Evaluation of potential regulatory elements identi®ed as DNase I hypersensitive sites in the CFTR gene Marios Phylactides 1 , Rebecca Rowntree 1 , Hugh Nuthall 1 , David Ussery 2 , Ann Wheeler 1 and Ann Harris 1 1 Paediatric Molecular Genetics, Institute of Molecular Medicine, Oxford University, John Radclie Hospital, UK; 2 Center for Biological Sequence Analysis, Biocentrum DTU, Technical University of Denmark, Lyngby, Denmark The cystic ®brosis transmembrane conductance regulator (CFTR) gene s hows a complex pattern of expression, with temporal and spatial regulation that is not accounted for b y elements in the p romoter. One approach to identifying t he regulatory elements for CFTR is the mapping of DNase I hypersensitive sites (DHS) within the locus. We previously identi®ed at least 12 clusters of DHS across the CF TR gene and here further evaluate DHS in introns 2, 3, 10, 16, 17a, 18, 20 and 21 to assess their function al importance in regulation of CFTR gene expression. Transient transfections of enhan- cer/reporter c onstructs containing the DHS regions showed that those in introns 20 and 21 augmented the activity of the CFTR promoter. Structural analysis of the DNA sequence at the DHS suggested that only the one intron 21 might be caused by inherent DN A structures. Cell speci®city of the DHS suggested a role for the DHS in introns 2 and 18 in CFTR expression in some pancreatic duct cells. Finally, regulatory elements at the DHS in intron s 1 0 a nd 18 may contribute to upregulation of CFTR gene transcription by forskolin and mitomycin C, respectively. These data support a model of r egulation of expression of the CFTR gene in which multiple elements contribute to t ightly co-ordinated expression in vivo. Keywords: CFTR; regulation; DNase I hypersensitive s ites. The cystic ®brosis transmembrane conductance regulator (CFTR) gene s hows a tightly regulated pattern o f t emporal and s patial expression though the elements responsible for this remain poorly characterized. We previously identi®ed DNase I hypersensitive sites (DHS) across 400 kb ¯anking the CFTR gene in order to locate potential regulatory elements [1±5]. These DHS lie 5¢ tothegeneat)79.5 and )20.9 kb with respect to the translational start site; in introns 1, 2, 3, 10, 16, 17a, 18, 20 and 21; and 3¢ to t he gene at +5.4 to +7.4 and +15.6 kb (Fig. 1). DHS are often, but not always, a ssociated with regulatory elements in chrom- atin. As we have identi®ed multiple clusters of DHS, it is possible that not all o f these represent important regulatory elements for the CFTR gene. The aim of t his work w as to evaluate the regions of th e CFTR gene containing the DHS to identify those containing i mportant regulatory elements. In vitro analyses of the DHS regions have included evaluation in enhancer/repor ter gene c onstructs where luciferase activity is driven by the CFTR basal p romoter and DNA ¯anking the DHS is inserted into the e nhancer site of the v ector. The results suggest that in addition to the DHS in intron 1 (at 185 + 10 kb) which was previously shown to increase CFTR promoter activity [2], the DHS in intron 20 (at 4005 + 4 kb) also augments promoter activity and the DHS in intron 21 (at 4 095 +7.2 kb) has modest enhancer activity. The majority o f the DHS were i nitially identi®ed in the Caco2 colon carcinoma cell line [5]. We have now looked f or tissue-speci®c regulatory elements by analysing chromatin structure a t these DHS in two pancreatic a denocarcinoma cell lines Capan1 [6] and NP31 [7] and an airway epithelial cell line Calu3 [8], all of which express CFTR,toseeifany showed cell type speci®city. The pancreatic lines show a different predominance of DHS than the C aco2 cell line, while the intensity of DHS in Calu3 chromatin is very weak. Finally we evaluated the effect of known activators of CFTR transcription, including forskolin [9] and mitomy- cin C [10] on the DHS. MATERIALS AND METHODS Cell culture The f ollowing cell lines w ere analysed: the colon carcinoma Caco2 [6], the pancreatic adenocarcinomas Capan1 [6] and NP31 [7] and the Calu3 lung adenocarcinoma [8]. Transient transfection assays Transient expression constructs were generated using the pGL3 Basic vector (Promega). A 787-bp fragment (245) [2] spanning the CFTR basal promoter (from )820 to )33 with respect to the ATG translational start codon) was cloned into NheIandBglII sites of the promoter multiple clon ing site of pGL3B in the correct orientation for driving transcription of the luciferase gene. The regions spanning the DHS identi®ed in intron 2 (P1.8, ac000111: 46769±48558), 10 (B1.5, ac000111: 111755± 113258 and H2.1, ac000111: 120380±122527), 16±17a Correspondence to A. Harris, Paediatric Molecular Genetics, Institute of Molecular Medicine, Oxford University, John Radclie Hospital, Oxford, OX3 9DS, UK. Fax: + 44 1865 222626, E-mail: aharris@molbiol.ox.ac.uk Abbreviations: CFTR, cystic ®brosis transmembrane conductance regulator; DHS, DNase I hypersensitive sites. (Received 3 0 August 2 001, revised 8 November 2001, accepted 16 November 200 1) Eur. J. Biochem. 269, 553±559 (2002) Ó FEBS 2002 (E4.5, ac000111 : 147607 to ac000061 : 3003, including intron 17a) 20 (Bg2.6, ac000061 : 35259±37869) and 21 (PE1.9, ac000061: 49458±51381) were cloned i nto the BamHI restriction site in the ÔenhancerÕ segment of the vector. The orientation of each fragment with respect to the 245 promoter fragment was veri®ed and further experiments carried out on those orientated 5¢)3¢ with respect to the vector backbone. In all transfection experiments the pGL3B-245 constructs were cotransfected with one-quarter or one-tenth the amount of DNA of pCMVbGal as a transfection c ontrol, using FuGene 6 (Roche). Luciferase and b-galactosidase assays were carried out by standard procedures using Promega reporter lysis buffer and luciferas e assay reagent and a lumine scent b-ga lactosidase detect ion kit (Clo ntech). Luminescence was measured i n a calibrated Turner TD 20e luminometer. Each transfection experiment was carried out a minimum of four times with individual c onstructs being assayed in triplicate in each experiment. Results are expressed as relative lucife rase activity, with the p GL3B- 245 CFTR promoter construct a ctivity equal to 1 , corrected for t ransfection e f®ciency as measured by b- galactosidase activity. Statistical analysis was performed using nonpaired t-tests assum ing unequal variance (Welch) using software available at http://www.graphpad.com. DNA structure determination Structural analysis of the CFTR sequ ence was made using two contigs that c over the en tire coding region (GenBank accession numbers ac000111 and ac000061). DNA struc- tural atlases were constructed as described previously [11]. DNase I hypersensitivity assays Chromatin from a panel of cell types was probed for DNase I hypersensitive regions by standard methods [2]. Nuclei were treated in parallel aliquots by digestion with DNase I (0i, 0, 20, 40, 80 and 160 U of DNase I; FPLCpure, Pharmacia; 1 U is equal to approximately 0.3 kunitz units for approximately 10±15 min). Sample 0i was kept on ice w hilst the remaining samples were incubated a t 37 °C. To ensure that each batch of DNase I digested chromatin was adequately digested, they were evaluated with the RA 2.2 probe that detects a constitutive DHS in the a globin locus [12]. Probes for the DHS in introns 1 (185 + 10 kb), H4.0; introns 2 ( 296 + 4.4 kb) and 3 (405 + 0.7 kb), F34L; intron 10 (1716 + 13.2 kb, + 13.7 k b and + 23 kb), 116/117 and BT1.2; i ntron 16 and 17a (3120 + 3kb and 3271 + 0.7 kb) E1.9 ; i ntron 20 (4005 + 4 kb) EB1.4 and intron 21 (4095 + 7.2 kb) H2.2 were described previously [1,5]. All cell types studied were test ed for CF TR mRNA expression by RT-PCR at the time of isolation of nuclei for chromatin analysis [1]. Treatment with activators of CFTR transcription Caco2, Capan1 and Calu3 cell lines were treated with Forskolin (10 l M in dimethylsulfoxide, 8 h), mitomicin C (0.25 l M , 4 h) by addition to the c ulture medium. Nuclei were then immediately p rocessed for DNase I hypersensi- tivity assays as above. For all experiments, RNA was extracted from an aliquot of cells to evaluate CFTR mRNA expression by RT-PCR at the time of i solation of nuclei for chromatin analysis [1]. RESULTS Transient transfections DNA fragments of between 1.5 and 4.5 kb were cloned into the enhancer site of pGL3B-245, containing 787 bp of the CFTR basal promoter, an d contructs a ssayed for enhancer activity f ollowing transient t ransfection i nto C aco2 colon carcinoma cells and MCF7 breast c arcinoma cells (Fig. 2 ). A 2.6-kb region of DNA en compassing the D HS region in intron 20 showed a 4 .4-fold enhancement of luciferase activity over the promoter only construct in Caco2 cells (P < 0.0001). A region of 1.9 kb of DNA encompassing the DHS region in intron 21 showed a 1.5-fold enhancement of luciferase activity in Caco2 (P £ 0.005); no other DHS region contained sequences that enhanced lucifer ase expression in Caco2 cells. N o DHS region enhanced CFTR promoter activity in MCF7 cells that do not express CFTR. In fact, many of the constructs showed reduced luciferase expression (P < 0.0007 to p < 0.002) in comparison to the CFTR promoter only construct i n MCF cells. Structure determination The regions encompassing each DHS as described in the transient assays were evaluated for structural motifs which might cause inherent DNase I hypersensitivity. Of Fig. 1. Diagram of the CFTR locus showing D Nase I hyperse nsitive sites. Numbers immediately above the line denote exons. Numbers above the arrows denote the individual D HS as de®ned p reviously [1,2,4,5]. 554 M. Phylactides et al. (Eur. J. Biochem. 269) Ó FEBS 2002 particular interest are two structural parameters, the ÔDNase I s ensitivityÕ model of Brukner et al. [14] and t he presence of alternating p yrimidine (Y) purine (R) tracts of 10 bp in length or longer. The results for the DNA sequence in ac000061 including the regions around the DHS 20 (Bg2.6) a nd DHS 21 (PE1.9) are shown in Fig. 3. Predicted DHS (based on the structural p roperties of the naked DNA sequence) are shown in lane C, where the darker blue regions represent predicted hypersensitive sites. For example there a re two predicted hypersensitive sites, which lie just 3 ¢ to the Bg2.6 region that encompasses t he DHS 20. These two predicted DHS also correspond to long YR tracts (blue bands in lane D). Note that these regions do not easily correlate with areas e xpected to melt readily or with higher AT content (red regions in lanes E and F). DHS 2/3 (P1.8), DHS 10a,b (B1.5), DHS 10C (H2.1), DHS 16/17a (E4.5), and DHS 20 (Bg2.6) did not contain r egions expected to be hypersensitive to DNase I, based on either the Brukner DNase I sensitivity model or the presence of YR tracts. Only DHS21 (PE1.9) encompasses a region predicted to be sensitive t o DNase I, based on structural properties of the DNA sequence alone. Fig. 2. Transient transfection e xperiments: the DHS in introns 20 (Bg2.6) and 21 (PE1.9) augment the activity of t he CFTR p romoter in Caco2 cells. The bar chart shows the luciferase activities for e ach construct relati ve to pGL3B-245 (CFTR promoter only co nstruc t) in Caco2 and MCF-7 cells. Luciferase activi- ties were no rmalized for transfection e cien- cies by cotransfection with pCMV/b. Each bar is the average of at least four transfection experiments, with each sample assa yed in triplicate, and standard errors of t he mean are shown. Fig. 3. DNA atlas of ac000061 showing a region of predicted DNase I hypersensitivity close to the DHS in intron 21, around 51 kb. Thelanesareas annotated in the ®gure. Lanes A and B are based on annotations from the GenBank ®le, and the DNase hyperse nsitivity sites marked in black are the experimentally determined DHS regions. Lane C (ÔDNase I sen sitivityÕ) is based on the trinucleotide model of B rukner et al. [14] smoothed over a 330-bp window, and lane D is the density of YR tracts of at least 10 bp in length, smoothed over a 165-bp window. Lane E is the stacking energy, in kcalámol )1 , based on the dinucleotid e model of Ornstein et al. [20]; on this scale, the red regions are expected to melt m ore readily. Lane F is the AT content, ranging from 20% (turquoise) to 80% (red). Ó FEBS 2002 Evaluation of CFTR DNase I hypersensitive sites (Eur. J. Biochem. 269) 555 Tissue speci®city of DHS elements In previous experiments we e valuated DHS i n the Caco2 colon carcinoma cell line and performed a preliminary screen for these DHS in other cell line s [5]. To loo k for tissue-speci®c DHS in the pancreatic duct, the pancreatic cell lines Capan1 and NP31 w ere evaluated further. DHS in airway epithelial cells were investigated further in t he airway carcinoma cell line Calu3. Of particular interest were the DHS in introns 2 and 18, which w ere s trongly evident in Capan1 in comparison to Caco2 chromatin (Fig. 4). The DHS in introns 2 and 3 were detected as subbands of 4.5 and 3.4 kb, respectively, when Caco2 chromatin was hybridized with the F34L probe (Fig. 4 B). In contrast, in C apan1 cells the DHS in intron 2 (4.5-kb f ragment) is much more evident a nd the DHS in intron 3 is not detectable (Fig. 4A). T he DHS in introns 16, 17, 18 are detected with a single probe (E1.9) and they appear as subfragments of the 24-kb genomic fragment at 5 , 7 and 17 kb, respectively. In Caco2 cells the DHS in introns 16 and 17 are of similar intensity but the DHS in intron 18 is less evident ( Fig. 5B). In contrast, the DHS in intron 18 is more prominent than those in introns 16 and 17 (Fig. 5A) in chromatin from Capan1 cells. The DHS in i ntrons 1, 10a,b (very weak) and 20 were also e vident in Capan1 cells (data not shown). Evaluation of DHS in another pancreatic adenocarcinoma cell line NP-31 revealed the DHS in introns 2, 10c, 17a, and 20 though o ther DHS were either extremely weak or nondetectable (data not shown). Extensive analysis o f chromatin from the Calu3 cell line revealed a paucity of DHS, with only the DHS i n introns 1, 16,17, 18 and 2 0 being detectable (data not shown). In addition to the 4005 + 4 kb DHS detected in intron 20 in chromatin from Caco2, two novel intron 20 DHS were seen in Calu3 chromatin, detected as 3.8 and 3.3-kb subfrag- ments of the 24.5-kb genomic fragment detected by the EB1.4 probe. These correspond to DHS at 4005 + 6.7 k b and 4005 + 7.2 kb (Fig. 6 ). Activation of CFTR expression Chromatin was extracted from untreated Caco2 and Capan1 cells or after incubation with forskolin or mitomy- cinCandthendigestedwithDNaseI.Toensurethat control and drug-treated samples of chromatin were equally digested th ey were evaluated with t he RA 2.2 p robe that detects a constitutive DNase I hypersensitive site in the a globin locus [12]. Subsequently the intensity of the DHS in introns 1, 2/3, 10a,b, 16/17/18 and 20 were compared i n drug-treated and control samples on Southern blots of chromatin. The intensity of the signal derived from the genomic band and the DHS-derived band were determined using a phosphorimager and IMAGEQUANT 5.12 software (Molecular Dynamics). In all cases where preliminary data showed increased intensity of a DHS, the experiment was repeated to show that it was a consistent observation. The only DHS that consistently s howed an increase in intensity following exposure to activators of CFTR transcription were DHS10a, b, f ollowing forskolin activation in Caco2 cells (Fig. 7A) and DHS 18 in Capan1 cells following mitomycin C act ivation (Fig. 7B). The bar charts show the intensity ratios of the DNase I derived subfragments to genomic fragments for forskolin/mitomicin C treated and control c hromatin processed s imultaneously. H ence if the drug treatment were having no effect on t he intensity o f the DHS then the two bars in each pair would be of the same height. F or both panels A and B the increasing amounts o f DNase show a proportionate increase in the intensity of the DHS fragments in the control samples. In Fig. 7A the forskolin-treated chromatin shows a relative increase in the intensity of the DHS10a (1716 + 13.2 kb) appearing at 20 U of DNase I but being more evident after 40 U of DNase I (ratio forskolin-treated/control  1.7 : 1.15). The effect of mitomycin C on DHS 18 (3600 + 7 kb) in Capan cells is shown in Fig. 7B w here ratios of genomic/DNase I Fig. 4 . The DHS in intron 2 (296 + 4.4 kb) is prominent in Capan1 pancreatic adenocarcinoma cells. Southern blots of DNase I digested (A) Capan1 and (B) Caco2 chromatin cleaved with BamHI and hybridized with the F34L prob e. In each panel, lanes 1 (stored on ice) and lanes 2 ( incubated at 37 °C) show chromatin not treated with DNase I a nd lanes 3±6 show chromatin prepared from nuclei with increasing amounts of DNase I (20, 40, 80 and 160 U , respectively). A 1-kb ladder (Life Technologies) was u sed for size markers. Fig. 5. The DHS in intron 18 (360 0 + 7 kb) is prominent in Capan1 pancreatic adeno- carcinoma cells. Southern blots of DNase I digested (A) Capan1 and (B) Caco2 chromatin cleaved with BamHIandhybridizedwiththe CE1.9 p robe. In each panel, lanes 1 (stored on ice) and lanes 2 (incubated a t 37 °C) show chromatin not treated with DN ase I an d lanes 3±6 show chromatin prepared f rom nuclei with increasing amou nts of DNase I (20, 40, 80 and 160 U, respectively). A 1-kb ladder (Life Technologies) was used for size markers. 556 M. Phylactides et al. (Eur. J. Biochem. 269) Ó FEBS 2002 treated fragment a re greater t han in the control c hromatin, most prominently at 20 U (ratio, 0.81 : 0.28). DISCUSSION Our c urrent model for tissue speci®c and temporal regula- tion of th e CFTR gene predicts that co-operation of many different regulatory elements may con tribute t o CF TR expression in the chromatin environment in vivo. DHS are often, though not always, associated with regulatory elements. We have previously identi®ed DHS both 5 ¢ and 3¢ to t he CFTR gene and within at least nine introns. The aim of the current experiments was to evaluate the role of individual intragenic DHS in regulation of CFTR expres- sion. The ®rst series of experiments evaluated potential enhancer activity of the intragenic DHS in transient transfection of reporter/enhancer constructs. We have shown previously t hat the 185 + 1 0 kb DHS in intron 1 augments CFTR promoter activity in transient transfections of Caco2 cells [2] and that removal of 32 bp at the predicted core of the DHS abolished this activity [15]. Analysis of the DHS in introns 2 , 3, 10, 16, 17a, 20 and 21 s howed that the 4005 + 4 kb DHS in intron 20 and the 4095 + 7.2 kb DHS in intron 21 both augmented the activity of the CFTR promoter in Caco2 cells. Neither construct affected CFTR prom oter activity in MCF7 cells that do not express CFTR. These data suggest that while the DHS in introns 20 and 21 m ay contain t issue-speci®c enhancer sequences, the remaining DHS are n ot associated with enhancer function. Due to the inherent limitations of transient transfection assays, further in vivo analysis will be required to evaluate the role of the intron 20 and 21 DHS in CFTR expression in chromatin. The DNA sequ ence within the intragenic DHS was evaluated to search for speci®c motifs that might cause inherent DNase I hypersensitivity b ased on bent or easily melted DNA. Generation of a DNA atlas for each of the t wo contigs covering the CFTR gene (ac000111 and ac000061) enabled the prediction of DNase sensitivity and YR steps that predict ease of melting. Although t here are many areas of predicted sequence-based DNase I hypersensitivity within the gene, the only one that corresponds to the DHS that we have evaluated here is that in intron 21. (The region of the intron 1 at 185 + 10 kb also shows some inherent DNase I sensitivity.) These data suggest that the DHS that we have observed, with the e xception of that in in tron 21, are not structural artefacts induced by DNA sequence a lone. Our model f or regulation of expression of the CFTR gene would predict that individual d ifferentiated cell types would show a speci®c set of DHS that might differ from other cell types. The Caco 2 intestinal carcinoma cell line t hat we u sed Fig. 7. EectofactivationofCFTR transcription on DHS. The charts show the eect of (A) forskolin on DHS10a (1716 + 13.2 kb) in Caco2 cells and (B) mitomycin on DHS 18 (36 00 + 7 kb) in Cap an1 cells. Charts show the r atio of the f ragmen t intensities of the D HS-derived subfragment to the genomic fragment on phosphorimages of Southern blots in control and forskolin/mitomycin C -treated cells. Fig. 6. Novel DHS in intron 20 at 4005 + 6.7 kb and 4005 + 7.2 k b in Calu3 chromatin. Southern b lot of DNase I d igested Calu3 chromatin cleaved with BamHI and hybridized with t he EB1.4 probe. In each panel, lanes 1 (stored on ice) and lanes 2 ( incubated at 37 °C) show chromatin that had not been treated with DNase I and lanes 3±6 show chromatin p repared from nuclei with increasing amounts of DNase I (20, 40, 80 and 160 U, respectively). A 1-kb ladder (Life Technologies) was used f or size markers . Ó FEBS 2002 Evaluation of CFTR DNase I hypersensitive sites (Eur. J. Biochem. 269) 557 initially to search for DHS due to its high level of endogenous CFTR transcription e xhibits at least 12 DHS or clusters of DHS lying 5¢, within the gene and 3¢. Several of these DHS have only b een seen in Caco2 c hromatin and fewer DHS are e vident in the other cell lines that we have examined. A mong other cell t ypes, s uch a s p ancreatic a nd airway epithelial cells we have not found consistent pro®les of DHS. For example the Capan1 and NP31 pancreatic adenocarcinoma cell lines that both express CFTR mRNA (the former at very low levels) show different DHS. Features of the Capan1 line were the strong DHS in intr on 2 a nd intron 18. Although NP31 showed the intron 2 DHS that in intron 18 was not evident and the DHS in intron 10 at 1716 + 23 kb was strong. The role of th ese DHS in CFTR expression in the pancreas w arrants further evaluation. In the airway cell line Calu3, that expresses a high level of CFTR mRNA, very few DHS were evident. This could be a genuine feature of this cell line or b e due to only a small percentage of cells in the culture expressing high levels of CFTR, which would then only contribute a small part of the chromatin sample m aking DHS hard to detect. One disadvantage of analysing carcinoma cell lines is that some of the DHS we observe may be the result of these lines showing aberrant gene expression following tumorigenesis, rather than normal endogenous CF TR expression. How- ever, it i s not possible to obtain suf®cient chromatin from primary cells from pancreas and airway epithelium to evaluate DHS. Our model for regulation of CFTR transcription also predicts that agents that activate CFTR transcription would act at certain regulatory elements assoc iated with DHS but not others, depending on their p roperties and role in CFTR transcription. It is known that chemicals which increase intracellular cAMP cause an increase in CFTR protein expression in cell membranes and activation of chloride secretion. The increased CFTR protein has been shown in part to be the result of transcriptional a ctivation of CFTR [9]. It is known that cAMP response e lements are present i n the CFTR promoter [16±18] but also in other predicted regulatory elements [4]. Here we have shown that fors- kolin (an inducer of intracellular cAMP) reproducibly enhances the DHS in intron 10 of the CFTR gene at 1716 + 13.2 kb in CaCO 2 cells. Analysis of the sequence around this DHS has shown a cluster of CREB and CREB-related motifs between ac000111 : 111 936±112 125 which a re undergoing further a nalysis to evaluate their potential role in CFTR expression. Noncytotoxic doses of mitomycin C, a DNA cross- linking reagent have been shown to preferentially alter the expression of inducible genes [19]. Mitomycin C was also shown to induce CF TR mRNA and protein levels in colon carcinoma cells lines [10]. We have shown t hat activation of the Capan1 p ancreatic adenocarcinoma cells by a l ow dose of mitomycin C reproducibly enhanced the intensity of the DHS in intron 1 8 in c omparison t o nonactivated cells. I t i s possible that activation of a potential regu latory element sited at this DHS plays a role in pancreatic expression o f CFTR. This would be consistent with our data on the cell- speci®c expression of this DHS. The d ata presented here con®rm t he complexity of the regulation of expression of the CFTR gene. Elements within the CFTR promoter are known t o be i nadequate to explain the tissue-speci®c and temporal regulation of CFTR. We have previously shown t hat t he DHS a t 1 85 + 10 kb in intron 1 of the CFTR gene augments intestinal expression of the gene in vivo, both in human colon carcinoma cells and in transgenic mice [15]. It is probable t hat several other DHS that we have identi®ed contain regulatory elements that have speci®c roles in co-ordinating CFTR expression in vivo. 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Biochem. 269) 559 . promoter multiple clon ing site of pGL3B in the correct orientation for driving transcription of the luciferase gene. The regions spanning the DHS identi®ed in intron. to identifying t he regulatory elements for CFTR is the mapping of DNase I hypersensitive sites (DHS) within the locus. We previously identi®ed at least