Title: How transcription disrupts genome 3D organization Abstract: The 3D packaging of chromatin within the nucleus plays an important role in regulating gene expression. While the principles of how the genome is folded in the nucleus are increasingly well understood, we know remarkably little about the mechanisms that drive dynamic changes in genome 3D structure, e.g. during differentiation. We recently discovered that influenza A infection inhibits transcription termination, resulting in read-through transcription often extends hundreds of kilobases past the 3’ ends of genes. Transcription of these regions disrupts local cohesin-mediated chromatin interactions, leads to chromatin decompaction and frequently induces switching of previously inactive genome regions to the active compartment. These compartment changes occur in a matter of hours, and in the absence of epigenetic changes, which usually are found to correlate with compartment association of a locus. Further analysis of genic transcription revealed that cohesin loss is closely temporally linked to RNA polymerase II arrival at cohesin binding sites, suggesting that RNA polymerase II itself is involved in displacing cohesin from chromatin. To study these phenomena in detail, we will comprehensively characterize the epigenetic and transcriptional effects of pervasive read-through in degron-tagged cell lines as an alternative model for transcription-induced genome 3D structure changes, and test the contribution of gene structure to delimiting where RNA polymerase II transcription changes genome organization. In a separate set of experiments, we will use a candidate approach to identify proteins involved in transcription-associated cohesin unloading. These studies will help reveal how transcription influences chromatin interaction and increase our knowledge of the mechanisms that dynamically reorganize genome 3D organization.