Article7 December 2017free access Source DataTransparent process Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins Gordana Wutz Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Csilla Várnai orcid.org/0000-0003-0048-9507 Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK Search for more papers by this author Kota Nagasaka Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author David A Cisneros Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Roman R Stocsits Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Wen Tang Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Stefan Schoenfelder Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK Search for more papers by this author Gregor Jessberger Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Matthias Muhar Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author M Julius Hossain orcid.org/0000-0003-3303-5755 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Nike Walther Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Birgit Koch Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Moritz Kueblbeck Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Jan Ellenberg Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Johannes Zuber Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Peter Fraser Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK Department of Biological Science, Florida State University, Tallahassee, FL, USA Search for more papers by this author Jan-Michael Peters Corresponding Author [email protected] orcid.org/0000-0003-2820-3195 Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Gordana Wutz Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Csilla Várnai orcid.org/0000-0003-0048-9507 Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK Search for more papers by this author Kota Nagasaka Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author David A Cisneros Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Roman R Stocsits Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Wen Tang Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Stefan Schoenfelder Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK Search for more papers by this author Gregor Jessberger Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Matthias Muhar Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author M Julius Hossain orcid.org/0000-0003-3303-5755 Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Nike Walther Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Birgit Koch Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Moritz Kueblbeck Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Jan Ellenberg Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Johannes Zuber Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Peter Fraser Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK Department of Biological Science, Florida State University, Tallahassee, FL, USA Search for more papers by this author Jan-Michael Peters Corresponding Author [email protected] orcid.org/0000-0003-2820-3195 Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Author Information Gordana Wutz1,‡, Csilla Várnai2,‡, Kota Nagasaka1,‡, David A Cisneros1,†,‡, Roman R Stocsits1, Wen Tang1, Stefan Schoenfelder2, Gregor Jessberger1, Matthias Muhar1, M Julius Hossain3, Nike Walther3, Birgit Koch3, Moritz Kueblbeck3, Jan Ellenberg3, Johannes Zuber1, Peter Fraser2,4 and Jan-Michael Peters *,1 1Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria 2Nuclear Dynamics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK 3Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany 4Department of Biological Science, Florida State University, Tallahassee, FL, USA †Present address: The Laboratory for Molecular Infection Medicine Sweden (MIMS) and Department of Molecular Biology, Umeå University, Umeå, Sweden ‡These authors contributed equally to this work *Corresponding author. Tel: +43 1797303000; E-mail: [email protected] EMBO J (2017)36:3573-3599https://doi.org/10.15252/embj.201798004 See also: J Gassler et al (December 2017) and JHI Haarhuis & BD Rowland (December 2017) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL. Synopsis Spatial organization of mammalian genomes facilitates gene regulation and replication. Hi-C data reveal that cohesin binding at CTCF sites controls long-range chromatin interactions, supporting a model in which chromatin loop extrusion drives genome 3D architecture. Cohesin suppresses compartments but is required for TADs and loops. CTCF and PDS5 proteins define TAD boundaries and loop anchors. WAPL and PDS5 proteins define the length of loops. Cohesin appears to form long-range contacts by loop extrusion. Introduction Duplicated DNA molecules become physically connected with each other during DNA replication. This sister chromatid cohesion is essential for bi-orientation of chromosomes on the mitotic or meiotic spindle and thus enables their symmetrical segregation during cell division (Dewar et al, 2004). Cohesion is mediated by cohesin complexes (Guacci et al, 1997; Michaelis et al, 1997; Losada et al, 1998) which are thought to perform this function by entrapping both sister DNA molecules inside a ring structure that is formed by the cohesin subunits SMC1, SMC3, and SCC1 (also known as RAD21 and Mcd1) (Haering et al, 2008). Cohesin is present at centromeres and on chromosome arms (reviewed in Peters et al, 2008). At centromeres, cohesin resists the pulling force of spindle microtubules, a function that is required both for stabilization of microtubule–kinetochore attachments and for chromosome bi-orientation. On chromosome arms, however, the precise location of cohesin would not be expected to matter if cohesin's only function was to mediate cohesion. But contrary to this expectation, cohesin is enriched at thousands of well-defined loci on chromosome arms. In mammalian genomes, ~90% of these are defined by binding sites for CCCTC binding factor (CTCF) (Parelho et al, 2008; Wendt et al, 2008). CTCF is a zinc-finger protein that has been implicated in various aspects of gene regulation, such as insulating gene promoters from distant enhancers (reviewed in Wendt & Peters, 2009). Early work revealed that this enhancer-blocking activity is required for imprinted gene expression at the H19-IGF2 locus (Bell & Felsenfeld, 2000; Hark et al, 2000) and indicated that CTCF mediates this function by creating allele-specific chromatin loops (Kurukuti et al, 2006; Splinter et al, 2006). Remarkably, cohesin is also required for CTCF's enhancer blocking activity at the H19-IGF2 locus (Wendt et al, 2008) and the chicken β-globin locus (Parelho et al, 2008), as well as for regulation of other genes reviewed in (Wendt & Peters, 2009; Dorsett & Merkenschlager, 2013). Importantly, these requirements have been identified in G1-phase of the cell cycle and in post-mitotic cells, in which cohesin is also present, indicating that they are not an indirect effect of cohesin's role in sister chromatid cohesion but reflect an independent function of cohesin (Pauli et al, 2008; Wendt et al, 2008). Because cohesin is required for CTCF-dependent gene regulation events that are thought to be mediated by chromatin looping, and because cohesin is well known to be able to physically connect distinct DNA elements to generate cohesion, it has been proposed that cohesin can also generate or stabilize chromatin loops (Wendt et al, 2008; Wendt & Peters, 2009). According to this hypothesis, cohesin would not only be able to connect two sister DNA molecules in trans to generate cohesion, but would also be able to connect regions on the same chromatid in cis. This hypothesis has been supported by chromatin conformation capture (3C) experiments which indicated that long-range chromosomal cis-interactions at the apolipoprotein gene cluster, the γ-interferon, and H19-IGF2 loci (Hadjur et al, 2009; Mishiro et al, 2009; Nativio et al, 2009) as well as enhancer–promoter interactions (Kagey et al, 2010) are dependent on cohesin. Evidence for a role of cohesin in chromatin structure has also come from experiments in which the cohesin-associated protein WAPL was depleted. WAPL can release cohesin from DNA (Gandhi et al, 2006; Kueng et al, 2006; Tedeschi et al, 2013), presumably by opening the cohesin ring (Kueng et al, 2006; Chan et al, 2012; Huis in ‘t Veld et al, 2014), and WAPL depletion, therefore, increases residence time and amounts of cohesin on DNA (Kueng et al, 2006; Tedeschi et al, 2013). Remarkably, this causes mild but global compaction of chromatin (Tedeschi et al, 2013), indicating that the effects of cohesin on chromatin architecture are widespread and not confined to a few loci. Unexpectedly, WAPL depletion also causes a dramatic intra-nuclear re-localization of cohesin. Cohesin is normally detectable in most regions of interphase chromatin (Losada et al, 1998; Sumara et al, 2000), but after WAPL depletion, it accumulates in axial structures called “vermicelli” which are thought to extend along the entire length of interphase chromosome territories (Tedeschi et al, 2013). Vermicelli are reminiscent of axial elements in mitotic and meiotic chromosomes. These structures contain condensin and meiosis-specific cohesin complexes, respectively, and are thought to form the base of chromatin loops, into which chromatin fibers are organized during chromosome condensation (Earnshaw & Laemmli, 1983; Saitoh et al, 1994; Klein et al, 1999; Blat et al, 2002; Ono et al, 2003; Yeong et al, 2003). It has, therefore, been proposed that vermicelli represent cohesin complexes that are located at the base of loops in interphase chromatin, and that vermicelli become detectable only after WAPL depletion because increased residence time and amounts of cohesin lead to the formation or stabilization of more chromatin loops than normally (Tedeschi et al, 2013). Interestingly, both chromatin compaction and accumulation of cohesin axial structures are predicted to occur in WAPL-depleted cells if one assumes that cohesin forms chromatin loops via a hypothetical loop extrusion process (Fudenberg et al, 2016). According to this idea, two distal chromosomal elements would be brought into direct proximity by a loop extrusion factor such as cohesin which would processively extrude a chromatin loop (Riggs, 1990; Nasmyth, 2001; Alipour & Marko, 2012; Gruber, 2014; Sanborn et al, 2015; Fudenberg et al, 2016). Since WAPL depletion increases cohesin's residence time on DNA, cohesin would extrude longer loops and cohesin complexes would come into closer proximity than normally, resulting in chromatin compaction and vermicelli formation, respectively. In silico modeling of DNA folding has confirmed these predictions (Fudenberg et al, 2016). Importantly, the loop extrusion hypothesis can also explain the “CTCF convergence rule” (Rao et al, 2014; Vietri Rudan et al, 2015; de Wit et al, 2015). This describes the unexpected phenomenon that CTCF binding sites that form the base of chromatin loops are typically oriented toward each other. The DNA sequences with which CTCF can associate are not palindromic (symmetrical), that is, could be positioned relative toward each other in convergent, tandem, or divergent orientations. If CTCF sites formed loops by random association, they would be expected to occur in convergent, tandem, and divergent orientations with frequencies of 25, 50, and 25%, respectively. However, high-resolution genome-wide chromatin conformation capture experiments coupled with DNA sequencing (“Hi-C”) (Lieberman-Aiden et al, 2009) have revealed that most chromatin loops contain convergent CTCF sites (Rao et al, 2014; Vietri Rudan et al, 2015; de Wit et al, 2015). This would be expected if these sites were brought into proximity by loop extrusion, but cannot be explained if these sites found each other by random diffusion (Sanborn et al, 2015; Fudenberg et al, 2016). Based on these observations and considerations, it has been proposed that CTCF sites function as directional boundary elements during loop extrusion (Sanborn et al, 2015; Fudenberg et al, 2016). However, for cohesin to be able to form extended loops in WAPL-depleted cells, as proposed by Fudenberg et al (2016), these boundaries would have to be permeable to some extent. The notion that CTCF sites can function as boundary elements for loop extrusion is consistent with the observation that cohesin and CTCF co-localize (Parelho et al, 2008; Wendt et al, 2008), and that many of these sites either form the base of chromatin loops (Rao et al, 2014), or are enriched at the boundaries of more complex chromatin structures, called topologically associating domains (TADs) (Dixon et al, 2012; Nora et al, 2012). These are covering chromosomal regions, typically up to 1 Mb in length, in which long-range chromosomal cis-interactions occur with increased frequencies. At the level of individual cells, TADs may represent loops that are in the process of being extruded to various degrees. In support of this idea, it has been proposed based on single-nucleus Hi-C experiments that TADs emerge from averaging interactions in large cell populations (Flyamer et al, 2017). Importantly, genome-editing experiments in which CTCF sites were deleted or inverted indicate that these are indeed required for the formation of TAD boundaries (Nora et al, 2012; Narendra et al, 2015; Sanborn et al, 2015; de Wit et al, 2015). The hypothesis that cohesin contributes to the formation of TADs and loops by mediating loop extrusion is also consistent with the observation that cohesin can move along DNA, both in vitro (Davidson et al, 2016; Kanke et al, 2016; Stigler et al, 2016) and in vivo (Lengronne & Pasero, 2014; Busslinger et al, 2017) and is constrained during these movements by CTCF (Davidson et al, 2016; Busslinger et al, 2017). In contrast, cohesin and CTCF are not thought to contribute to chromatin organization at levels above TADs and loops (i.e., covering larger genomic regions), at which Hi-C experiments have identified higher-order structures called A and B compartments (Lieberman-Aiden et al, 2009). Both of these exist in different sub-types (Rao et al, 2014; Nagano et al, 2017) and are thought to represent euchromatic and heterochromatic regions, respectively. However, even though the above-mentioned observations indicate that cohesin and CTCF may have important roles in the formation of TADs and loops, previous Hi-C experiments found that inactivation of cohesin or CTCF had only modest effects on chromatin organization (Seitan et al, 2013; Sofueva et al, 2013; Zuin et al, 2014). It is, therefore, incompletely understood whether cohesin and CTCF have important roles in chromatin organization, and if so, whether these are mediated by loop extrusion. We have, therefore, further analyzed the roles of cohesin and CTCF in chromatin organization by using microscopic imaging and Hi-C experiments. For this purpose, we have either inactivated cohesin or CTCF by conditional proteolysis (Nishimura et al, 2009), or stabilized cohesin on DNA by depleting WAPL and its binding partners PDS5A and PDS5B (Kueng et al, 2006). Our experiments indicate that cohesin is required for the formation and/or maintenance of TADs and loops but suppresses compartments, that WAPL and PDS5 proteins antagonize these functions, and that CTCF is required for the formation of “sharp” boundaries between TADs and the formation of loops by defined loop anchors. We also show that co-depletion of WAPL, PDS5A, and PDS5B enables cohesin to condense chromosomes to an extent that is normally only seen in early mitosis by creating long-range chromosomal cis-interactions that are reminiscent of the ones observed in mitotic chromosomes (Naumova et al, 2013; Nagano et al, 2017). These observations are consistent with the loop extrusion hypothesis. Unexpectedly, however, our results indicate that PDS5 proteins, which are thought to cooperate with WAPL in releasing cohesin from DNA (Rowland et al, 2009; Shintomi & Hirano, 2009; Ouyang et al, 2016), have a function in chromatin loop formation that is distinct from the role of WAPL and that may be required for CTCF's ability to function as a boundary element for loop extrusion. Cohesin, CTCF and WAPL, but not PDS5 proteins, have also been inactivated in other recent Hi-C studies which were deposited on bioRxiv (Kubo et al, 2017) or published in peer-reviewed journals during preparation or after submission of our manuscript (Gassler et al, 2017; Haarhuis et al, 2017; Rao et al, 2017; Schwarzer et al, 2017). We compare and contrast the results of these as well as the earlier studies (Seitan et al, 2013; Sofueva et al, 2013; Zuin et al, 2014) with ours in the Discussion. Results Acute cohesin inactivation by auxin-mediated degradation To analyze cohesin's role in chromatin structure, we modified all SCC1 alleles in HeLa cells by CRISPR-Cas9-mediated genome editing to encode proteins in which SCC1 is C-terminally fused to monomeric enhanced green fluorescent protein (mEGFP) and an auxin-inducible degron (AID; Fig 1A). To enable auxin-dependent recognition of SCC1-mEGFP-AID by SCF ubiquitin ligases, we stably expressed the Oryza sativa F-box transport inhibitor response-1 auxin receptor protein (Tir1) in these cells. In immunoblotting experiments, only SCC1-mEGFP-AID but no untagged SCC1 could be detected, confirming that all SCC1 alleles had been modified (Fig 1B). These experiments also revealed that mEGFP-AID tagging reduced SCC1 levels (Fig 1B), consistent with the finding that C-terminal tagging compromises SCC1 function in mice (Tachibana-Konwalski et al, 2010). Cells expressing only SCC1-mEGFP-AID nevertheless proliferated at similar rates as wild-type cells, indicating that cohesin complexes containing this fusion protein can perform their essential cellular functions and are present in copy numbers sufficient for this. Figure 1. Chromatin organization changes upon auxin-induced SCC1 degradation Genotype analysis of parental HeLa cells (WT), homozygous SCC1-mEGFP cells (GFP), and homozygous SCC1-mEGFP-AID cells (GFP-AID). Genomic PCR products were generated with the primers that are designed external to the homology arm which was used for inserting mEGFP or mEGFP-AID encoding sequences downstream of the SCC1 gene. This resulted in fusion proteins with mEGFP or mEGFP-AID tags C-terminal to the SCC1 gene. Immunoblotting analysis of whole-cell extracts from parental HeLa WT cells, SCC1-mEGFP cells, SCC1-mEGFP-AID (−) cells (i.e., not expressing Tir1), and SCC1-mEGFP-AID cells expressing Tir1 (+). α-Tubulin: loading control. Time course live-cell imaging of SCC1-mEGFP-AID cells after auxin treatment. SCC1-mEGFP-AID cells with (+) or without (−) Tir1 were imaged after addition of auxin. Scale bar indicates 20 μm. Quantification of nuclear GFP signal over time after auxin addition to SCC1-mEGFP-AID cells with (+) or without (−) Tir1. Normalized nuclear GFP signals are plotted over time after addition of Auxin into −Tir1 and +Tir1 cells (mean ± SD). n = 9 cells per condition. Chromatin fractionation and immunoblot analysis of auxin-treated SCC1-mEGFP-AID cells expressing Tir1. At the indicated time points after auxin addition, whole-cell extracts, the chromatin pellet fraction, and the supernatant fraction were analyzed by immunoblotting, using antibodies against the proteins indicated on the left. Intra-chromosomal contact frequency distribution as a function of genomic distance, at 0 (black), 15 (blue), and 180 min (cyan) after auxin addition to SCC1-mEGFP-AID cells expressing Tir1. Coverage-corrected Hi-C contact matrices of chromosome 4, at 0 (left), 15 (center), and 180 min (right) after auxin addition to SCC1-mEGFP-AID cells expressing Tir1. The corresponding compartment signal tracks at 250 kb bin resolution are shown above the matrices. The matrices were plotted using Juicebox. Long-range (> 2 Mb) intra-chromosomal contact enrichment between bins with varying compartment signal strength from most B-like (1) to most A-like (50). Average contact enrichment around loops after auxin addition to SCC1-mEGFP-AID cells expressing Tir1, for the 82 × 600 kb long loops identified in G1 control HeLa cells. The matrices are centered (0) around the halfway point of the loop anchor coordinates. For the same conditions as in (G–I), coverage-corrected Hi-C contact matrices in the 88–94.5 Mb region of chromosome 12, plotted using Juicebox. Source data are available online for this figure. Source Data for Figure 1 [embj201798004-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Auxin addition reduced GFP fluorescence intensity to “background” levels in 20 min in cells expressing Tir1, but not in cells lacking Tir1 (Fig 1C and D). Immunoblotting experiments confirmed that loss of fluorescence intensity was caused by SCC1-mEGFP-AID degradation and showed that it resulted in concomitant release of the cohesin subunits SMC1 and SMC3 from chromatin, whereas CTCF and the cohesin loading complex NIPBL-MAU2 remained chromatin-associated (Figs 1E and EV1A). Click here to expand this figure. Figure EV1. Chromatin organization changes upon auxin-induced Scc1 degradation for 15 and 180 min Same chromatin fractionation experiment as shown in Fig 1E. Note that the amount of cohesin loading complex, NIPBL, and MAU2, on chromatin were largely unchanged after SCC1 degradation. Aggregate TAD analysis for SCC1-mEGFP-AID cells expressing Tir1, at 0 (left), 15 (center), and 180 min (right) after auxin addition. Average coverage-corrected Hi-C contact matrices are shown centered around the 166 × 500–550 kb long TADs identified in the control-depleted G1 cells. Average insulation score around TAD boundaries identified for the control-depleted G1 cells, at 0 (black), 15 (blue), and 180 min (cyan) after auxin addition. Dashed lines show the average insulation score around the +1 Mb shifted boundaries as control. Number of loops identified by HiCCUPS, at 0, 15, and 180 min after auxin addition. Colors are the same as in (C). Total contact counts around loops after auxin addition, for all 750 kb–6 Mb long loops identified by HiCCUPS in G1 control. Source data are available online for this figure. Download figure Download PowerPoint Cohesin inactivation strengthens compartments but weakens TADs and loops To analyze the effects of cohesin inactivation on chromatin structure, we synchronized SCC1-mEGFP-AID cells in G1-phase by double-thymidine arrest–release, treated them for 0, 15, or 180 min with auxin, and measured chromatin interactions genome-wide by Hi-C (Fig 1F–J). In a separate experiment, we analyzed the same cells after 0 and 120 min of auxin treatment (Fig EV2; all Hi-C libraries analyzed in this study and their properties are listed in Table EV1). All genome-wide interaction matrices were analyzed for the presence of five types of patterns which have been observed in Hi-C data from mammalian genomes: cis-trans interaction ratios, distance-dependent interaction frequencies at the genomic level, compartments, TADs, and point interactions (“loops”) at specific genomic positions (Lajoie et al, 2015). Importantly, none of these patterns differed significantly between Hi-C matrices obtained from cells collected at the 0 min time points of the two experiments, indicating that changes observed in auxin-treated cells were caused by cohesin degradation and not inter-experimental variation. Click here to expand this figure. Figure EV2. Chromatin organization changes upon auxin-induced Scc1 degradation for 15 and 120 min Coverage-corrected Hi-C contact matrices of chromosome 4, at 0 and 120 min after auxin addition in SCC1-mEGFP-AID cells. The corresponding compartment signal tracks at 250 kb bin resolution are shown above the matrices. The matrices were plotted using Juicebox. For the same conditions, coverage-corrected Hi-C contact matrices in the 89–97-Mb region of chromosome 12, plotted by using Juicebox. Inter-chromosomal contact enrichment between 250 kb bins with varying compartment strength from most B-like (1) to most A-like (50), in SCC1-mEGFP-AID control cells in a replicate experiment, at 0 and 120 min after auxin addition. Aggregate TAD analysis for CTCF-mEGFP-AID cells expressing Tir1, 0 (left) and 120 min (right) after auxin addition. Average coverage-corrected Hi-C contact matrices are shown centered around the 166 × 500–550 kb long TADs identified in the control-depleted HeLa cells. Average contact enrichment around loops after auxin addition in SCC1-mEGFP-AID cells, for the 82 × 600 kb long loops identified by HiCCUPS in G1 control. The matrices are centered (0) around the halfway point of the loop anchor coordinates. Total contact counts around loops after auxin addition in SCC1-mEGFP-AID cells, for all 750 kb–6 Mb long loops identified by HiCCUPS in G1 control. Download figure Download PowerPoint Cis-trans interaction ratios were high in all cases, with inter-chromosomal interactions only representing 5–15%, indicating that all Hi-C libraries were of high quality. However, trans interactions were consistently higher in cohesin-depleted cells (Table EV1). Contact frequencies were affected differently by cohesin inactivation, depending on genomic distances between contacting loci. Cohesin depletion gradually decreased contact frequencies at 100 kb–1 Mb distances, while the frequency of long-range (> 10 Mb) interactions increased (Fig 1F). Cohesin inactivation might therefore enable more contacts with distant parts of chromosomes and even neighboring chromosomes, possibly because chromatin structure becomes more flexible under these conditions. For reasons explained in the next paragraph, we do not believe that these changes are caused by technical artifacts, such as increased “noise” in the Hi-C libr
