CFTR Promoter Research Articles

The Secondary Bile acid, Lithocholic Acid, Inhibits Cl− Secretion Across Colonic Epithelial Cells in Vitro

Lithocholic acid (LCA) is a secondary bile acid formed in the colon by bacterial metabolism of chenodeoxycholic acid. Our previous studies have shown the bile acid to be protective under conditions of colonic inflammation. Here, we sought to determine if LCA might also play a role in regulating fluid and electrolyte transport. T84 cell monolayers were mounted in Ussing chambers for measurements of Cl− secretion, the primary driving force for fluid secretion into the colon. qRT-PCR and Western blotting were used to analyze mRNA and protein expression. Results were expressed as mean ± SEM and data were analyzed by one way ANOVA and Tukey’s post hoc test or by mixed-effects analysis and Dunnett's post hoc test. To assess the effects of LCA on CFTR promoter activity, we used a luciferase promoter/reporter system, where HEK293 cells were transfected with a plasmid containing the firefly luciferase gene under control of the CFTR promoter. Co-transfections were carried out with 2 additional plasmids expressing either the nuclear bile acid receptor, FXR, or its dimerization partner, RXR. Pretreatment of T84 cell monolayers with LCA inhibited subsequent Cl− secretory responses to the cAMP-dependent agonist, forskolin (FSK; 10 μM), in a concentration (1 – 10 μM) and time-dependent (3 - 24 hrs) manner. Maximal effects of LCA were observed at a concentration of 10 μM after treatment for 24 hrs, when responses to FSK were reduced to 50.9 ± 8.5% of those in controls (n = 6; p < 0.01). LCA (10 μM; 24 hrs) also inhibited responses to the Ca2+-dependent secretagogues, thapsigargin (2 μM) and histamine (100 μM), by 59.4 ± 2.4% (n = 4; p < 0.001) and 52.2 ± 1.9% (n = 5; p < 0.001), respectively. In further experiments, using nystatin-permeabilized T84 monolayers to isolate apical Cl− conductances, LCA (10 μM; 24 hrs) reduced FSK-stimulated responses to 72.7 ± 6.6% (n = 17; p < 0.001) of those in control cells. Analysis of CFTR expression, the primary exit pathway for Cl− in colonic epithelial cells, revealed that LCA treatment reduced mRNA and protein expression of the channel to 0.65 ± 0.05 fold (n = 7; p < 0.01) and 0.43 ± 0.06 fold (n = 6; p < 0.001), respectively. In CFTR promoter assays, LCA (10 μM) reduced CFTR promoter activity to 0.7 ± 0.02 fold of that in control cells (n = 5; p < 0.01), with co-expression of FXR being required for this effect to be observed. Finally, while LCA activated both FXR and the vitamin D receptor (VDR) in T84 cells, its effects in downregulating CFTR expression and Cl− conductances were mimicked by the FXR agonist, GW4064, but not by the VDR agonist, calcitriol. In conclusion, LCA, at physiologically relevant concentrations, inhibits Cl− secretion across colonic epithelial cells, likely through a mechanism involving FXR activation and inhibition of CFTR expression. These data add to the growing pool of knowledge regarding important regulatory actions of LCA in the colon and highlight its potential role as a target for the treatment of intestinal disorders. This work was supported by a grant from Science Foundation Ireland. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

DNA Methylation-Mediated Low Expression of CFTR Stimulates the Progression of Lung Adenocarcinoma.

In recent years, the mortality rate of lung adenocarcinoma (LUAD) is persistently increasing, which has already caused a huge impact on human living standards. Hence, there is an urgent need to probe the molecular mechanism of LUAD progression, so as to disclose prognostic and diagnostic markers for patients with LUAD. Methylation 450K data and mRNA expression data of LUAD were obtained via bioinformatics analysis to screen methylation-driven genes. The expression of the target gene was detected through qRT-PCR, while the methylation level was evaluated via methylation-specific PCR (MSP). The impact of the gene on cell proliferation, migration, invasion, apoptosis and cell cycle was measured through CCK-8, wound healing, Transwell invasion assay, and flow cytometry. CFTR was defined by bioinformatics analysis as the target gene for this study. qRT-PCR revealed that CFTR was lowly expressed in LUAD cells. MSP displayed that the CFTR promoter region in LUAD cells was hypermethylated, and demethylation could pronouncedly increase the level of CFTR mRNA in LUAD cells. Cell biological functional experiments exhibited that CFTR hindered cell proliferation, migration, and invasion, fostered cell apoptosis of LUAD, and blocked the cell cycle in G2-M phase. CFTR was hypermethylated in LUAD, which mediated the low expression of CFTR in LUAD to stimulate the progression of LUAD.

CFTR Functions as a Tumor Suppressor and Is Regulated by DNA Methylation in Colorectal Cancer.

PurposeCystic fibrosis transmembrane conductance regulator (CFTR) was shown to be downregulated or silenced in carcinomas and acts as a candidate tumor suppressor gene. However, the function of CFTR gene in colorectal cancer (CRC) is still unclear. This aim of this study was to investigate the CFTR promoter methylation status and its impact on the expression and functional role of CFTR in CRC development.Patients and MethodsCFTR expression in CRC tissues and CRC cell lines was detected via quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The promoter methylation status of CFTR was measured using methylation-specific PCR (MSP). colony formation, transwell, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to evaluate the effect of CFTR overexpression in CRC cell lines.ResultsqRT-PCR and IHC results indicated that CFTR expression was downregulated in the CRC tissues compared to the adjacent normal tissues. The promoter methylation status of CFTR was further analyzed in 70 CRC specimens. MSP validation showed methylation of CFTR promoter in 62.2% (45/70) of CRC tissues. The methylation of CFTR promoter was significantly associated with age (P=0.013) and lymph node metastasis (P=0.026) in CRC tissues. Results of transwell, MTT, and colony formation assays showed that CFTR overexpression inhibited the migration, invasion, and proliferation of CRC cells.ConclusionCFTR expression was downregulated in CRC and promoter methylation may be responsible for this downregulation. Overexpression of CFTR may suppress CRC tumor growth by inhibiting the proliferation, migration, and invasion of CRC cells. CFTR promoter methylation was significantly correlated with lymph node metastasis; thus, CFTR may be a potential marker for lymph node metastasis of CRC.

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further exvivo and invivo editing.

HIV Tat Protein Induces the TGF‐β Signaling Pathway and Suppresses the CFTR Biogenesis and Activity by microRNA‐Regulated Gene‐Silencing Mechanism

The advent of combination antiretroviral therapy (cART) has led to a dramatic decline in morbidity and mortality from HIV/AIDS. HIV infected subjects are still six times more likely to contract pneumonia compared to non‐infected age matched controls. Mortality, following an episode of bacterial pneumonia was also four times higher in HIV infected subjects compared to non‐infected controls. Thus, pneumonia can also contribute to HIV progression. Understanding the pathophysiological mechanisms that lead to microbial colonization of the airways in HIV infected patients is therefore important to public health. HIV Tat and CS can suppress CFTR function, which is a critical determinant of ASL depth and this can lead to depressed MCC and consequent microbial colonization. Our study showed that HIV‐infected cells in the airway namely alveolar macrophages, other immune cells or bronchial epithelial cells can serve as reservoirs and a source of HIV proteins like Tat which upregulates TGF‐beta mRNA in primary human bronchial epithelial cells with a corresponding decrease in CFTR mRNA. Chromatin immunoprecipitation with RNA Polymerase II demonstrated that transcription from the CFTR promoter is unaffected and blocking the miRNA processing pathway with Aurin Tricarboxylic acid (ATA) restore CFTR inhibition caused by TGF‐ beta. Our data showed that HIV infection suppress CFTR mRNA and function and blocking TGF‐beta signaling rescues this. Together this posits a strong role for (TGF‐beta induced) miRNA in Tat mediated CFTR gene silencing.Support or Funding InformationFlight Attendant Medical Research Institute (FAMRI)HypothesisFigure 1HIV Tat suppresses CFTR via common TGF‐beta signalingFigure 2

The detection and significance of cystic fibrosis transmembrane conductance regulator gene promoter mutations in Chinese patients with congenital bilateral absence of the vas deferens

ObjectiveMutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) gene can be involved in the development of congenital bilateral absence of the vas deferens (CBAVD). This study was aimed at investigating mutations in the promoter region of the CFTR gene and its associated effects on CFTR transcription in Chinese patients with CBAVD. MethodsTo identify CFTR promoter region mutations in Chinese CBAVD patients, fragments 1.4 kb upstream of the ATG start codon of the CFTR gene were sequenced in 66 Chinese patients with CBAVD and compared to the corresponding sequences from 60 healthy subjects and sequence data present in the NCBI database. The relationship between the mutations and gene regulation was explored using Transfac analysis and a phylogenetic footprint method. Plasmids were constructed by incorporating statistically significant variant sequences. The effects of the mutations on CFTR transcription were investigated using a dual luciferase reporter gene assay. Resultsa total of six point mutations, which included c.-150G > T, c.-205 T > C, c.-245C > T, c.-871G > T, c.-966 T > G and c.-1062G > C, and one deletion mutation, namely c.-861delT, were identified in the promoter region of the CFTR gene in Chinese CBAVD patients. Among them, c.-966 T > G had the highest frequency and presented in either a homozygous or heterozygous mutation state. The frequency of G/G genotype in the CBAVD group was 33/66 (50.00%), while the T/G and T/T genotypes had frequencies of 18/66 (27.27%) and 15/66 (22.73%), respectively. A significant difference was found between the CBAVD and control group (P < 0.01). The locus for this variant was found in a conserved sequence. The Transfac tool showed that transcription factors EHF and STAT3, which are closely associated with reproduction, can bind to the sequence containing the variant locus. Two types of plasmid vectors, one carrying the G/G variant and the other the wild type T/T sequences, were constructed and respectively transfected into human cervical cancer cells (HeLa), human renal epithelial cells (HEK-293), and human colon cancer cells (SW480). It was found that the homozygous c.-966 T > G mutation significantly reduced CFTR transcription efficiency by 18.75%–35.50%. ConclusionMutations in the promoter region of the CFTR gene in Chinese CBAVD patients are different from those found in comparable Caucasian patients. The homozygous c.-966 T > G mutation state had the highest frequency, which reduced the CFTR transcriptional level and showed significant tissue-specificity.

Retinoic acid promotes stem cell differentiation and embryonic development by transcriptionally activating CFTR

Retinoic acid (RA) plays a pivotal role in many cellular processes; however, the signaling mechanisms mediating the effect of RA are not fully understood. Here, we show that RA transcriptionally upregulates cystic fibrosis transmembrane conductance regulator (Cftr) by promoting the direct binding of its receptor RARα to Cftr promoter in mouse spermatogonia and embryonic stem (ES) cells. The RA/CFTR pathway is involved in the differentiation of spermatogonia and organogenesis during the embryo development of Xenopus laevis. Loss of CFTR by siRNA-mediated knockdown blunts the RA-induced spermatogonial differentiation. Overexpression of CFTR mimics the effect of RA on the induction of spermatogonial differentiation or restores the developmental defects induced by the knockdown of RARα in spermatogonial cells and Xenopus laevis. Analysis of the human database shows that the expression of CFTR positively correlates with RARα in brain tissues, stem cells as well as cancers, supporting the role of RA/CFTR pathway in various developmental processes in humans. Together, our study discovers an essential role of CFTR in mediating the RA-dependent signaling for stem cell differentiation and embryonic development.

The HDAC inhibitor SAHA does not rescue CFTR membrane expression in Cystic Fibrosis

The development of suitable Cystic Fibrosis (CF) models for preclinical bench tests of therapeutic candidates is challenging. Indeed, the validation of molecules to rescue the p.Phe508del-CFTR channel (encoded by the Cystic Fibrosis Transmembrane conductance Regulator gene carrying the p.Phe508del mutation) requires taking into account their overall effects on the epithelium. Suberoylanilide Hydroxamic Acid (SAHA), a histone deacetylase inhibitor (HDACi), was previously shown to be a CFTR corrector via proteostasis modulation in CFTR-deficient immortalized cells. Here, we tested SAHA effects on goblet cell metaplasia using an ex vivo model based on the air-liquid interface (ALI) culture of differentiated airway epithelial cells obtained by nasal scraping from CF patients and healthy controls. Ex vivo epithelium grew successfully in ALI cultures with significant rise in the expression of CFTR and of markers of airway epithelial differentiation compared to monolayer cell culture. SAHA decreased CFTR transcript and protein levels in CF and non-CF epithelia. Whereas SAHA induced lysine hyperacetylation, it did not change histone modifications at the CFTR promoter. SAHA reduced MUC5AC and MUC5B expression and inhibited goblet epithelial cell differentiation. Similar effects were obtained in CF and non-CF epithelia. All the effects were fully reversible within five days from SAHA withdrawal. We conclude that, ex vivo, SAHA modulate the structure of airway epithelia without specific effect on CFTR gene and protein suggesting that HDACi cannot be useful for CF treatment.

Deep resequencing of CFTR in 762 F508del homozygotes reveals clusters of non-coding variants associated with cystic fibrosis disease traits.

Extensive phenotypic variability is commonly observed in individuals with Mendelian disorders, even among those with identical genotypes in the disease-causing gene. To determine whether variants within and surrounding CFTR contribute to phenotypic variability in cystic fibrosis (CF), we performed deep sequencing of CFTR in 762 patients homozygous for the common CF-causing variant, F508del. In phase 1, ~200 kb encompassing CFTR and extending 10 kb 5′ and 5 kb 3′ of the gene was sequenced in 486 F508del homozygotes selected from the extremes of sweat chloride concentration. In phase 2, a 510 kb region, which included the entire topologically associated domain of CFTR, was sequenced in 276 F508del homozygotes drawn from extremes of lung function. An additional 163 individuals who carried F508del and a different CF-causing variant were sequenced to inform haplotype construction. Region-based burden testing of both common and rare variants revealed seven regions of significance (α=0.01), five of which overlapped known regulatory elements or chromatin interactions. Notably, the −80 kb locus known to interact with the CFTR promoter was associated with variation in both CF traits. Haplotype analysis revealed a single rare recombination event (1.9% frequency) in intron 15 of CFTR bearing the F508del variant. Otherwise, the majority of F508del chromosomes were markedly similar, consistent with a single origin of the F508del allele. Together, these high-resolution variant analyses of the CFTR locus suggest a role for non-coding regulatory motifs in trait variation among individuals carrying the common CF allele.

Targeted Integration of a Super-Exon into the CFTR Locus Leads to Functional Correction of a Cystic Fibrosis Cell Line Model.

In vitro disease models have enabled insights into the pathophysiology of human disease as well as the functional evaluation of new therapies, such as novel genome engineering strategies. In the context of cystic fibrosis (CF), various cellular disease models have been established in recent years, including organoids based on induced pluripotent stem cell technology that allowed for functional readouts of CFTR activity. Yet, many of these in vitro CF models require complex and expensive culturing protocols that are difficult to implement and may not be amenable for high throughput screens. Here, we show that a simple cellular CF disease model based on the bronchial epithelial ΔF508 cell line CFBE41o- can be used to validate functional CFTR correction. We used an engineered nuclease to target the integration of a super-exon, encompassing the sequences of CFTR exons 11 to 27, into exon 11 and re-activated endogenous CFTR expression by treating CFBE41o- cells with a demethylating agent. We demonstrate that the integration of this super-exon resulted in expression of a corrected mRNA from the endogenous CFTR promoter and used short-circuit current measurements in Ussing chambers to corroborate restored ion transport of the repaired CFTR channels. In conclusion, this study proves that the targeted integration of a large super-exon in CFTR exon 11 leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all CFTR mutations located downstream of the 5’ end of exon 11.

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation.

Detection of promoter region of CFTR gene in Chinese congenital bilateral absence of vas deferens

To detect the variants in the promoter region of the CFTR gene in congenital bilateral absence of vas deferens (CBAVD). A total of 11 CBAVD patients and 50 healthy men as control were enrolled in the study from May 2013 to January 2015. Sanger sequencing was performed in the promoter region of 3.8 kb of the CFTR gene on the PCR products. The genome sequence of the CFTR gene was compared and analyzed with the website of NCBI and Cystic Fibrosis Mutation Database. Also, Transfac and phylogenetic footprinting method was used to investigate the relationship between the promoter region variants and the transcription factors function components. SNP of c.-8G > C (n = 1) and c.-966T > G (n = 7), as well as one single nucleotide variant of c.-195C > A (n = 1) were identified in the promoter region of the CBAVD patients, of which c.-195C > A was in the conserved domains of the promoter region. A single nucleotide variant within the conserved region of CFTR promoter is detected in Chinese CBAVD. And further functional study should be performed to explore the relationship between the variants in CFTR promoter and Chinese CBAVD.

Phosphorylated C/EBPβ Influences a Complex Network Involving YY1 and USF2 in Lung Epithelial Cells

The promoter of the cystic fibrosis transmembrane conductance regulator gene CFTR is tightly controlled by regulators including CCAAT/enhancer binding proteins (C/EBPs). We previously reported that the transcription factors YY1 and USF2 affect CFTR expression. We can now demonstrate that C/EBPβ, a member of the CCAAT family, binds to the CFTR promoter and contributes to its transcriptional activity. Our data reveal that C/EBPβ cooperates with USF2 and acts antagonistically to YY1 in the control of CFTR expression. Interestingly, YY1, a strong repressor, fails to repress the CFTR activation induced by USF2 through DNA binding competition. Collectively, the data strongly suggest a model by which USF2 functionally interacts with YY1 blocking its inhibitory activity, in favour of C/EBPβ transactivation. Further investigation into the interactions between these three proteins revealed that phosphorylation of C/EBPβ influences the DNA occupancy of YY1 and favours the interaction between USF2 and YY1. This phosphorylation process has several implications in the CFTR transcriptional process, thus evoking an additional layer of complexity to the mechanisms influencing CFTR gene regulation.

Cftr controls lumen expansion and function of Kupffer’s vesicle in zebrafish

Regulated fluid secretion is crucial for the function of most organs. In vertebrates, the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a master regulator of fluid secretion. Although the biophysical properties of CFTR have been well characterized in vitro, little is known about its in vivo role during development. Here, we investigated the function of Cftr during zebrafish development by generating several cftr mutant alleles using TAL effector nucleases. We found that loss of cftr function leads to organ laterality defects. In zebrafish, left-right (LR) asymmetry requires cilia-driven fluid flow within the lumen of Kupffer's vesicle (KV). Using live imaging we found that KV morphogenesis is disrupted in cftr mutants. Loss of Cftr-mediated fluid secretion impairs KV lumen expansion leading to defects in organ laterality. Using bacterial artificial chromosome recombineering, we generated transgenic fish expressing functional Cftr fusion proteins with fluorescent tags under the control of the cftr promoter. The transgenes completely rescued the cftr mutant phenotype. Live imaging of these transgenic lines showed that Cftr is localized to the apical membrane of the epithelial cells in KV during lumen formation. Pharmacological stimulation of Cftr-dependent fluid secretion led to an expansion of the KV lumen. Conversely, inhibition of ion gradient formation impaired KV lumen inflation. Interestingly, cilia formation and motility in KV were not affected, suggesting that fluid secretion and flow are independently controlled in KV. These findings uncover a new role for cftr in KV morphogenesis and function during zebrafish development.

Molecular mechanisms controlling CFTR gene expression in the airway

The low levels of CFTR gene expression and paucity of CFTR protein in human airway epithelial cells are not easily reconciled with the pivotal role of the lung in cystic fibrosis pathology. Previous data suggested that the regulatory mechanisms controlling CFTR gene expression might be different in airway epithelium in comparison to intestinal epithelium where CFTR mRNA and protein is much more abundant. Here we examine chromatin structure and modification across the CFTR locus in primary human tracheal (HTE) and bronchial (NHBE) epithelial cells and airway cell lines including 16HBE14o- and Calu3. We identify regions of open chromatin that appear selective for primary airway epithelial cells and show that several of these are enriched for a histone modification (H3K4me1) that is characteristic of enhancers. Consistent with these observations, three of these sites encompass elements that have cooperative enhancer function in reporter gene assays in 16HBE14o- cells. Finally, we use chromosome conformation capture (3C) to examine the three-dimensional structure of nearly 800 kb of chromosome 7 encompassing CFTR and observe long-range interactions between the CFTR promoter and regions far outside the locus in cell types that express high levels of CFTR.

Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene.

Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements.

An insulator element 3′ to the CFTR gene binds CTCF and reveals an active chromatin hub in primary cells

Regulation of expression of the CFTR gene is poorly understood. Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin. We previously mapped DNase I hypersensitive sites (DHS) in 400 kb spanning the CFTR locus including a cluster of sites close to the 3′-end of the gene. Here we focus on a DHS at +6.8 kb from the CFTR translation end-point to evaluate its potential role in regulating expression of the gene. This DHS, which encompasses a consensus CTCF-binding site, was evident in primary human epididymis cells that express abundant CFTR mRNA. We show by DNase I footprinting and electophoretic mobility shift assays that the cis-regulatory element within this DHS binds CTCF in vitro. We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo. Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub.

A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter

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.

Transforming Growth Factor‐β1 Inhibits CFTR‐dependent cAMP‐stimulated Alveolar Epithelial Fluid Transport: Critical Role of Interleukin‐8

β‐adrenergic agonist‐dependent stimulation of the lung fluid clearance is an important mechanism that protects the lung from alveolar flooding. In this study we hypothesized that critical mediators of acute lung injury (ALI), such as transforming growth factor‐β1 (TGF‐β1) and interleukin‐8 (IL‐8), could directly antagonize the epithelial response to β‐adrenergic agonists. Short circuit current experiments revealed that TGF‐β1 inhibits CFTR‐specific β‐agonist‐stimulated vectorial Cl‐transport across the apical membrane of primary rat and human alveolar epithelial type II (ATII). TGF‐β1 also significantly decreased β‐agonist‐stimulated cAMP production resulting in the inhibition of the CFTR promoter activity and gene expression in ATII cells. We found that this TGF‐β1‐dependent inhibition required IL‐8 and was mediated by desensitization the β‐adrenergic receptor through both TGF‐β1 and IL‐8 signaling pathways. Consistent with the in vitro results, we showed that TGF‐β1 requires IL‐8 to inhibit the β‐agonist‐stimulated fluid transport across the distal airspace epithelium in vivo in rats. In summary, the results show for the first time that TGF‐β1 and IL‐8 inhibit the stimulated vectorial chloride transport across the distal lung epithelium, thus impairing the β‐adrenergic‐dependent protective mechanism against alveolar flooding.NIH P50HL074005 (JFP), ALA Award and T32 GM008440 (JR)