High-throughput Oligopaint screen identifies druggable 3D genome regulators

Although the molecular rules governing chromatin folding are being quickly elucidated, relatively few proteins regulating genome organization have been identified. To address this, we developed HiDRO (high-throughput labeling of DNA or RNA with optimized Oligopaint probes). Combined with automated image analysis, we used HiDRO to conduct a novel FISH-based screen of ~3,000 druggable genes that alter inter-TAD interactions. We isolated several enzymes that alter the integrity of architectural boundaries, including our top hit Glycogen synthase kinase alpha (GSK3A). We demonstrated using chemical and genetic inhibition that this ubiquitously expressed kinase enhances chromatin looping by regulating the chromatin binding of cohesin regulators. Finally, our data demonstrate that a GSK3A inhibitor can rescue both chromatin misfolding and gene misexpression at a sensitive locus after partial cohesin loss. These results illustrate a noncanonical role for GSK3A in cohesin regulation and highlight the broader utility of HiDRO-based screening to identify novel genome organizing factors. Overall design: RAD21and CTCF ChIP-seq was performed for three biological replicates each of control siRNA or GSK3A siRNA treated HCT-116 cells. After input normalization, bigwig files from each replicate as well as a bigwig file subsampled from all three replicates were produced. Bed files of peaks were generated from each bigwig. In situ Hi-C was performed for two biological replicate each of control siRNA, GSK3A siRNA, WAPL siRNA or PDS5A siRNA treated HCT-116 cells.