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

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Date
2009-04
Authors
Ott, Christopher J
Suszko, Magdalena
Blackledge, Neil P
Wright, Jane E
Crawford, Gregory E
Harris, Ann
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140
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Abstract
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.
Type
Journal article
Subject
Base Pairing
Base Sequence
Binding Sites
Cell Line
Cystic Fibrosis Transmembrane Conductance Regulator
DNA Footprinting
Deoxyribonuclease I
Enhancer Elements, Genetic
Gene Expression Regulation
Genes, Reporter
Hepatocyte Nuclear Factor 1
Humans
Introns
Molecular Sequence Data
Organ Specificity
Promoter Regions, Genetic
Protein Binding
Transcription Factors
Transcription, Genetic
Transfection
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https://hdl.handle.net/10161/10684
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Scholars@Duke

Crawford

Gregory E. Crawford

Professor in Pediatrics
My research involves identifying gene regulatory elements across the genome to help us understand how chromatin structure dictates cell function and fate. For the last 30 years, mapping chromatin accessible sites has been the gold standard method to identify the location of active regulatory elements, including promoters, enhancers, silencers, and locus control regions. I have developed technologies that can identify most DNase I hypersensitive sites from potentially any cell type from any speci
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