A cohesin traffic pattern genetically linked to gene regulation [CRISPR screens]

Cohesin-mediated loop extrusion is blocked at specific cis-elements, including CTCF sites, producing patterns of loops and domain boundaries along chromosomes. Here, we explore such cis-elements, and their role in gene regulation. We find that transcription termination sites of active genes form cohesin- and RNA polymerase II-dependent domain boundaries that do not accumulate cohesin. At these sites, cohesin is first stalled and then rapidly unloaded. Start sites of transcriptionally active genes form cohesin-bound boundaries, as shown before, but are cohesin-independent. Together with cohesin loading possibly at enhancers, these sites create a pattern of cohesin traffic that guides enhancer-promoter interactions. Disturbing this traffic pattern, by removing CTCF, renders cells sensitive to knock-out of genes involved in transcription initiation, such as the SAGA complexes, and RNA processing such DEAD/H-Box RNA helicases. Without CTCF, these factors are less efficiently recruited to active promoters. We engineered a HAP1-derived human cell line in which CTCF could efficiently be removed using an auxin-inducible degron system. We performed genome-wide CRISPR screens in HAP1-CTCFdegron-Tir1 cells (that expressed reduced levels of CTCF) and compared the results to similar screens performed in HAP1-CTCF-degron cells (that express higher levels of CTCF). We also performed screens in the presence of different concentrations of auxin to further reduce the CTCF protein level (NT, 25uM and 500uM auxin). The screens were performed in three replicates.