Cohesin-independent STAG proteins interact with RNA and R-loops and promote complex loading
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This study provides evidence that the Stromalin Antigen (SA) proteins known to ubiquitously interact with cohesins, retain their capacity to bind CTCF and chromatin in the absence of RAD21 cohesin component. Authors imply that SA has an independent function in addition to its joint role with RAD21 and CTCF, providing experiments that make them suggest that SA proteins organize around RNA:DNA regions in the absence of cohesin, contributing to R-loop regulation and linking chromatin on structure to cohesin loading. The paper is a nice piece of work of interest to readers in the field of cohesin biology and genome organization. However, additional, experiments would be required to strengthen some of the conclusions.
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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, 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.
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eLife assessment
This study provides evidence that the Stromalin Antigen (SA) proteins known to ubiquitously interact with cohesins, retain their capacity to bind CTCF and chromatin in the absence of RAD21 cohesin component. Authors imply that SA has an independent function in addition to its joint role with RAD21 and CTCF, providing experiments that make them suggest that SA proteins organize around RNA:DNA regions in the absence of cohesin, contributing to R-loop regulation and linking chromatin on structure to cohesin loading. The paper is a nice piece of work of interest to readers in the field of cohesin biology and genome organization. However, additional, experiments would be required to strengthen some of the conclusions.
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Reviewer #1 (Public Review):
To explore possible functions of SA proteins in the absence of cohesin, authors use conditional AII-dependent proteins SA1 and SA2, after whose degradation they observe the phenotypes just indicated. 3D analysis shows that SA proteins cluster at specific regions. In addition, it is shown that SA proteins not only interact with CTCF after RAD21 degradation but with other F/YXF-motif containing proteins such as CHD6, MCM3 or HRNPUL2 as determined by ChIP. Mass spectrometry of proteins co-immunoprecipitated with SA1 reveals 136 interactor proteins that include a number of chromatin remodeling factors, transcription factors and RNA binding proteins including factors involved in RNA processing and modification, ribosome biogenesis and translation. After these results, authors perform CLIP to show that SA1 protein …
Reviewer #1 (Public Review):
To explore possible functions of SA proteins in the absence of cohesin, authors use conditional AII-dependent proteins SA1 and SA2, after whose degradation they observe the phenotypes just indicated. 3D analysis shows that SA proteins cluster at specific regions. In addition, it is shown that SA proteins not only interact with CTCF after RAD21 degradation but with other F/YXF-motif containing proteins such as CHD6, MCM3 or HRNPUL2 as determined by ChIP. Mass spectrometry of proteins co-immunoprecipitated with SA1 reveals 136 interactor proteins that include a number of chromatin remodeling factors, transcription factors and RNA binding proteins including factors involved in RNA processing and modification, ribosome biogenesis and translation. After these results, authors perform CLIP to show that SA1 protein binds RNA in the absence of cohesin. Different analysis using RNH, mainly IF and IP and the S9.6 antibody, are used to conclude that SA1 binds to R-loop regions. The authors conclude that SA proteins are loaded to chromatin via NIPBL/mMAu complex at RNA:DNA hybrid regions. Further analyses suggest that SA proteins stabilize RNA via interaction with other RNA-binding proteins, some of which have been shown by other authors to be enriched at R loop-containing regions, a property that localizes to exon 32 in SA2. The manuscript provides a large amount of work that has been put together in a large collaboration to bring new roles for SA in RNA metabolism, even though this is not investigated.
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Reviewer #2 (Public Review):
In the manuscript by Porter et al., the authors describe a putative role for the STAG proteins (SA1 and SA2), not as part of the cohesin complex, but in isolation and in particular at R-loops where they contribute to R-loop regulation, linking chromatin structure and cohesin loading.
My major concern is rather general: " the role of SA1 and SA2 proteins (or cohesion subunits) its only highlighted upon acute depletion of RAD21 (cohesin subunit that holds together the complex)". I am not sure that this context is recapitulated in living cells. I.e is there a particular phase of the cell cycle where RAD21 is acutely depleted or targeted for specific degradation? How do we know that we are not looking at remnants of a complex (cohesin) that has been partially targeted by IIA mediated degradation? Is the RNA …
Reviewer #2 (Public Review):
In the manuscript by Porter et al., the authors describe a putative role for the STAG proteins (SA1 and SA2), not as part of the cohesin complex, but in isolation and in particular at R-loops where they contribute to R-loop regulation, linking chromatin structure and cohesin loading.
My major concern is rather general: " the role of SA1 and SA2 proteins (or cohesion subunits) its only highlighted upon acute depletion of RAD21 (cohesin subunit that holds together the complex)". I am not sure that this context is recapitulated in living cells. I.e is there a particular phase of the cell cycle where RAD21 is acutely depleted or targeted for specific degradation? How do we know that we are not looking at remnants of a complex (cohesin) that has been partially targeted by IIA mediated degradation? Is the RNA binding of SA1 an SA2 CTCF-independent (as CTCF encompasses an RNA binding domain?).
Is there any proof that in untreated cells (ie before depletion) SA1 AND SA2 are chromatin bound independently of Rad21 (and /or SMC1-3)? Overall, it is a nice manuscript, but I am not sure whether the IAA-dependent degradation of a single subunit of pentameric complex is the right tool to assess whether other subunits of the same complex work independently.
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Reviewer #3 (Public Review):
Porter, Li et al. investigate the roles of SA1 and SA2 in cohesin loading, and as well as roles that are independent of the cohesin ring. Using co-IP and imaging approaches, they show that both SA1 and SA2 interact with CTCF and they use auxin-induced degradation of Rad21 to show that this is only partially dependent on cohesin. The authors next use IP followed by mass spectrometry to identify additional SA binding partners, which include many RNA binding proteins including factors involved in RNA modification, export, splicing, and translation. Unlike the interaction with CTCF, these interactions are enhanced in cohesin depletion conditions. In fact, CLIP experiments show that SA binds RNA directly, in an R-loop-dependent manner. This co-localisation of SA with R-loops is confirmed by STORM.
To address …
Reviewer #3 (Public Review):
Porter, Li et al. investigate the roles of SA1 and SA2 in cohesin loading, and as well as roles that are independent of the cohesin ring. Using co-IP and imaging approaches, they show that both SA1 and SA2 interact with CTCF and they use auxin-induced degradation of Rad21 to show that this is only partially dependent on cohesin. The authors next use IP followed by mass spectrometry to identify additional SA binding partners, which include many RNA binding proteins including factors involved in RNA modification, export, splicing, and translation. Unlike the interaction with CTCF, these interactions are enhanced in cohesin depletion conditions. In fact, CLIP experiments show that SA binds RNA directly, in an R-loop-dependent manner. This co-localisation of SA with R-loops is confirmed by STORM.
To address whether SA proteins are involved in cohesin loading, the authors measure chromatin-bound cohesin levels after auxin washoff in the presence and absence of NIPBL and SA. They find that SA knockdown has a comparable impact on cohesin binding to chromatin compared to NIPBL knockdown, and that combining the knockdowns reduces cohesin loading further. This newly synthesised cohesin co-localises with R-loop domains by STORM, and this localisation is sensitive to RNAse H. The authors propose that SA promotes cohesin loading at R-loops, and that SA1 is the main contributor to this. Finally, they provide evidence that differential usage of a conserved exon between SA1 and SA2 may be responsible for differences between SA1 and SA2 in this system, as SA2 with this exon included has higher RBP binding and is more enriched at R-loops.
This paper provides convincing evidence that SA proteins associate with R-loops and various RNA-binding proteins, suggesting that they may have a cohesin-independent role related to RNA processing or R-loops specifically. Additionally, the paper provides evidence for a NIPBL-independent role of SA proteins at cohesin loading, which may occur at R-loops. These results will be of broad interest in relation to chromatin organisation and the role of SA proteins/cohesin in cancer.
Overall, the experiments are thorough and well-controlled, including some nice validations such as the use of siRNA-mediated cohesin depletion and a different cell line to confirm the SA-CTCF interactions. In many cases STORM imaging is used to provide complementary evidence to western blots / IP experiments.
However, one weakness is that imaging approaches can only address co-localisation. Although the vast majority of cohesin complexes will be bound to DNA, imaging approaches cannot distinguish between chromatin-bound and unbound nuclear proteins. For example, although cohesin co-localises with R-loops and SA after auxin washoff, and this is dependent on R-loops, it is not possible to tell from imaging whether this cohesin is chromatin bound and whether this is bound to specific genomic loci that contain R-loops or just associated with them in 3D space. Therefore it would be preferable to have a clearer distinction in terminology depending on whether the evidence discussed can demonstrate chromatin binding (e.g. chromatin fractionation experiments), or just co-localisation.
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