A promoter-dependent upstream activator augments CFTR expression in diverse epithelial cell types
The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an anion-selective channel found in epithelial cell membranes. Mutations in CFTR cause cystic fibrosis (CF), an inherited disorder that impairs epithelial function in multiple organs. Most men with CF are infertile due to loss of intact genital ducts. Here we investigated a novel epididymis-selective cis-regulatory element (CRE), located within a peak of open chromatin at -9.5 kb 5' to the CFTR gene promoter. Activation of the -9.5 kb CRE alone by CRISPRa had no impact on CFTR gene expression. However, CRISPRa co-activation of the -9.5 kb CRE and the CFTR gene promoter in epididymis cells significantly augmented CFTR mRNA and protein expression when compared to promoter activation alone.  This increase was accompanied by enhanced chromatin accessibility at both sites. Furthermore, the combined CRISPRa strategy activated CFTR expression in other epithelial cells that lack open chromatin at the -9.5kb site and in which the locus is normally inactive. However, the -9.5 kb CRE does not function as a classical enhancer of the CFTR promoter in transient reporter gene assays.  These data provide a novel mechanism for activating/augmenting CFTR expression, which may have therapeutic utility for mutations that perturb CFTR transcription. Overall design: Single guide RNAs (sgRNAs) targeting the -9.5 Kb DHS and the -44 Kb DHS were designed using Benchling. Two sgRNAs were used for each DHS. The sgRNA targeting CFTR promoter was described elsewhere (Mendenhall, 2021, Sharma et al., 2021).  sgRNAs were cloned into the pSPgRNA plasmid (Addgene #47108 (Perez-Pinera et al., 2013)) and transfected with the SP-dCas9-VPR plasmid (Addgene #63798, a gift from George Church (Chavez et al., 2015))  into HEP cells (Coleman and Harris 1991) with Lipofectamine 3000 (ThermoFisher). Cells were processed for ATAC-seq 48h post-transfection. Omni-ATAC was performed as described in Corces et al. 2017.