The regulated expression of large human genes can depend on long-range interactions to establish appropriate three-dimensional structures across the locus. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encompasses 189 kb of genomic DNA, shows a complex pattern of expression with both spatial and temporal regulation. The flanking loci, ASZ1 and CTTNBP2, show very different tissue-specific expression. The mechanisms governing control of CFTR expression remain poorly understood, although they are known to involve intronic regulatory elements. Here, we show a complex looped structure of the CFTR locus in cells that express the gene, which is absent from cells in which the gene is inactive. By using chromatin conformation capture (3C) with a bait probe at the CFTR promoter, we demonstrate close interaction of this region with sequences in the middle of the gene about 100 kb from the promoter and with regions 3 to the locus that are about 200 kb away. We show that these interacting regions correspond to prominent DNase I hypersensitive sites within the locus. Moreover, these sequences act cooperatively in reporter gene constructs and recruit proteins that modify chromatin structure. The model for CFTR gene expression that is revealed by our data provides a paradigm for other large genes with multiple regulatory elements lying within both introns and intergenic regions. We anticipate that these observations will enable original approaches to designing regulated transgenes for tissue-specific gene therapy protocols.cis-acting elements ͉ enhancer:promoter interactions ͉ regulation of expression ͉ cystic fibrosis transmembrane conductance regulator U nderstanding the three-dimensional organization of individual loci within the human genome and how this relates to the regulation of gene expression is the focus of intense study. Many new technologies are being developed to locate and classify the functional elements of the human genome (1). These elements may contribute to the transcription of ubiquitously expressed genes and are also likely responsible for regulating genes with expression that is temporally and spatially controlled. Cis-acting regulatory elements located within noncoding regions of genomic DNA can influence the organization of chromosomes and the transcriptional activity of genes. These cis sequences include distal enhancers that may reside large distances from the gene promoters they control. Variations in these enhancer/promoter interactions and the nuclear localization of the genes they regulate are thought to be contributing factors in the diversity of transcriptional profiles between different cell types. Moreover, they are important in adjusting these profiles throughout cellular differentiation and development (2-7). These long-range associations are facilitated by the looping of chromatin, whereby regulatory elements come together with requisite nuclear factors to function within ''transcriptional hubs'' where transcriptional activity is coordinated (8).Here, we present...

CFTR modulator theratyping is a novel and rapidly evolving field that has the potential to identify rare CFTR variants that are responsive to approved drugs or drugs in development.

Although loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl channel function is common to all epithelia in cystic fibrosis (CF) patients, the severity of disease varies in different organs. We hypothesized that differences in disease severity in CF relate to

Primary airway epithelial cells grown in air-liquid interface differentiate into cultures that resemble native epithelium morphologically, express ion transport similar to those in vivo, and secrete cytokines in response to stimuli. Comparisons of cultures derived from normal and cystic fibrosis (CF) individuals are difficult to interpret due to genetic differences besides CFTR. The recently discovered CFTR inhibitor, CFTRinh-172, was used to create a CF model with its own control to test if loss of CFTR-Cl− conductance alone was sufficient to initiate the CF inflammatory response. Continuous inhibition of CFTR-Cl− conductance for 3–5 days resulted in significant increase in IL-8 secretion at basal ( P = 0.006) and in response to 109 Pseudomonas ( P = 0.0001), a fourfold decrease in Smad3 expression ( P = 0.02), a threefold increase in RhoA expression, and increased NF-κB nuclear translocation upon TNF-α/IL-1β stimulation ( P < 0.000001). CFTR inhibition by CFTRinh-172 over this period does not increase epithelial sodium channel activity, so lack of Cl− conductance alone can mimic the inflammatory CF phenotype. CFTRinh-172 does not affect IL-8, IL-6, or granulocyte/macrophage colony-stimulating factor secretion in two CF phenotype immortalized cell lines: 9/HTEo− pCEP-R and 16HBE14o− AS, or IL-8 secretion in primary CF cells, and inhibitor withdrawal abolishes the increased response, so CFTRinh-172 effects on cytokines are not direct. Five-day treatment with CFTRinh-172 does not affect cells deleteriously as evidenced by lactate dehydrogenase, trypan blue, ciliary activity, electron micrograph histology, and inhibition reversibility. Our results support the hypothesis that lack of CFTR activity is responsible for the onset of the inflammatory cascade in the CF lung.