Cohesin-independent STAG proteins interact with RNA and R-loops and 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 the localization of cohesin 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, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, RNA binding proteins and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse 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

“…In our experiments, we did not observe any influence of Rad21 on CTCF-SA interactions, suggesting that SAs are stabilized by CTCF ZFs independently of other cohesin subunits. In agreement with this, Rad21-independent colocalization of CTCF and SA have also been observed in vivo 23 . The direct recruitment of SA by CTCF ZFs as well as CTCF's high stability on CBSs and ability to perform secondary RNA capture could therefore regulate SA1 and SA2's different functions in transcription regulation and genome architecture 71,72,75 , possibly also by cohesin-independent interactions 23 .…”

“…In contrast, when we repeated this experiment with SAs, which colocalize with R-loops in vivo 23 , the presence of transcription bubbles on DNA curtains led to a complete change in binding behavior (Figure S4G,H). Both SA1 and SA2 were no longer enriched on AT-rich regions (Figure S4I) and instead accumulated at transcription bubbles, with a higher lifetime compared to DNA (Figure S4J).…”

“…Both CTCF and RNA can stabilize SA-DNA binding independent of cohesin SA colocalizes with R-loops in vivo, possibly by a direct interaction with RNA, or with other RNAbinding proteins 23 . Our experiments show that in the absence of RNA, SA preferentially interacts with AT-rich DNA-regions, with below-average occupancy on CBSs, fast binding dynamics and low salt stability (Figure 3F-I).…”

“…(Figure S4G-J). SA-RNA interactions could therefore facilitate their localization to R-loops and to active genes 23,[70][71][72] , as well as recruitment of cohesin-SA to CTCF sites 69 . However, unlike CTCF, DNA-bound SA cannot recruit RNA.…”

Cohesin composition and dosage independently affect early development in zebrafish

“…Several molecular studies show that STAG1 and STAG2 paralogues have distinct roles in 3D genome organization, but overlapping roles in the cell cycle [16, 65–69]. Moreover, STAG subunits can be detected at specific locations on DNA independently of the rest of the cohesin complex [67, 70]. Deficiency in STAG2 leads to the upregulation of STAG1 and the substitution of STAG1 for STAG2 in the cohesin complex such that STAG2 mutation leads to altered cohesin composition [71, 72].…”