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. 2022 Dec 5;54(12):1907–1918. doi: 10.1038/s41588-022-01232-7

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a, Clonal mESC lines containing random TetO arrays flanked by 3 × CTCF motifs and expressing TetR-tdTomato. Constructs were integrated using piggyBac transposition in mESCs allowing auxin-inducible degradation of GFP-tagged RAD21, WAPL or CTCF. ITR, inverted terminal repeats. b, Representative images of RAD21-AID-eGFP cells containing 3 × CTCF-TetO imaged before or after 90 min of auxin treatment (exposure time eGFP and tdTomato: 50 ms, deconvolved, maximum intensity projection, bicubic interpolation, n = 3 replicates). c, Left, time series of TetR-tdTomato signal over 30 min (maximum intensity projection, time interval dt = 10 s, color-coded for intensity changes over time). Right, magnification with overlay of TetR-tdTomato signal with reconstructed trajectories of individual TetO arrays. d, Left, cell motion is approximated as the average roto-translational motion of TetO signals within the same nucleus. Right, MSD averaged over trajectories within one nucleus (mean ± s.e.m.) before (cyan, n = 77) and after (blue, n = 77) cell motion and localization error correction. Green, radial MSD of pairs of operator arrays within the same nucleus (mean ± s.e.m., n = 491 pairs). e, Left, MSD (mean ± s.e.m.) in mESC lines before (blue, 310 cells, 13,537 trajectories) or after (red, 271 cells, 11,082 trajectories) Cre-mediated removal of 3 × CTCF sites. Three replicates per cell line and three lines per condition were analyzed and merged here and in all following MSD graphs. P values (two-sided Student’s t-test) for all panels shown in Extended Data Fig. 2e. Right, schematic representation of Cre-mediated removal of CTCF sites. f, Left, same as in e but in mESC lines with 3 × CTCF-TetO arrays, before (blue, 323 cells, 9,829 trajectories) or after (red, 365 cells, 12,495 trajectories) CTCF degradation (6 h of auxin treatment). Right, schematic representation of auxin-induced CTCF degradation. g, MSD (mean ± s.e.m.) of 3 × CTCF-TetO insertions before (blue, 310 cells, 13,537 trajectories) or after (red, 240 cells, 8,788 trajectories) RAD21 degradation (90 min of auxin). h, MSD (mean ± s.e.m.) of 3 × CTCF-TetO before (blue, 336 cells, 6,687 trajectories) or after (red, 350 cells, 6,717 trajectories) WAPL degradation (24 h of auxin). i, Fold changes in generalized diffusion coefficients (D) and scaling exponents (α) in untreated cells compared with cells where degradation of CTCF, RAD21 and WAPL or removal of CTCF motifs (3 × CTCF) occurred.

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