CROSS-SPECIES CHARACTERIZATION OF THE PROMOTER REGION OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR GENE REVEALS MULTIPLE LEVELS OF REGULATION

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is highly conserved within vertebrate species, Its pattern of expression in vivo seems to be tightly regulated both developmentally and in a t issue-specific manner, but shows differences with species, To identify transcriptional regulatory elements in the CFTR promoter region, we h ave used a combined approach based both on the analysis of the chromat in structure in vivo in rat tissues and on evolutionary clues (i.e. ph ylogenetic footprinting), In CFTR-expressing tissues, 15 DNase I-hyper sensitive sites were identified within a 36 kb region encompassing exo n 1. Eleven of them are clustered in a 3.5 kb region that exhibits ele ven phylogenetic footprints observed when comparing sequences from eig ht mammalian species representing four orders (Primates, Artiodactylia , Lagomorpha and Rodentia), Comparison of the two sets of data allows the identification of two types of regulatory elements. Some are conse rved between species, such as a non-consensus cAMP response element (C RE) and a PMA-responsive element (TRE) located respectively at positio ns -0.1 and -1.3 kb relative to ATG, Some are species-specific element s such as a 300 bp purine-pyrimidine (Pu.Py) stretch that is present o nly in rodents. Analysis of protein/DNA interactions in vitro with rat tissue protein extracts on the conserved elements revealed that the T RE site binds a specific heterodimeric complex composed of Fra-2, Jun D and a protein immunologically related to Jun/CRE-binding protein in the duodenum, whereas the CRE-like site binds ATF-1 ubiquitously. Func tional analysis in Caco-2 cells showed that the CRE-like site supports a high basal transcriptional activity but is not able by itself to in duce a response to cAMP, whereas the TRE site acts as a weak transacti vator stimulated by PMA. Lastly, we found that the rodent-specific Pu. Py stretch confers nuclease S1 hypersensitivity under conditions of ac idic pH and supercoiling, This indicates a non-B DNA conformation and thus reinforces the biological significance of non-random Pu.Py strand asymmetry in the regulation of transcription. Thus the tight transcri ptional regulation of CFTR expression involves the combination of mult iple regulatory elements that act in the chromatin environment in vivo . Some of them are conserved throughout evolution, such as the CRE-lik e element, which is clearly involved in the basal level of transcripti on; others are species-specific.