SCHULTE, P.M.*; SINGER, T.D.; Univ. of British Columbia; Univ. of Waterloo: Comparative genomic sequence analysis of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) has a highly regulated expression pattern, but the sequences regulating its transcription remain poorly characterized. We have used a genomics approach (phylogenetic footprinting) to characterize regulatory elements involved in CFTR expression. We cloned and sequenced the CFTR gene of a euryhaline teleost, the killifish, Fundulus heteroclitus, and compared it to teleost and mammalian homologues (Fugu rubripes, Tetradon nigroviridis, human and mouse). Although the non-coding sequences were generally poorly conserved, several important regulatory elements were identified. A DNase I hypersensitive site in the first intron of hCFTR, which correlates with hCFTR expression, was conserved in both mCFTR and FuguCFTR but was absent from kfCFTR. Four transcriptional elements were common to all the CFTR homologues examined: a CCAAT element, cAMP responsive element (CRE), Sp1 element, and glucocorticoid responsive element (GRE). We also cloned and sequenced the CFTR promoter from 8 additional Fundulus species with varying salinity tolerances. Although the sequences were very similar, a number of important differences emerged, including lack of conservation of many of the elements that were conserved between F. heteroclitus and Fugu rubripes. We are currently using transient transfection analyses to determine the functional significance of these changes. We conclude that while genomic sequence comparisons can help to identify some highly conserved elements, the pattern of conservation is not always maintained as additional taxa are added. These results point out the advantages and limitations of phylogenetic footprinting as an approach for discovering novel regulatory elements.