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• W3044693806 abstract "•The majority of promoter-anchored contacts are lost upon rapid degradation of cohesin or CTCF•A significant minority of promoter contacts are retained, and some are gained•Cohesin-independent promoter contacts preferentially engage active enhancers•Loss of cohesin-dependent promoter-enhancer links associates with transcriptional changes It is currently assumed that 3D chromosomal organization plays a central role in transcriptional control. However, depletion of cohesin and CTCF affects the steady-state levels of only a minority of transcripts. Here, we use high-resolution Capture Hi-C to interrogate the dynamics of chromosomal contacts of all annotated human gene promoters upon degradation of cohesin and CTCF. We show that a majority of promoter-anchored contacts are lost in these conditions, but many contacts with distinct properties are maintained, and some new ones are gained. The rewiring of contacts between promoters and active enhancers upon cohesin degradation associates with rapid changes in target gene transcription as detected by SLAM sequencing (SLAM-seq). These results provide a mechanistic explanation for the limited, but consistent, effects of cohesin and CTCF depletion on steady-state transcription and suggest the existence of both cohesin-dependent and -independent mechanisms of enhancer-promoter pairing. It is currently assumed that 3D chromosomal organization plays a central role in transcriptional control. However, depletion of cohesin and CTCF affects the steady-state levels of only a minority of transcripts. Here, we use high-resolution Capture Hi-C to interrogate the dynamics of chromosomal contacts of all annotated human gene promoters upon degradation of cohesin and CTCF. We show that a majority of promoter-anchored contacts are lost in these conditions, but many contacts with distinct properties are maintained, and some new ones are gained. The rewiring of contacts between promoters and active enhancers upon cohesin degradation associates with rapid changes in target gene transcription as detected by SLAM sequencing (SLAM-seq). These results provide a mechanistic explanation for the limited, but consistent, effects of cohesin and CTCF depletion on steady-state transcription and suggest the existence of both cohesin-dependent and -independent mechanisms of enhancer-promoter pairing. DNA regulatory elements such as enhancers play key roles in transcriptional control, particularly for developmental and stimulus-response genes (Long et al., 2016Long H.K. Prescott S.L. Wysocka J. Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution.Cell. 2016; 167: 1170-1187Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar; Shlyueva et al., 2014Shlyueva D. Stampfel G. Stark A. Transcriptional enhancers: from properties to genome-wide predictions.Nat. Rev. Genet. 2014; 15: 272-286Crossref PubMed Scopus (625) Google Scholar). Many enhancers are located large distances (up to megabases) away from their target promoters and are brought into physical proximity with them through DNA looping interactions (Göndör and Ohlsson, 2018Göndör A. Ohlsson R. Enhancer functions in three dimensions: beyond the flat world perspective.F1000Res. 2018; 7: 681Crossref Scopus (8) Google Scholar). 3D chromosomal architecture and its regulators are, therefore, thought to be important for transcriptional control. The cohesin complex and regulatory proteins that control cohesin-DNA interactions are critical for shaping chromosomal architecture (Merkenschlager and Nora, 2016Merkenschlager M. Nora E.P. CTCF and Cohesin in Genome Folding and Transcriptional Gene Regulation.Annu. Rev. Genomics Hum. Genet. 2016; 17: 17-43Crossref PubMed Google Scholar). According to the current understanding, cohesin is involved in the extrusion of DNA loops in interphase nuclei (Davidson et al., 2019Davidson I.F. Bauer B. Goetz D. Tang W. Wutz G. Peters J.-M. DNA loop extrusion by human cohesin.Science. 2019; 366: 1338-1345Crossref PubMed Scopus (94) Google Scholar; Fudenberg et al., 2016Fudenberg G. Imakaev M. Lu C. Goloborodko A. Abdennur N. Mirny L.A. Formation of Chromosomal Domains by Loop Extrusion.Cell Rep. 2016; 15: 2038-2049Abstract Full Text Full Text PDF PubMed Google Scholar; Kim et al., 2019Kim Y. Shi Z. Zhang H. Finkelstein I.J. Yu H. Human cohesin compacts DNA by loop extrusion.Science. 2019; 366: 1345-1349Crossref PubMed Scopus (88) Google Scholar; Sanborn et al., 2015Sanborn A.L. Rao S.S.P. Huang S.-C. Durand N.C. Huntley M.H. Jewett A.I. Bochkov I.D. Chinnappan D. Cutkosky A. Li J. et al.Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes.Proc. Natl. Acad. Sci. USA. 2015; 112: E6456-E6465Crossref PubMed Scopus (636) Google Scholar), additionally to its well-characterized role in holding sister chromatids together from DNA replication until mitosis (Uhlmann, 2016Uhlmann F. SMC complexes: from DNA to chromosomes.Nat. Rev. Mol. Cell Biol. 2016; 17: 399-412Crossref PubMed Google Scholar). Extruding DNA loops are confined by chromatin boundary elements; in particular, by the binding of zinc-finger protein CTCF to its two recognition motifs on the DNA in convergent orientation (Rao et al., 2014Rao S.S.P. Huntley M.H. Durand N.C. Stamenova E.K. Bochkov I.D. Robinson J.T. Sanborn A.L. Machol I. Omer A.D. Lander E.S. Aiden E.L. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping.Cell. 2014; 159: 1665-1680Abstract Full Text Full Text PDF PubMed Scopus (2333) Google Scholar; Vietri Rudan et al., 2015Vietri Rudan M. Barrington C. Henderson S. Ernst C. Odom D.T. Tanay A. Hadjur S. Comparative Hi-C reveals that CTCF underlies evolution of chromosomal domain architecture.Cell Rep. 2015; 10: 1297-1309Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). These loops underpin the formation of topologically associated domains (TADs) (Dixon et al., 2012Dixon J.R. Selvaraj S. Yue F. Kim A. Li Y. Shen Y. Hu M. Liu J.S. Ren B. Topological domains in mammalian genomes identified by analysis of chromatin interactions.Nature. 2012; 485: 376-380Crossref PubMed Scopus (2941) Google Scholar; Nora et al., 2012Nora E.P. Lajoie B.R. Schulz E.G. Giorgetti L. Okamoto I. Servant N. Piolot T. van Berkum N.L. Meisig J. Sedat J. et al.Spatial partitioning of the regulatory landscape of the X-inactivation centre.Nature. 2012; 485: 381-385Crossref PubMed Scopus (1382) Google Scholar; Sexton et al., 2012Sexton T. Yaffe E. Kenigsberg E. Bantignies F. Leblanc B. Hoichman M. Parrinello H. Tanay A. Cavalli G. Three-dimensional folding and functional organization principles of the Drosophila genome.Cell. 2012; 148: 458-472Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar) and substructures such as insulated neighborhoods (INs) (Hnisz et al., 2016Hnisz D. Day D.S. Young R.A. Insulated Neighborhoods: Structural and Functional Units of Mammalian Gene Control.Cell. 2016; 167: 1188-1200Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar; Nuebler et al., 2018Nuebler J. Fudenberg G. Imakaev M. Abdennur N. Mirny L.A. Chromatin organization by an interplay of loop extrusion and compartmental segregation.Proc. Natl. Acad. Sci. USA. 2018; 115: E6697-E6706Crossref PubMed Scopus (150) Google Scholar; Szabo et al., 2019Szabo Q. Bantignies F. Cavalli G. Principles of genome folding into topologically associating domains.Sci. Adv. 2019; 5: eaaw1668Crossref PubMed Scopus (76) Google Scholar). Depletion of cohesin leads to rapid dissolution of TADs, whereas inactivation of CTCF reduces the strength of TAD boundaries (Gassler et al., 2017Gassler J. Brandão H.B. Imakaev M. Flyamer I.M. Ladstätter S. Bickmore W.A. Peters J.-M. Mirny L.A. Tachibana K. A mechanism of cohesin-dependent loop extrusion organizes zygotic genome architecture.EMBO J. 2017; 36: 3600-3618Crossref PubMed Scopus (99) Google Scholar; Nora et al., 2017Nora E.P. Goloborodko A. Valton A.-L. Gibcus J.H. Uebersohn A. Abdennur N. Dekker J. Mirny L.A. Bruneau B.G. Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.Cell. 2017; 169: 930-944.e22Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar; Rao et al., 2017Rao S.S.P. Huang S.-C. Glenn St Hilaire B. Engreitz J.M. Perez E.M. Kieffer-Kwon K.-R. Sanborn A.L. Johnstone S.E. Bascom G.D. Bochkov I.D. et al.Cohesin Loss Eliminates All Loop Domains.Cell. 2017; 171: 305-320.e24Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar; Schwarzer et al., 2017Schwarzer W. Abdennur N. Goloborodko A. Pekowska A. Fudenberg G. Loe-Mie Y. Fonseca N.A. Huber W. Haering C.H. Mirny L. Spitz F. Two independent modes of chromatin organization revealed by cohesin removal.Nature. 2017; 551: 51-56Crossref PubMed Scopus (344) Google Scholar; Wutz et al., 2017Wutz G. Várnai C. Nagasaka K. Cisneros D.A. Stocsits R.R. Tang W. Schoenfelder S. Jessberger G. Muhar M. Hossain M.J. et al.Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins.EMBO J. 2017; 36: 3573-3599Crossref PubMed Scopus (189) Google Scholar). In contrast, the higher order levels of chromosomal organization, such as A/B compartments that broadly separate active and inactive chromatin, are not reduced upon cohesin and CTCF depletion (Gassler et al., 2017Gassler J. Brandão H.B. Imakaev M. Flyamer I.M. Ladstätter S. Bickmore W.A. Peters J.-M. Mirny L.A. Tachibana K. A mechanism of cohesin-dependent loop extrusion organizes zygotic genome architecture.EMBO J. 2017; 36: 3600-3618Crossref PubMed Scopus (99) Google Scholar; Nora et al., 2017Nora E.P. Goloborodko A. Valton A.-L. Gibcus J.H. Uebersohn A. Abdennur N. Dekker J. Mirny L.A. Bruneau B.G. Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.Cell. 2017; 169: 930-944.e22Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar; Rao et al., 2017Rao S.S.P. Huang S.-C. Glenn St Hilaire B. Engreitz J.M. Perez E.M. Kieffer-Kwon K.-R. Sanborn A.L. Johnstone S.E. Bascom G.D. Bochkov I.D. et al.Cohesin Loss Eliminates All Loop Domains.Cell. 2017; 171: 305-320.e24Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar; Schwarzer et al., 2017Schwarzer W. Abdennur N. Goloborodko A. Pekowska A. Fudenberg G. Loe-Mie Y. Fonseca N.A. Huber W. Haering C.H. Mirny L. Spitz F. Two independent modes of chromatin organization revealed by cohesin removal.Nature. 2017; 551: 51-56Crossref PubMed Scopus (344) Google Scholar; Seitan et al., 2013Seitan V.C. Faure A.J. Zhan Y. McCord R.P. Lajoie B.R. Ing-Simmons E. Lenhard B. Giorgetti L. Heard E. Fisher A.G. et al.Cohesin-based chromatin interactions enable regulated gene expression within preexisting architectural compartments.Genome Res. 2013; 23: 2066-2077Crossref PubMed Scopus (199) Google Scholar; Sofueva et al., 2013Sofueva S. Yaffe E. Chan W.-C. Georgopoulou D. Vietri Rudan M. Mira-Bontenbal H. Pollard S.M. Schroth G.P. Tanay A. Hadjur S. Cohesin-mediated interactions organize chromosomal domain architecture.EMBO J. 2013; 32: 3119-3129Crossref PubMed Scopus (239) Google Scholar; Vian et al., 2018Vian L. Pękowska A. Rao S.S.P. Kieffer-Kwon K.-R. Jung S. Baranello L. Huang S.-C. El Khattabi L. Dose M. Pruett N. et al.The Energetics and Physiological Impact of Cohesin Extrusion.Cell. 2018; 175: 292-294Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar; Wutz et al., 2017Wutz G. Várnai C. Nagasaka K. Cisneros D.A. Stocsits R.R. Tang W. Schoenfelder S. Jessberger G. Muhar M. Hossain M.J. et al.Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins.EMBO J. 2017; 36: 3573-3599Crossref PubMed Scopus (189) Google Scholar; Zuin et al., 2014Zuin J. Dixon J.R. van der Reijden M.I.J.A. Ye Z. Kolovos P. Brouwer R.W.W. van de Corput M.P.C. van de Werken H.J.G. Knoch T.A. van IJcken W.F.J. et al.Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells.Proc. Natl. Acad. Sci. USA. 2014; 111: 996-1001Crossref PubMed Google Scholar). Chromosomal domains such as TADs and INs constrain promoter-enhancer interactions (Bonev and Cavalli, 2016Bonev B. Cavalli G. Organization and function of the 3D genome.Nat. Rev. Genet. 2016; 17: 772Crossref PubMed Scopus (2) Google Scholar; Sun et al., 2019Sun F. Chronis C. Kronenberg M. Chen X.-F. Su T. Lay F.D. Plath K. Kurdistani S.K. Carey M.F. Promoter-Enhancer Communication Occurs Primarily within Insulated Neighborhoods.Mol. Cell. 2019; 73: 250-263.e5Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), albeit incompletely (Freire-Pritchett et al., 2017Freire-Pritchett P. Schoenfelder S. Várnai C. Wingett S.W. Cairns J. Collier A.J. García-Vílchez R. Furlan-Magaril M. Osborne C.S. Fraser P. et al.Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells.eLife. 2017; 6: e21926Crossref PubMed Scopus (45) Google Scholar; Javierre et al., 2016Javierre B.M. Burren O.S. Wilder S.P. Kreuzhuber R. Hill S.M. Sewitz S. Cairns J. Wingett S.W. Várnai C. Thiecke M.J. et al.BLUEPRINT ConsortiumLineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters.Cell. 2016; 167: 1369-1384.e19Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), and perturbations of their boundaries can lead to gene misregulation in development and in cancer (Lupiáñez et al., 2016Lupiáñez D.G. Spielmann M. Mundlos S. Breaking TADs: How Alterations of Chromatin Domains Result in Disease.Trends Genet. 2016; 32: 225-237Abstract Full Text Full Text PDF PubMed Google Scholar; Robson et al., 2019Robson M.I. Ringel A.R. Mundlos S. Regulatory Landscaping: How Enhancer-Promoter Communication Is Sculpted in 3D.Mol. Cell. 2019; 74: 1110-1122Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). However, genes sharing these domains may have different regulatory wiring and expression patterns (Hnisz et al., 2016Hnisz D. Day D.S. Young R.A. Insulated Neighborhoods: Structural and Functional Units of Mammalian Gene Control.Cell. 2016; 167: 1188-1200Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar; Schoenfelder and Fraser, 2019Schoenfelder S. Fraser P. Long-range enhancer-promoter contacts in gene expression control.Nat. Rev. Genet. 2019; 20: 437-455Crossref PubMed Scopus (124) Google Scholar). In addition, active enhancers might themselves function as TAD boundary elements (Barrington et al., 2019Barrington C. Georgopoulou D. Pezic D. Varsally W. Herrero J. Hadjur S. Enhancer accessibility and CTCF occupancy underlie asymmetric TAD architecture and cell type specific genome topology.Nat. Commun. 2019; 10: 2908Crossref PubMed Scopus (13) Google Scholar; Bonev et al., 2017Bonev B. Mendelson Cohen N. Szabo Q. Fritsch L. Papadopoulos G.L. Lubling Y. Xu X. Lv X. Hugnot J.-P. Tanay A. Cavalli G. Multiscale 3D Genome Rewiring during Mouse Neural Development.Cell. 2017; 171: 557-572.e24Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar), and contacts between superenhancers recover more rapidly upon reversal of cohesin depletion compared with other chromosomal loops (Rao et al., 2017Rao S.S.P. Huang S.-C. Glenn St Hilaire B. Engreitz J.M. Perez E.M. Kieffer-Kwon K.-R. Sanborn A.L. Johnstone S.E. Bascom G.D. Bochkov I.D. et al.Cohesin Loss Eliminates All Loop Domains.Cell. 2017; 171: 305-320.e24Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). These observations suggest that factors acting in cis to DNA regulatory elements are involved in establishing their chromosomal interactions. The interplay between higher order domains and specific regulatory chromosomal interactions such as those between enhancers and their target promoters is not fully understood. Consistent with a role for cohesin in shaping gene regulatory architecture, its depletion prevents adequate activation of inducible genes (Cuartero et al., 2018Cuartero S. Weiss F.D. Dharmalingam G. Guo Y. Ing-Simmons E. Masella S. Robles-Rebollo I. Xiao X. Wang Y.-F. Barozzi I. et al.Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation.Nat. Immunol. 2018; 19: 932-941Crossref PubMed Scopus (52) Google Scholar). Surprisingly however, steady-state gene expression levels are less affected upon architectural protein depletion (Busslinger et al., 2017Busslinger G.A. Stocsits R.R. van der Lelij P. Axelsson E. Tedeschi A. Galjart N. Peters J.-M. Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl.Nature. 2017; 544: 503-507Crossref PubMed Scopus (159) Google Scholar; Haarhuis et al., 2017Haarhuis J.H.I. van der Weide R.H. Blomen V.A. Yáñez-Cuna J.O. Amendola M. van Ruiten M.S. Krijger P.H.L. Teunissen H. Medema R.H. van Steensel B. et al.The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension.Cell. 2017; 169: 693-707.e14Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar; Nora et al., 2017Nora E.P. Goloborodko A. Valton A.-L. Gibcus J.H. Uebersohn A. Abdennur N. Dekker J. Mirny L.A. Bruneau B.G. Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.Cell. 2017; 169: 930-944.e22Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar; Rao et al., 2017Rao S.S.P. Huang S.-C. Glenn St Hilaire B. Engreitz J.M. Perez E.M. Kieffer-Kwon K.-R. Sanborn A.L. Johnstone S.E. Bascom G.D. Bochkov I.D. et al.Cohesin Loss Eliminates All Loop Domains.Cell. 2017; 171: 305-320.e24Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar; Remeseiro et al., 2012Remeseiro S. Cuadrado A. Gómez-López G. Pisano D.G. Losada A. A unique role of cohesin-SA1 in gene regulation and development.EMBO J. 2012; 31: 2090-2102Crossref PubMed Scopus (86) Google Scholar; Schwarzer et al., 2017Schwarzer W. Abdennur N. Goloborodko A. Pekowska A. Fudenberg G. Loe-Mie Y. Fonseca N.A. Huber W. Haering C.H. Mirny L. Spitz F. Two independent modes of chromatin organization revealed by cohesin removal.Nature. 2017; 551: 51-56Crossref PubMed Scopus (344) Google Scholar; Seitan et al., 2013Seitan V.C. Faure A.J. Zhan Y. McCord R.P. Lajoie B.R. Ing-Simmons E. Lenhard B. Giorgetti L. Heard E. Fisher A.G. et al.Cohesin-based chromatin interactions enable regulated gene expression within preexisting architectural compartments.Genome Res. 2013; 23: 2066-2077Crossref PubMed Scopus (199) Google Scholar; Sofueva et al., 2013Sofueva S. Yaffe E. Chan W.-C. Georgopoulou D. Vietri Rudan M. Mira-Bontenbal H. Pollard S.M. Schroth G.P. Tanay A. Hadjur S. Cohesin-mediated interactions organize chromosomal domain architecture.EMBO J. 2013; 32: 3119-3129Crossref PubMed Scopus (239) Google Scholar; Tedeschi et al., 2013Tedeschi A. Wutz G. Huet S. Jaritz M. Wuensche A. Schirghuber E. Davidson I.F. Tang W. Cisneros D.A. Bhaskara V. et al.Wapl is an essential regulator of chromatin structure and chromosome segregation.Nature. 2013; 501: 564-568Crossref PubMed Scopus (146) Google Scholar; Zuin et al., 2014Zuin J. Dixon J.R. van der Reijden M.I.J.A. Ye Z. Kolovos P. Brouwer R.W.W. van de Corput M.P.C. van de Werken H.J.G. Knoch T.A. van IJcken W.F.J. et al.Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells.Proc. Natl. Acad. Sci. USA. 2014; 111: 996-1001Crossref PubMed Google Scholar). This suggests that gene expression may be maintained by mechanisms independent of these architectural proteins, but whether this involves continued input from enhancers remains unclear. The effects of architectural protein depletion on 3D chromosomal architecture were typically analyzed using Hi-C. While this is a powerful method for global detection of chromosomal conformation (Lieberman-Aiden et al., 2009Lieberman-Aiden E. van Berkum N.L. Williams L. Imakaev M. Ragoczy T. Telling A. Amit I. Lajoie B.R. Sabo P.J. Dorschner M.O. et al.Comprehensive mapping of long-range interactions reveals folding principles of the human genome.Science. 2009; 326: 289-293Crossref PubMed Scopus (3578) Google Scholar), the complexity of Hi-C sequencing libraries limits the coverage and resolution of data obtained using this technology, making the robust analysis of specific enhancer-promoter interactions challenging. Combining Hi-C with sequence capture (Capture Hi-C) makes it possible to mitigate this limitation by selectively enriching Hi-C libraries for interactions involving, at least on one end, regions of interest such as gene promoters (Mifsud et al., 2015Mifsud B. Tavares-Cadete F. Young A.N. Sugar R. Schoenfelder S. Ferreira L. Wingett S.W. Andrews S. Grey W. Ewels P.A. et al.Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C.Nat. Genet. 2015; 47: 598-606Crossref PubMed Scopus (412) Google Scholar; Sahlén et al., 2015Sahlén P. Abdullayev I. Ramsköld D. Matskova L. Rilakovic N. Lötstedt B. Albert T.J. Lundeberg J. Sandberg R. Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution.Genome Biol. 2015; 16: 156Crossref PubMed Scopus (64) Google Scholar; Schoenfelder et al., 2015aSchoenfelder S. Furlan-Magaril M. Mifsud B. Tavares-Cadete F. Sugar R. Javierre B.-M. Nagano T. Katsman Y. Sakthidevi M. Wingett S.W. et al.The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.Genome Res. 2015; 25: 582-597Crossref PubMed Google Scholar). The fact that this approach does not depend on proteins bound to either interaction partner makes it particularly suitable for studying interactions where these proteins are either unknown or ectopically depleted. Here, we use Capture Hi-C to study the effects of architectural protein depletion on promoter interactions. We show that, while a majority of promoter interactions dissolve upon cohesin and CTCF depletion, large numbers of such contacts remain unaffected, and some are gained in these conditions. Interactions that are lost, gained, and maintained upon cohesin depletion have distinct properties with respect to localization within TADs, interaction distance, and the identity of associated proteins. We further demonstrate that changes in the levels of newly synthesized transcripts of specific genes upon cohesin depletion (as measured by SLAM sequencing [SLAM-seq]) associate with changes in the connectivity of their active enhancers. These results provide a mechanistic explanation for the limited but significant effects of cohesin and CTCF perturbations on gene expression and suggest the existence of alternative mechanisms supporting promoter-enhancer interactions. To study the effects of architectural protein depletion on promoter interactions, we took advantage of HeLa cells, in which all alleles of either cohesin subunit SCC1 or CTCF were tagged with a minimized auxin-inducible degron (AID) (Morawska and Ulrich, 2013Morawska M. Ulrich H.D. An expanded tool kit for the auxin-inducible degron system in budding yeast.Yeast. 2013; 30: 341-351Crossref PubMed Scopus (137) Google Scholar) and an mEGFP reporter (mEGFP-SCC1-AID and mEGFP-CTCF-AID cells, respectively). Additionally, these cells stably express Oryza sativa Tir1 protein required for proteasome targeting of AID-tagged proteins (Nishimura et al., 2009Nishimura K. Fukagawa T. Takisawa H. Kakimoto T. Kanemaki M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells.Nat. Methods. 2009; 6: 917-922Crossref PubMed Scopus (629) Google Scholar). We previously showed that SCC1 and CTCF are rapidly degraded in these cell lines within 20 min of auxin treatment (Wutz et al., 2017Wutz G. Várnai C. Nagasaka K. Cisneros D.A. Stocsits R.R. Tang W. Schoenfelder S. Jessberger G. Muhar M. Hossain M.J. et al.Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins.EMBO J. 2017; 36: 3573-3599Crossref PubMed Scopus (189) Google Scholar). We performed high-resolution Promoter Capture Hi-C (PCHi-C) in G1-synchronized, auxin-treated mEGFP-SCC1-AID and mEGFP-CTCF-AID cells and auxin-untreated controls. In addition, to compare the effects of depletion of these proteins with cell-cycle effects, we performed PCHi-C in intact HeLa cells synchronized in G1, G2, and mitosis. We also analyzed cells in which the cohesin release factor WAPL was depleted by RNAi (Tedeschi et al., 2013Tedeschi A. Wutz G. Huet S. Jaritz M. Wuensche A. Schirghuber E. Davidson I.F. Tang W. Cisneros D.A. Bhaskara V. et al.Wapl is an essential regulator of chromatin structure and chromosome segregation.Nature. 2013; 501: 564-568Crossref PubMed Scopus (146) Google Scholar; Wutz et al., 2017Wutz G. Várnai C. Nagasaka K. Cisneros D.A. Stocsits R.R. Tang W. Schoenfelder S. Jessberger G. Muhar M. Hossain M.J. et al.Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins.EMBO J. 2017; 36: 3573-3599Crossref PubMed Scopus (189) Google Scholar). We did not profile Tir1-expressing cells without AID-tagged proteins or the effects of auxin treatment alone, since previous studies had shown that these factors did not significantly contribute to the observed cohesin and CTCF depletion phenotypes (Nora et al., 2017Nora E.P. Goloborodko A. Valton A.-L. Gibcus J.H. Uebersohn A. Abdennur N. Dekker J. Mirny L.A. Bruneau B.G. Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.Cell. 2017; 169: 930-944.e22Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar; Rao et al., 2017Rao S.S.P. Huang S.-C. Glenn St Hilaire B. Engreitz J.M. Perez E.M. Kieffer-Kwon K.-R. Sanborn A.L. Johnstone S.E. Bascom G.D. Bochkov I.D. et al.Cohesin Loss Eliminates All Loop Domains.Cell. 2017; 171: 305-320.e24Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar; Wutz et al., 2017Wutz G. Várnai C. Nagasaka K. Cisneros D.A. Stocsits R.R. Tang W. Schoenfelder S. Jessberger G. Muhar M. Hossain M.J. et al.Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins.EMBO J. 2017; 36: 3573-3599Crossref PubMed Scopus (189) Google Scholar). Two biological replicates of each condition were sequenced, aligned, and filtered using the HiCUP pipeline (Wingett et al., 2015Wingett S. Ewels P. Furlan-Magaril M. Nagano T. Schoenfelder S. Fraser P. Andrews S. HiCUP: pipeline for mapping and processing Hi-C data.F1000Res. 2015; 4: 1310Crossref PubMed Scopus (150) Google Scholar) to a median coverage of ~95 M valid read pairs each, with the overall coverage across conditions of ~1.5 billion valid read pairs. Given the ~17-fold enrichment for interactions involving ~22,000 baited promoter fragments compared with conventional Hi-C, the combined coverage of promoter interactions in our dataset is equivalent to that achievable with ~25 billion Hi-C read pairs. Using the CHiCAGO pipeline (Cairns et al., 2016Cairns J. Freire-Pritchett P. Wingett S.W. Várnai C. Dimond A. Plagnol V. Zerbino D. Schoenfelder S. Javierre B.-M. Osborne C. et al.CHiCAGO: robust detection of DNA looping interactions in Capture Hi-C data.Genome Biol. 2016; 17: 127Crossref PubMed Google Scholar), we detected, on average, ~100,000 significant interactions between promoters and promoter-interacting regions (PIRs) in unperturbed cells (see Figures 1A and 6A for examples). The numbers of significant interactions were, however, markedly lower in architectural-protein-depleted cells compared with controls, with a particularly strong effect for cohesin (118,074 SCC1 control and 61,702 SCC1 depleted, respectively; Figure S1). Clustering of interactions based on CHiCAGO scores identified 13 coherent clusters denoted A to M (Figure 1B; see STAR Methods for details). Clusters B, C, and F were characterized by loss of the interaction signal upon cohesin and CTCF depletion, while interactions in cluster D were sensitive to degradation of cohesin but not CTCF. Notably, all of these four clusters also showed loss of promoter interaction signal in mitosis, in which the majority of these proteins are thought to be released from chromosomes. In addition, promoter interactions in these clusters appeared generally stronger in unmodified HeLa cells compared with AID-modified, but auxin-untreated, cells (“control” in Figure 1B). This is presumably due to the baseline residual activity of the degron system toward AID-tagged proteins without the auxin treatment reported previously (Wutz et al., 2017Wutz G. Várnai C. Nagasaka K. Cisneros D.A. Stocsits R.R. Tang W. Schoenfelder S. Jessberger G. Muhar M. Hossain M.J. et al.Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins.EMBO J. 2017; 36: 3573-3599Crossref PubMed Scopus (189) Google Scholar). Promoter interactions in clusters A, E, and J were maintained upon cohesin and CTCF depletion (Figure 1B). Some of these interactions showed sensitivity to WAPL depletion and were lost in mitosis (cluster A), while others were generally retained in all analyzed conditions (clusters E and J). Finally, distinct subsets of promoter interactions were gained depending on the depleted protein (SCC1, cluster K; CTCF, cluster G). Jointly, these results point to both the shared and specific effects of cohesin and CTCF depletion on promoter wiring. In addition, they suggest that large numbers of promoter interactions are retained in these conditions, and these interactions appear generally stable throughout interphase. Guided by the exploratory observations from cluster analysis, we next sought to define subsets of promoter interactions that are lost, maintained, or gained upon cohesin and CTCF" @default.
• W3044693806 created "2020-07-29" @default.
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• W3044693806 date "2020-07-01" @default.
• W3044693806 modified "2023-10-12" @default.
• W3044693806 title "Cohesin-Dependent and -Independent Mechanisms Mediate Chromosomal Contacts between Promoters and Enhancers" @default.
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