Molecular Mechanism of Directional CTCF Recognition of a Diverse Range of Genomic Sites

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 the crystal structures of four human CTCF-CBS complexes of the protocadherin (Pcdh) clusters, and 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 2-3, and module 1, respectively, with ZF8 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 and lack of 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 Examination of CTCF binding sites in 293T cell.

CCCTC结合因子（CTCF）是一种保守的三维基因组架构蛋白，其通过11个锌指（zinc finger, ZF）结构域与大量CTCF结合位点（CTCF-binding site, CBS）内四个模块的特定序列元件进行方向性结合，从而保障全基因组范围内染色质环化相互作用的正常进行。本研究解析了原钙粘蛋白（protocadherin, Pcdh）基因簇中四个人类CTCF-CBS复合物的晶体结构，并揭示：CTCF对原钙粘蛋白增强子与启动子的同源CBS进行方向性结合的机制，是通过将其ZF3、ZF4-7以及ZF9-11分别嵌入CBS所在的DNA大沟中，从而依次特异性识别模块4、模块2-3与模块1；其中ZF8作为间隔元件，适配模块1与模块2之间的可变间距。此外，与模块2-3中心位置的不对称腺嘌呤（"A"）碱基形成的接触，对于识别缺乏模块1且核心序列对称的CBS具有关键作用。进一步而言，CTCF可耐受简并CBS序列中特定位置的碱基变异，这使得其能够在全基因组范围内结合多种不同的CBS。综上，这些复合物结构为解析CTCF在整个人类基因组中与同源位点多价结合的方向性、多样性、灵活性、动态性及保守性的分子机制提供了重要见解；本研究同时对293T细胞中的CTCF结合位点进行了检测。