Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus.
The regulated expression of large human genes can depend on long-range interactions to establish appropriate three-dimensional structures across the locus. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encompasses 189 kb of genomic DNA, shows a complex pattern of expression with both spatial and temporal regulation. The flanking loci, ASZ1 and CTTNBP2, show very different tissue-specific expression. The mechanisms governing control of CFTR expression remain poorly understood, although they are known to involve intronic regulatory elements. Here, we show a complex looped structure of the CFTR locus in cells that express the gene, which is absent from cells in which the gene is inactive. By using chromatin conformation capture (3C) with a bait probe at the CFTR promoter, we demonstrate close interaction of this region with sequences in the middle of the gene about 100 kb from the promoter and with regions 3' to the locus that are about 200 kb away. We show that these interacting regions correspond to prominent DNase I hypersensitive sites within the locus. Moreover, these sequences act cooperatively in reporter gene constructs and recruit proteins that modify chromatin structure. The model for CFTR gene expression that is revealed by our data provides a paradigm for other large genes with multiple regulatory elements lying within both introns and intergenic regions. We anticipate that these observations will enable original approaches to designing regulated transgenes for tissue-specific gene therapy protocols.
Ott
Christopher J
CJ
Human Molecular Genetics Program, Children's Memorial Research Center, Northwestern University Feinberg School of Medicine, 2300 Children's Plaza #211, Chicago, IL 60614, USA.
Blackledge
Neil P
NP
Kerschner
Jenny L
JL
Leir
Shih-Hsing
SH
Crawford
Gregory E
GE
Cotton
Calvin U
CU
Harris
Ann
A
D002478:Cells, Cultured; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D051537:Hepatocyte Nuclear Factor 1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007438:Introns; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D050880:p300-CBP Transcription Factors / Q000235:genetics / Q000378:metabolism
D051537:Hepatocyte Nuclear Factor 1; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D050880:p300-CBP Transcription Factors