An ectopic enhancer restores CFTR expression through de novo chromatin looping.

Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by both ubiquitous and cell-type selective cis-regulatory elements (CREs). These CREs include extragenic and intronic enhancers that bind lineage-specific transcription factors, and architectural protein-marked structural elements. Deletion of the airway-selective -35 kb enhancer in 16HBE14o- lung epithelial cells was shown earlier to disrupt normal enhancer-promoter looping at the CFTR locus and nearly abolish its expression. Using a 16HBE14o- clone that lacks the endogenous -35 kb CRE, we explore the impact of relocating the functional core of this element to an ectopic site in intron 1. The -35 kb sequence establishes a de novo enhancer signature in chromatin at the insertion site, and augments CFTR expression, albeit not fully restoring WT levels. The relocated -35 kb enhancer also initiates de novo chromatin contacts with the CFTR promoter and other known CFTR CREs. These results are broadly relevant to therapeutic gene editing.