[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(4):e60211. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hereditary factor VII (FVII) deficiency is a rare autosomal recessive disorder. Deleterious mutations that prevent the synthesis of any amount of functional FVII have been associated with life-threatening haemorrhage in neonates. Here we report two infants, of Maghrebian origin, who suffered a fatal spontaneous cerebral haemorrhage. Investigation of the molecular basis for their severe FVII deficiency revealed novel mutations in a homozygous state within the F7 gene promoter: a single nucleotide substitution (c.-65G>C) and a 2bp deletion (c.-60_-59delTT). To determine whether these promoter variants were responsible for the FVII deficiency, computer-assisted sequence analyses were performed. The data predicted a disrupted binding of both HNF4 and COUP-TF transcription factors with each variant. Concordantly, experimental results revealed an altered HNF4-induced transactivation in the promoter mutated variants. The execution of functional tests is critical to ensuring a complete understanding of the effect of any promoter mutant on FVII deficiency. Only then can an accurate molecular diagnosis be made and further genetic counselling and prenatal diagnosis be offered.
Thrombosis and Haemostasis 05/2012; 108(2):277-83. · 6.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: In European populations, large rearrangements contribute to approximately 2% of CF mutations. Here, we reported a novel duplication, the CFTRdup2, identified in a patient heterozygous for Phe508del and suffering from a mild CF. Using a combination of functional tests, we studied the impact of duplication/deletion on CFTR expression. We showed that the copy number variations of exon 2, in addition to abolishing the rate of the mature CFTR protein, affect the CFTR mRNA levels. These data illustrate the importance to perform functional analysis to better understand the molecular basis responsible for cystic fibrosis. Determining the impact of deletions or duplications is relevant for a more comprehensive diagnosis and prognosis of patients.
[show abstract][hide abstract] ABSTRACT: In monogenic diseases, the presence of several sequence variations in the same allele may complicate our understanding of genotype-phenotype relationships. We described new alterations identified in a cystic fibrosis (CF) patient harboring a 48C>G promoter sequence variation associated in cis of a 3532AC>GTA mutation and in trans with the F508del mutation. Functional analyses including in vitro experiments confirmed the deleterious effect of the 3532GTA frameshift mutation through the creation of a premature termination codon. The analyses also revealed that the 48G promoter variant has a negative effect on both transcription and mRNA level, thus demonstrating the importance of analyzing all mutations or sequence variations with potential impact on CF transmembrane conductance regulator processing, even when the two known disease-causing mutations have already been detected. Our results emphasize the need to perform, wherever possible, functional studies that may greatly assist the interpretation of the disease-causing potential of rare mutation-associated sequence variations.
European journal of human genetics: EJHG 08/2011; 20(2):180-4. · 3.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: Congenital bilateral absence of the vas deferens (CBAVD), a frequent cause of obstructive azoospermia, is generated by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite extensive testing for point mutations and large rearrangements, a small proportion of alleles still remains unidentified in CBAVD patients.
Mutation scanning analysis of microsatellite variability in the CFTR gene identified two undescribed 4 bp sequence repeats (TAAA)(6) and (TAAA)(8) in intron 9 in two CBAVD patients heterozygote for either the -33G→A promoter transition or the classical [TG12T5] CBAVD mutation. This study explores the putative impact of this promoter variant by using a combination of web based prediction tools, reporter gene assays, and DNA/proteins interaction analyses. Results of transiently transfected vas deferens cells with either the -33G wild-type or the -33A variant CFTR directed luciferase reporter gene confirmed that the -33A variant, which alters the FOXI1 (Forkhead box I1) binding, significantly decreases the CFTR promoter activity. It was also investigated whether regulatory elements located within the intronic tetrarepeat might influence the CFTR expression. There was evidence that both the (TAAA)(6) and the (TAAA)(8) alleles modulate the CFTR transcription and the binding affinity for FOX transcription factors, involved in the chromatin architecture.
As the vas deferens seems to be one of the tissues most susceptible to a reduction in the normal CFTR transcripts levels, and as two mild mutations are sufficient to induce CBAVD phenotype, these findings raise the possibility that these uncommon variants may be a novel cause of CBAVD.
Journal of Medical Genetics 10/2010; 48(3):152-9. · 5.70 Impact Factor