CCCTC-binding Factor N-terminal Domain Regulates Clustered Protocadherin Gene Expression by Enhancing Cohesin Processivity

CTCF (CCCTC-binding factor) instructs 3D genome folding by anchoring or forestalling cohesin loop extrusion, but the exact mechanism remains obscure. Here, using clustered protocadherins (cPcdh) as model genes, we report that CTCF assists or facilitates cohesin loop extrusion by enhancing its processivity. Specifically, we show that, compared with the Pcdh alpha and gamma clusters, the Pcdh beta cluster is greatly affected upon CTCF Y226A/F228A mutation in the N-terminal domain. Given the long-range distance of the Pcdh beta cluster from the distal enhancer, this finding has interesting implications in CTCF regulation of cohesin processivity along the linear chromatin during DNA loop extrusion. In particular, the effect on cohesin processivity upon CTCF Y226A/F228A mutation is conspicuously similar to that of WAPL overexpression, suggesting that, in addition to the general view of blocking or forestalling cohesin, CTCF may also enhances or facilitates cohesin loop extrusion. Here, through CRISPR editing of the YDF motif—the key cohesin interacting moiety—of the CTCF N-terminal domain (NTD) in N2a cells in vitro and in mice in vivo, in conjunction with RNA-seq, ChIP-seq, 4C, and Hi-C analyses, we found that members of the Pcdh beta cluster are highly susceptible to the perturbed cohesin processivity owing to their distal genomic arrangement relative to the downstream super-enhancer region. Our data broaden the molecular logic that generates Pcdh isoform diversity required for neural circuit assembly via a unique 3D genome organization.