PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection.

Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells.This work was supported by the Spanish Ministry of Economy and Competitiveness and FEDER Grants BFU2013-48481-R and BFU2016-79841-R (to A. L.) and BFU2016-80402-R (to J. M.) and by FPI "Severo Ochoa" fellowships (to C. M. and M. R.-T.). This work was also supported by funding from Boehringer Ingelheim Fonds (to M. R.-T.). The authors declare that they have no conflicts of interest with the contents of this article.S

Xenopus HJURP and condensin II are required for CENP-A assembly

Chromatin structure imposed by condensin II at centromeres enables xHJURP-mediated incorporation of CENP-A

Heterochromatin Protein 1 (HP1) Proteins Do Not Drive Pericentromeric Cohesin Enrichment in Human Cells

Sister chromatid cohesion mediated by cohesin is essential for accurate chromosome segregation. Classical studies suggest that heterochromatin promotes cohesion, but whether this happens through regulation of cohesin remains to be determined. Heterochromatin protein 1 (HP1) is a major component of heterochromatin. In fission yeast, the HP1 homologue Swi6 interacts with cohesin and is required for proper targeting and/or stabilization of cohesin at the centromeric region. To test whether this pathway is conserved in human cells, we have examined the behavior of cohesin in cells in which the levels of HP1 alpha, beta or gamma (the three HP1 proteins present in mammalian organisms) have been reduced by siRNA. We have also studied the consequences of treating human cells with drugs that change the histone modification profile of heterochromatin and thereby affect HP1 localization. Our results show no evidence for a requirement of HP1 proteins for either loading of bulk cohesin onto chromatin in interphase or retention of cohesin at pericentric heterochromatin in mitosis. However, depletion of HP1gamma leads to defects in mitotic progression

Different NIPBL requirements of cohesin-STAG1 and cohesin-STAG2.

Cohesin organizes the genome through the formation of chromatin loops. NIPBL activates cohesin's ATPase and is essential for loop extrusion, but its requirement for cohesin loading is unclear. Here we have examined the effect of reducing NIPBL levels on the behavior of the two cohesin variants carrying STAG1 or STAG2 by combining a flow cytometry assay to measure chromatin-bound cohesin with analyses of its genome-wide distribution and genome contacts. We show that NIPBL depletion results in increased cohesin-STAG1 on chromatin that further accumulates at CTCF positions while cohesin-STAG2 diminishes genome-wide. Our data are consistent with a model in which NIPBL may not be required for chromatin association of cohesin but it is for loop extrusion, which in turn facilitates stabilization of cohesin-STAG2 at CTCF positions after being loaded elsewhere. In contrast, cohesin-STAG1 binds chromatin and becomes stabilized at CTCF sites even under low NIPBL levels, but genome folding is severely impaired.We are grateful to R.G. Syljuasen (Oslo U.H.) and Lola Martinez (Flow Cytometry Unit, CNIO) for advice on the flow cytometry protocol, Diego Megias (Confocal Microscopy Unit) for analysis of microscopy images, Alvaro Quevedo for his contribution to initial ChIP-seq and RNA-seq analyses and the rest of the members of the Chromosome Dynamics and DNA Replication groups at CNIO for helpful discussions. We also thank K. Shirahige (Tokyo University) for the ESCO1 antibody, J. Mendez (CNIO) for MCM3 and ORC2 antibodies, and E. de Alava ( IBIS) for the A673 cell line. Thiswork has been funded by grant PID2019-106499RB-I00 from Agencia Estatal de Investigacion (AEI/10.13039/501100011033), Ministerio de Ciencia e Innovacion, to A.L. D.A.G. is the recipient of FPI fellowship BES-2017-080051 and D.G.-L. is supported by a grant from the Spanish Association against Cancer (AECC).N

Specific contributions of cohesin-SA1 and cohesin-SA2 to TADs and polycomb domains in embryonic stem cells

Cohesin exists in two variants carrying either STAG/SA1 or SA2. Here we have addressed their specific contributions to the unique spatial organization of the mouse embryonic stem cell genome, which ensures super-enhancer-dependent transcription of pluripotency factors and repression of lineage-specification genes within Polycomb domains. We find that cohesin-SA2 facilitates Polycomb domain compaction through Polycomb repressing complex 1 (PRC1) recruitment and promotes the establishment of long-range interaction networks between distant Polycomb-bound promoters that are important for gene repression. Cohesin-SA1, in contrast, disrupts these networks, while preserving topologically associating domain (TAD) borders. The diverse effects of both complexes on genome topology may reflect two modes of action of cohesin. One, likely involving loop extrusion, establishes overall genome arrangement in TADs together with CTCF and prevents excessive segregation of same-class compartment regions. The other is required for organization of local transcriptional hubs such as Polycomb domains and super-enhancers, which define cell identity.This work was funded by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (FEDER) (grant BFU2016-79841-R to A.L.), Comunidad de Madrid (contract PEJD-2016/BMD-3190 to G.M.-S.), Centro de Excelencia Severo Ochoa to CNIO (SEV-2015-0510), and the National Institute of Health Carlos III (ISCIII). The work of Y.C. and M.A.M.-R. was partially supported by the European Research Council (ERC) under the Seventh Framework Programme FP7/2007–2013 (ERC grant agreement 609989) and the European Union’s Horizon 2020 research and innovation program (grant agreement 676556). M.A.M.-R. also acknowledges support from the Spanish Ministry of Economy and Competitiveness (BFU2017-85926-P and the Centro de Excelencia Severo Ochoa to Center for Genomic Regulation), and the Generalitat de Catalunya (AGAUR grant SGR468 and CERCA Programme)

Additional file 5 of Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

Additional file 5: Movie S3. RAD21-GFP iFRAP

Additional file 4 of Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

Additional file 4: Movie S2. STAG2-GFP iFRAP

Specific contributions of cohesin-SA1 and cohesin-SA2 to TADs and polycomb domains in embryonic stem cells

Cohesin exists in two variants carrying either STAG/SA1 or SA2. Here we have addressed their specific contributions to the unique spatial organization of the mouse embryonic stem cell genome, which ensures super-enhancer-dependent transcription of pluripotency factors and repression of lineage-specification genes within Polycomb domains. We find that cohesin-SA2 facilitates Polycomb domain compaction through Polycomb repressing complex 1 (PRC1) recruitment and promotes the establishment of long-range interaction networks between distant Polycomb-bound promoters that are important for gene repression. Cohesin-SA1, in contrast, disrupts these networks, while preserving topologically associating domain (TAD) borders. The diverse effects of both complexes on genome topology may reflect two modes of action of cohesin. One, likely involving loop extrusion, establishes overall genome arrangement in TADs together with CTCF and prevents excessive segregation of same-class compartment regions. The other is required for organization of local transcriptional hubs such as Polycomb domains and super-enhancers, which define cell identity.This work was funded by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (FEDER) (grant BFU2016-79841-R to A.L.), Comunidad de Madrid (contract PEJD-2016/BMD-3190 to G.M.-S.), Centro de Excelencia Severo Ochoa to CNIO (SEV-2015-0510), and the National Institute of Health Carlos III (ISCIII). The work of Y.C. and M.A.M.-R. was partially supported by the European Research Council (ERC) under the Seventh Framework Programme FP7/2007–2013 (ERC grant agreement 609989) and the European Union’s Horizon 2020 research and innovation program (grant agreement 676556). M.A.M.-R. also acknowledges support from the Spanish Ministry of Economy and Competitiveness (BFU2017-85926-P and the Centro de Excelencia Severo Ochoa to Center for Genomic Regulation), and the Generalitat de Catalunya (AGAUR grant SGR468 and CERCA Programme)

Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization.

Two variant cohesin complexes containing SMC1, SMC3, RAD21 and either SA1 (also known as STAG1) or SA2 (also known as STAG2) are present in all cell types. We report here their genomic distribution and specific contributions to genome organization in human cells. Although both variants are found at CCCTC-binding factor (CTCF) sites, a distinct population of the SA2-containing cohesin complexes (hereafter referred to as cohesin-SA2) localize to enhancers lacking CTCF, are linked to tissue-specific transcription and cannot be replaced by the SA1-containing cohesin complex (cohesin-SA1) when SA2 is absent, a condition that has been observed in several tumors. Downregulation of each of these variants has different consequences for gene expression and genome architecture. Our results suggest that cohesin-SA1 preferentially contributes to the stabilization of topologically associating domain boundaries together with CTCF, whereas cohesin-SA2 promotes cell-type-specific contacts between enhancers and promoters independently of CTCF. Loss of cohesin-SA2 rewires local chromatin contacts and alters gene expression. These findings provide insights into how cohesin mediates chromosome folding and establish a novel framework to address the consequences of mutations in cohesin genes in cancer.We thank Y. Cuartero and J. Quilez (4D Genome-CRG) for technical help with the Hi-C experiments, D. Rico (Newcastle University), F.X. Real (CNIO) and M. Manzanares (CNIC) for comments on the manuscript, T. Hirano (RIKEN) and H. Yu (UT Southwestern) for reagents, and M. Quintela (CNIO) for MCF10A cells. This work has been supported by the Spanish Ministry of Economy and Competitiveness and FEDER funds (grant no. BFU2013-48481-R (A.L.), BFU2016-79841-R (A.L.) and BFU2013-47736-P (M.A.M.-R.), fellowship no. BES-2014-069166 (M.D.K.), and Centro de Excelencia Severo Ochoa grant no. SEV-2015-0510 (to CNIO) and SEV-2012-0208 (to CRG), the European Research Council (FP7/2010-2015, ERC grant agreement 609989; M.A.M.-R.), the EU Horizon 2020 Research and Innovation Program (agreement 676556; M.A.M.-R.), the CERCA Programme-Generalitat de Catalunya (M.A.M.-R.) and the La Caixa Foundation (PhD fellowship to A.K.).S

Xenopus Shugoshin 2 regulates the spindle assembly pathway mediated by the chromosomal passenger complex

Shugoshins (Sgo) are conserved proteins that act as protectors of centromeric cohesion and as sensors of tension for the machinery that eliminates improper kinetochore–microtubule attachments. Most vertebrates contain two Sgo proteins, but their specific functions are not always clear. Xenopus laevis Sgo1, XSgo1, protects centromeric cohesin from the prophase dissociation pathway. Here, we report the identification of XSgo2 and show that it does not regulate cohesion. Instead, we find that it participates in bipolar spindle assembly. Both Sgo proteins interact physically with the Chromosomal Passenger Complex (CPC) containing Aurora B, a key regulator of mitosis, but the functional consequences of such interaction are distinct. XSgo1 is required for proper localization of the CPC while XSgo2 positively contributes to its activation and the subsequent phosphorylation of at least one key substrate for bipolar spindle assembly, the microtubule depolymerizing kinesin MCAK (Mitotic Centromere-Associated Kinesin). Thus, the two Xenopus Sgo proteins have non-overlapping functions in chromosome segregation. Our results further suggest that this functional specificity could rely on the association of XSgo1 and XSgo2 with different regulatory subunits of the PP2A complex