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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 promoter of the gene. We previously identified DNase I hypersensitive sites (DHSs) 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 investigated further the DHS at -20.5 kb to evaluate its potential function in the regulation 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 responsible for about 60% of wild-type levels of transcription from the YAC transgene when expressed in Caco2 cells. DNase I footprinting showed several regions 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 transcription factors involved. Two purine/pyrimidine mirror repeat elements within 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.

Original publication

DOI

10.1046/j.1432-1327.1999.00872.x

Type

Journal article

Journal

Eur J Biochem

Publication Date

12/1999

Volume

266

Pages

431 - 443

Keywords

Amino Acid Motifs, Base Sequence, Binding Sites, Chromosomes, Artificial, Yeast, Cloning, Molecular, Cystic Fibrosis Transmembrane Conductance Regulator, DNA, Superhelical, Deoxyribonuclease I, Electrophoresis, Agar Gel, Exons, Gene Deletion, Gene Expression Regulation, Humans, Models, Genetic, Molecular Sequence Data, Nucleic Acid Conformation, Plasmids, Protein Biosynthesis, Purines, Pyrimidines, RNA, Messenger, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Sequence Homology, Nucleic Acid, Single-Strand Specific DNA and RNA Endonucleases, Transcription, Genetic, Transgenes, Tumor Cells, Cultured