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.
Ott
Christopher J
CJ
Children's Memorial Research Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
Suszko
Magdalena
M
Blackledge
Neil P
NP
Wright
Jane E
JE
Crawford
Gregory E
GE
Harris
Ann
A
D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D051537:Hepatocyte Nuclear Factor 1 / Q000378:metabolism; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003850:Deoxyribonuclease I