A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter.
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 articleSubject
Base PairingBase 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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Show full item recordScholars@Duke
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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