CTCF Binding Polarity Determines Chromatin Looping

CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure. [Display omitted] •CTCF binding polarity determines chromatin looping•Inverted or disengaged CTCF sites do not necessarily form new chromatin loops•Cohesin recruitment to CTCF sites is independent of loop formation•Phenocopied linear but altered 3D chromatin landscape can affect gene expression CCCTC-binding factor (CTCF) shapes the three-dimensional genome. Here, de Wit et al. provide direct evidence for CTCF binding polarity playing an underlying role in chromatin looping. Cohesin association persists, but inverted CTCF sites fail to loop, which can sometimes lead to long-range changes in gene expression.