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Guided nuclear exploration increases CTCF target search efficiency

Abstract

The enormous size of mammalian genomes means that for a DNA-binding protein the number of nonspecific, off-target sites vastly exceeds the number of specific, cognate sites. How mammalian DNA-binding proteins overcome this challenge to efficiently locate their target sites is not known. Here, through live-cell single-molecule tracking, we show that CCCTC-binding factor, CTCF, is repeatedly trapped in small zones that likely correspond to CTCF clusters, in a manner that is largely dependent on an internal RNA-binding region (RBRi). We develop a new theoretical model called anisotropic diffusion through transient trapping in zones to explain CTCF dynamics. Functionally, transient RBRi-mediated trapping increases the efficiency of CTCF target search by ~2.5-fold. Overall, our results suggest a ‘guided’ mechanism where CTCF clusters concentrate diffusing CTCF proteins near cognate binding sites, thus increasing the local ON-rate. We suggest that local guiding may allow DNA-binding proteins to more efficiently locate their target sites.

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Fig. 1: spaSPT reveals anisotropic CTCF diffusion in the nucleus.
Fig. 2: A model wherein CTCF diffusion in the nucleus is governed by its interaction with trapping zones can explain the experimental data.
Fig. 3: Anisotropy and nuclear distribution of ΔRBRi-CTCF.
Fig. 4: Direct evidence that TTZs correspond to CTCF clusters.
Fig. 5: RBRi-guided CTCF target search mechanism.
Fig. 6: Model.

Data availability

Raw and processed SPT data is freely available at Zenodo: https://zenodo.org/record/2208323. All cell lines will be provided upon request.

Code availability

Raw code as well as a detailed description of how the data was analyzed is available on GitLab: https://gitlab.com/anders.sejr.hansen/anisotropy. The code for localization and tracking is also available on GitLab: https://gitlab.com/tjian-darzacq-lab/SPT_LocAndTrack. The code for performing Brownian motion simulations (Supplementary Figs. 2a–c and 3) is likewise available on GitLab: https://gitlab.com/tjian-darzacq-lab/simSPT. Finally, the PALM-analysis code is also available on GitLab: https://gitlab.com/anders.sejr.hansen/palm_pipeline/.

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Acknowledgements

We thank L. Lavis for generously providing JF dyes, M. Woringer for insightful discussions and help with simSPT, A. Tangara and A. Robles for microscope assembly and maintenance, G. Dailey for help and assistance with cloning, K. Heydari at the Li Ka Shing Facility for flow cytometry assistance and A. deHart, L. Witowsky, A. Manford, L. Dahal and A. Basil Heckert for discussions and help with fluorescence polarization experiments. We thank A. Seeber, K. Dao Duc, D. McSwiggen and other members of the Tjian and Darzacq laboratories for comments on the manuscript. This work was performed in part at the CRL Molecular Imaging Center, supported by the Gordon and Betty Moore Foundation. A.S.H. was a postdoctoral fellow of the Siebel Stem Cell Institute and is supported by a National Institutes of Health (NIH) NIGMS K99 Pathway to Independence Award (no. K99GM130896). This work was supported by NIH grant nos. UO1-EB021236 and U54-DK107980 (X.D.), the California Institute of Regenerative Medicine grant no. LA1–08013 (X.D.) and by the Howard Hughes Medical Institute (003061, R.T.).

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Contributions

A.S.H., A.A., R.T. and X.D. conceived of the project. A.S.H. and A.A. conceived of the ADTZ model. A.S.H. performed the experiments, developed anisotropy analysis pipeline, analyzed the experimental data and performed Brownian motion simulations. A.A. developed the theoretical framework, performed and analyzed model simulations. A.S.H. and C.C. generated the C59D2 ΔRBRi-Halo-CTCF mESC line. C.C. performed in vitro CTCF binding assays. A.S.H. and A.A. drafted the manuscript and all authors edited the manuscript. R.T. and X.D. supervised the project. A.S.H. and A.A. contributed equally to this project.

Corresponding authors

Correspondence to Robert Tjian or Xavier Darzacq.

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Supplementary information

Supplementary Information

Supplementary Tables 2 and 3, Figs. 1–15 and Notes 1–3.

Reporting Summary

Supplementary Table 1

Comparison of vbSPT and Spot-On. Effect of HMM (vbSPT) on filtering out bound population.

Supplementary Video 1

Comparison of vbSPT and Spot-On. Effect of HMM (vbSPT) on filtering out bound population.

Supplementary Video 2

Single Halo-CTCF protein exhibiting anomalous diffusion inside the mESC nucleus.

Supplementary Video 3

Single Halo-CTCF protein exhibiting anomalous diffusion inside the mESC nucleus.

Supplementary Video 4

Single Halo-CTCF protein exhibiting anomalous diffusion inside the mESC nucleus.

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Hansen, A.S., Amitai, A., Cattoglio, C. et al. Guided nuclear exploration increases CTCF target search efficiency. Nat Chem Biol 16, 257–266 (2020). https://doi.org/10.1038/s41589-019-0422-3

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