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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).

Nuclear architecture is decisive for the assembly of transcriptional responses. However, how chromosome organization is dynamically modulated to permit rapid and transient transcriptional changes in response to environmental challenges remains unclear. Here we show that hyperosmotic stress disrupts different levels of chromosome organization, ranging from A/B compartment changes to reduction in the number and insulation of topologically associating domains (TADs). Concomitantly, transcription is greatly affected, TAD borders weaken, and RNA Polymerase II runs off from hundreds of transcription end sites. Stress alters the binding profiles of architectural proteins, which explains the disappearance of local chromatin organization. These processes are dynamic, and cells rapidly reconstitute their default chromatin conformation after stress removal, uncovering an intrinsic organization. Transcription is not required for local chromatin reorganization, while compartment recovery is partially transcription-dependent. Thus, nuclear organization in mammalian cells can be rapidly modulated by environmental changes in a reversible manner. ; The study was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-64437-P and FEDER, BFU2014-52125-REDT, and BFU2014-51672-REDC to F.P.; BFU2017-85152-P and FEDER to E.d.N.), the Catalan Government (2017 SGR 799), the Fundación Botín, and the Banco Santander through its Santander Universities Global Division to F.P. and the Unidad de Excelencia Maria de Maeztu, MDM-2014-0370. F.P. is recipient of an ICREA Acadèmia (Generalitat de Catalunya). M.B. received the support of the European Research Council (ERC Synergy Grant 4D Genome 609989) and CERCA.

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. ; We thank Luciano di Croce for providing the Ring1B antibody as well as for comments on the manuscript. We also thank the Genomics and Proteomics Units at CNIO and the 4DG Unit at CRG for technical support. This work wasfunded 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 ...

In breast cancer cells, some topologically associating domains (TADs) behave as hormonal gene regulation units, within which gene transcription is coordinately regulated in response to steroid hormones. Here we further describe that responsive TADs contain 20- to 100-kb-long clusters of intermingled estrogen receptor (ESR1) and progesterone receptor (PGR) binding sites, hereafter called hormone-control regions (HCRs). In T47D cells, we identified more than 200 HCRs, which are frequently bound by unliganded ESR1 and PGR. These HCRs establish steady long-distance inter-TAD interactions between them and organize characteristic looping structures with promoters in their TADs even in the absence of hormones in ESR1+-PGR+ cells. This organization is dependent on the expression of the receptors and is further dynamically modulated in response to steroid hormones. HCRs function as platforms that integrate different signals, resulting in some cases in opposite transcriptional responses to estrogens or progestins. Altogether, these results suggest that steroid hormone receptors act not only as hormone-regulated sequence-specific transcription factors but also as local and global genome organizers. ; We received funding from the European Research Council under the European Union's Seventh Framework Program (FP7/2007–2013)/ERC Synergy grant agreement 609989 (4DGenome). The content of this manuscript reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. We acknowledge support of the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013–2017' and Plan Nacional (SAF2016-75006-P), as well as support of the CERCA Programme/Generalitat de Catalunya.

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium. ; Fundacion Cientifica de la Asociacion Espanola Contra el Cancer (to F.X.R., E.L., in part); V.P. is supported by INSERM, the Fondation Toulouse Cancer Sante and Pierre Fabre Research Institute as part of the Chair of Bioinformatics in Oncology of the CRCT; Bioinfo4women programme at the Barcelona Supercomputing Center; European Union's H2020 Framework Programme through the ERC [609989 to M.A.M.-R., in part]; Spanish Ministerio de Ciencia, Innovaci ' on y Universidades [BFU2017-85926P to M.A.M.-R.]; C.N.I.O. is supported by Ministerio de Ciencia, ...

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium. ; Fundación Científica de la Asociación Española Contra el Cáncer (to F.X.R., E.L., in part); V.P. is supported by INSERM, the Fondation Toulouse Cancer Santé and Pierre Fabre Research Institute as part of the Chair of Bioinformatics in Oncology of the CRCT; Bioinfo4women programme at the Barcelona Supercomputing Center; European Union's H2020 Framework Programme through the ERC [609989 to M.A.M.-R., in part]; Spanish Ministerio de Ciencia, Innovación y Universidades [BFU2017-85926-P to M.A.M.-R.]; C.N.I.O. is supported by Ministerio de Ciencia, Innovación y Universidades as a Centro de Excelencia Severo Ochoa [SEV-2015-0510]; C.R.G. acknowledges support from 'Centro de Excelencia Severo Ochoa 2013–2017' [SEV-2012-0208]; Spanish ministry of Science and Innovation to the EMBL partnership and the CERCA Programme/Generalitat de Catalunya (to C.R.G.); C.R.G. also acknowledges support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, the Generalitat de Catalunya through Departament de Salut and Departament d'Empresa i Coneixement; Spanish Ministry of Science and Innovation with funds from the European Regional Development Fund (ERDF) corresponding to the 2014–2020 Smart Growth Operating Program (to C.N.A.G.). Funding for open access charge: Own funds.

© 2019 The Author(s). ; Mammalian gametogenesis involves dramatic and tightly regulated chromatin remodeling, whose regulatory pathways remain largely unexplored. Here, we generate a comprehensive high-resolution structural and functional atlas of mouse spermatogenesis by combining in situ chromosome conformation capture sequencing (Hi-C), RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) of CCCTC-binding factor (CTCF) and meiotic cohesins, coupled with confocal and super-resolution microscopy. Spermatogonia presents well-defined compartment patterns and topological domains. However, chromosome occupancy and compartmentalization are highly re-arranged during prophase I, with cohesins bound to active promoters in DNA loops out of the chromosomal axes. Compartment patterns re-emerge in round spermatids, where cohesin occupancy correlates with transcriptional activity of key developmental genes. The compact sperm genome contains compartments with actively transcribed genes but no fine-scale topological domains, concomitant with the presence of protamines. Overall, we demonstrate how genome-wide cohesin occupancy and transcriptional activity is associated with three-dimensional (3D) remodeling during spermatogenesis, ultimately reprogramming the genome for the next generation. ; This work was supported by the Ministry of Economy and Competitiveness ( BFU2017-89408-R to A.M.P.; BFU2013-47736-P and BFU2017-85926-P to M.A.M.-R.; and CGL2014-54317-P and CGL2017-83802-P to A.R.-H.) and the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) ( DI2015 to A.R.-H. and R.A.C., as well as SGR468 to M.A.M-R.). Work at CRG, BIST, and UPF was in part funded by the Spanish Ministry of Economy and Competitiveness , 'Centro de Excelencia Severo Ochoa 2013-2017' (SEV-2012-0208), and 'Centro de Excelencia María de Maeztu 2016-2019.' CIC-IBMCC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia , co-funded by Junta de Castilla y León ( CSI239P18 ) and the European Regional Development Fund ( CLC–2017–01 ). C.V. is supported by a FPI predoctoral fellowship from the Ministry of Economy and Competitiveness ( BES-2015-072924 ). A.P.-G. is supported by a 'Doctorats Industrials' predoctoral fellowship ( AGAUR ). H.H. is a Miguel Servet ( CP14/00229 ) researcher funded by the Spanish Institute of Health Carlos III (ISCIII), the Agencia Estatal de Investigación (AEI), and FEDER ( SAF2017-89109-P ). C.M. is an Asociación Española Contra el Cáncer (AECC) postdoctoral fellow. M.A.M.-R. acknowledges support by the European Research Council under the 7th Framework Program FP7/2007-2013 (ERC grant agreement 609989 ) and the European Union's Horizon 2020 Research and Innovation Programme (grant agreement 676556 ). A.M.P. and A.R.-H. also acknowledge support from MeioNet ( BFU2015-71786-REDT ).

Lamins (A/C and B) are major constituents of the nuclear lamina (NL). Structurally conserved lamina-associated domains (LADs) are formed by genomic regions that contact the NL. Lamins are also found in the nucleoplasm, with a yet unknown function. Here we map the genome-wide localization of lamin B1 in an euchromatin-enriched fraction of the mouse genome and follow its dynamics during the epithelial-to-mesenchymal transition (EMT). Lamin B1 associates with actively expressed and open euchromatin regions, forming dynamic euchromatin lamin B1-associated domains (eLADs) of about 0.3 Mb. Hi-C data link eLADs to the 3D organization of the mouse genome during EMT and correlate lamin B1 enrichment at topologically associating domain (TAD) borders with increased border strength. Having reduced levels of lamin B1 alters the EMT transcriptional signature and compromises the acquisition of mesenchymal traits. Thus, during EMT, the process of genome reorganization in mouse involves dynamic changes in eLADs ; This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) FIS/FEDER (PI15/00396; CPII14/0006), Ministerio de Economía y Competitividad (MINECO) (SAF2013-40922-R1; FPU14/0407; BFU2016-75008-P), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional-FEDER (SAF2016-76461-R), Generalitat de Catalunya (2014 SGR 32), Fundació FERO, Fundació La Marató TV3, and La Caixa Foundation. We also thank the Advanced Light Microscopy Unit at the CRG for their assistance and the Cellex Foundation for providing research facilities and equipment. M.A.M.-R. acknowledges funding from the European Research Council under the 7th Framework Program (FP7/2010-2015, ERC grant agreement 609989), the European Union's Horizon 2020 research and innovation program (agreement 676556), the Spanish Ministry of Economy and Competitiveness (BFU2013-47736-P), and the Centro de Excelencia Severo Ochoa 2013-2017 (SEV-2012-0208) to the CRG

The spatial folding of chromosomes inside the nucleus has regulatory effects on gene expression, yet the impact of genome reshuffling on this organization remains unclear. Here, we take advantage of chromosome conformation capture in combination with single-nucleotide polymorphism (SNP) genotyping and analysis of crossover events to study how the higher-order chromatin organization and recombination landscapes are affected by chromosomal fusions in the mammalian germ line. We demonstrate that chromosomal fusions alter the nuclear architecture during meiosis, including an increased rate of heterologous interactions in primary spermatocytes, and alterations in both chromosome synapsis and axis length. These disturbances in topology were associated with changes in genomic landscapes of recombination, resulting in detectable genomic footprints. Overall, we show that chromosomal fusions impact the dynamic genome topology of germ cells in two ways: (i) altering chromosomal nuclear occupancy and synapsis, and (ii) reshaping landscapes of recombination. ; This work was supported by the Ministry of Economy and Competitiveness (CGL2014-54317-P and CGL2017-83802-P to A.R.-H., BFU2013-47736-P and BFU2017-85926-P to M.A.M.-R. and CGL2010-15243 to J.V.) and the Agència de Gestió d'Ajuts Universitaris i de Recerca, AGAUR (DI2015 and SGR1215 to A.R-H. as well as SGR468 to M.A.M.-R.). C.V. and L.A.-G. were supported by FPI predoctoral fellowships from the Ministry of Economy and Competitiveness (BES-2015-072924 and PRE-2018-083257). L.M.-G. was supported by an FPU predoctoral fellowships from the Ministry of Science, Innovation and University (FPU18/03867). M.A.M.-R. acknowledges support by the European Research Council under the 7th Framework Program FP7/2007-2013 (ERC grant agreement 609989) and the European Union's Horizon 2020 research and innovation program (grant agreement 676556). A.R.-H. also acknowledges support from MeioNet (BFU2015-71786-REDT).

Mammalian gametogenesis involves dramatic and tightly regulated chromatin remodeling, whose regulatory pathways remain largely unexplored. Here, we generate a comprehensive high-resolution structural and functional atlas of mouse spermatogenesis by combining in situ chromosome conformation capture sequencing (Hi-C), RNA sequencing (RNA-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) of CCCTC-binding factor (CTCF) and meiotic cohesins, coupled with confocal and super-resolution microscopy. Spermatogonia presents well-defined compartment patterns and topological domains. However, chromosome occupancy and compartmentalization are highly re-arranged during prophase I, with cohesins bound to active promoters in DNA loops out of the chromosomal axes. Compartment patterns re-emerge in round spermatids, where cohesin occupancy correlates with transcriptional activity of key developmental genes. The compact sperm genome contains compartments with actively transcribed genes but no fine-scale topological domains, concomitant with the presence of protamines. Overall, we demonstrate how genome-wide cohesin occupancy and transcriptional activity is associated with three-dimensional (3D) remodeling during spermatogenesis, ultimately reprogramming the genome for the next generation. ; This work was supported by the Ministry of Economy and Competitiveness (BFU2017-89408-R to A.M.P.; BFU2013-47736-P and BFU2017-85926-P to M.A.M.-R.; and CGL2014-54317-P and CGL2017-83802-P to A.R.-H.) and the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) (DI2015 to A.R.-H. and R.A.C., as well as SGR468 to M.A.M-R.). Work at CRG, BIST, and UPF was in part funded by the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017' (SEV-2012-0208), and 'Centro de Excelencia María de Maeztu 2016-2019.' CIC-IBMCC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia, co-funded by Junta de Castilla y León (CSI239P18) and the European Regional Development Fund (CLC–2017–01). C.V. is supported by a FPI predoctoral fellowship from the Ministry of Economy and Competitiveness (BES-2015-072924). A.P.-G. is supported by a 'Doctorats Industrials' predoctoral fellowship (AGAUR). H.H. is a Miguel Servet (CP14/00229) researcher funded by the Spanish Institute of Health Carlos III (ISCIII), the Agencia Estatal de Investigación (AEI), and FEDER (SAF2017-89109-P). C.M. is an Asociación Española Contra el Cáncer (AECC) postdoctoral fellow. M.A.M.-R. acknowledges support by the European Research Council under the 7th Framework Program FP7/2007-2013 (ERC grant agreement 609989) and the European Union's Horizon 2020 Research and Innovation Programme (grant agreement 676556). A.M.P. and A.R.-H. also acknowledge support from MeioNet (BFU2015-71786-REDT).