A negatively-charged region within carboxy-terminal domain maintains proper CTCF DNA binding

As an essential regulator of higher-order chromatin structures, CTCF is a highly-conserved DNA binding protein with a central DNA-binding domain of 11 tandem zinc fingers, which are flanked by N- and C-terminal domains of intrinsically disordered regions. The N-terminal domain interacts with cohesin complex and the central zinc finger domains recognize a large range of diverse genomic sites. However, the function of unstructured C-terminal domain remains unknown. Here we report that CRISPR deletion of the entire C-terminal domain of alternating charge blocks decreases CTCF DNA binding but deletion of C-terminal fragment of 116 amino acids results in increased CTCF DNA binding and aberrant gene regulation. Through a series of genetic targeting experiments, in conjunction with ChIP-seq and ATAC-seq, we uncovered a negatively-charged region responsible for weakening CTCF DNA binding and chromatin accessibility. Thus, the unstructured C-terminal domain plays an intricate role in maintaining proper CTCF-DNA interactions and 3D genome organization.