The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex temporal and spatial pattern of expression that is controlled by multiple cis-acting elements interacting with the basal promoter. Although significant progress has been made towards understanding these genomic elements, there have been no reports of post-transcriptional regulation of CFTR by miRNAs (microRNAs). In the present study, we identify two miRNAs, hsa-miR-145 and hsa-miR-494, which regulate CFTR expression by directly targeting discrete sites in the CFTR 3′ UTR (untranslated region). We show that at least 12 miRNAs are capable of repressing endogenous CFTR mRNA expression in the Caco-2 cell line. Ten of these also inhibit expression of a reporter construct containing the CFTR 3′ UTR in one or more cell lines, and five repress endogenous CFTR protein expression in Caco-2 cells. Moreover, at least three are expressed in primary human airway epithelial cells, where CFTR expression is maintained at low levels in comparison with intestinal cell lines. Three of the miRNAs that target CFTR, hsa-miR-384, hsa-miR-494 and hsa-miR-1246, also inhibit expression of a reporter carrying the Na⁺–K⁺–Cl⁻ co-transporter SLC12A2 [solute carrier family 12 (Na⁺–K⁺–Cl⁻ transporters), member 2] 3′ UTR, suggesting that these miRNAs may play a more general role in regulating chloride transport in epithelial cells.

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.

The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with high-mannose structures (Hex_5-9HexNAc₂) and complex/hybrid-type glycans (NeuAc_0-3Fuc_0-3Hex_3-8HexNAc_3-7). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N‐glycans on membrane-bound and secreted forms of the native molecule.

Region-specific transcriptional profiling of tissues and cultured epithelial cells from the human epididymis will predict functional specialization along the duct. We identified the molecular signature driving functions of the caput, corpus and cauda epithelium, and determined how these differ to establish the regional differentiation of the organ. The epithelium lining the human male genital ducts has a critical role in fertility. In particular, it controls the luminal environment in the epididymis, which is required for normal sperm maturation and reproductive competence. Studies in many animal species have largely informed our understanding of the molecular basis of epididymis function. However, there are substantial differences between species. Using RNA sequencing on biological replicates, we described gene expression profiles for tissue from each region of the epididymis and cultured epithelial cells derived from these regions. Bioinformatic tools were then utilized to identify differentially expressed genes (DEGs) between tissues and cells from the caput, corpus and cauda. The data showed that the caput is functionally divergent from the corpus and cauda, which have very similar transcriptomes. Interrogation of DEGs using gene ontology process enrichment analyses showed that processes of ion transport, response to hormone stimulus and urogenital tract development are more evident in the caput, while defense response processes are more important in the corpus/cauda. Consistent with these regional differences in epididymis function, we observed differential expression of transcription factors in the caput and corpus/cauda. Cultured caput, corpus and cauda cells may not faithfully represent the same cells in the intact organ, due to loss of hormonal signals from the testis and communication from other cell types. Our data provide a molecular characterization that will facilitate advances in understanding human epididymis epithelium biology in health and disease. They may also reveal the mechanisms coordinating epididymis luminal environment and sperm maturation. Data deposited at http://www.ncbi.nlm.nih.gov/geo/GSE72986. This work was supported by the National Institutes of Health: R01HD068901 (PI: A.H.). The authors declare no conflict of interest.

Genes can maintain spatiotemporal expression patterns by long‐range interactions between cis‐acting elements. The cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed primarily in epithelial cells. An element located within a DNase I‐hyper‐sensitive site (DHS) 10 kb into the first intron was previously shown to augment CFTR promoter activity in a tissue‐specific manner. Here, we reveal the mechanism by which this element influences CFTR transcription. We employed a high‐resolution method of mapping DHS using tiled microarrays to accurately locate the intron 1 DHS. Transfection of promoter‐reporter constructs demonstrated that the element displays classical tissue‐specific enhancer properties and can independently recruit factors necessary for transcription initiation. In vitro DNase I footprinting analysis identified a protected region that corresponds to a conserved, predicted binding site for hepatocyte nuclear factor 1 (HNF1). We demonstrate by electromobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) that HNF1 binds to this element both in vitro and in vivo. Moreover, using chromosome conformation capture (3C) analysis, we show that this element interacts with the CFTR promoter in CFTR‐expressing cells. These data provide the first insight into the three‐ dimensional (3D) structure of the CFTR locus and confirm the contribution of intronic cis‐acting elements to the regulation of CFTR gene expression.

Irreversible respiratory obstruction resulting from progressive airway damage, inflammation and fibrosis is a feature of several chronic respiratory diseases, including cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). The cytokine transforming growth factor β (TGF-β) has a pivotal role in promoting lung fibrosis and is implicated in respiratory disease severity. In the present study, we show that a previously uncharacterized miRNA, miR-1343, reduces the expression of both TGF-β receptor 1 and 2 by directly targeting their 3′-UTRs. After TGF-β exposure, elevated intracellular miR-1343 significantly decreases levels of activated TGF-β effector molecules, pSMAD2 (phosphorylated SMAD2) and pSMAD3 (phosphorylated SMAD3), when compared with a non-targeting control miRNA. As a result, the abundance of fibrotic markers is reduced, cell migration into a scratch wound impaired and epithelial-to-mesenchymal transition (EMT) repressed. Mature miR-1343 is readily detected in human neutrophils and HL-60 cells and is activated in response to stress in A549 lung epithelial cells. miR-1343 may have direct therapeutic applications in fibrotic lung disease.