GSE167887
Cohesin-independent STAG proteins interact with RNA and localise to R-loops to promote complex loading
Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in cohesins localization to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin and interact with CTCF, as well as a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA and are localised to endogenous R-loops where they act to suppress R-loop formation. Our results place SA proteins on chromatin upstream of the cohesin complex and reveal a role for SA in cohesin loading at R-loops which is independent of NIPBL, the canonical cohesin loader. We propose that SA takes advantage of this structural R-loop platform to link cohesin loading and chromatin structure with diverse genome functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease. Overall design: RAD21mAC cells were treated for 4hrs with EtOH or IAA.   Two biological replicates were prepared for each condition (B1 or B2).  For ChIP-seq, chromatin was extracted and immunoprecipitation with antibodies was performed. All IPs in one biologocal replicate samples were processed together.  ChIP-seq libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina kit and using NEBNext Multiplex Oligos.  Libraries were amplified with 6-8 cycles of PCR. Libraries were sequenced on the Illumina HiSeq2500 platform, 80bp single-end reads.  For DRIP-seq, cells were fractionated to isolate chromatin, nucleic acids were isolated and RNA:DNA hybrids were immunoprecipitated with s9.6 antibody.  DRIP-seq libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina kit and using NEBNext Multiplex Oligos. Libraries were sequenced on the Illumina NovaSeq platform, 100bp paired-end reads.