Abstract
CTCF, a conserved 3D genome architecture protein, determines proper genome-wide chromatin looping interactions through directional binding to specific sequence elements of four modules within numerous CTCF-binding sites (CBSs) by its 11 zinc fingers (ZFs). Here, we report four crystal structures of human CTCF in complex with CBSs of the protocadherin (Pcdh) clusters. We show that directional CTCF binding to cognate CBSs of the Pcdh enhancers and promoters is achieved through inserting its ZF3, ZFs 4-7, and ZFs 9-11 into the major groove along CBSs, resulting in a sequence-specific recognition of module 4, modules 3 and 2, and module 1, respectively; and ZF8 serves as a spacer element for variable distances between modules 1 and 2. In addition, the base contact with the asymmetric “A” in the central position of modules 2-3, is essential for directional recognition of the CBSs with symmetric core sequences but lacking module 1. Furthermore, CTCF tolerates base changes at specific positions within the degenerated CBS sequences, permitting genome-wide CTCF binding to a diverse range of CBSs. Together, these complex structures provide important insights into the molecular mechanisms for the directionality, diversity, flexibility, dynamics, and conservation of multivalent CTCF binding to its cognate sites across the entire human genome.
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Acknowledgements
We thank the staffs from BL18U1 and BL19U1 beamlines of National Center for Protein Science Shanghai (NCPSS) at Shanghai Synchrotron Radiation Facility (SSRF), and staffs of BL-17U1 beamline at SSRF for assistance with data collection. We thank Dr Lingling Chen (Institute of Biochemistry and Cell Biology, Shanghai) for providing the human CTCF construct. We thank Hongjie Zhang at the IBP radioactive isotope laboratory for the guidance in handling radiolabeled chemicals. We also thank Dr Torsten Juelich for critical reading of the manuscript and linguistic assistance. This work was supported by the Ministry of Science and Technology of China (2017YFA0504203 and 2014CB910102), the National Natural Science Foundation of China (31630015, 91440201, 31571335, 31400640, 31630039, 91640118 and 31470820), the Science and Technology Commission of Shanghai Municipality (14JC1403601), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08010203).
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Supplementary information
Supplementary information, Figure S1
Orientation of diverse CBS repertoires conserved in the promoter and enhancer regions of the three human Pcdh gene clusters. (PDF 952 kb)
Supplementary information, Figure S2
Crystal structure of CTCF ZFs 4-8 bound the DNA HS5-1a. (PDF 759 kb)
Supplementary information, Figure S3
Crystal structure of CTCF ZFs 4-8 bound the eCBS of the Pcdhα8 promoter. (PDF 634 kb)
Supplementary information, Figure S4
Three types of diverse CBSs in the human genome revealed by ChIP-nexus. (PDF 547 kb)
Supplementary information, Figure S5
Crystal structure of CTCF ZFs 6-11 bound to DNA γb7CSE, which contains module 1 and modules 2-3. (PDF 480 kb)
Supplementary information, Figure S6
In vivo mapping of CTCF binding borders by ChIP-nexus. (PDF 1771 kb)
Supplementary information, Figure S7
Molar mass determination of CTCF-HS5-1aR by static light scattering. (PDF 509 kb)
Supplementary information, Table S1
Crystallographic data collection and refinement statistics. (PDF 145 kb)
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Yin, M., Wang, J., Wang, M. et al. Molecular mechanism of directional CTCF recognition of a diverse range of genomic sites. Cell Res 27, 1365–1377 (2017). https://doi.org/10.1038/cr.2017.131
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DOI: https://doi.org/10.1038/cr.2017.131
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