Cohesin-independent STAG proteins interact with RNA and localise to R-loops to promote complex loading

Abstract

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. This way, SA takes advantage of the 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.

Single-molecule imaging reveals a direct role of CTCF’s zinc fingers in SA interaction and cluster-dependent RNA recruitment

“…Most recent data suggest that CTCF blocks loop extrusion via interaction of a conserved domain in CTCF’s N‐terminus with cohesin subunits SA and Rad21 21 . However, CTCF has also been shown in vivo to colocalize with SA in the absence of Rad21 23 . The exact mechanism of CTCF’s interaction with SA remains unknown.…”

“…CBS-bound CTCF ZFs recruit SA by a Rad21-independent interaction, increasing SA’s residence time. CTCF can therefore influence cohesin-independent functions of SAs in the nucleus 23 . SAs may then recruit other cohesin subunits, facilitating TAD-formation.…”

“…This orientation-dependent arrest of loops is determined by a direct interaction of cohesin subunits SA and Rad21 with the CTCF N-terminus 21,22 . However, in ‐ vivo studies revealed that SAs remain associated with CTCF even in the absence of cohesin 23 , suggesting a cohesin-independent interaction between CTCF and SA. Another study showed SA to be the only cohesin subunit directly interacting with CTCF, dependent on CTCF’s C-terminus, in contrast to the Rad21-dependent interaction with its N-terminus 21,24 .…”

CTCF and R-loops are boundaries of cohesin-mediated DNA looping

“…R-loops also form genome-wide during transcription via hybridization of the nascent transcript with the template DNA strand. Cohesin binds RNA via its STAG1/2 subunits in vitro (Pan et al, 2020), and STAG1/2 proteins are enriched at R-loops in cells (Porter et al, 2021). Moreover, apoCas9 doesn’t block cohesin translocation, suggesting that R-loops may impede cohesin directly.…”

STAG2 promotes the myelination transcriptional program in oligodendrocytes

“…Second, loop extrusion by STAG2-cohesin may promote transcription elongation by regulating transcription-coupled pre-mRNA processing. For example, STAG2 has been shown to interact with RNA-DNA hybrid structures termed R-loops in vitro and in cells (Pan et al, 2020;Porter et al, 2021). R-loops formed between the nascent pre-mRNA and the DNA template impede transcription elongation and need to be suppressed (Moore and Proudfoot, 2009).…”