Chromatin folding contributes to the regulation of genomic processes such as gene activity. Existing conformation capture methods characterize genome topology through analysis of pairwise chromatin contacts in populations of cells but cannot discern whether individual interactions occur simultaneously or competitively. Here we present multi-contact 4C (MC-4C), which applies Nanopore sequencing to study multi-way DNA conformations of individual alleles. MC-4C distinguishes cooperative from random and competing interactions and identifies previously missed structures in subpopulations of cells. We show that individual elements of the β-globin superenhancer can aggregate into an enhancer hub that can simultaneously accommodate two genes. Neighboring chromatin domain loops can form rosette-like structures through collision of their CTCF-bound anchors, as seen most prominently in cells lacking the cohesin-unloading factor WAPL. Here, massive collision of CTCF-anchored chromatin loops is believed to reflect ‘cohesin traffic jams’. Single-allele topology studies thus help us understand the mechanisms underlying genome folding and functioning.

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We thank the Utrecht Sequencing Facility for providing sequencing data and service, E. Schijlen for help with initial Pacific Biosciences sequencing, and D. Leyton-Puig for help with imaging. We thank N. Geijsen and P. Shang (Hubrecht Institute) for providing Cas9 protein. This work was supported by an NWO VIDI grant (639.072.715) to J.d.R. and an NWO/CW TOP grant (714.012.002) and NWO VICI grant (724.012.003) to W.d.L. and by the NIH Common Fund Program, grant U01CA200147, as a Transformative Collaborative Project Award (TCPA, TCPA-2017-DE-LAAT).

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Author notes

  1. These authors contributed equally: Amin Allahyar, Carlo Vermeulen.


  1. Center for Molecular Medicine, University Medical Center, Utrecht University, Utrecht, the Netherlands

    • Amin Allahyar
    • , Roy Straver
    • , Ivo J. Renkens
    • , Wigard P. Kloosterman
    •  & Jeroen de Ridder
  2. Delft Bioinformatics Lab, Faculty of Electrical Engineering, Mathematics and Computer Science, Delft University of Technology, Delft, the Netherlands

    • Amin Allahyar
  3. Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands

    • Carlo Vermeulen
    • , Britta A. M. Bouwman
    • , Peter H. L. Krijger
    • , Marjon J. A. M. Verstegen
    • , Geert Geeven
    • , Melissa van Kranenburg
    • , Mark Pieterse
    •  & Wouter de Laat
  4. Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, the Netherlands

    • Judith H. I. Haarhuis
    •  & Benjamin D. Rowland
  5. Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands

    • Kees Jalink
  6. Oncode Institute and Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, the Netherlands

    • Hans Teunissen
    •  & Elzo de Wit


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A.A. designed and performed the computational analysis, prepared corresponding plots and wrote the methods and Supplementary Information sections. C.V. and B.A.M.B. designed and performed experiments. C.V. wrote the manuscript and designed figures. P.H.L.K., M.J.A.M.V., M.v.K., M.P. and H.T. performed ‘C’ methods experiments. R.S. implemented the pipeline in Python. J.H.I.H. generated ∆WAPL cell lines and prepared microscopic slides for super-resolution imaging. K.J. guided acquisition and analyzed super-resolution microscopy data. I.J.R. performed and W.P.K. designed and supervised MinION sequencing experiments. B.D.R. supervised the generation of ∆WAPL cell lines and preparation of microscopic slides for super-resolution imaging. G.G. and E.d.W. helped with computational analysis. E.d.W. performed data analysis on the ΔWAPL Hi-C data. J.d.R. designed and supervised the computational analyses and pipelines and cowrote the manuscript. W.d.L. conceived and supervised the study and wrote the manuscript.

Competing Interests

C.V., B.A.M.B., P.H.L.K., M.J.A.M.V. and G.G. are shareholders of Cergentis. E.d.W. is cofounder and shareholder of Cergentis. W.d.L. is founder and shareholder of Cergentis. J.d.R. is cofounder and shareholder of Cyclomics.

Corresponding authors

Correspondence to Jeroen de Ridder or Wouter de Laat.

Supplementary information

  1. Supplementary Figures

    Supplementary Figures 1–16

  2. Reporting Summary

  3. Supplementary Table 1

    Statistics of the MC-4C experiments. Shown, per experiment, are the total number of reads sequenced per experiment (Raw read column), number of reads with more than one fragment—excluding VP—in the region of interest (Informative), number of reads with at least one unique molecular identifier fragment allowing a check for PCR duplication (Has far cis/trans UMI), number of independent alleles after removing PCR duplicates (PCR filtered unique reads), and the number of MinION sequencing runs that were pooled for each experiment (Sequence runs). The numbers in parentheses are percentage of reads remaining after each step of filtering compared to total number of reads sequenced

  4. Supplementary Table 2

    Primers used in this study. The primers used for each individual viewpoint, and the coordinates of each region of interest. FW and RV primers are used for MC-4C PCR; A and B refer to the FW primer used to synthesize the gRNA for the upstream and downstream neighboring fragment (respectively) of the viewpoint; VP is the primer used to synthesize the gRNA designed on the viewpoint fragment

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