title: Analysis of a DNase I hypersensitive site located -20.9 kb upstream of the CFTR gene.
abstract: 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.
authors.affiliation: Paediatric Molecular Genetics, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
authors.forename: H N
authors.initials: HN
authors.lastname: Nuthall
authors.forename: G
authors.initials: G
authors.lastname: Vassaux
authors.forename: C
authors.initials: C
authors.lastname: Huxley
authors.forename: A
authors.initials: A
authors.lastname: Harris
mesh_terms: D020816:Amino Acid Motifs; D001483:Base Sequence; D001665:Binding Sites; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics*; D004278:DNA, Superhelical / Q000648:ultrastructure; D003850:Deoxyribonuclease I / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D005091:Exons; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014158:Transcription, Genetic; D019076:Transgenes; D014407:Tumor Cells, Cultured
chemical_list: C505032:CFTR protein, human; D004278:DNA, Superhelical; D011687:Purines; D011743:Pyrimidines; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases