The cystic fibrosis transmembrane conductance regulator (CFTR) gene exhibits a tightly regulated pattern of expression in human epithelial cells. The mechanism of this regulation is complex and is likely to involve a number of genetic elements that effect temporal and spatial expression. To date none of the elements that have been identified in the CFTR promoter regulate tissue-specific expression. We have identified a putative regulatory element within the first intron of the CFTR gene at 181+10kb. The region containing this element was first identified as a DNase I hypersensitive site that was present in cells that express the CFTR gene but absent from cells not transcribing CFTR. In vitro analysis of binding of proteins to this region of DNA sequence by gel mobility shift assays and DNase I footprinting revealed that some proteins that are only present in CFTR-expressing cells bound to specific elements, and other proteins that bound to adjacent elements were present in all epithelial cells irrespective of their CFTR expression status. When assayed in transient expression systems in a cell line expressing CFTR endogenously, this DNA sequence augmented reporter gene expression through activation of the CFTR promoter but had no effect in nonexpressing cells.
The cystic fibrosis transmembrane conductance regulator gene (CFTR) exhibits a complex pattern of expression that shows temporal and spatial regulation, although the control mechanisms are not fully known. We have mapped DNase-I-hypersensitive sites (DHSs) flanking the CFTR gene with the aim of identifying potential regulatory elements. We previously characterized DHSs at -79.5 and -20.9 kb with respect to the CFTR translational start site and a regulatory element in the first intron of the gene at 185+10 kb. We have now mapped five DHSs lying 3′ to the CFTR gene at 4574+5.4, +6.8, +7.0, +7.4 and +15.6 kb that show some degree of tissue specificity. The DHSs are seen in chromatin extracted from human primary epithelial cells and cell lines; the presence of the +15.6 kb site is tissue-specific in transgenic mice carrying a human CFTR yeast artificial chromosome. Further analysis of the 4574+15.6 kb DHS implicates the involvement of CCAAT-enhancer-binding protein (C/EBP), cAMP-response-element-binding protein (CREB)/activating transcription factor (ATF) and activator protein 1 (AP-1) family transcription factors at this regulatory element.
Background: The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex pattern of expression. The regulatory elements conferring tissue-specific and temporal regulation are thought to lie mainly outside the promoter region. Previously, we identified DNase I hypersensitive sites (DHS) that may contain regulatory elements associated with the CFTR gene at -79.5 and at -20.5 kb with respect to the ATG and at 10 kb into the first intron. Materials and Methods: In order to evaluate these regulatory elements in vivo we examined these DHS in a human CFTR gene that was introduced on a yeast artificial chromosome (YAC) into transgenic mice. The 310 kb human CFTR YAC was shown to restore the phenotype of CF-null mice and so is likely to contain most of the regulatory elements required for tissue-specific expression of CFTR. Results: We found that the YAC does not include the -79.5 kb region. The DHS at -20.5 kb is present in the chromatin of most tissues of the transgenic mice, supporting its non-tissue-specific nature. The DHS in the first intron is present in a more restricted set of tissues in the mice, although its presence does not show complete concordance with CFTR expression. The intron 1 DHS may be important for the higher levels of expression found in human pancreatic ducts and in lung submucosal glands. Conclusion: These data support the in vivo importance of these regulatory elements.
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