Genome folding by loop extrusion and compartmentalization
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This document discusses the mechanisms of genome folding, particularly focusing on loop extrusion and compartmentalization that influence chromosomal organization and gene expression. It presents evidence for the dynamic structure of chromosomal domains, emphasizing the role of the cohesin complex in mediating loop extrusion and the insulation of topologically associated domains (TADs). The findings highlight the functional significance of chromatin loop size and the factors determining chromatin compartmentalization independent of local TAD architecture.
![Modeling of compartmentalization
Comp.Stength = Sumi [XXi/toti]
Martin Falk
MIT
phase separation
in a block copolymer](https://image.slidesharecdn.com/loopextrusionquizextrusionceesinvertedv2-180330151805/85/Genome-folding-by-loop-extrusion-and-compartmentalization-36-320.jpg)
Genome folding by loop extrusion and compartmentalization
- 1. Leonid Mirny ! leonid@mit.edu Genome folding by loop extrusion and compartmentalization !
- 2. Inac%ve Ac%ve A/B Compartments 0.1-1 Mb Topological Associa%on Domains (TADs) Nora et al. Nature 2012; Dixon et al Nature 2012 2-3 fold Multiple levels of organization: compartments and domains Chr4: Mb Rao et al. Cell 2014 active inactive Irina Solovei, LMU
- 3. HiGlass.io by Nils Gehlenborg, Peter Karpedjiev, Nezar Abdennur, and others Harvard University and MIT HiGlass: Web-based Visual Exploration and Analysis of Genome Interaction Maps https://www.biorxiv.org/content/early/2017/10/30/121889
- 4. Outline Compartments and TADs 1. Emerging evidence of loop extrusion! 2. Compartmentalization by phase separation 3. Phase separation vs extrusion Emerging Evidence of Chromosome Folding by Loop Extrusion https://www.biorxiv.org/content/early/2018/02/16/264648
- 5. Model 36 domains=10MbC D Loop extrusion with boundaries => TADs e Formation of Chromosomal Domains by Loop Extrusion! bioRxiv Aug 14 (2015)! Fudenberg, Imakaev et al.! DOI: 10.1101/024620 Article Formation of Chromosomal Domains by Loop (2016)
- 6. Model 36 domains=10MbC D Loop extrusion with boundaries => TADs genomic distance, s (bp) 104 105 1 normalized C 10-2 D e Formation of Chromosomal Domains by Loop Extrusion! bioRxiv Aug 14 (2015)! Fudenberg, Imakaev et al.! DOI: 10.1101/024620 Article Formation of Chromosomal Domains by Loop (2016)
- 7. contact frequency 46Mb 50Mb 48Mb C i ii iii iv iv iviv chr5, NPC (Bonev et al., 2017) How does loop extrusion make TADs/flames/peaks etc? corner peakflame (track)insulation peak gridi ii iii iv C Scanning! <10% <10%
- 8. b 00.0100.0200.035 ContactProbability(simulations) = 13 5’-GGCGGAGACCACAAGGTGGCGCCAGATCCC-3’ 17.417.6 1 kb resolution CTCF RAD21 SMC3 Chr 1 Chr1 17.6 Mb17.4 0 0.5 1 1.5 2 2.5 3 3.5 4 Number of PeaksD Forwardmotif FoldChange 0 0.5 1.0 1.5 0% 20% 40% 60% 80% 100% Percentage of peak loci bound YY1 CTCF RAD21 (2%)(3%)(3%)(92%) CCACNAGGTGGCAGconsensus x 1000 CTCF anchor (arrowhead indicates motif orientation) Loop domain Ordinary domain 290 Kb 110 Kb 190 Kb 350 Kb 270 Kb 130 Kb 450 Kb 170 Kb F Figure 6. Many Loops Demarcate Contact Domains; The Vast Majority of Loops Are Anchored at a Pair of Convergent CTCF/RAD21/SMC3 Binding Sites (A) Histograms of corner scores for peak pixels versus random pixels with an identical distance distribution. (B) Contact matrix for chr4:20.55 Mb–22.55 Mb in GM12878, showing examples of transitive and intransitive looping behavior. (C) Percent of peak loci bound versus fold enrichment for 76 DNA-binding proteins. (D) The pairs of CTCF motifs that anchor a loop are nearly all found in the convergent orientation. (legend continued on next page) 1674 Cell 159, 1665–1680, December 18, 2014 ª2014 Elsevier Inc. Border-to-border loops cannot reproduce Hi-C data
- 9. b 00.0100.0200.035 ContactProbability(simulations) = 13 5’-GGCGGAGACCACAAGGTGGCGCCAGATCCC-3’ 17.417.6 1 kb resolution CTCF RAD21 SMC3 Chr 1 Chr1 17.6 Mb17.4 0 0.5 1 1.5 2 2.5 3 3.5 4 Number of PeaksD Forwardmotif FoldChange 0 0.5 1.0 1.5 0% 20% 40% 60% 80% 100% Percentage of peak loci bound YY1 CTCF RAD21 (2%)(3%)(3%)(92%) CCACNAGGTGGCAGconsensus x 1000 CTCF anchor (arrowhead indicates motif orientation) Loop domain Ordinary domain 290 Kb 110 Kb 190 Kb 350 Kb 270 Kb 130 Kb 450 Kb 170 Kb F Figure 6. Many Loops Demarcate Contact Domains; The Vast Majority of Loops Are Anchored at a Pair of Convergent CTCF/RAD21/SMC3 Binding Sites (A) Histograms of corner scores for peak pixels versus random pixels with an identical distance distribution. (B) Contact matrix for chr4:20.55 Mb–22.55 Mb in GM12878, showing examples of transitive and intransitive looping behavior. (C) Percent of peak loci bound versus fold enrichment for 76 DNA-binding proteins. (D) The pairs of CTCF motifs that anchor a loop are nearly all found in the convergent orientation. (legend continued on next page) 1674 Cell 159, 1665–1680, December 18, 2014 ª2014 Elsevier Inc. TAD ≠ border-to-border loop Border-to-border loops cannot reproduce Hi-C data
- 10. Domain — systems of actively extruded loops youtube mirnylab http://mirnylab.mit.edu/projects/emerging-evidence-for-loop-extrusion/ https://www.youtube.com/watch?v=8FW6gOx5lPI
- 11. increased LEF density & processivity WT LEF depletion weakened barriers genomic separation s, bp WT 10 0 10 1 P(s) 10 2 10 5 10 6 genomic separation s, bp 10 0 10 1 10 2 10 5 10 6 genomic separation s, bp 10 0 10 1 10 2 10 5 10 6 genomic separation s, bp 10 0 10 1 10 2 10 5 10 6 0 0 0 300kb A B C D Testing loop extrusion ! Predictions
- 12. Testing loop extrusion ! Experiments control 46Mb 51Mb 46Mb 51Mb 19Mb 23Mb1 Mb 1 Mb 1 Mb chr5 chr5 chr11 contactfrequency 0.008 0 Elphege Nora Benoit G. Bruneau UCSF Francois Spitz! Wibke Schwarzer! ! Article The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension Judith H.I. Haarhuis,1,6 Robin H. van der Weide,2,6 Vincent A. Blomen,3 J. Omar Ya´ n˜ ez-Cuna,2 Mario Amendola,2,7 Marjon S. van Ruiten,1 Peter H.L. Krijger,4 Hans Teunissen,2 Rene´ H. Medema,1 Bas van Steensel,2 Thijn R. Brummelkamp,3,5 Elzo de Wit,2,* and Benjamin D. Rowland1,8,* 1Division of Cell Biology, the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands 2Division of Gene Regulation, the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands 3Division of Biochemistry, the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands 4Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands 5Cancer Genomics Center, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands 6These authors contributed equally 7Present address: UMR_S951, Genethon, 91000 Evry, France 8Lead Contact *Correspondence: e.d.wit@nki.nl (E.d.W.), b.rowland@nki.nl (B.D.R.) http://dx.doi.org/10.1016/j.cell.2017.04.013 SUMMARY The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin’s DNA release factor the SCC2/SCC4 complex (also known as NIPBL and respectively), and DNA release is driven by cohesin’s anta WAPL (Ciosk et al., 2000; Gandhi et al., 2006; Kueng et al., The cohesin complex consists of three core subunits, SMC3, and SCC1 (also known as RAD21 or Mcd1), that to form a ring-shaped structure that can entrap DNA ins lumen (Haering et al., 2008). WAPL drives cohesin’s r from chromatin by opening up a distinct DNA exit gate interface connecting cohesin’s SMC3 and SCC1 subunits oue¨ t et al., 2016; Murayama and Uhlmann, 2015). In the ab ARTICLE doi:10.1038/nature24281 Two independent modes of chromatin organization revealed by cohesin removal Wibke Schwarzer1 *, Nezar Abdennur2 *, Anton Goloborodko3 *, Aleksandra Pekowska4 , Geoffrey Fudenberg5 , Yann Loe-Mie6,7 , Nuno A Fonseca8 , Wolfgang Huber4 , Christian H. Haering9 , Leonid Mirny3,5 & Francois Spitz1,4,6,7 Imaging and chromosome conformation capture studies have revealed several layers of chromosome organization, Article Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization Elphe` ge P. Nora,1,2,* Anton Goloborodko,3 Anne-Laure Valton,4 Johan H. Gibcus,4 Alec Uebersohn,1,2,7 Nezar Abdennur,3 Job Dekker,4 Leonid A. Mirny,3 and Benoit G. Bruneau1,2,5,6,8,* 1Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA 2Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA 94158, USA 3Institute for Medical Engineering and Science and Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 4Howard Hughes Medical Institute, Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605-0103, USA 5 regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF rein- states proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromo- somes emerges independently of proper insulation of TADs. Furthermore, our data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to het- erochromatin spreading. Beyond defining the func- tions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization. INTRODUCTION Chromosomes meet the dual challenge of packaging DNA into the nucleus and, at the same time, enabling access to genetic in- formation. Decades of work on chromosome organization have tackled the link between chromosome structure and genetic Potts et al., 20 but here we foc Mammalian c Euchromatin c rich regions (G is condensed, g highlights the ical, biochemic somes. Chromo belonging to tw vealed by high- (3C), with chro loci of the same chromosomes ( on linear genom types forms a d with regional c 2013; Bonev an ment contains B compartmen lamina-associat et al., 2015), wh At a more loc megabase segm relatively insulat cally associatin et al., 2012). Th by the binding et al., 2012; Phi zinc-finger nucl 930 Cell 169, 930–944, May 18, 2017 ª 2017 Elsevier Inc.
- 13. Testing loop extrusion ! Experiments control 46Mb 51Mb 46Mb 51Mb 19Mb 23Mb1 Mb 1 Mb 1 Mb chr5 chr5 chr11 contactfrequency 0.008 0
- 14. Testing loop extrusion ! Experiments control 46Mb 51Mb 46Mb 51Mb 19Mb 23Mb1 Mb 1 Mb 1 Mb chr5 chr5 chr11 contactfrequency 0.008 0 perturbation 100 10 1 10 2 10 3 10 4 104 105 106 107 genomic separation s, bp 100 10 1 10 2 10 3 10 4 104 105 106 107 Control 100 10 1 10 2 10 3 10 4 104 105 106 107 ControlControl genomic separation s, bpgenomic separation s, bp contactfrequency,P(s) 46Mb 51Mb 46Mb 51Mb 19Mb 23Mb
- 15. Testing loop extrusion ! Experiments control 46Mb 51Mb 46Mb 51Mb 19Mb 23Mb1 Mb 1 Mb 1 Mb chr5 chr5 chr11 contactfrequency 0.008 0 perturbation 100 10 1 10 2 10 3 10 4 104 105 106 107 genomic separation s, bp 100 10 1 10 2 10 3 10 4 104 105 106 107 Control 100 10 1 10 2 10 3 10 4 104 105 106 107 ControlControl genomic separation s, bpgenomic separation s, bp contactfrequency,P(s) 46Mb 51Mb 46Mb 51Mb 19Mb 23Mb
- 16. Same results from direct targeting of cohesinArticle Cohesin Loss Eliminates All Loop Domains Suhas S.P. Rao,1,2,3 Su-Chen Huang,1,2 Brian Glenn St Hilaire,1,2,4 Jesse M. Engreitz,5 Elizabeth M. Perez,5 Kyong-Rim Kieffer-Kwon,6 Adrian L. Sanborn,1,4,7 Sarah E. Johnstone,5,8 Gavin D. Bascom,9 Ivan D. Bochkov,1,2 Xingfan Huang,1,10 Muhammad S. Shamim,1,2,10,11 Jaeweon Shin,1,10 Douglass Turner,1,12 Ziyi Ye,1,10 Arina D. Omer,1,2 James T. Robinson,1,5,12 Tamar Schlick,9,13,14 Bradley E. Bernstein,5,8 Rafael Casellas,6,15 Eric S. Lander,5,16,17 and Erez Lieberman Aiden1,2,4,5,10,18,* 1The Center for Genome Architecture, Baylor College of Medicine, Houston, TX 77030, USA 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA 3Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA 4Center for Theoretical Biological Physics, Rice University, Houston, TX 77030, USA 5Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA 6Lymphocyte Nuclear Biology, NIAMS, NIH, Bethesda, MD 20892, USA 7Department of Computer Science, Stanford University, Stanford, CA 94305, USA 8Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA 9Department of Chemistry, New York University, New York, NY 10003, USA 10Departments of Computer Science and Computational and Applied Mathematics, Rice University, Houston, TX 77030, USA 11Medical Scientist Training Program, Baylor College of Medicine, Houston, TX 77030, USA 12Department of Medicine, University of California, San Diego, La Jolla, CA 92037, USA 13Courant Institute of Mathematical Sciences, New York University, New York, NY 10012, USA 14NYU-ECNU Center for Computational Chemistry, NYU Shanghai, Shanghai 200062, China 15Center of Cancer Research, NCI, NIH, Bethesda, MD 20892, USA 16Department of Biology, MIT, Cambridge, MA 02139, USA 17Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA 18Lead Contact *Correspondence: erez@erez.com https://doi.org/10.1016/j.cell.2017.09.026 SUMMARY The human genome folds to create thousands of intervals, called ‘‘contact domains,’’ that exhibit enhanced contact frequency within themselves. ‘‘Loop domains’’ form because of tethering between two loci—almost always bound by CTCF and cohe- sin—lying on the same chromosome. ‘‘Compartment domains’’ form when genomic intervals with similar histone marks co-segregate. Here, we explore the ef- fects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains (Wendt et al., 2008) and lie at the anchors of loops (Rao et al., 2014; Splinter et al., 2006) and the boundaries of contact do- mains (also called ‘‘topologically constrained domains,’’ ‘‘topo- logically associated domains,’’ or ‘‘physical domains’’) (Dixon et al., 2012; Lieberman-Aiden et al., 2009; Nora et al., 2012; Rao et al., 2014). This suggests that these proteins help regulate genome folding (Merkenschlager and Nora, 2016). Consistent with this, deletion of CTCF sites interferes with loop and contact domain formation (Guo et al., 2015; Sanborn et al., 2015; de Wit et al., 2015). However, initial, low-resolution experiments exam- ining genome-wide depletion of CTCF and cohesin observed only limited effects, reporting that compartments and contact domains still appear to be present (Seitan et al., 2013; Sofueva et al., 2013; Zuin et al., 2014). These results have made it difficult A = 42 = 114 = 111 134.6133.842.140.8 42.140.8 91.995.8 91.9 95.8 Mb 134.6133.8 HCT116-RAD21-mAC - auxin HCT116-RAD21-mAC + auxin, 6hr Chr4 Chr 4 Chr 1 Chr1Chr8 Chr 8 134.6 Mb133.8 Chr 8 = 38 = 111 42.1 Mb40.8 Chr 4 = 107 91.9 95.8 Mb Chr 1 E H3K27ac H3K4me3 H3K4me1 NIPBL Fast + auxin, 6hr withdraw, 20 Withdraw 68.467.6Mb68.2 = 107 = 34= 116 Chr18Chr14 0 7 0 25 0 100 0 50 Chr18 Rao et al., 2017, Cell 171, 305–320 October 5, 2017 ª 2017 Elsevier Inc. https://doi.org/10.1016/j.cell.2017.09.026 Article Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins 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 & Jan-Michael Peters1,* Abstract Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compart- ments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chro- matin 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 func- tion. 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 extru- sion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL. 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 (re- viewed 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 cohe- sin’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 Published online: December 7, 2017 Article A mechanism of cohesin-dependent loop extrusion organizes zygotic genome architecture Johanna Gassler1,† , Hugo B Brandão2,† , Maxim Imakaev3,4 , Ilya M Flyamer5 , Sabrina Ladstätter1 , Wendy A Bickmore5 , Jan-Michael Peters6 , Leonid A Mirny2,3,* & Kikuë Tachibana1,** Abstract Introduction Published online: December 7, 2017 Zp3-Cre ∆/∆ ∆/∆fl/flfl/fl B I R T H + B A 1.43 Scc1fl combined 1.14 A B 1.03 Scc1∆ combined 1.03 Average loop -100kb 0 kb +90kb activeinactive TAD Average TAD Com A B -100kb 0 kb +90kb TAD activeinactivee The EMBO Journal Published online: December 7, 2017 G 190 Mb0 Mb Chromosome 4 0 min auxin 190 15 min auxin 180 min auxin J 0 10 kb 100 kb 1 Mb 10 Mb100 Mb c genomic distance 88 Mb 94.5 Mbchromosome 12 8 0 min auxin 15 min auxin 180 min auxin a-tubulin H3 0 20 40 0.0 0.2 time (min) nor 10 30 H I log2(observed/expected) 1.6 0.8 0 -0.8 -1.6 1 50 0 min auxin 15 min auxin 180 min auxin 1 50 -450 kb 0 450 kb 0 min auxin 15 min auxin 180 min auxin chromosome 4 471 377 5 6 2 0 -2 log2enrichment Figure 1.
- 17. SMC is a motor! Cite as: M. Ganji et al., Science 10.1126/science.aar7831 (2018). REPORTS The spatial organization of chromosomes is of paramount importance to cell biology. Members of the SMC family of protein complexes, including condensin, cohesin, and the Smc5/6 complex, play vital roles in restructuring genomes during the cellular life cycle (1–3). The principles by which SMC complexes achieve these fundamental tasks are still incompletely understood. Models based on random cross- linking of DNA by pairwise interactions or conformational changes in the DNA superhelicity have been proposed (4, 5). An alternative hypothesis suggested that SMC protein com- plexes bind to small loops in the genome to then processive- ly enlarge them (6). More recently, the idea emerged that condensin can start and subsequently extrude DNA loops, which would elegantly explain how condensin mediates the formation of mitotic chromosomes structures observed in electron micrographs and deduced from Hi-C experiments (7, 8). Indeed, polymer simulations showed that loop extru- sion can, in principle, result in the efficient disentanglement and compaction of chromatin fibers (9–11). The recent dis- covery that condensin exhibits DNA translocase activity (12) was consistent with, but did not provide conclusive evidence for (13), DNA loop extrusion. In this Report, we visualize the formation of DNA loops by the Saccharomyces cerevisiae condensin complex in real (ATP), we observed the accumulation of fluorescence densi- ty at one spot along the length of the DNA (Fig. 1, D and E, fig. S1, and movie S2). This finding shows that condensin induces local compaction of DNA. To visualize the compacted DNA structures in the imag- ing plane of the microscope, we applied flow at a large angle with respect to the double-tethered DNA. This revealed that the bright spots were made up of extended pieces of DNA, consistent with single large DNA loops (Fig. 1, F and G, fig. S2, and movie S3). Importantly, we observed no DNA loop formation by wild-type condensin in the absence of either ATP or Mg2+ , when we replaced ATP by the non- hydrolyzable analogs ATP S or AMPPNP, or when we used a mutant condensin that is unable to bind ATP. Condensin hence creates DNA loops in a strictly ATP-hydrolysis- dependent manner, either by gradually extruding DNA or by randomly grabbing and linking two DNA loci. To distinguish between these two possibilities, we moni- tored the looping process by real-time imaging of the DNA while applying constant flow. This revealed the gradual ap- pearance of an initially weak increase in fluorescence inten- sity at a local spot that grew into an extended loop over time (Fig. 2A, fig. S3, and movies S4 and S5), providing direct visual evidence of loop extrusion and ruling out the random Real-time imaging of DNA loop extrusion by condensin Mahipal Ganji,1 Indra A. Shaltiel,2 * Shveta Bisht,2 * Eugene Kim,1 Ana Kalichava,1 Christian H. Haering,2 † Cees Dekker1 † 1 Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, Netherlands. 2 Cell Biology and Biophysics Unit, Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. *These authors contributed equally to this work. †Corresponding author. Email: christian.haering@embl.de (C.H.H.); c.dekker@tudelft.nl (C.D.) It has been hypothesized that Structural Maintenance of Chromosomes (SMC) protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. Here, we provide unambiguous evidence for loop extrusion by directly visualizing the formation and processive extension of DNA loops by yeast condensin in real-time. We find that a single condensin complex is able to extrude tens of kilobase pairs of DNA at a force-dependent speed of up to 1,500 base pairs per second, using the energy of ATP hydrolysis. Condensin-induced loop extrusion is strictly asymmetric, which demonstrates that condensin anchors onto DNA and reels it in from only one side. Active DNA loop extrusion by SMC complexes may provide the universal unifying principle for genome organization. Cite as: M. Ganji et al., Science 10.1126/science.aar7831 (2018). REPORTS First release: 22 February 2018 www.sciencemag.org (Page numbers not final at time of first release) 1 The spatial organization of chromosomes is of paramount importance to cell biology. Members of the SMC family of protein complexes, including condensin, cohesin, and the Smc5/6 complex, play vital roles in restructuring genomes during the cellular life cycle (1–3). The principles by which SMC complexes achieve these fundamental tasks are still incompletely understood. Models based on random cross- linking of DNA by pairwise interactions or conformational changes in the DNA superhelicity have been proposed (4, 5). An alternative hypothesis suggested that SMC protein com- plexes bind to small loops in the genome to then processive- ly enlarge them (6). More recently, the idea emerged that condensin can start and subsequently extrude DNA loops, which would elegantly explain how condensin mediates the formation of mitotic chromosomes structures observed in electron micrographs and deduced from Hi-C experiments (7, 8). Indeed, polymer simulations showed that loop extru- sion can, in principle, result in the efficient disentanglement and compaction of chromatin fibers (9–11). The recent dis- covery that condensin exhibits DNA translocase activity (12) was consistent with, but did not provide conclusive evidence for (13), DNA loop extrusion. In this Report, we visualize the formation of DNA loops by the Saccharomyces cerevisiae condensin complex in real time (Fig. 1A). We tethered both ends of a double-stranded 48.5-kilobase pair (kbp) -DNA molecule to a passivated surface (14, 15), using flow to adjust the DNA end-to-end length to a distance much shorter than its contour length (Fig. 1B). We then imaged DNA after staining with Sytox Orange (SxO; Fig. 1C and movie S1). Upon flushing in 1 nM of condensin (12) and 5 mM of adenosine triphosphate (ATP), we observed the accumulation of fluorescence densi- ty at one spot along the length of the DNA (Fig. 1, D and E, fig. S1, and movie S2). This finding shows that condensin induces local compaction of DNA. To visualize the compacted DNA structures in the imag- ing plane of the microscope, we applied flow at a large angle with respect to the double-tethered DNA. This revealed that the bright spots were made up of extended pieces of DNA, consistent with single large DNA loops (Fig. 1, F and G, fig. S2, and movie S3). Importantly, we observed no DNA loop formation by wild-type condensin in the absence of either ATP or Mg2+ , when we replaced ATP by the non- hydrolyzable analogs ATP S or AMPPNP, or when we used a mutant condensin that is unable to bind ATP. Condensin hence creates DNA loops in a strictly ATP-hydrolysis- dependent manner, either by gradually extruding DNA or by randomly grabbing and linking two DNA loci. To distinguish between these two possibilities, we moni- tored the looping process by real-time imaging of the DNA while applying constant flow. This revealed the gradual ap- pearance of an initially weak increase in fluorescence inten- sity at a local spot that grew into an extended loop over time (Fig. 2A, fig. S3, and movies S4 and S5), providing direct visual evidence of loop extrusion and ruling out the random cross-linking model. The extruded loops were in general stable (fig. S4), but occasionally disrupted spontaneously in a single step (Fig. 2A and movie S6). Such a single-step dis- ruption suggests that the DNA loop had been extruded by a single condensin unit that spontaneously let go of the loop, instead of a multi-step relaxation of the loop due to multiple units. Real-time imaging of DNA loop extrusion by condensin Mahipal Ganji,1 Indra A. Shaltiel,2 * Shveta Bisht,2 * Eugene Kim,1 Ana Kalichava,1 Christian H. Haering,2 † Cees Dekker1 † 1 Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, Netherlands. 2 Cell Biology and Biophysics Unit, Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. *These authors contributed equally to this work. †Corresponding author. Email: christian.haering@embl.de (C.H.H.); c.dekker@tudelft.nl (C.D.) It has been hypothesized that Structural Maintenance of Chromosomes (SMC) protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. Here, we provide unambiguous evidence for loop extrusion by directly visualizing the formation and processive extension of DNA loops by yeast condensin in real-time. We find that a single condensin complex is able to extrude tens of kilobase pairs of DNA at a force-dependent speed of up to 1,500 base pairs per second, using the energy of ATP hydrolysis. Condensin-induced loop extrusion is strictly asymmetric, which demonstrates that condensin anchors onto DNA and reels it in from only one side. Active DNA loop extrusion by SMC complexes may provide the universal unifying principle for genome organization. onFebruary23,2018http://science.sciencemag.org/Downloadedfrom ~8 Kb/min for two motors ~40Kb/min on nucleosomal fiber g. 2. peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not.http://dx.doi.org/10.1101/137711doi:preprint first posted online May. 13, 2017; Speed =4 Kb/min Cite as: T. Terakawa et al., Science 10.1126/science.aan6516 (2017). REPORTS First release: 7 September 2017 www.sciencemag.org (Page numbers not final at time of first release) 1 Structural maintenance of chromosomes (SMC) complexes are the major organizers of chromosomes in all living organ- isms (1, 2). These protein complexes play essential roles in sister chromatid cohesion, chromosome condensation and segregation, DNA replication, DNA damage repair, and gene expression. A distinguishing feature of SMC complexes is their large ring-like architecture, the circumference of which is made up of two SMC protein coiled-coil proteins and a sin- gle kleisin subunit (Fig. 1A) (1–4). The ~50-nm long antipar- allel coiled-coils are connected at one end by a stable dimerization interface, referred to as the hinge domain, and at the other end by globular ATP-binding cassette (ABC) fam- ily ATPase domains (5). The ATPase domains are bound by a protein of the kleisin family, along with additional accessory subunits, which vary for different types of SMC complexes (Fig. 1A). The relationship between SMC structures and their functions in chromosome organization is not completely un- derstood (6), but many models envision that the coiled-coil domains allow the complexes to topologically embrace DNA (1–4). Given the general resemblance to myosin and kinesin, some early models postulated that SMC proteins might be mechanochemical motors (7–10). SMC complexes are thought to regulate genome architec- ture by physically linking distal chromosomal loci, but how these bridging interactions might be formed remains un- known (1, 2, 11). An early model suggested that many three- dimensional (3D) features of eukaryotic chromosomes might be explained by DNA loop extrusion (Fig. 1B) (12, 13), and re- cent polymer dynamics simulations have shown that loop ex- trusion can recapitulate the formation of topologically associating domains (TADs), chromatin compaction, and sis- ter chromatid segregation (14–18). This loop extrusion model assumes a central role for SMC complexes in actively creating the DNA loops (11, 12). Similarly, it has been proposed that prokaryotic SMC proteins may structure bacterial chromo- somes through an active loop extrusion mechanism (19–21). Yet, the loop extrusion model remains hypothetical, in large part because the motor activity that is necessary for driving loop extrusion could not be identified (11). Indeed, the ab- sence of an identifiable motor activity in SMC complexes in- stead has lent support to alternative models in which DNA loops are not actively extruded, but instead are captured and stabilized by stochastic pairwise SMC binding interactions to bridge distal loci (22). To help distinguish between possible mechanisms of SMC protein-mediated chromosomal organization, we examined the DNA-binding properties of condensin (23). We overex- pressed the five subunits of the condensin complex in bud- ding yeast and purified the complex to homogeneity (Fig. 1C and fig. S1). Electron microscopy images confirmed that the complexes were monodisperse (Fig. 1D). As previously de- scribed for electron micrographs of immunopurified Xenopus laevis or human condensin (24), we observed electron density that presumably corresponds to the two HEAT-repeat subu- The condensin complex is a mechanochemical motor that translocates along DNA Tsuyoshi Terakawa,1 * Shveta Bisht,2 * Jorine M. Eeftens,3 * Cees Dekker,3 † Christian H. Haering,2 † Eric C. Greene1 † 1 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA. 2 Cell Biology and Biophysics Unit, Structural and Computational Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. 3 Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, Netherlands. *These authors contributed equally to this work. †Corresponding author. Email: c.dekker@tudelft.nl (C.D.); christian.haering@embl.de (C.H.H.); ecg2108@cumc.columbia.edu (E.C.G.) Condensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. Here, we use single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of ATP hydrolysis-dependent translocation along double-stranded DNA. Condensin’s translocation activity is rapid and highly processive, with individual complexes traveling an average distance of 10 kilobases at a velocity of ~60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ~50-nanometer coiled-coil subunits, suggestive of a translocation mechanism that is distinct from any reported DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation. onNovember1,2017http://science.sciencemag.org/Downloadedfrom Speed =36 Kb/min Theory: speed ~100-200Kb per cohesin residence time (5-20min) => 10-40Kb/min One-sided!?
- 18. …but one-sided cannot compact chromosomes! ? S.cerevisiae mammals
- 19. Conclusions 1. Strong experimental support of the loop extrusion by cohesin, hindered by CTCF !
- 20. Outline Compartments and TADs 1. Emerging evidence of loop extrusion 2. Compartmentalization by phase separation! 3. Phase separation vs extrusion Chromatin Organization by an Interplay of Loop Extrusion and Compartmental Segregation https://www.biorxiv.org/content/early/2017/10/03/196261 Heterochromatin drives organization of conventional and inverted nuclei https://www.biorxiv.org/content/early/2018/01/09/244038
- 21. Kikue Tachibana-Konwalski Ilya M. Flyamer ! Johanna Gassler! IBMA, Vienna! ! ! ! ! ! Maxim Imakaev MIT Hugo Brandao Harvard Biophysics Single-nucleus Hi-C Flyamer, Gassler, Imakaev et al. Nature 2017! a TADs compartments yesyes yes no Compartments and TADs are formed by separate mechanisms b active inactive 0.0 0.5 Log enrichment -80kbinactive 0.5 Log enrichment 0.32 0.88 Effective contact probability -80kb loop +70kb TAD 0.0 0.8 Log enrichment Compartmentalization Average loop Average TAD active
- 22. TADs (but not compartments) are cohesin-dependent
- 23. TADs (but not compartments) are cohesin-dependent
- 24. Compartments and TADs are formed by separate mechanisms Compartments become stronger and fragmented in the absence of loop extrusion A B 1.5 1.5 35.0 47.5 60.035.0 47.5 60.0 35.0 47.5 60.0 1.5 1.5 TAM (control)
- 25. Fine compartments match epigenetic state better than wild-type compartments C D E Eigenvector 1.5 - -1.5 _ -0- H3K4me3 2 - 0 _ H3K4me1 2 - 0 _ H3K36me3 2 - 0 _ H3K27me3 2 - 0 _ 25.0 30.0 35.0 10 Mb H3K27ac 2 - 0 _ Activity chr15 20.0 30.0 40.0 20.0 30.0 40.0 20.0 30.0 40.0 55.0 70.0 72.0 88.0 103.5 B C TAM chr17 1.5 1.5
- 26. Conclusions 1. Strong experimental support of the loop extrusion by cohesin, hindered by CTCF 2. TAD and compartment formation — separate mechanisms 3. Innate compartments (associated with histone marks) are partially suppressed by loop extrusion A 1.5 1.5 35.0 47.5 60.035.0 47.5 60.0 35.0 47.5 60.0 1.5 1.5 TAM
- 27. Published online 04 August 2014 Nucleic Acids Research, 2014, Vol. 42, No. 15 9553–9561 doi: 10.1093/nar/gku698 Modeling epigenome folding: formation and dynamics of topologically associated chromatin domains Daniel Jost1 , Pascal Carrivain2 , Giacomo Cavalli2,* and C´edric Vaillant1,* 1 Laboratoire de Physique, Ecole Normale Sup´erieure de Lyon, CNRS UMR 5672, Lyon 69007, France and 2 Institute of Human Genetics, CNRS UPR 1142, Montpellier 34000, France Received April 1, 2014; Revised July 02, 2014; Accepted July 19, 2014 ABSTRACT Genomes of eukaryotes are partitioned into domains of functionally distinct chromatin states. These do- mains are stably inherited across many cell gener- ations and can be remodeled in response to devel- opmental and external cues, hence contributing to the robustness and plasticity of expression patterns and cell phenotypes. Remarkably, recent studies in- dicate that these 1D epigenomic domains tend to fold into 3D topologically associated domains forming specialized nuclear chromatin compartments. How- ever, the general mechanisms behind such compart- mentalization including the contribution of epige- netic regulation remain unclear. Here, we address the question of the coupling between chromatin fold- ing and epigenome. Using polymer physics, we ana- lyze the properties of a block copolymer model that accounts for local epigenomic information. Consid- ering copolymers build from the epigenomic land- scape of Drosophila, we observe a very good agree- ment with the folding patterns observed in chro- mosome conformation capture experiments. More- over, this model provides a physical basis for the existence of multistability in epigenome folding at sub-chromosomal scale. We show how experiments are fully consistent with multistable conformations where topologically associated domains of the same epigenomic state interact dynamically with each other. Our approach provides a general framework to improve our understanding of chromatin folding during cell cycle and differentiation and its relation to epigenetics. INTRODUCTION Gene expression is regulated by many sets of proteins that associate with the genome in a cell-type and condition- specific manner at specific regulatory elements including proximal promoters, enhancers and repressors. The packag- ing of eukaryotic DNA into chromatin contributes to this regulation via the modulation of the accessibility and speci- ficity of regulators to their nucleic sites. Locally, the chro- matin state is characterized by various features like the nu- cleosome positioning, the covalent modifications of DNA and histones tails and the insertion of histone variants. This pattern of chromatin states along the genome, the so-called ‘epigenome’, is itself regulated by the combined action of different specialized chromatin regulators like chromatin re- modelers, modifying enzymes and histone chaperones. The general picture that emerges from the genome-wide high-resolution profiling of structural and functional chro- matin marks obtained in various organisms and cell types (1–4), is that eukaryotic genomes are linearly organized into distinct epigenomic domains. These domains extend over few kilobases up to few megabases, are characterized by a specific type of chromatin and are isolated from their neighborhood by boundary elements such as insulators. Euchromatin, less condensed, early replicating and con- taining most active genes, is generally distinguished from heterochromatin, typically highly condensed, late replicat- ing and inhibitory to transcriptional machinery. In many higher eukaryotes, from plants to mammals, statistical anal- yses of hundreds of chromatin marks have identified only a small number of main chromatin types (1,3,5,6), typi- cally four or five, covering the well-known constitutive HP1- like heterochromatin or the facultative (developmentally regulated) Polycomb-like heterochromatin but also a less- characterized ultra-repressive heterochromatin enriched in genes that are expressed in very few tissues, the so-called void or black chromatin (1,7). Interestingly, within epigenomic domains, regulatory se- quences such as enhancers may be located far from the tar- get genes and multiple elements that are distributed over large regions may collaborate or compete for the regula- tion of individual genes or gene clusters. This implies the existence of long-range mechanisms where regulatory ele- ments could act over large genomic distances up to hun- dreds of kilobases or more. A possible mechanism regu- lating such long-range effects is the linear spreading of a *To whom correspondence should be addressed. Tel: +33 4 72 72 86 34; Fax: +33 4 72 72 89 50; Email: cedric.vaillant@ens-lyon.fr Correspondence may also be addressed to Giacomo Cavalli. Tel: +33 4 34 35 99 70; Fax: +33 4 34 35 99 01; Email: giacomo.cavalli@igh.cnrs.fr C⃝ The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Mechanism of compartmentalization Johannes Nübler Many problems associated with it are not solved yet, and the potential of its practical use is far from being exhausted. Recall that a block copolymer is a chain consisting of consecutively joined blocks, each of which constitutes a long homopolymer chain. For example, the chemical structure of a two-block copolymer is A--...--A--B--...--B. The number of blocks in the molecule, just as the number of links in the block, can be arbitrary. What happens when a sufficiently concentrated solution or melt is made from the chains of a block copolymer? From previous subsections, we know that in a typical case, the chains of poly-A and poly-B (or in our case, the blocks) are incompatible. A phase separation in such a system, .however, is impossible because of the covalent bonding of the blocks into common chains. As a result, the phase separation, which is impossible on the some of the whole system, occurs on a certain limited length scale defined by the size of the blocks. The arising microdomain structure is schematically shown in Figure 5.1. If the total amount of one of the components (e.g., A) is relatively small, then the corresponding phase enriched with A component (the A phase) occupies a small fraction of the total volume, and it constitutes a system of spherically shaped micelles scattered like "islets>, in a °’sea" of the phase enriched with B component (the B phase). On increasing the fraction of A links, the spherically shaped micelles become cylindric ones piercing the B phase like reinforcing wires. On further increasing the A fraction, a lamellar (or layer), structure appears, with A and B phases laid out in alternating planar layers. Finally, on still further increasing the fraction of A links, the so-called in~zerse phases emerge: first the cylindric phase (B phase cylinders piercing the A phase), then the spheric one ( B "’raisins" in the A "’pudding"). To conclude, it should be noted that the microdomain (or micellar) structures are typical not only for block copolymers but also for systems consisting of the so-called diphilic molecules. One of their blocks has a low molecular weight, but because of its thermodynamic properties, it cannot mix withthe other block. Examples include phospholipid molecules consisting of a hydrophilic "head" and a polymeric (usually not very long) "tail." The dissolution of such mole- h ~ FIGUItE 5.1. Microdomain structure in a melt of block copolymers. (a), Spheric A phase micelles in massive B phase. (b), Cylindric micelles. (c), Alternating planar lamellae. cu (a 2 .o ( o t t " Statistical Physics of Macromolecules 1991 (micro)phase separation extrusion segregation counteracts native compartmentalization contact frequency low high no loop extrusion (nA+nB) -1 (nA-nB) (nA+nB) counts,a.u. A B Chromatin Organization by an Interplay of Loop Extrusion and Compartmental Segregation https://www.biorxiv.org/content/early/2017/10/03/196261
- 28. …..add loop extrusion on top loop extrusion compartmental segregation counteracts native compartmentalization no loop extrusion -1 1(nA-nB) (nA+nB) -1 1(nA-nB) (nA+nB) counts,a.u. 0.0 0.18 A B Eattr(kBT) 5 MB equiv.5 MB equiv. 50 MB equiv. simul
- 29. …..add loop extrusion on top 5 MB equiv. 5 MB equiv. 5 MB equiv. loop extrusion compartmental segregation counteracts native compartmentalization no loop extrusion -1 1(nA-nB) (nA+nB) -1 1(nA-nB) (nA+nB) counts,a.u. 0.0 0.18 A B Eattr(kBT) 50 MB equiv. simul compaction 1 ofile rr. 1 50 MB equiv. modelB loop extrusion compartmental segregation counteracts simulationC example snapshot A/B number difference -1 1(nA-nB) (nA+nB) counts,a.u.u. 0.0 A B
- 30. Active mixing by loop extrusion suppresses small compartments compartments Nuebler et al., bioRxiv (2017) Hi-C Fig. 2 92Mb 97Mb72Mb 122Mbchr6 comp.profile autocorr. WTNipbl experiments (Schwarzer 2017)
- 31. Active mixing by loop extrusion suppresses small compartments compartments Nuebler et al., bioRxiv (2017) Hi-C Fig. 2 small compartments are “erased” by loop extrusion 92Mb 97Mb72Mb 122Mbchr6 comp.profile autocorr. WTNipbl experiments (Schwarzer 2017)
- 32. Active mixing by loop extrusion suppresses small compartments compartments Nuebler et al., bioRxiv (2017) Hi-C Fig. 2 small compartments are “erased” by loop extrusion 92Mb 97Mb72Mb 122Mbchr6 comp.profile autocorr. WTNipbl experiments (Schwarzer 2017)
- 33. Attractions: A-A B-B (direct or mediated, e.g. HP1) B-Lamina ! - Can one disentangle these contributions? - Which ones are more important for compartmentalization? Mechanism of compartmentalization Irina Solovei, LMU Yana Fedorova, Plovidv U Hard to disentangle in conventional nuclei Heterochromatin drives organization of conventional and inverted nuclei https://www.biorxiv.org/content/early/2018/01/09/244038
- 34. Mechanism of compartmentalization igures and Figure Cap ons: inverted conventionalconventionalinverted Irina Solovei, LMU
- 35. Mechanism of compartmentalization igures and Figure Cap ons: inverted conventionalconventionalinverted Irina Solovei, LMU
- 36. Modeling of compartmentalization Comp.Stength = Sumi [XXi/toti] Martin Falk MIT phase separation in a block copolymer
- 41. Mechanism of compartmentalization CC<BC<BB<AB<AA BC<CC<BB<AB<AA AA<AB<BB<BC<CC ….720…. Correct order: AA<AB<BB<BC<CC; AA≈0! BB is the only free parameter
- 42. Correct order: AA<AB<BB<BC<CC; AA≈0! BB is the only free parameter Mechanism of compartmentalization Figure 3. Polymer model reproduces micro Hi-C features a, Our approach is to: define a mechanistic mo interactions; simulate an ensemble of configurati dynamics; and compare these configurations to Hi- Correct order: AA<AB<BB<BC<CC; AA≈0! BB = 0.5-0.6 kT
- 43. Mechanism of compartmentalization AA<AB<BB<BC<CC; AA≈0! ! ! BB = 0.5-0.6 kT! Lamina-B = 0.2 kT
- 44. Attractions: A-A B-B B-Lamina Mechanism of compartmentalization <— for compartmentalization <— for positioning in the nucleus
- 45. Attractions: A-A B-B B-Lamina Mechanism of compartmentalization <— for compartmentalization <— for positioning in the nucleus Figures and Figure Cap ons: inverted is the default state of the nucleus!!
- 46. Summary Active loop extrusion! by cohesin! ! ! ! ! ! 1. Loop extrusion can be universal mechanism! • fold domains • compacts chromosomes 2. Compartments are formed by heterochromatin interactions, and positioned in space interactions with lamina Active loop extrusion! by cohesin! ! ! ! ! blocked by CTCF! 1. Fundenberg J, Abnennur N, et al bioRxiv 2018 2. Gibcus, Samejima, Goloborodko A et al Science 2018 3. Falk M, et al., bioRxiv 2018 4. Nueber J et al., bioRxiv 2018 http://HiGlass.io II
- 47. Emerging Evidence of Chromosome Folding by Loop Extrusion https://www.biorxiv.org/content/early/2018/02/16/264648 HiGlass: Web-based Visual Exploration and Analysis of Genome Interaction Maps https://www.biorxiv.org/content/early/2017/10/30/121889 Chromatin Organization by an Interplay of Loop Extrusion and Compartmental Segregation https://www.biorxiv.org/content/early/2017/10/03/196261 Heterochromatin drives organization of conventional and inverted nuclei https://www.biorxiv.org/content/early/2018/01/09/244038 bioRxiv
- 48. Maxim Imakaev MIT Nezar Abdennur MIT Comp/Sys Biology NSF, NIH: Center of Structure and Physics of the Genome Hugo Brandao Harvard Biophysics Ed Banigan MIT Aafke Van den Berg MIT Carolyn Lu MIT senior Martin Falk MIT Job Dekker UMass Medical ! ! John Marko Northwestern U. Francois Spitz Institut Pasteur Elphege Nora Benoit G. Bruneau UCSF Kick Tachibana- Konwalski IBMA, Vienna Bill Earnshaw U of Edinburgh Irina Solovei, LMU Geoff Fudenberg UCSF Anton Goloborodko MIT Physics Johannes Nübler MIT
- 49. 2-8 molecules (or motors) of cohesin per Mb each consume ATP at a rate of 2 per sec per motor < 1e5 ATP/sec! ! Fibroblast ATP production: 1e9 ATP/sec, ! ! hence the fraction consumed by cohesins < 0.0001 (very modest: ~1% of the NIH budget in US GDP).
- 50. How can SMCs extrude loops? 2. From translocation to extrusion
- 51. How can SMCs extrude loops? 2. From translocation to extrusion Figure 4. a. Walking as a possible mechanism of SMC translocation, with SMC arms in yellow and orang kleisin in blue, creating a shackled walker. possible implementation of two-sided
- 52. Mechanism of loop extrusion long-range interactions are formed by 1D process number of sites L ) 1: At time t = 0, M motile element pairs are dispersed randomly, each pair initially occupying adjacent sites of this lattice. The DNA-binding motile elements (referred to below as ‘motors’) then move along the DNA with rates independent of position; steps that move a motor away from its partner (‘forward’ steps that extrude a DNA loop) occur at a rate r+ and steps that move a motor back toward its partner (‘reverse steps’ that retract the loop) occur at a rate rÀ (Figure 1). We suppose the motion to be directed by energy gained from ATP hydrolysis, with r+ > rÀ (when r+ ¼ rÀ there is 1D diffu- sion of each motor; when rÀ > r+, the motors are driven together which is not of interest here). The motor pairs are assumed to have left/right symmetry, i.e. the left and right motors move with the same rates. increment Át to the event is distributed over the 0 Át 1 exponentially, with probability distr PðÁtÞ ¼ ReÀRt : The actual realization of Át is from this continuous distribution. Which of the K tions actually occurs is determined from their prob distribution pi ¼ ri=R: This second, discrete, distrib be used to select which of the K candidates actually Once Át and i are determined, the state of the sy changed, and time is increased to t+Át: The algor then repeated to propagate the system forward from to event, for as many transition steps as one r (or for as long a total time as is required). The r a series of transition events, distributed in time acc to the rates that define the model. There is no tim cretization; events can occur separated by arbitraril Figure 1. Schematic drawing of machine positions on the lattice as time progresses; lattice model equivalent is sketched below each pan dumbbell shapes (and arrows in the lattice sketch) depict enzymes and green lines show DNA. Panel (a) depicts the starting point and the pro of infinitely processive machines, while Panel (b) shows machines with lower processivity (disassociation rate is still relatively small, Panel (c) depicts a single step, with ATP binding, hydrolysis and release associated with extrusion of a small amount of DNA. Nucleic Acids Research, 2 Self-organization of domain structures by DNA-loop-extruding enzymes Elnaz Alipour1, * and John F. Marko2, * 1 Center for Cell Analysis and Modeling, University of Connecticut Health Sciences Center, Farmington, CT 06030 and 2 Departments of Physics and Astronomy and Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA Received June 1, 2012; Revised August 17, 2012; Accepted September 13, 2012 ABSTRACT The long chromosomal DNAs of cells are organized into loop domains much larger in size than individual DNA-binding enzymes, presenting the question of how formation of such structures is controlled. We present a model for generation of defined chromo- somal loops, based on molecular machines consist- ing of two coupled and oppositely directed motile elements which extrude loops from the double helix along which they translocate, while excluding one another sterically. If these machines do not dissoci- ate from DNA (infinite processivity), a disordered, exponential steady-state distribution of small loops is obtained. However, if dissociation and rebinding of the machines occurs at a finite rate (finite processivity), the steady state qualitatively changes to a highly ordered ‘stacked’ configuration with sup- pressed fluctuations, organizing a single large, stable loop domain anchored by several machines. The size of the resulting domain can be simply regulated by boundary elements, which halt the progress of the extrusion machines. Possible real- izations of these types of molecular machines are discussed, with a major focus on structural main- tenance of chromosome complexes and also with discussion of type I restriction enzymes. This mech- anism could explain the geometrically uniform folding of eukaryote mitotic chromosomes, through nucleoids. It has been proposed that chromosomes might simply occupy maximum-entropy conformations, in the manner of confined random-coil polymers (1,2). However, sequence position analyses reveal DNA to be spatially ordered. Chromosomes of Escherichia coli (3–5) and Caulobacter crescentus (6) have loci precisely pos- itioned inside the cell, with fluctuations too small to be consistent with random-polymer statistics (7). In eukary- ote cells, interphase chromosomes in differentiated cells occupy distinct territories (8). Furthermore, analyses of DNA juxtapositions inside eukaryote nuclei reveal that loci up to tens of megabases apart along chromosomes are positioned near one another in the nucleus (9,10), with statistical properties inconsistent with random- polymer organization (10). Detailed characterizations of specific cases of in cis gene regulation also indicate that chromosomes have a well-defined ‘loop domain’ organization, with specific but distant sequences along the same chromosome pos- itioned to be near one another (11). It is thought that ‘chromatin-bridging’ proteins (12) somehow stabilize these loop structures, but the processes by which sequence-defined chromatin loops are established and maintained are unknown. Strong correlations of juxtaposed DNA sequences are especially clear during eukaryote mitosis, when chromo- somes are compactly folded, following their replication. Chromosomes are ‘condensed’ by folding along their length into linear paired-chromatid noodle-like structures, with a well-defined thickness and strikingly uniform struc- tural and mechanical properties (13). As mitotic chromo- Nucleic Acids Research, 2012, 1–11 doi:10.1093/nar/gks925 Nucleic Acids Research Advance Access published October 15, 2012 atMITLibrariesonNovember13,2012http://nar.oxfordjournals.org/Downloadedfrom Marko 2013 Nasmyth 2001 on July 13, 2010rstb.royalsocietypublishing.orgDownloaded from doi: 10.1098/rstb.1990.0012 , 285-2973261990Phil. Trans. R. Soc. Lond. B A. D. Riggs Folding and Enhancer Function Memory, and Type 1 DNA Reeling Could Aid Chromosome DNA Methylation and Late Replication Probably Aid Cell References http://rstb.royalsocietypublishing.org/content/326/1235/285#related-urls Article cited in: Rapid response http://rstb.royalsocietypublishing.org/letters/submit/royptb;326/1235/285 Respond to this article Email alerting service hereright-hand corner of the article or click Receive free email alerts when new articles cite this article - sign up in the box at the top http://rstb.royalsocietypublishing.org/subscriptionsgo to:Phil. Trans. R. Soc. Lond. BTo subscribe to on July 13, 2010rstb.royalsocietypublishing.orgDownloaded from Riggs 1990