A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter.


Genes can maintain spatiotemporal expression patterns by long-range interactions between cis-acting elements. The cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed primarily in epithelial cells. An element located within a DNase I-hypersensitive site (DHS) 10 kb into the first intron was previously shown to augment CFTR promoter activity in a tissue-specific manner. Here, we reveal the mechanism by which this element influences CFTR transcription. We employed a high-resolution method of mapping DHS using tiled microarrays to accurately locate the intron 1 DHS. Transfection of promoter-reporter constructs demonstrated that the element displays classical tissue-specific enhancer properties and can independently recruit factors necessary for transcription initiation. In vitro DNase I footprinting analysis identified a protected region that corresponds to a conserved, predicted binding site for hepatocyte nuclear factor 1 (HNF1). We demonstrate by electromobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) that HNF1 binds to this element both in vitro and in vivo. Moreover, using chromosome conformation capture (3C) analysis, we show that this element interacts with the CFTR promoter in CFTR-expressing cells. These data provide the first insight into the three- dimensional (3D) structure of the CFTR locus and confirm the contribution of intronic cis-acting elements to the regulation of CFTR gene expression.







Gregory E. Crawford

Professor in Pediatrics

My primary research interest is understanding how the genome is regulated.  The human genome contains approximately 25,000 genes, which are encoded in ~2% of the genome. The overarching goal of my research program is to identify and characterize how these genes are turned on and off in different cell types, tissues, development states, environmental responses, diseases, and individuals. By understanding where all gene regulatory elements are located, how they work to regulate gene expression, and how non-coding variants within these regions affect function, my research program can address a number of important basic and clinical questions.

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