Analysis of a DNase I hypersensitve site located - 20.9 kb upstream of theCFTR gene

The Cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a tightly regulated pattern of expression with spatial and temporal control. The regulatory elements achieving this appear to lie outside the basal pro moter of the gene. We previously identified DNase I hypersensitive sites (D HSs) at -79.5 kb and -20.5 kb with respect to the CFTR translational start site which may contain important regulatory elements. We have now investiga ted further the DHS at -20.5 kb to evaluate its potential function in the r egulation of CFTR expression. Finer mapping revealed that the DHS lies at - 20.9 kb, Deletion of the DHS from a 310-kb yeast artificial chromosome (YAC ) containing the human CFTR :gene has shown that this site may be responsib le for about 60% of wild-type levels of transcription from the YAC transgen e when expressed in Caco2 cells. DNase I footprinting showed several region s of protection within the -20.9:kb region with nuclear extracts from Caco2 cells, but not with extracts from lymphoblastoid cells, which do not show the DHS. Matches to several transcription factor-binding sites were found, but supershift analysis with specific antibodies did not identify the trans cription factors involved. Two purine/pyrimidine mirror repeat elements wit hin the -20.9-kb DHS were shown not to adopt non-B-DNA conformations. Thus, we provide evidence for a role for the -20.9 kb DHS in the transcriptional regulation of the CFTR gene, although the mechanisms mediating this effect remain unclear.