Cross-talk between enhancers, structural elements and activating transcription factors maintains the 3D architecture and expression of the CFTR gene.. Cross-talk between enhancers, structural elements and activating transcription factors maintains the 3D architecture and expression of the CFTR gene.

Here we examine the mechanisms by which multiple cis-regulatory elements (CREs) at the CFTR locus coordinate its expression in intestinal epithelial cells. Using CRISPR/Cas9 to remove CREs, individually and in tandem, followed by assays of gene expression and higher-order chromatin structure (4C-seq), we reveal the cross-talk and dependency of two cell-specific intronic enhancers. The results also suggest an internal monitoring mechanism at the locus whereby alternative enhancers may be recruited when the normal sites are lost. Of note, activating transcription factors such as FOXA2 may contribute to this mechanism. Also, by deleting the 5' topologically-associating domain (TAD) boundary, we illustrate its relative contribution to normal mechanisms of CFTR gene expression and 3D gene architecture. Overall design: Analysis of the 3D chromatin architecture at the CFTR locus to determine interaction frequencies between elements which lead to changes in CFTR gene regulatory patterns.