A balance between activating and repressive histone modifications regulates cystic fibrosis transmembrane conductance regulator (CFTR) expression in vivo

The genetic mechanisms that regulate CFTR, the gene responsible for cystic fibrosis, have been widely investigated in cultured cells. However, mechanisms responsible for tissue-specific and time-specific expression are not completely elucidated in vivo. Through the survey of public databases, we found that the promoter of CFTR was associated with bivalent chromatin in human embryonic stem (ES) cells. In this work, we analyzed fetal (at different stages of pregnancy) and adult tissues and showed that, in digestive and lung tissues, which expressed CFTR, H3K4me3 was maintained in the promoter. Histone acetylation was high in the promoter and in two intronic enhancers, especially in fetal tissues. In contrast, in blood cells, which did not express CFTR, the bivalent chromatin was resolved (the promoter was labeled by the silencing mark H3K27me3). Cis-regulatory sequences were associated with lowly acetylated histones. We also provide evidence that the tissue-specific expression of CFTR is not regulated by dynamic changes of DNA methylation in the promoter. Overall, this work shows that a balance between activating and repressive histone modifications in the promoter and intronic enhancers results in the fine regulation of CFTR expression during development, thereby ensuring tissue specificity.     

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.     

A survey of detergents for the purification of stable,active human cystic fibrosis transmembrane conductance regulator (CFTR)

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1 mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C₁₂E₈, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2–3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1 mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6–9 days in MNG or Chaps, and 12–17 days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.     

Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR)

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active. Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states. These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein. In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter. The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered.     

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator

Proteomic analysis has proved to be an important tool for understanding the complex nature of genetic disorders, such as cystic fibrosis (CF), by defining the cellular protein environment (proteome) associated with wild-type and mutant proteins. Proteomic screens identified the proteome of CF transmembrane conductance regulator (CFTR), and provided fundamental information to studies designed for understanding the crucial components of physiological CFTR function. Simultaneously, high-throughput screens for small-molecular correctors of CFTR mutants provided promising candidates for therapy. The majority of CF cases are caused by nucleotide deletions (ΔF508 CFTR; >75%), resulting in CFTR misfolding, or insertion of premature termination codons (～10%), leading to unstable mRNA and reduced levels of truncated dysfunctional CFTR. In this article, we review recent results of proteomic screens, developments in identifying correctors for the most frequent CFTR mutants, and comment on how integration of the knowledge gained from these studies may aid in finding a cure for CF and a number of other genetic disorders.     

Role of DNA methylation and histone modifications in structural maintenance of heterochromatin domains (chromocenters)

Effects of DNA methylation inhibitor; 5-azacytidine (5-aza-C); and histone acetylation inhibitor, trichostatine A (TSA), on the structure of pericentric heterochromatin of L929 mouse cells have been studied. 5-aza-C treatment for 48 h resulted in the transformation of ovoid chromocenters into elongated structures in 85% of cells. Hypotonic treatment of these cells reveals tandemly arranged DAPI-positive globules that are well distinguishable by light microscopy. Similar globular units can be observed in hypotonic-treated control cells. TSA treatment for 48 h causes dramatic decrease in HP1α content in cells. In 25% of treated cells chromocenters became highly decondensed and can not be reliably detected by light and electron microscopy. 85% cells demonstrate globular chromocenters with low HP1α content. Hypotonic treatment induces transformation of compact chromocenters into ring-like structures that can be either single or clustered. Rings are formed by uniform fiber in which no globular subunits are detected. The data obtained are discussed concerning several mechanisms of heterochromatin structure maintenance and the role of epigenetic factors.     

Functional genomic responses to cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR(delta508) in the lung

Cystic fibrosis (CF), a common lethal pulmonary disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that disturbs fluid homeostasis and host defense in target organs. The effects of CFTR and delta508-CFTR were assessed in transgenic mice that 1) lack CFTR expression (Cftr-/-); 2) express the human delta508 CFTR (CFTR(delta508)); 3) overexpress the normal human CFTR (CFTR(tg)) in respiratory epithelial cells. Genes were selected from Affymetrix Murine Gene-Chips analysis and subjected to functional classification, k-means clustering, promoter cis-elements/modules searching, literature mining, and pathway exploring. Genomic responses to Cftr-/- were not corrected by expression of CFTR(delta508). Genes regulating host defense, inflammation, fluid and electrolyte transport were similarly altered in Cftr-/- and CFTR(delta508) mice. CFTR(delta508) induced a primary disturbance in expression of genes regulating redox and antioxidant systems. Genomic responses to CFTR(tg) were modest and were not associated with lung pathology. CFTR(tg) and CFTR(delta508) induced genes encoding heat shock proteins and other chaperones but did not activate the endoplasmic reticulum-associated degradation pathway. RNAs encoding proteins that directly interact with CFTR were identified in each of the CFTR mouse models, supporting the hypothesis that CFTR functions within a multiprotein complex whose members interact at the level of protein-protein interactions and gene expression. Promoters of genes influenced by CFTR shared common regulatory elements, suggesting that their co-expression may be mediated by shared regulatory mechanisms. Genes and pathways involved in the response to CFTR may be of interest as modifiers of CF.     

Successful targeting of the mouse cystic fibrosis transmembrane conductance regulator gene in embryonal stem cells

We wish to construct a mouse model for the human inherited disease cystic fibrosis. We describe here the successful targeting in embryonal stem cells of the murine homologue (Cftr) of the cystic fibrosis transmembrane conductance regulator gene, as the first critical step towards this end. The targeting event precisely disrupts exon 10, the site of the major mutation in patients with cystic fibrosis. The targeted cells are pluripotent and competent to form chimaeras.     

A rare DNA variant in exon 15 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

We describe a rare single-strand conformation polymorphism that occurs at nucleotide 3030 (G or A) in exon 15 of the cystic fibrosis transmembrane conductance regulator gene.     

Pharmacogenomics of the cystic fibrosis transmembrane conductance regulator (CFTR) and the cystic fibrosis drug CPX using genome microarray analysis

BACKGROUND: Cystic fibrosis (CF) is the most common lethal recessive disease affecting children in the U.S. and Europe. For this reason, a number of ongoing attempts are being made to treat the disease either by gene therapy or pharmacotherapy. Several phase 1 gene therapy trials have been completed, and a phase 2 clinical trial with the xanthine drug CPX is in progress. The protein coded by the principal CFTR mutation, DeltaF508-CFTR, fails to traffic efficiently from the endoplasmic reticulum to the plasma membrane, and is the pathogenic basis for the missing cAMP-activated plasma membrane chloride channel. CPX acts by binding to the mutant DeltaF508-CFTR and correcting the trafficking deficit. CPX also activates mutant CFTR channels. The comparative genomics of wild-type and mutant CFTR has not previously been studied. However, we have hypothesized that the gene expression patterns of human cells expressing mutant or wild-type CFTR might differ, and that a drug such as CPX might convert the mutant gene expression pattern into one more characteristic of wild-type CFTR. To the extent that this is true, a pharmacogenomic profile for such corrective drugs might be deduced that could simplify the process of drug discovery for CF. MATERIALS AND METHODS: To test this hypothesis we used cDNA microarrays to study global gene expression in human cells permanently transfected with either wild-type or mutant CFTR. We also tested the effects of CPX on global gene expression when incubated with cells expressing either mutant or wild-type CFTR. RESULTS: Wild-type and mutant DeltaF508-CFTR induce distinct and differential changes in cDNA microarrays, significantly affecting up to 5% of the total genes in the array. CPX also induces substantial mutation-dependent and -independent changes in gene expression. Some of these changes involve movement of gene expression in mutant cells in a direction resembling expression in wild-type cells. CONCLUSIONS: These data clearly demonstrate that cDNA array analysis of cystic fibrosis cells can yield useful pharmacogenomic information with significant relevance to both gene and pharmacological therapy. We suggest that this approach may provide a paradigm for genome-based surrogate endpoint testing of CF therapeutics prior to human administration.     

Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH

Potentiators are molecules that increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Some potentiators can also inhibit CFTR at higher concentrations. The activating binding site is thought to be located at the interface of the dimer formed by the two nucleotide-binding domains. We have hypothesized that if binding of potentiators involves titratable residues forming salt bridges, then modifications of cytosolic pH (pH(i)) would alter the binding affinity. Here, we analyzed the effect of pH(i) on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pH(i) does modify CFTR maximum current (I(m)) and half-activation concentration (K(d)): I(m) = 127.7, 185.5, and 231.8 μA/cm(2) and K(d) = 32.7, 56.6 and 71.9 μm at pH 6, 7.35, and 8, respectively. We also found that the genistein apparent dissociation constant for activation (K(a)) increased at alkaline pH(i), near cysteine pK (K(a) = 1.83, 1.81 and 4.99 μm at pH(i) 6, 7.35, and 8, respectively), suggesting the involvement of cysteines in the binding site. Mutations of cysteine residues predicted to be within (Cys-491) or outside (Cys-1344) the potentiator-binding site showed that Cys-491 is responsible for the sensitivity of potentiator binding to alkaline pH(i). Effects of pH(i) on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pH(i) on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.     

Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants

A complex involving Derlin-1 and p97 mediates the retrotranslocation and endoplasmic reticulum (ER)-associated degradation of misfolded proteins in yeast and is used by certain viruses to promote host cell protein degradation (Romisch, K. (2005) Annu. Rev. Cell Dev. Biol. 21, 435-456; Lilley, B. N., and Ploegh, H. L. (2004) Nature 429, 834-840; Ye, Y., Shibata, Y., Yun, C., Ron, D., and Rapoport, T. A. (2004) Nature 429, 841-847). We asked whether the components of this pathway are involved in the endoplasmic reticulum-associated degradation of the mammalian integral membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), a substrate for the ubiquitin-proteasome system. We report that Derlin-1 and p97 formed complexes with CFTR in human airway epithelial cells. Derlin-1 interacted with nonubiquitylated CFTR, whereas p97 associated with ubiquitylated CFTR. Exogenous expression of Derlin-1 led to its co-localization with CFTR in the ER where it reduced wild type (WT) CFTR expression and efficiently degraded the disease-associated CFTR folding mutants, DeltaF508 and G85E (>90%). Consistent with this, Derlin-1 also reduced the amount of WT or DeltaF508 CFTR appearing in detergent-in-soluble aggregates. An approximately 70% knockdown of endogenous Derlin-1 by RNA interference increased the steady-state levels of WT and DeltaF508 CFTR by 10-15-fold, reflecting its significant role in CFTR degradation. Derlin-1 mediated the degradation of N-terminal CFTR fragments corresponding to the first transmembrane domain of CFTR, but CFTR fragments that incorporated additional domains were degraded less efficiently. These findings suggest that Derlin-1 recognizes misfolded, nonubiquitylated CFTR to initiate its dislocation and degradation early in the course of CFTR biogenesis, perhaps by detecting structural instability within the first transmembrane domain.     

Estrogen,through estrogen receptor 1, regulates histone modifications and chromatin remodeling during spermatogenesis in adult rats

Estrogen receptors (ESR1 and ESR2) play crucial roles in various processes during spermatogenesis. To elucidate individual roles of ESRs in male fertility, we developed in vivo selective ESR agonist administration models. Adult male rats treated with ESR1 and ESR2 agonist for 60 days show spermatogenic defects leading to reduced sperm counts and fertility. While studying epigenetic changes in the male germ line that could have affected fertility, we earlier observed a decrease in DNA methylation and its machinery upon ESR2 agonist treatment. Here, we explored the effects on histone modifications, which could contribute to decreased male fertility upon ESR agonist administration. ESR1 agonist treatment affected testicular levels of histone modifications associated with active and repressed chromatin states, along with heterochromatin marks. This was concomitant with deregulation of corresponding histone modifying enzymes in the testis. In addition, there was increased retention of histones along with protamine deficiency in the caudal spermatozoa after ESR1 agonist treatment. This could be due to the observed decrease in several chromatin remodeling proteins implicated in mediating histone-to-protamine exchange during spermiogenesis. The activating and repressing histone marks in spermatozoa, which play a critical role in early embryo development, were deregulated after both the ESR agonist treatments. Together, these epigenetic defects in the male germ line could affect the spermatozoa quality and lead to the observed decrease in fertility. Our results thus highlight the importance of ESRs in regulating different epigenetic processes during spermatogenesis, which are crucial for male fertility.     

Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)

P-glycoprotein (P-gp, ABCB1) is an ATP-dependent drug pump. Each of its two homologous halves contains a transmembrane domain (TMD) that has six transmembrane (TM) segments and a nucleotide-binding domain (NBD). Determining how the two halves interact may provide insight into the folding of P-gp as the drug-binding pocket and nucleotide-binding sites are predicted to be at the interface between the two halves. Here, we present evidence for NBD1-TMD2 and NBD2-TMD1 interactions. We also show that TMD-NBD interactions in immature and mature P-gp can be affected by the presence of a processing mutation. We found that the NBD-TMD mutants L443C(NBD1)/S909C(TMD2) and A266C(TMD1)/F1086C(NBD2) could be cross-linked at 0 degrees C with oxidant (copper phenanthroline). Cross-linking was inhibited by vanadate-trapping of nucleotide. The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Expression of the processing mutants in the presence of a pharmacological chaperone (cyclosporin A), however, resulted in the expression of mature (170 kDa) protein at the cell surface that could be cross-linked. Similarly, CFTR mutants A274C(TMD1)/L1260C(NBD2) and V510C(NBD1)/A1067C(TMD2) could be cross-linked at 0 degrees C with copper phenanthroline. Introduction of DeltaF508 mutation in these mutants, however, resulted in the synthesis of immature CFTR that could not be cross-linked. These results suggest that establishment of NBD interactions with the opposite TMD is a key step in folding of ABC transporters.     

The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate

Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified regions in the CFTR map, not accounted for by Sav1866, which were potential locations for the regulatory region as well as the channel gate. In this analysis, we were aided by the fact that the unit cell was composed of two molecules not related by crystallographic symmetry. We also identified regions in the fitted Sav1866 model that were missing from the map, hence regions that were either disordered in CFTR or differently organized compared with Sav1866. Apart from the N and C termini, this indicated that in CFTR, the cytoplasmic end of transmembrane helix 5/11 and its associated loop could be partly disordered (or alternatively located).     

A large deletion causes apparent homozygosity for the D1152H mutation in the cystic fibrosis transmembrane regulator (CFTR) gene

We report the case of a patient with an apparent homozygosity for the D1152H mutation located in exon 18 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The parents had no personal history of cystic fibrosis (CF) and referred to our laboratory after the diagnosis of fetal bowel hyperechogenicity. The proband presented with meconium ileus and normal sweat chloride test. Sequencing of the CFTR exon 18 together with quantitative genomic assays, such as real-time PCR and the multiplex ligation probe amplification (MLPA) techniques, were performed and revealed that the father was heterozygous for the D1152H mutation and the mother carried a large deletion of the CFTR gene encompassing the genomic sequence including the same mutation. The child inherited D1152H from his father and the large deletion of the CFTR gene from his mother. We suggest that D1152H likely acts as a mild mutation with a dominant effect on the severe deletion of exon 18, considering that after 3 years of clinical examinations the child shows no classical signs and symptoms of CF. Not testing for large deletions in subjects with apparent homozygosity for a mutated CFTR allele could lead to the misidentification of CFTR mutation carrier status.     

The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular Cl- concentration

Activation of the CFTR Cl^– channel inhibits epithelial Na⁺ channels (ENaC), according to studies on epithelial cells and overexpressing recombinant cells. Here we demonstrate that ENaC is inhibited during stimulation of the cystic fibrosis transmembrance conductance regulator (CFTR) in Xenopus oocytes, independent of the experimental set-up and the magnitude of the whole-cell current. Inhibition of ENaC is augmented at higher CFTR Cl^– currents. Similar to CFTR, ClC-0 Cl^– currents also inhibit ENaC, as well as high extracellular Na⁺ and Cl^– in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and seems to be mediated by Cl^–.