New insights into the cis-regulation of the CFTR gene in pancreatic cells

Background Spatial organization of the genome is fundamental for ensuring accurate gene expression. This process depends on the communication between gene promoters and distal cis-regulatory elements (CREs), which together make up 8% of the human genome and are supported by the chromatin structure. It is estimated that over 90% of disease-associated variants are located in the non-coding region of the genome and may affect CRE. For the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a complete understanding of tissue-specific CFTR expression and regulation is missing, in particular in the pancreas. Mechanistic insights into tissue-specific expression may provide clarity on the clinical heterogeneity observed in Cystic Fibrosis and CFTR-related disorders.

To understand the role of 3D chromatin architecture in establishing tissue-specific expression of the CFTR gene, we mapped chromatin interactions via circular chromosome conformation capture (4C) and epigenomic regulation through H3K27ac and DNase Hypersensitive site I (DHS) in Capan-1 pancreatic cells. Candidate regulatory regions are validated by luciferase reporter assay and CRISPR-knock out.

We identified active regulatory regions not only around the CFTR gene but also outside the topologically associating domain (TAD). By performing functional assays, we validated our targets and revealed a cooperative effect of the −44 kb, −35 kb, +15.6 kb and 37.7 kb regions, which share common predicted transcription factor (TF) motifs. Comparative 3D genomic analysis and functional assays using the Caco-2 intestinal cell line revealed the presence of tissue-specific CREs.

By studying the chromatin architecture of the CFTR locus in Capan-1 cells, we demonstrated the involvement of multiple CREs upstream and downstream of the CFTR gene. We also extend our analysis to compare intestinal and pancreatic cells and provide information on the tissue-specificity of CRE. These findings highlight the importance of expanding the search for causative variants beyond the gene coding sequence but also by considering the tissue-specific 3D genome. Competing Interest Statement The authors have declared no competing interest. List of abbreviations - 4C - circular chromosome conformation capture - ABC - activity-by-contact - ATAC - assay for transposase-accessible chromatin - CBAVD - congenital bilateral absence of vas deferens - CF - cystic fibrosis - CFTR - cystic fibrosis transmembrane conductance regulator - CFTR-RD - CFTR-related diseases - cCRE - candidate cis-regulatory elements - CRE - cis-regulatory elements - CRISPR - clustered regularly interspaced short palindromic repeats - CTCF - CCCTC-binding factor - DHS - DNase I hypersensitive site - CUT&RUN - Cleavage Under Targets & Release Using Nuclease - gRNA - guide RNA - PTM - post-translational modifications - TAD - topologically associating domain - TF - transcription factor - TSS - transcription start site