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Multi-feature clustering of CTCF binding creates robustness for loop extrusion blocking and Topologically Associating Domain boundaries.

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Topologically Associating Domains (TADs) separate vertebrate genomes into insulated regulatory neighborhoods that focus genome-associated processes. TADs are formed by Cohesin-mediated loop extrusion, with many TAD boundaries consisting of clustered binding sites of the CTCF insulator protein. Here we determine how this clustering of CTCF binding contributes to the blocking of loop extrusion and the insulation between TADs. We identify enrichment of three features of CTCF binding at strong TAD boundaries, consisting of strongly bound and closely spaced CTCF binding peaks, with a further enrichment of DNA-binding motifs within these peaks. Using multi-contact Nano-C analysis in cells with normal and perturbed CTCF binding, we establish that individual CTCF binding sites contribute to the blocking of loop extrusion, but in an incomplete manner. When clustered, individual CTCF binding sites thus create a stepwise insulation between neighboring TADs. Based on these results, we propose a model whereby multiple instances of temporal loop extrusion blocking create strong insulation between TADs.


Acknowledgements: We thank Alessandra Montecucco (IGM-CNR, Pavia, Italy) and members of the Noordermeer lab for useful discussion. We thank Joke van Bemmel, Edith Heard, Elphège P. Nora, Benoit Bruneau, Maxim Greenberg, Ning Qing Liu, and Elzo de Wit for sharing of mESC lines. We thank Benoit Moindrot (I2BC, Gif-sur-Yvette, France) for help with Western blotting. This work has benefited from the facilities and expertise of the high-throughput sequencing core facility of the I2BC (Gif-sur-Yvette, France). D.N. and E.F.J. benefit from collaborative funding from the Agence Nationale pour la Recherche (ANR-21-CE12-0034-01) / National Science Foundation (NSF). D.N. and D.H. benefit from collaborative funding from PlanCancer (19CS145-00). D.N. was further supported by the Agence Nationale pour la Recherche (ANR-18-CE12-0022-02, ANR-17-CE12-0001-02, ANR-16-TERC-0027-01, ANR-14-ACHN-0009-01), the Fondation Bettencourt Schueller, the Fondation de Coopération Scientifique Campus Paris-Saclay (2015-0980i) and Oxford Nanopore Technologies (MinION Early Access Program and Direct-RNA Early Access Program). S.G. and A.P. were supported by postdoctoral fellowships from the Fondation pour la Recherche Médicale (Post-doctorat en France - SPF201909009328 and SPF201909009284). Cryo-EM experiments were supported by the CNRS network Microscopie Electronique et Sonde Atomique (METSA, FR CNRS 3507) to D.N. and the Investissements d’Avenir LabEx PALM (ANR-10-LABX-0039-PALM) to A.L. J.M.L. was supported by the National Institute of Health (F31HD102084). E.F.J. was supported by the National Institute of Health (R35GM128903 and U01DA052715). D.H. was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation program (882673).

Funder: Fondation Bettencourt Schueller (Bettencourt Schueller Foundation); doi:

Funder: Fondation de Coopération Scientifique Campus Paris-Saclay (Foundation for Scientific Cooperation Paris-Saclay Campus); doi:


Binding Sites, CCCTC-Binding Factor, Cluster Analysis, Protein Domains

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Nat Commun

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Springer Science and Business Media LLC
Fondation pour la Recherche Médicale (Foundation for Medical Research in France) (SPF201909009328, SPF201909009284)