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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.

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Fig. 1: Relocation of the -35 kb enhancer partially restores CFTR expression in 16HBE14o cells.
Fig. 2: Relocation of the CFTR -35kb enhancer element creates open chromatin at the insertion site in intron 1.
Fig. 3: The -35kb enhancer influences 3D structure of the CFTR locus independent of its location.
Fig. 4: The -35kb enhancer establishes an enhancer chromatin signature independent of genomic location.
Fig. 5: Model to summarize the impact on CFTR locus 3D structure of relocating the -35kb enhancer to a site in intron 1.

Data availability

Data are deposited at NCBI GEO accession GSE203560.


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We thank Drs. Shih-Hsing Leir and Tom Kelley for helpful discussion and the Epithelial Cell Core at the CWRU CF Center (Cystic Fibrosis Foundation RDP R447-CR11) for Ussing chamber measurements. Also, Dr. Vian Peshdary for reagent generation, and the CWRU School of Medicine Genomics Core for sequencing.


This work was supported by the Cystic Fibrosis Foundation (Davis19XX0) and NIH HL094585 and HD068901 (both to AH).

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Conceptualization: JLK and AH. Methodology: JLK and AH. Validation: JLK and AP. Formal analysis: JLK and AP. Investigation: JLK, NV, and MDW. Data curation: AP. Writing – original draft: JLK and AH. Writing – review & editing: JLK and AH. Visualization: JLK. Supervision: AH. Project administration: AH. Funding acquisition: AH.

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Correspondence to Ann Harris.

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Kerschner, J.L., Paranjapye, A., Vaghela, N. et al. An ectopic enhancer restores CFTR expression through de novo chromatin looping. Gene Ther (2022).

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