CFTR Gene Regulation in Human Pancreatic Duct, Bile Duct and Sweat Gland Epithelial Cells

Abstract

Epithelial cells at many sites in the body are affected by the inherited disorder cystic fibrosis (CF). The lung was the major focus of research until recently, when effective therapeutics became available for most people with CF. There is now renewed interest in CF aetiology in other locations in the body, where the regulatory mechanisms for the CF transmembrane conductance regulator (CFTR) gene are less well-characterised. The definition of the genomic elements controlling CFTR expression and their associated transcription factors is important for the design of gene-based therapies. Here we identify the cis-regulatory elements (CREs) associated with the CFTR locus by open chromatin mapping in pancreatic adenocarcinoma cell lines, primary human pancreatic and bile duct (cholangiocyte) organoids and single cells from tissues, as well as sweat gland coil and duct epithelial cells. We show that broadly these cell types use a combination of CREs that were characterised previously either in airway or intestinal epithelial cells, though not occurring together in these two cell lineages. Moreover, the chromatin structure of the CFTR locus in pancreatic cell lines is consistent with earlier models. We also use bioinformatic tools to predict the transcription factor network in these rare cell lineages from open chromatin peaks genome-wide.

Keywords: ATAC‐seq/snATAC‐seq; bile duct/cholangiocyte; cis‐regulatory elements; cystic fibrosis transmembrane conductance regulator gene; pancreatic duct; sweat gland.

FIGURE 1

CFTR protein is expressed at a low level in Capan‐1 pancreatic adenocarcinoma cells but is absent from BxPC‐3 cells. The western blot shows protein extracted from BxPC‐3, Capan‐1, HT‐29 and NP31 cells. CFTR protein is detected with Ab596 compared to the β‐tubulin loading control.

FIGURE 2

Open chromatin maps at the CFTR locus in pancreatic adenocarcinoma cells compared to intestinal (Caco‐2) and airway (Calu‐3) epithelial cells. Open chromatin maps generated by Omni‐ATAC‐seq of BxPC‐3, Capan‐1, NP31, Caco‐2 and Calu‐3 cell lines are shown. Each track shows merged data from 2 technical replicates. The locations of ASZ1, CFTR and CTTNBP2 are shown at the top of the figure. Key DHS identified at the CFTR locus in all cells, and other cell‐selective DHS of interest are marked below the gene track in black. This manuscript uses legacy nomenclature for the CFTR gene to be consistent with our earlier work (see Table 1 for conversion to RefSeq). TAD boundaries are shown by blue boxes at −80.1 and +48.9 kb. Airway selective DHS at −35, −44 and +36.6 kb are shown by teal boxes. Intestinal selective DHS at intron 10a,b, intron 10c, intron 11, intron 20, intron 23 and +15.6 kb are shown by brown boxes.

FIGURE 3

Open chromatin maps at the CFTR locus in primary pancreatic duct cell and cholangiocyte organoids compared to intestinal (Caco‐2) and airway (Calu‐3) epithelial cells. Open chromatin maps generated by Omni‐ATAC‐seq on pancreas organoids, HES‐derived cholangiocyte organoids and Caco‐2 and Calu‐3 cell lines are shown. Each track shows merged data from 2 technical replicates. The locations of ASZ1, CFTR and CTTNBP2 are shown at the top of the figure. Key DHS identified at the CFTR locus in all cells, and other cell‐selective DHS of interest are marked below the gene track in black. TAD boundaries, airway‐selective DHS and intestinal DHS are marked by blue, teal and brown boxes, respectively, as described in the Figure 2 legend. Open chromatin peaks described in the results section are marked by arrowheads.

FIGURE 4

Open chromatin maps at the CFTR locus in human sweat gland coil and duct epithelial cells compared to intestinal (Caco‐2) and airway (Calu‐3) epithelial cells. Open chromatin maps generated by Omni‐ATAC‐seq on human sweat gland coil and duct epithelial cells and Caco‐2 and Calu‐3 cell lines are shown. Each track shows merged data from 2 technical replicates. The locations of ASZ1, CFTR and CTTNBP2 are shown at the top of the figure. Key DHS identified at the CFTR locus in all cells, and other cell‐selective DHS of interest are marked below the gene track in black. TAD boundaries, airway selective DHS and intestinal DHS are marked by blue, teal and brown boxes, respectively, as described in the Figure 2 legend. Open chromatin peaks described in the results section are marked by grey and red arrowheads.

FIGURE 5

CFTR expression levels correlate with active and repressive histone modifications at the gene promoter and at specific CREs across the locus in pancreatic adenocarcinoma cell lines. (A) Map of active histone (H3K27Ac) modifications generated by ChIP‐seq, aligned to the same ATAC‐seq peak tracks shown in Figure 2 in NP31 and Capan‐1 cells. An individual ChIP‐seq experiment is illustrated for each line, below which are shown IDR peaks from replicate experiments. (B) ChIP for H3K27ac in (i) BxPC‐3; (iii) NP31; (v) Capan‐1 cells. ChIP for H3K27me3 in (ii) BxPC‐3; (iv) NP31; (vi) Capan‐1. All ChIP datasets are normalised to percent input. n = 3. Enrichment level statistics: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05 using a two‐way ANOVA with Sidak's multiple comparison test.

FIGURE 6

Open chromatin maps at the CFTR locus in key cell types from human pancreas and liver tissue by snATAC‐seq. (A) Pancreatic duct cell, acinar cell and beta cell snATAC‐seq compared to ATAC‐seq from Capan‐1 cells and pancreatic organoids (Figure 2). (B) Cholangiocyte, hepatocyte and liver endothelial cell snATAC‐seq compared to ATAC‐seq from cholangiocyte organoids (Figure 3). Data from [70, 71]. Key: Blue box: TAD boundary; teal box and arrows: Airway CREs; brown box and arrows: intestinal CREs, as described in the discussion. Blue arrows: Promoter; red arrows: Intron 15 CRE; purple arrows: +6.8 kb CTCF‐binding insulator element. Legacy nomenclature.