Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus Christopher J. Otta, Neil P. Blackledgea,1,2, Jenny L. Kerschnera,1, Shih-Hsing Leira, Gregory E. Crawfordb, Calvin U. Cottonc, and Ann Harrisa,3 aHuman Molecular Genetics Program, Children’s Memorial Research Center, Northwestern University Feinberg School of Medicine, 2300 Children’s Plaza #211, Chicago, IL 60614; bInstitute for Genome Sciences and Policy, Duke University, Durham, NC 27708; and cDepartments of Pediatrics, Physiology, and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106 Edited by Bing Ren, University of California–San Diego School of Medicine, La Jolla, CA, and accepted by the Editorial Board September 22, 2009 (received for review January 28, 2009) The regulated expression of large human genes can depend on conformation to facilitate expression. The CFTR gene encom- long-range interactions to establish appropriate three-dimensional passes 189 kb at human chromosome 7q31.2 and mutations structures across the locus. The cystic fibrosis transmembrane within it cause the common genetic disease cystic fibrosis (CF) conductance regulator (CFTR) gene, which encompasses 189 kb of (9). CFTR encodes a membrane-associated chloride ion channel genomic DNA, shows a complex pattern of expression with both that is expressed at the highest levels in chloride-secreting spatial and temporal regulation. The flanking loci, ASZ1 and epithelial cells of the small intestine, pancreas, and male genital CTTNBP2, show very different tissue-specific expression. The mech- duct, and at lower levels in the respiratory epithelium and certain anisms governing control of CFTR expression remain poorly un- other sites (10–14). The CFTR promoter has been characterized derstood, although they are known to involve intronic regulatory as a ‘‘housekeeping-like’’ promoter and does not posses the elements. Here, we show a complex looped structure of the CFTR regulatory elements responsible for the diverse expression pro- file of the gene (15–17). Thus, it is likely that elements outside locus in cells that express the gene, which is absent from cells in the basal promoter region contribute to its diverse expression which the gene is inactive. By using chromatin conformation profile. We previously used classical methods of chromatin capture (3C) with a bait probe at the CFTR promoter, we demon- analysis to map potential regulatory elements in a number of cell strate close interaction of this region with sequences in the middle lines that express the CFTR gene and identified several func- of the gene about 100 kb from the promoter and with regions 3؅ tionally important elements (18–23). In this study we aimed to to the locus that are about 200 kb away. We show that these use recent technologies to comprehensively map potential reg- interacting regions correspond to prominent DNase I hypersensi- ulatory elements of the CFTR locus and to establish their tive sites within the locus. Moreover, these sequences act cooper- mechanism of action. Moreover, we used a number of human atively in reporter gene constructs and recruit proteins that modify primary cell types relevant to CF pathology, in addition to chromatin structure. The model for CFTR gene expression that is evaluating pertinent cell lines. revealed by our data provides a paradigm for other large genes Using high-resolution tiled microarrays, we detected multiple with multiple regulatory elements lying within both introns and intronic and extragenic DNase I hypersensitive sites (DHS), intergenic regions. We anticipate that these observations will regions of open chromatin that are depleted of nucleosomes and enable original approaches to designing regulated transgenes for are often associated with gene regulatory elements (24). We tissue-specific gene therapy protocols. demonstrate that several of these DHS regions bind both tissue-specific and general transcription factors and also possess cis-acting elements ͉ enhancer:promoter interactions ͉ regulation cooperative enhancer activity in vitro. Moreover, we show that of expression ͉ cystic fibrosis transmembrane conductance regulator in vivo, these enhancers interact directly with the CFTR pro- moter region. The recent advances in methodology to evaluate regulatory elements in the human genome in vivo, combined nderstanding the three-dimensional organization of indi- with a biological approach to the expression and function of Uvidual loci within the human genome and how this relates CFTR, have enabled us to perform an in-depth study of the to the regulation of gene expression is the focus of intense study. organization of the entire CFTR locus. We demonstrate the Many new technologies are being developed to locate and properties of key regulatory elements for CFTR and show a classify the functional elements of the human genome (1). These transcriptionally active human gene adopting distinct conforma- elements may contribute to the transcription of ubiquitously tions in different cell types. expressed genes and are also likely responsible for regulating genes with expression that is temporally and spatially controlled. Results Cis-acting regulatory elements located within noncoding regions Detection of DNase I Hypersensitive Sites Across the CFTR Locus. We of genomic DNA can influence the organization of chromo- used DNase-chip to identify DHS in a number of cell types somes and the transcriptional activity of genes. These cis se- quences include distal enhancers that may reside large distances Author contributions: C.J.O., N.P.B., J.L.K., and A.H. designed research; C.J.O., N.P.B., J.L.K., from the gene promoters they control. Variations in these S.-H.L., and A.H. performed research; G.E.C. and C.U.C. contributed new reagents/analytic enhancer/promoter interactions and the nuclear localization of tools; C.J.O., N.P.B., J.L.K., and A.H. analyzed data; and C.J.O. and A.H. wrote the paper. the genes they regulate are thought to be contributing factors in Conflict of interest statement: A patent application is pending to protect the subject matter the diversity of transcriptional profiles between different cell of this manuscirpt. types. Moreover, they are important in adjusting these profiles This article is a PNAS Direct Submission. B.R.is a guest editor invited by the Editorial Board. throughout cellular differentiation and development (2–7). Data deposition: The sequence reported in this paper has been deposited in the GEO These long-range associations are facilitated by the looping of database (accession no. GSE18735). chromatin, whereby regulatory elements come together with 1N.P.B. and J.L.K. contributed equally to this work. requisite nuclear factors to function within ‘‘transcriptional 2Current address: Department of Biochemistry, Oxford University, Oxford, U.K. hubs’’ where transcriptional activity is coordinated (8). 3To whom correspondence should be addressed. E-mail: [email protected]. Here, we present evidence that the cystic fibrosis transmem- This article contains supporting information online at www.pnas.org/cgi/content/full/ brane conductance regulator (CFTR) locus adopts a looped 0900946106/DCSupplemental. 19934–19939 ͉ PNAS ͉ November 24, 2009 ͉ vol. 106 ͉ no. 47 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900946106 Downloaded by guest on September 26, 2021 Fig. 1. Identification of DHS within the CFTR locus in cell types relevant to CF pathology. (A) Averaged DNase-chip hybridization data from three (Caco2, skin fibroblasts), two (HT29, primary tracheal epithelial cells and NHBE cells), or a single (primary epididymis) experiment was analyzed with ACME statistical software (38). A major DHS was identified at the CFTR promoter (Pr) in all cells that express the gene; several specific DHS of interest were detected, including those in intron 1 (Int1) and intron 11 (Int11). A DHS at ϩ15.6 kb 3Ј to the CFTR translational stop site (20, 30) was seen in several cell types, and at ϩ48.9 kb a ubiquitous DHS (Ubiq) is marked in the last intron of the CTTBP2 gene. The zero point of the x axis represents the beginning of the first CFTR exon. The y axis for each DHS track represents –log10(P-value) between 0 and16 as determined by ACME. (B) CFTR mRNA levels measured by qRT-PCR; each value is relative to the amount of detected skin fibroblast transcript. Error bars represent SEM, n ϭ 3. relevant to CFTR expression, including primary human tracheal element in vitro and in vivo (19, 25–27) was detected in both and bronchial epithelial cells, primary human fetal epididymis Caco2 and HT29 cells. Additional cell-line specific DHS are epithelial cells, and the human colon carcinoma cell lines Caco2 evident in intron 10 (1716 ϩ 13.2 kb) in Caco2 cells and intron and HT29, all of which express CFTR. We also evaluated human 18 (3600 ϩ 1.6 kb) in HT29. The intron 10 site encompasses two skin fibroblasts that do not express CFTR to provide an example closely spaced DHS at 1716 ϩ 13.2 kb and ϩ13.7 kb, that were of the chromatin structure of the transcriptionally inactive CFTR characterized in our previous work (22, 23, 28). Also of interest locus. Three DNase-chip experiments were performed on inde- are the DHS located –35 kb and –44 kb with respect to the CFTR pendent cultures of skin fibroblasts and Caco2 cells, two exper- translational start site in primary epididymis cells. These up- iments were carried out on primary tracheal and NHBE (bron- stream DHS are closer to the neighboring gene ASZ1 (ankyrin chial and tracheal) epithelial cells and HT29 cells, and one repeat, SAM, and basic leucine zipper) and may be involved in experiment evaluated primary epididymis cells (Fig. 1A). The its regulation. DNase hypersensitivity tracks represent averaged data where The relative CFTR expression levels in each cell type were multiple experiments were performed. The data demonstrate determined using RNA isolated at the same time they were that each cell type has a specific and unique pattern of DHS harvested for DNase-chip (Fig.