Role of Non-coding RNAs in Cystic Fibrosis

Cystic Fibrosis (CF) is a common autosomal recessive disorder, caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. CFTR gene expression is tightly controlled by transcriptional and post-transcriptional regulatory factors, resulting in complex spatial and temporal expression patterns. Here, we describe an overview of the findings about the contribution of ncRNAs, especially miRNAs, in physiological CFTR gene expression and in CF. Determination of mechanisms governing its expression is essential for developing new CF therapies. ncRNAs, including lncRNAs and miRNAs, could also contribute to CF progression and severity and their dysregulation in CF opens new perspectives for patient follow-up and treatment

New Molecular Diagnosis Approaches — From the Identification of Mutations to their Characterization

Molecular diagnosis of cystic fibrosis is based on the detection of mutation in the CFTR gene, identified in 1989. During the past 20 years, thanks to evolutions of diagnostic techniques, our knowledge of mutation spectrum and pathophysiological mechanisms involved in the disease has significantly improved. Sanger sequencing and quantitative methods greatly contributed to the identification of the 2,000 sequence variations reported worldwide in CFTR. We are now entering the new technological age with the generalisation of Next Generation Sequencing (NGS) technologies in diagnostics laboratories. These high throughput approaches allow scanning for the entire CFTR locus, including deep intronic regions, and in parallel other candidate genes that possibly influence the clinical evolution of patients. However, this powerful technology poses new challenge in test interpretation. In this chapter, we review the current and new technologies used in molecular diagnostics of cystic fibrosis, particularly NGS approaches. We also present current and new bioinformatics tools available for the interpretation of variants and in vitro/ex vivo and in vivo techniques that can be used to improve the characterization of the functional impact of CFTR variations

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

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Current and future molecular approaches in the diagnosis of cystic fibrosis

International audienceCystic Fibrosis is among the first diseases to have general population genetic screening tests and one of the most common indications of prenatal and preimplantation genetic diagnosis for single gene disorders. During the past twenty years, thanks to the evolution of diagnostic techniques, our knowledge of CFTR genetics and pathophysiological mechanisms involved in cystic fibrosis has significantly improved. Areas covered: Sanger sequencing and quantitative methods greatly contributed to the identification of more than 2,000 sequence variations reported worldwide in the CFTR gene. We are now entering a new technological age with the generalization of high throughput approaches such as Next Generation Sequencing and Droplet Digital PCR technologies in diagnostics laboratories. These powerful technologies open up new perspectives for scanning the entire CFTR locus, exploring modifier factors that possibly influence the clinical evolution of patients, and for preimplantation and prenatal diagnosis. Expert commentary: Such breakthroughs would, however, require powerful bioinformatics tools and relevant functional tests of variants for analysis and interpretation of the resulting data. Ultimately, an optimal use of all those resources may improve patient care and therapeutic decision-making

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

International audienceThe 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

Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis

International audienceThe CFTR gene displays a tightly regulated tissue-specific and temporal expression. Mutations in this gene cause cystic fibrosis (CF). In this study we wanted to identify trans-regulatory elements responsible for CFTR differential expression in fetal and adult lung, and to determine the importance of inhibitory motifs in the CFTR-3'UTR with the aim of developing new tools for the correction of disease-causing mutations within CFTR. We show that lung development-specific transcription factors (FOXA, C/EBP) and microRNAs (miR-101, miR-145, miR-384) regulate the switch from strong fetal to very low CFTR expression after birth. By using miRNome profiling and gene reporter assays, we found that miR-101 and miR-145 are specifically upregulated in adult lung and that miR-101 directly acts on its cognate site in the CFTR-3'UTR in combination with an overlapping AU-rich element. We then designed miRNA-binding blocker oligonucleotides (MBBOs) to prevent binding of several miRNAs to the CFTR-3'UTR and tested them in primary human nasal epithelial cells from healthy individuals and CF patients carrying the p.Phe508del CFTR mutation. These MBBOs rescued CFTR channel activity by increasing CFTR mRNA and protein levels. Our data offer new understanding of the control of the CFTR gene regulation and new putative correctors for cystic fibrosis

Should diffuse bronchiectasis still be considered a CFTR-related disorder?

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Phosphorylation of C/EBPβ affects the YY1 DNA occupancy and favours YY1/USF2 interaction.

<p>(A) Left panel: ChIP experiments were performed using NAF-treated Beas2B cells when indicated. Protein extracts were immunoprecipitated IP with the indicated antibodies. Input (IN), corresponds to total lysate used as a control for PCR amplification of total DNA and served to normalize <i>CFTR</i> amplification as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060211#s2" target="_blank">Materials and Methods</a> Section. CFTR, represents <i>CFTR</i> promoter amplification and negative control, ChIP analysis of <i>CFTR</i> sequence which lacks both C/EBPβ and YY1 binding motifs. Right panel: DNA from immunoprecipitates and input DNA were analyzed by quantitative PCR. Data are defined as fold enrichment relative to input chromatin and specific binding was expressed as a function of non-specific (NS) antibody (anti-HA) binding set as 1. (B) Beas2B cells were treated when indicated with NAF and protein extracts were immunoprecipitated with either an anti-YY1 (lanes 2 and 3) or an irrelevant antibody (lane 4). Immunoprecipitated proteins were then analyzed by western blotting using either an anti-USF2a or an anti-YY1 antibody. Lane 1 corresponds to whole cell extracts used for IP.</p

Schematic model depicting the potential mechanism that might contribute to regulation of the <i>CFTR</i> gene.

<p>In this model, coloured bubbles correspond to the transcription factors characterized in this study. Double arrow shows the functional antagonism between C/EBPβ and YY1. The upper representation corresponds to the transcriptional activation of the <i>CFTR</i> gene when C/EBPβ is phosphorylated and the lower representation corresponds to the decrease of the transcription following overexpression of a C/EBPβ form not phosphorylatable on its 235T residue.</p

NAF treatment stimulates the <i>CFTR</i> activity induced by C/EBPβ.

<p>(A) Left panel: When indicated, Beas2B cells were incubated in the presence of NAF (left panel) at the indicated concentrations. Immunoblots showing either CFTR or LaminA/C expression are represented below the graph. Right panel: Endogenous mRNA level of either <i>CFTR</i> or <i>C/EBPβ</i> following NAF incorporation at indicated concentrations. (B) Upper panel: Combinatorial effect of C/EBPβ and NAF treatment. Cells were transfected either with C/EBPβ plasmid after NAF treatment (left panel) or with LAPT235A expression vector (right panel). Middle panel: Representative immunoblots are shown below the graphs. Lower panel: Endogenous <i>CFTR</i> mRNA level. *P<0.05.</p