptome regulatory proteins’ role in the regulation of developmental gene expression as they relate to human developmental disorders. This proposal also represents the first application of Oligopaint-based Hi-FISH that isolated novel cohesin regulators. This will work permit us to study how cohesin proteins control genes during active human cell development by merging genomic, imaging, and cell-based assays to characterize the molecular basis of a developmental syndrome. Experiments in this grant proposal are strongly motivated by the premise that a detailed elaboration of the molecular and cellular mechanisms of cohesin activity and genome folding will not only provide better insight into the workings of this complex cellular program, but may also profoundly change the way chromatin- misfolding disorders, including CdLS, are treated in the future.