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      Depletion of the Chromatin Looping Proteins CTCF and Cohesin Causes Chromatin Compaction: Insight into Chromatin Folding by Polymer Modelling

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          Abstract

          Folding of the chromosomal fibre in interphase nuclei is an important element in the regulation of gene expression. For instance, physical contacts between promoters and enhancers are a key element in cell-type–specific transcription. We know remarkably little about the principles that control chromosome folding. Here we explore the view that intrachromosomal interactions, forming a complex pattern of loops, are a key element in chromosome folding. CTCF and cohesin are two abundant looping proteins of interphase chromosomes of higher eukaryotes. To investigate the role of looping in large-scale (supra Mb) folding of human chromosomes, we knocked down the gene that codes for CTCF and the one coding for Rad21, an essential subunit of cohesin. We measured the effect on chromosome folding using systematic 3D fluorescent in situ hybridization (FISH). Results show that chromatin becomes more compact after reducing the concentration of these two looping proteins. The molecular basis for this counter-intuitive behaviour is explored by polymer modelling usingy the Dynamic Loop model (Bohn M, Heermann DW (2010) Diffusion-driven looping provides a consistent framework for chromatin organization. PLoS ONE 5: e12218.). We show that compaction can be explained by selectively decreasing the number of short-range loops, leaving long-range looping unchanged. In support of this model prediction it has recently been shown by others that CTCF and cohesin indeed are responsible primarily for short-range looping. Our results suggest that the local and the overall changes in of chromosome structure are controlled by a delicate balance between short-range and long-range loops, allowing easy switching between, for instance, open and more compact chromatin states.

          Author Summary

          Folding of chromosomes in interphase nuclei of higher eukaryotes is a key element in regulating gene expression. The mechanisms that control chromatin folding are largely unknown. We have shown earlier that looping is a fundamental aspect of large-scale chromatin structure. Two abundant looping proteins are known: CTCF and cohesin. Here we combine quantitative fluorescent in situ hybridisation experiments in human cells with polymer modelling to unravel mechanisms of chromatin folding. We show that chromatin becomes more compact after depletion of looping proteins. This is remarkable, since polymer models describing chromatin predict decompaction. We present a polymer model that shows that specific biologically relevant looping regimes give rise to this behaviour. Importantly, chromosome conformation capture studies of mammalian chromatin support such looping regimes. Our results indicate that the local and overall compaction of the chromatin is defined by a subtle balance between short and long range loops; this may explain cell cycle and genome activity dependent structural transitions of chromatin.

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          Most cited references27

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          Architectural protein subclasses shape 3D organization of genomes during lineage commitment.

          Understanding the topological configurations of chromatin may reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here, we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3D interactions that undergo marked reorganization at the submegabase scale during differentiation. Distinct combinations of CCCTC-binding factor (CTCF), Mediator, and cohesin show widespread enrichment in chromatin interactions at different length scales. CTCF/cohesin anchor long-range constitutive interactions that might form the topological basis for invariant subdomains. Conversely, Mediator/cohesin bridge short-range enhancer-promoter interactions within and between larger subdomains. Knockdown of Smc1 or Med12 in embryonic stem cells results in disruption of spatial architecture and downregulation of genes found in cohesin-mediated interactions. We conclude that cell-type-specific chromatin organization occurs at the submegabase scale and that architectural proteins shape the genome in hierarchical length scales. Copyright © 2013 Elsevier Inc. All rights reserved.
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            Cohesin mediates transcriptional insulation by CCCTC-binding factor.

            Cohesin complexes mediate sister-chromatid cohesion in dividing cells but may also contribute to gene regulation in postmitotic cells. How cohesin regulates gene expression is not known. Here we describe cohesin-binding sites in the human genome and show that most of these are associated with the CCCTC-binding factor (CTCF), a zinc-finger protein required for transcriptional insulation. CTCF is dispensable for cohesin loading onto DNA, but is needed to enrich cohesin at specific binding sites. Cohesin enables CTCF to insulate promoters from distant enhancers and controls transcription at the H19/IGF2 (insulin-like growth factor 2) locus. This role of cohesin seems to be independent of its role in cohesion. We propose that cohesin functions as a transcriptional insulator, and speculate that subtle deficiencies in this function contribute to 'cohesinopathies' such as Cornelia de Lange syndrome.
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              Cohesins functionally associate with CTCF on mammalian chromosome arms.

              Cohesins mediate sister chromatid cohesion, which is essential for chromosome segregation and postreplicative DNA repair. In addition, cohesins appear to regulate gene expression and enhancer-promoter interactions. These noncanonical functions remained unexplained because knowledge of cohesin-binding sites and functional interactors in metazoans was lacking. We show that the distribution of cohesins on mammalian chromosome arms is not driven by transcriptional activity, in contrast to S. cerevisiae. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer blocking function and boundary-element activity. We find cohesins at most CTCF sites and show that CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions and, because CTCF binding is sensitive to DNA methylation, allows cohesin positioning to integrate DNA sequence and epigenetic state.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Comput Biol
                PLoS Comput. Biol
                plos
                ploscomp
                PLoS Computational Biology
                Public Library of Science (San Francisco, USA )
                1553-734X
                1553-7358
                October 2014
                9 October 2014
                : 10
                : 10
                : e1003877
                Affiliations
                [1 ]Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands
                [2 ]Institute for Theoretical Physics, Heidelberg University, Heidelberg, Germany
                [3 ]Van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlands
                [4 ]Department for Molecular Biotechnology, University of Gent, Gent, Belgium
                CNAG - Centre Nacional d'Anàlisi Genòmica and CRG - Centre de Regulació Genòmica, Spain
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: RvD DWH MTD HJ. Performed the experiments: MTD HJ. Analyzed the data: MTD HJ. Contributed reagents/materials/analysis tools: EMMM. Wrote the paper: MTD RvD HJ DWH.

                Article
                PCOMPBIOL-D-14-00485
                10.1371/journal.pcbi.1003877
                4191888
                25299688
                4d240720-9f3e-4bd9-a95c-a8f6679e7d18
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 March 2014
                : 18 August 2014
                Page count
                Pages: 12
                Funding
                This work was supported by a grant of the Dutch Foundation for Fundamental Research on Matter (FOM) as part of the research programme Physics of the Genome (Grant 07DNAA11 to RvD). Hansjoerg Jerabek was funded by the Heidelberg Graduate School of Mathematical and Computational Methods for the Sciences and the Institute for Theoretical Physics. The Netherlands Organization for Scientific Research (NWO) is acknowledged for a grant for using supercomputing facilities (Grant MP-190-12 to RvD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biophysics
                Cell Biology
                Computational Biology
                Systems Biology
                Research and Analysis Methods
                Computational Techniques
                Imaging Techniques
                Mathematical and Statistical Techniques
                Microscopy
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All data are fully available without restriction. Raw experimental data and computational data can be accessed via the repositories of University of Amsterdam ( http://dare.uva.nl) and Heidelberg University ( https://heidata.uni-heidelberg.de) using the manuscript author data. Direct link to the data: http://hdl.handle.net/11245/1.430734.

                Quantitative & Systems biology
                Quantitative & Systems biology

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