characterization of the cystic fibrosis transmembrane conductance

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268. No. 21, Issue of July 25, pp. 15912-15921,1993 Printed in U.S.A.

Characterization of the Cystic Fibrosis Transmembrane Conductance Regulator Promoter Region CHROMATIN CONTEXT AND TISSUE-SPECIFICITY*

(Received for publication, January 21, 1993, and in revised form, April 14, 1993)

James KohS, Thomas J. SferraSglI, and Francis S. CollinsSII**$$ From the Departments of $Human Genetics, 11 Internal Medicine, §Pediatrics and Communicable Diseases, and the **Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan 48109-0650

Expression of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) is tightly regulated. Using a panel of cell lines expressing different levels of CFTR mRNA, we investigated the mechanisms mediating control of CFTR transcription. In highly expressing cells, multiple sites of transcription initiation can be identified between positions -95 and +50 of the CFTR gene, and an alternatively initiated splice variant transcript is also present. Non- epithelial cell lines expressing very low levels of CFTR utilize a start site at -32. Promoter sequence elements from -83 to + 11 1 are at least partially responsible for dictating CFTR transcriptional tissue-specificity, while multiple elements located farther 5’ augment promoter strength. Analysis of the chromatin structure and methylation status of the CFTR promoter region reveals cell line differences which correlate with expression levels, suggesting that the physical context of the CFTR gene in vivo may contribute significantly to appropriate regulation of CFTR transcription. Taken together, these findings indicate that cellular control of CFTR gene expression is likely to be a complex function of several overlapping regulatory path- ways.

Cystic fibrosis is among the most common of the severe autosomal recessive diseases, occurring in all ethnic groups and geographic locations but with dramatically higher inci- dence among Caucasians of northern European descent, where carrier frequencies are approximately 1 in 25 (1, 2). Cystic fibrosis is a complex disorder characterized by patho- logical processes in multiple tissues and organ systems, sec- ondary to dysfunctional regulation of chloride transport across epithelial surfaces (3-5). The principal clinical mani- festations of cystic fibrosis include progressive respiratory failure, insufficient pancreatic exocrine function, gastrointestinal tract obstruction, and infertility. The successful posi-

* This work was supported in part by the Cystic Fibrosis Founda- tion (to J. K., T . J. S., and F. S. C.) and National Institutes of Health Grants DK 39690 and DK 42718 (to F. S. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Association of Medical School Pediatric Chairmen, Inc., (AMSPDC) Pediatric Scientist Development Program Fellow supported by National Institute of Child Health and Human Develop- ment Grant HD-22297.

$$ Investigator in the Howard Hughes Medical Institute. To whom correspondence should be addressed 4570 MSRB 11, Box 0650,1150 W. Medical Center Dr., Ann Arbor, MI 48109-0650. Tel.: 313-747- 3414: Fax: 313-763-4692.

tional cloning of the gene responsible for cystic fibrosis (6, 7 ) (known as the cystic fibrosis transmembrane conductance regulator, or CFTR)’ provides the opportunity to study how transcriptional regulation of the CFTR gene relates to cystic fibrosis pathology. Transcription of the CFTR mRNA is limited primarily to epithelial tissues, almost always in low abundance, and thus elucidation of the controlling elements which dictate such a restricted pattern of expression is a prerequisite for developing a rational model of the complex biological phenomena associated with cystic fibrosis.

CFTR gene expression clearly is not controlled by a “housekeeping-type’’ constitutive promoter. In situ probing of both rodent (8) and human (9)’ tissue sections demonstrates that the in vivo distribution of CFTR mRNA and protein is limited to specific subsets of epithelial cells. The steady state abundance of CFTR mRNA varies widely in different tissues and cultured cell lines (this paper, Refs. 11 and 12) and can be modulated in cultured cells by forskolin and CAMP (13), phorbol esters (14, 15), protein kinase C stimulants (16), and agents which influence intracellular divalent cation levels (17). CFTR transcription appears to be temporally and spa- tially regulated in the developing vas deferens (18), and in the rodent uterus the abundance of CFTR mRNA has been observed to fluctuate with the menstrual cycle (8). Despite the continuing accumulation of evidence indicating that CFTR gene expression is a tightly regulated, dynamic process, relatively little is known of the mechanisms which mediate CFTR transcriptional regulation.

Although two groups have previously evaluated reporter constructs in CFTR expressing cell lines, at present there is no consensus opinion on what sequences constitute the min- imal CFTR promoter (19,20). In this report we have analyzed CFTR transcription, methylation status of the gene, chromatin accessibility, and promoter strength in a panel of cell lines that express varying levels of CFTR mRNA. By primer extension, S1 nuclease protection, RNase protection, and anchored PCR assays, we define the sites of transcription initiation in the CFTR gene and identify a splice variant transcript resulting from alternative promoter usage. In experiments assessing methylation status and nuclease sensitivity in the CFTR promoter region, we demonstrate tissue- specific physical changes in the chromatin structure of the CFTR gene, including the appearance of several DNase I- hypersensitive sites which correlate with high level expression of CFTR. A comparative analysis of CFTR promoter activity as measured in transient transfection assays using luciferase

The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PCR, polymerase chain reaction; DHFR, dihydrofolate reductase; kb, kilobase(s); bp, base pair(s).

T. V. Strong and F. S. Collins, manuscript in preparation.

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reporter constructs indicates that tissue-specific transcription of CFTR is a t least partly dictated by proximal 5’ genomic sequences. Finally, from the incremental loss of promoter activity observed across a progressive 5’ deletion series, we infer that regulation of CFTR transcription is achieved through the coordinate, synergistic activity of multiple cis- and trans-acting factors which may individually contribute only modest effects.

MATERIALS AND METHODS

Cell Culture-The cell line T84 (ATCC CCL 248) was cultured in a medium consisting of a 1:l mixture of high glucose Dulbecco’s modified Eagle’s medium-Ham’s F-12 nutrient broth, supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. The cell lines CaCo2 (ATCC HTB 37) (kindly provided by Peter Traber, University of Pennsylvania), CFPAC (ATCC CRL 1918), and PANC- 1 (ATCC CRL 1469) were cultured in high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. HeLa cells (ATCC CCL 2) were cultured in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. B3.1.0 cells, a suspension culture of Epstein-Barr virus-immortalized B lymphoblasts, were grown in RPMI 1640 supplemented with 15% fetal calf serum and 1% penicillin/ streptomycin. All tissue culture media reagents were obtained from Life Technologies, Inc. Cells were maintained at 37 “C in a humidified atmosphere of 5% CO,, 95% room air.

RNase Protection Assays-Total RNA for all RNA based assays was extracted using the guanidinium isothiocyanate-CsC1 cushion technique described by Chirgwin et al. (21). A genomic DNA fragment extending from position -210 to +163 of the CFTR gene was subcloned into pBluescript I1 (Stratagene, La Jolla, CA) using standard methods. The plasmid template was linearized with XhoI and a run- off transcript was prepared using standard in vitro transcription conditions. Labelling was achieved by incorporation of 4 p1 of 20 mCi/ml (2000 Ci/mmol) CTP supplemented with 1.6 p1 of 100 p M unlabeled CTP in a final in vitro transcription reaction volume of 20 pl. After DNase I digestion to destroy the plasmid template, the labeled RNA was ethanol precipitated and the 483 nucleotide antisense transcript was gel purified on a denaturing 6% polyacrylamide gel.

150,000 counts/min of antisense probe was combined with 30 pg of total RNA or 20 pg of yeast tRNA and allowed to hybridize overnight (16 h) at a temperature of 45 “C, in an 80% formamide solution prepared by Ambion, Inc. (Austin, TX). Annealed RNA duplexes were digested with 40 pg/ml RNase A and 2 pg/ml RNase T1 (Ambion, Inc) for 30 min at 37 “C. The RNase digestion was terminated and the reaction products were precipitated using a guanidinium/ethanol reagent and conditions provided by Ambion.

The conditions and procedures used to detect DHFR transcripts were exactly as described for CFTR, except that 15 pg of RNA were assayed in the reaction and the probe consisted of sequences subcloned into pBluescript I1 KS via RNA PCR, using the primers 5‘- AGACATGGTCTGGATAGTTGGTGG-3‘ and 5”CAATGTCAAG- GACTGGCAAGAGTG-3’, based upon published information (22). The DHFR template was linearized with BglII to prepare a 413- nucleotide run-off transcript.

Autoradiographs were exposed for 16 h at -70 “C, using Kodak XAR-5 film and a Cronex intensifying screen.

Anchored PCR-A primer extension reaction was performed using the aqueous hybridization protocol of Boorstein and Craig (23). 20 pg of total RNA isolated from the cell lines T84 or CFPAC was reverse transcribed from an antisense primer located in exon 2 of CFTR. The primer sequence is 5”ACTAGGATCCCCAATTTTTCAGATA- GATTGTCAGCAG-3’. The single-stranded cDNA reaction products were then purified over a Qiagen resin (Qiagen, Inc., Chatsworth, CA) column using conditions provided by the manufacturer. The cDNA population was “tailed” with multiple guanine nucleotides using terminal transferase (Boehringer Mannheim) in a cobalt buffer provided by the manufacturer. After 10 min of heat inactivation at 70 “C, one-fifth of the terminal transferase reaction was subsequently used as template in a polymerase chain reaction (PCR). The amplifying primers used were a poly(C) primer which defined the 5’ end of the PCR product, and either an antisense primer located in exon -la or an antisense primer located in exon 1 of CFTR. The primer sequences are 5’-GTGACTCGAGCCCCCCCCCCCCCCC-3’ (poly(C)primer),B’-TTAGTTTCAGGTTTAGGTGAGTGA-3’(exon

- 1 a primer), and 5‘-GTTTGGAGACAACGCTGGCCTTTTCC-3’ (exon 1 primer). PCR parameters were 94 “C for 1 min, 72 “C for 2 min for 30 cycles, followed by one cycle of 94 “C for 1 min, 72 “C for 10 min. The PCR products were directly subcloned into a “T-tailed” pBluescriptI1 SK+ vector (Stratagene, La Jolla, CAI prepared as described by Marchuk et al. (24). Recombinant clones initially identified by interruption of the vector 0-galactosidase gene were screened secondarily by hybridization to CFTR exon 1 or exon -la sequences. Positive clones were sequenced using standard methods.

RNA PCR-1 pg of total RNA isolated from the cell lines T84 or CFPAC was converted to cDNA with Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories) and random hexamer primers, under conditions provided with the enzyme. The cDNA was amplified in a polymerase chain reaction using a 3’ primer located in exon 2 of CFTR and a 5’ primer located in exon -la of CFTR. The sequence of these primers is as follows: 5”ACTAGGAT CCCCAATTTTTCAGATAGATTGTCAGCAG-3’ (exon 2 primer), 5’-ACTAGGATCCATTATCTCCTCTTACCTCCT-3’ (exon -la primer). The PCR parameters were 94 “C for 1 min, 60 “C for 1 min, 72 “C for 2 min for 30 cycles, followed by one cycle of 94 “C for 1 min, 72 “C for 10 min. One-fifth of the PCR reaction was loaded onto a 4% polyacrylamide gel (29:l acrylamide/bis ratio), electrophoresed, and visualized by staining with ethidium bromide. Negative control samples were treated in an identical manner, except for the omission of reverse transcriptase in the initial reaction.

Northern Blot Analysis-20 pg of total RNA isolated from con- fluent CaCo2 monolayers were resolved electrophoretically under denaturing conditions in a 1% agarose formaldehyde gel, using standard protocols. The RNA was transferred to a Nytran membrane (Schleicher and Schuell) by capillary blotting, and hybridized under standard conditions. The -la probe consisted of a cDNA fragment that extended from genomic position -816 to -663 relative to the exon 1 start site. The cystic fibrosis 4.6 probe represents the entire CFTR cDNA, described in earlier publications (25). The autoradiographs shown are each overnight exposures of the same Northern blot filter. The filter was first hybridized to the exon -la probe, then stripped, and following an overnight exposure of the stripped blot to verify complete removal of the -la probe, the blot was reprobed with CF4.6.

Methylation Assays-20 pg of genomic DNA isolated from a panel of cell lines was digested overnight at 37 “C with 7.5 units of EcoRI (all restriction enzymes were obtained from New England Biolabs, Beverly, MA) per pg of DNA. Following this initial digestion the DNA was phenol-extracted and ethanol-precipitated. The DNA was resuspended and digested either with HpaII or MspI at 5 units/mg.

gel in 1 X TBE buffer (89 mM Tris, 89 mM boric acid, 1 mM EDTA), The products of the restriction digest were resolved on a 1.4% agarose

and the DNA was transferred by capillary blotting onto a Nytran charged membrane filter. Probes were labeled to a specific activity of approximately lo9 counts/min/pg in a random hexamer-primed Kle- now reaction, with reagents and conditions provided in a kit purchased from Amersham Corp. Hybridization and washing conditions were based upon standard protocols provided by the Nytran membrane manufacturer, except that the nonspecific blocking DNA in the hybridization solution was sonicated human placental DNA (Sigma), at a final concentration of 100 pg/ml.

DNase Z Hypersensitivity Assays-Chromatin from a panel of cell lines was probed for hypersensitive regions using the indirect end- labeling procedure first described by Wu (26, 27). Isolation of nuclei, digestion with DNase I, and recovery of genomic DNA were performed as described (28). Briefly, 1-3 X 10’ cells were harvested and washed in a hypotonic buffer and then disrupted in the same hypotonic buffer containing 0.5% Nonidet P-40. The intact nuclei were collected by a gentle centrifugation step and then were exposed in parallel aliquots to digestion by a dilution series of DNase I concentrations. Following this light DNase I digestion, the reaction was terminated and the nuclei lysed by addition of a proteinase K-SDS solution. After an overnight incubation at 37 “C to allow complete proteolysis, the DNA was recovered by organic phase extraction and ethanol precipitation. In a typical experiment, 20 pg of genomic DNA/DNase I point was digested with 4-5 units of restriction enzyme/pg of DNA for 4-6 h. The reaction products were resolved on a 1% agarose gel in 1 X TBE buffer and transferred to Nytran-charged nylon membrane filters using standard conditions. Hybridization and washing conditions were as described above for the methylation studies.

Transfections and Reporter Assays-Transfections into the cell lines T84, PANC-1, and HeLa were performed via electroporation, using a BTX Transfector 300 instrument (Biotechnologies and Ex-

15914 CFTR Promoter

perimental Research, Inc., San Diego CA) with a 0.35-cm electrode gap in the sample cuvette. For transfection into T84, 3 x lo7 cells were concentrated in 400 pl of HeBS buffer (20 mM HEPES (pH 7.05), 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HP04, 6 mM glucose) (29) and electroporated at discharge settings of 200 volts pulse amplitude and 2400 microfarads capacitance. PANC-1 cells were concentrated to a density of 1.5 X lo7 cells/400 pl of HeBS, and electroporated at 200 volts pulse amplitude and 500 microfarads capacitance. HeLa cells were concentrated to a density of 1.5 X lo7 cells per 400 p1 of HeBS and electroporated at 200 volts pulse amplitude and 1300 microfarads capacitance. CaCo2 cells were plated at a density of approximately 2 X lo4 cells/cm2, allowed to grow for 24 h, and then transfected using a standard calcium phosphate precipitation protocol (30).

48 h after transfection, cells were harvested in phosphate-buffered saline with EDTA (5 mM), pelleted, and washed twice in a buffer consisting of 100 mM potassium phosphate, pH 7.8, and 1 mM dithiothreitol. The washed cells were then resuspended in 100 pl of the same buffer, and lysed by three freeze-thaw cycles. Luciferase assays were performed using reagents purchased from Promega (Mad- ison, WI), and activity was measured in an LKB model 1215 BioOrbit luminometer (Wallac, Inc., Gaithersburg, MD) set to measure light intensity integrated over a 30-s window. An enzyme-linked immu- nosorbant assay-based assay system (Boehringer Mannheim) was used for quantitation of chloramphenicol acetyltransferase protein in the transfected cell extracts; data were recorded in a Bio-Tek model EL312 microplate reader (BioTek Instruments, Inc., Winooski, VT). Standard curve determinations (? > 0.98) verified that chloramphenicol acetyltransferase protein concentrations and luciferase activity measurements were in the linear range of their respective assays. Normalized promoter activity represents the ratio of luciferase values to the internal control chloramphenicol acetyltransferase protein measurements.

All reporter plasmids were column-purified (Qiagen resin) using conditions recommended by the manufacturer (Qiagen, Inc., Chat- sworth, CA). Pilot experiments directly comparing double-banded CsCl gradient plasmid preparations with Qiagen-purified plasmids demonstrated that comparable transfection efficiencies were achieved with both protocols.

Construction of Reporter Plasmids-A 4.3-kb EcoRI genomic DNA fragment containing 5”flanking sequence from CFTR (7) was subcloned into pBluescript I1 KS (Stratagene, La Jolla, CA). The genomic region extending from -3.7 kb to +111 bp was subcloned into the luciferase reporter vector pA3LUC (31), kindly provided by William Wood (Genentech). The resulting construct, pA&F-3.7LUC, contains part of the pBluescript I1 KS & polylinker extending from the KpnI site to the EcoRI site at the 5’ end of the CFTR genomic DNA insert. To construct a vector to use as a promoterless control, pAsCF-3.7LUC was digested with HindIII (removing all of the CF genomic region) and religated. Nested deletions were constructed by digesting pA3CF- 3.7LUC with XhoI (a unique site in the polylinker) and unique sites within the CFTR genomic region. The ends were filled in by Klenow DNA polymerase using standard conditions and religated. One clone was constructed by the PCR using primers located at -83 and +111. The primers were designed to generate a product with an XhoI site at the 5’ end and a HindIII site at the 3’ end. Using these sites the PCR product was cloned into the XhoI and HindIII sites of pA3LUC. All cloning junctions and PCR-generated regions were sequenced.

The SV40-promoter/enhancer driven chloramphenicol acetyltransferase reporter construct, pCAT-Control (Promega, Madison, WI), was used as an internal control to normalize for transfection efficiency.

RESULTS

CFTR Transcription Start Sites-As an initial step in cha- racterizing the CFTR promoter, we mapped the sites of CFTR transcription initiation. Primer extension, S1 nuclease protection, anchored PCR, and ribonuclease protection assays consistently reveal multiple but discrete transcription initiation sites among different cell lines expressing CFTR mRNA. Fig. 1 is a representative example of a ribonuclease protection assay probing for CFTR transcripts in RNA isolated from a panel of cell lines; simultaneous probing for the dihydrofolate reductase (DHFR) transcript serves as a control for loading variability.

The cell line HeLa is a well described human epithelioid cervical carcinoma derivative which expresses very low quan- tities of CFTR mRNA (10) and as shown in Fig. 1 these low abundance transcripts initiate at position -32. Position +1, defined by Riordan and co-workers (6) in the original description of the CFTR cDNA, is located 121 bp upstream of the ATG translation initiation codon and corresponds to position +11 in the numbering system used by Chou and co-workers (19), and to position -50 in the numbering system used by Yoshimura and co-workers (20). The start site at +50 appears to be the major initiation point for CFTR transcription in highly expressing cell lines. B3.1.0 cells are an Epstein-Barr virus immortalized human lymphoblastoid line. CFTR transcripts are at low but clearly detectable levels in B3.1.0 RNA, and initiate at the same position used in HeLa cells. The PANC-1 cell line was established from a human pancreatic ductal epithelial adenocarcinoma. CFTR mRNA can be detected by RNA PCR from these cells (lo), but as can be seen in Fig. 1, the abundance of the transcript is extremely low, barely detectable in the assay shown despite an overloading of PANC-1 RNA relative to the other samples tested (compare the DHFR signal intensities). CaCo2 cells are derived from a human colonic epithelial adenocarcinoma and are thought to retain characteristics of gastrointestinal crypt progenitor cells, including the ability to respond to in uitro cell culture manipulations by undergoing morphological and biochemical changes which mimic in uiuo differentiation (32). T84 cells are a colonic epithelial adenocarcinoma derivative which express relatively high levels of CFTR mRNA, protein, and functional activity (33). The location and proportional usage of transcription initiation sites are similar in both T84 and CaCo2 cells (Fig. l) , with an approximately 5-fold greater abundance of CFTR mRNA in T84 cells, as assessed by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) scan- ning of the ribonuclease protection gel. The pattern and location of the multiple transcription start sites we observe are in general consistent with previously published data (20).

The data in Fig. 1 are supported by primer extension, S1 nuclease protection, and anchored PCR assays (data not shown). Theoretically, a subset of the multiple bands detected in the ribonuclease protection assay could arise from inter- mediate digestion products rather than actual start sites. However, since the same apparent start sites can be consistently identified by primer extension and S1 nuclease protection assays, both of which rely on 3’ end labeling, such a possibility is unlikely. Furthermore, the position and relative distribution of anchored PCR clone end points from exon 1- containing cDNA species confirms that the start sites identified by the ribonuclease protection assay are true termini rather than artifactual assignments.

From this analysis it is apparent that CFTR transcription initiates a t discrete, multiple sites in the promoter region. The steady state abundance of CFTR mRNA varies widely in the panel of cell lines tested, with a rank order of T84 > CaCo2 >> B.3.1.0 = HeLa > PANC-1. The two colonic epithelial cell lines, both relatively high expressors of CFTR mRNA, share similar patterns of start site usage. The low level expressors PANC-I, HeLa, and B.3.1.0 apparently limit transcription initiation to position -32.

Alternatiue Upstream CFTR Exons-Numerous alternatively spliced isoforms of the CFTR mRNA have been identified (10, 14, 34-36), suggesting an additional level of regulatory complexity in the control of CFTR gene expression. During the process of screening for cDNA clones which represent the 5’ terminus of the CFTR transcript, Rommens and co-workers isolated a population of alternatively spliced

CFTR Promoter

tRNA HeLa

B.31.0 CaCo-2

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PANC-1 T84 ’ 1 t ‘ C aF

A A -95 -32

A +50

v

DHFR

-95 -52 -32 +1 +25 +50

FIG. 1. Transcription start sites. An RNase protection assay was used to map the sites of CFTR transcription initiation. 30 pg of total RNA isolated from the indicated cell lines or 20 pg of yeast tRNA were allowed to hybridize to a T7 transcribed antisense riboprobe which spans the region from -210 to +163 bp. Principal sites of transcription initiation are indicated by bent arrows on the accompanying schematic diagram. The major start site used in T84 and CaCo2 cells is indicated by the taller arrow a t +50. The start site at +1 is the terminus tentatively assigned by primer extension in the original description of the CFTR cDNA (6). The open arrow indicates the transcription start site used in the cell lines HeLa and R3.1.0. In parallel control RNase protection assays using 15 pg of RNA, a 413- nucleotide antisense DHFR riboprobe prepared as described under “Materials and Methods” detects a 323-nucleotide species.

CFTR isoforms from a pancreatic cDNA library.2 These clones do not initiate at exon 1 of CFTR but alternatively include one or both of two previously uncharacterized upstream exons spliced directly into exon 2. Both the proximal (la) and distal (-la) upstream exons are located closely adjacent to exon 1, as depicted in Fig. 2 A . The canonical translation initiation codon of CFTR, located in exon 1, is excluded from the - la and la splice variant transcripts. Transcripts originating from exon -la would not be detected in the assay shown in Fig. 1.

To verify that the -la and la cDNA clones represented bona fide CFTR transcripts, we conducted RNA PCR assays using primers positioned in exon -la and exon 2 of CFTR, as indicated by the horizontal arrows in Fig. 2 A . The intron separating exons 1 and 2 is approximately 26 kb in length, effectively precluding amplification of contaminating genomic DNA. Shown in Fig. 2B are the reaction products generated in an RNA PCR experiment amplifying from random primed cDNA template prepared from T84 total RNA. Two reverse transcriptase-dependent bands are visible; subcloning and sequencing of these PCR products demonstrates that the larger band represents a CFTR cDNA species which splices from exons - la to l a to 2, and that the smaller band results from an exon - la to 2 splice. Furthermore, on a Northern blot of CaCo2 RNA, exon -la sequences hybridize to an mRNA which comigrates with the 6.5-kb transcript detected by the exon 1-initiated CFTR cDNA (Fig. 2C). Taken together, these data argue strongly for the existence of a sub- population of alternatively initiated CFTR transcripts.

To determine the start site of these upstream transcripts, we utilized an anchored PCR strategy to generate a large number of independently derived cDNA clones from T84 or CFPAC RNA. Sequence analysis of clones containing exon -la revealed a tightly focused distribution of end points (Fig. 2 0 ) . Taking into account the fact that no splice acceptor consensus sequences could be found in the -la region, our data are most consistent with the interpretation that CFTR transcription can initiate in exon -la, between nucleotides -868 and -794, with the predominant start site at nucleotide

Methylation in the CFTR Promoter Region-The promoter region of the CFTR gene is remarkably C + G-rich (19, 20) and contains an HTF island with six HpaII/MspI sites clus-

’ J. Koh, T. J. Sferra, and F. S. Collins, unpublished observations.

-815.

tered in a span of less than 1 kb. Since transcriptional activity often is correlated with methylation status ( 3 7 , we analyzed CFTR promoter methylation in several cell lines. Methylation at certain CpG dinucleotides can be detected by analyzing the restriction digest patterns produced by the isoschizomers HpaII and MspI. Both enzymes recognize the sequence CCGG, but HpaII does not cleave if the internal CpG dinucleotide is methylated. To probe for methylation near the CFTR promoter, we compared the ability of MspI and HpaII to cleave within a 4.3-kb genomic fragment encompassing the CFTR promoter region. This fragment contains all the transcription start sites described above.

The Southern blot in Fig. 3 was probed with a fragment derived from the 5’ end of the 4.3-kb genomic subclone. Subbands produced by HpaII cleavage at non-methylated sites will appear a t 3.6, 3.3, and 2.9 kb. The faint bands visible below 2.9 kb are not predicted by the restriction map of the CFTR promoter region and therefore probably represent cross-hybridization with non-CFTR sequences. In genomic DNA isolated from HeLa cells, HpaII does not cleave within the 4.3-kb interval tested, indicating that in this cell line all six MspI/HpaII sites in the CFTR promoter region are methylated. DNA from B3.1.0 cells displays a similar but distinct pattern, with only partial methylation a t one or more of the clustered MspI/HpaII sites located in exon la, generating a faint band at 3.3 kb. T84 and PANC-1 DNA are cleavable by HpaII at the most 5‘ site, and subsequent experiments with other probe combinations reveal a lack of methylation at the most 3’ site in both cell lines. DNA isolated from CFPAC cells demonstrates a different methylation pattern, with complete methylation at the -la site and partial methylation throughout the rest of the region.

The methylation data are summarized schematically in Fig. 4. This figure represents a compilation of data generated using multiple probes located a t different positions within the 4.3- kb EcoRI fragment. With the notable exception of PANC-1, the apparent degree of methylation in the CFTR promoter region correlates well with the abundance of CFTR mRNA detected in these cell lines. In HeLa and B3.1.0 cells, the CFTR promoter region is heavily methylated and very low levels of CFTR mRNA can be detected. In the CFPAC cell line, which expresses a moderate level of CFTR mRNA (data not shown), the CFTR promoter region is methylated to a lesser extent than in HeLa and B3.1.0. The CFTR promoter

15916 CFTR Promoter

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0 a 4 6 . 5 k b

28s-

18s-

D

0 0

FIG. 2. Exon -la. A, diagram of the proximal promoter region of CFTR. Boxes indicate exons, with the canonical translation initiation codon shown in exon 1. Horizontal arrows indicate the place- ment of PCR primers used in the RNA PCR experiment depicted below. Vertical lines adjacent to exon -la represent the indeterminate 5’ boundary of exon -la, where transcription of the splice variant transcript initiates. B, RNA PCR of splice variant CFTR mRNA. Two distinct cDNA species amplified from T84 RNA are indicated by arrowheads to the left of the gel photograph. The presence or absence of reverse transcriptase in the initial reaction is indicated by the + and -, respectively. Numbers to the right of the photograph indicate the migration of size standards, in base pairs. C, Northern blot of CaCo2 total RNA. The lane labeled - l a was probed with exon -la sequence from a T84 anchored PCR clone. The blot was then stripped and rehybridized with the CFTR cDNA clone CF4.6, to generate the signal shown in the lane labeled CF4.6. Arrowheads marked 18 S and 28 S indicate the location of ribosomal RNA species; the arrowhead marked 6.5 kb indicates the full-length CFTR transcript. D, a depiction of the end point distribution of independently derived anchored PCR cDNA clones. Each dot represents one such clone. Open dots denote clones generated from RNA isolated from the pancreatic adenocarcinoma cell line CFPAC; filled dots represent clones derived in the same manner from T84 RNA. The sequence shown in D is derived from the region indicated by the horizontal line beneath exon -la in A.

region appears free of methylation in the cell line T84, which expresses relatively high levels of CFTR mRNA. The situa- tion in the PANC-1 cell line appears paradoxical, since these cells express nearly undetectable levels of CFTR mRNA despite a lack of methylation in the CFTR promoter region.

Chromatin Structure of the CFTR Promoter Region-Using the indirect end-labeling technique developed by Wu and co- workers (26), we probed for nuclease hypersensitivity in a large genomic interval surrounding the CFTR transcription initiation sites. The probes and restriction map of the 26-kb region we scanned are depicted schematically in Fig. 5 . As an

independent positive control to verify appropriate DNase I digestion conditions, we tested each DNA series with a probe which detects the well characterized constitutive hypersensitive sites associated with the c-myc gene (28).

Five nuclease-hypersensitive sites were identified in the CFTR proximal promoter region in chromatin isolated from the cell line T84, while chromatin from B3.1.0, PANC-1, or HeLa cells lack these or any other hypersensitive sites in the 26-kb region analyzed. Shown in Fig. 6 is a comparative analysis of B3.1.0 and T84 chromatin structure in the CFTR promoter region. The probe used in this example, a 500-bp EcoRIIAccI genomic fragment indicated by the solid bar in Fig. 6A, detects a 3.3-kb fragment in genomic DNA cut with AccI, or a 4.3-kb fragment in genomic DNA digested with EcoRI. As demonstrated in Fig. 6B, the 3.3-kb AccI fragment in the context of either B3.1.0 or T84 chromatin is completely resistant to digestion by up to 50 units of DNase I, the highest concentration used. In contrast, the 4.3-kb EcoRI fragment detected by the same probe used in Fig. 6B is completely digested by 50 units of DNase I in both B3.1.0 and T84 nuclei, and in addition contains five distinct hypersensitive sites which appear only in the context of T84 chromatin (Fig. 6C). The T84-hypersensitive sites, indicated by the vertical arrows in Fig. 6A, are located approximately a t positions -900, -800, -400, -200, and +200. The fact that these hypersensitive sites are manifested in T84 DNA even in the absence of exogenous DNase I (the 0 lane in Fig. 6C) probably indicates high endogenous nuclease activity in this cell line: the c-myc hypersensitive sites are similarly revealed in T84 preparations (data not shown). The relative insensitivity of the 3.3-kb AccI fragment to DNase I digestion in both cell lines suggests that this fragment is configured as bulk chromatin, and lies outside the domain boundary which defines active or potentially active chromatin associated with the CFTR gene.

Promoter Activity in the 5”Flanking Region of CFTR-To assess CFTR promoter activity functionally, we constructed a series of reporter plasmids containing sequential deletions of CFTR 5”flanking sequences directing the expression of the firefly luciferase gene. We introduced the CFTR reporter constructs into HeLa, PANC-1, and T84 cells via electroporation, cotransfecting with an SV40 promoter-driven chloramphenicol acetyltransferase plasmid to control for transfection efficiency. The results of these experiments are plotted in Fig. 7.

CFTR promoter sequences are capable of directing a significant degree of tissue-specific gene expression. The reporter construct containing 3.7 kb of 5’-flanking sequence is at least 12 times more active in T84 than in HeLa cells, a result consistent with the relative steady state abundance of CFTR mRNA detected in these cell lines. Deletion to -2.5 kb does not significantly affect overall promoter strength or the relative promoter activity in T84 and HeLa cells. Progressively smaller deletion constructs are incrementally less active in both cell lines, but a construct containing as little as 83 bp of 5”flanking sequence retains the ability to distinguish between HeLa and T84 cell environments, directing transcription at a level 7-8-fold higher in T84 than in HeLa cells.

The degree of promoter activity observed in PANC-1 cells is discordant with the low level of CFTR mRNA detectable in this cell line. The construct containing 3.7 kb of 5”flanking sequence is twice as active in PANC-1 as in T84 cells. With progressive deletions this ratio widens; the construct containing 83 bp of 5“flanking sequence is five to six times as active in PANC-1 as in T84 cells.

In all three cell lines tested, the gradual loss of promoter activity with progressive 5‘ deletions indicates an absence of

CFTR Promoter 15917

“ “ P 83.1 .O T84 CFPAC PANCl HELA

M H M H M H M H M H Size

(kb) Marker 5.1 -

4.1 -

3.1 -

2.0 -

Probe: Acc 0.5

RI M HI HI HI M M

HI

I 2.9/f I 330 1 1 1 1 263 I 71 6 1 M RI

-la l a 1

FIG. 3. Methylation in the CFTR promoter region. Southern blot detecting methylation in the promoter region of CFTR. The line diagram indicates the location of HpaII (H)/MspI ( M ) sites within a 4.3-kilobase EcoRI fragment which encompasses the promoter region of CFTR. Distances between restriction sites are in base pairs, except for the 5’ EcoRI-to-HpaII/MspI interval, which is 2.9 kb. The location of the probe used in the experiment shown is indicated on the diagram by the line labeled Ace 0.5. Open boxes indicate CFTR exons -la, la, and 1.

H M M M U M M

H H H H H

.ra l a 1

CFPAC 41 ” u

FIG. 4. Summary of methylation in the CFTR promoter region. CFTR promoter region methylation in a panel of cell lines depicted in schematic summary form. The figure represents a compilation of data derived from multiple experiments with probes located a t different positions within the 4.3-kb EcoRI fragment. The line diagrams are drawn as in Fig. 3, with each line representing data from the indicated cell line. Filled shapes indicate methylated sites, and open shapes indicate unmethylated sites. Partial methylation is indicated by partly filled shapes, with the proportion of filling approx- imating the degree of methylation observed.

dominant positive or negative transcriptional control sequences. These data are at odds with a published report that describes a strong transcriptional repressor element in the CFTR promoter at positions -356 to -288 (19). In their study, Chou and co-workers transfected chloramphenicol acetyltransferase reporter constructs into the cell line CaCo2 and observed that deletions which eliminated sequences 5‘ to position -288 relieved a 4-fold repression effect, suggesting the presence of a negative regulatory element. Since we did not detect any evidence of transcriptional repression in the

R ‘I I

R RRR R F! I

F!

‘I A AA

’L V v v

R V V A R ARRR RA VR AV V A R

no 0 H 1 kb

FIG. 5. Genomic region scanned for DNase I-hypersensitive sites. The bottom line is a restriction map of a 26 kb interval that includes CFTR exons -la, la, and 1. The location of restriction enzyme sites for EcoRI ( R ) , AuaI ( V), and Ace1 ( A ) are shown. Open boxes below the line indicate CFTR exons -la, la, and 1. The three lines above the composite restriction map represent individual restriction maps for EcoRI, AccI, and AuaI, respectively, with Southern blot probes indicated by solid bars.

cell lines PANC-1, T84, or HeLa using reporter constructs with termini appropriately positioned to reveal the putative transcriptional silencer element, we decided to test our constructs in CaCo2 cells. To replicate the experimental conditions reported by Chou and co-workers, we transfected the CaCo2 monolayers via calcium phosphate precipitation. In Fig. 8, normalized promoter activity is expressed relative to a reference construct which contains 253 bp of 5”flanking sequence. The 5‘ end point of this construct is identical to the reference construct described by Chou and co-workers. As shown in Fig. 8, we find no evidence for a negative transcrip-

15918 CFTR Promoter

A A R R R R A It I 4 I ?

IllUU u - .a la 1

A A

R R

%a

63.1.0 T84 C

B 3.1.0 T84 0-0- * *)e.--

4.0- 4.0-

3.0-

I! 1 !$ !!I h < + 4 I

3.0- I

2.0- 2.0-

1.6- 1.6-

FIG. 6. DNase I-hypersensitive sites in the CFTR promoter proximal region. An example of DNase I hypersensitive site screening. A, the line drawing is a restriction site map for the enzymes AccI and EcoRI, used in the assays shown below. The solid bar indicates the probe used in the two blots; the 3.3-kb AccI fragment and 4.3-kb EcoRI fragment detected by the probe are shown as lines below the diagram. CFTR exons in this region are depicted as open boxes on the diagram. Vertical arrows indicate DNase I hypersensitive sites. R, lack of hypersensitivity in a 3.3-kb AccI fragment. Southern blot of B.3.1.0 and T84 DNA samples prepared as described under “Ma- terials and Methods,” digested with AccI, and probed with the fragment depicted as the solid bar in the diagram. The number 0 indicates a sample prepared from nuclei not treated with DNase I; arrows to the right indicate samples prepared from nuclei treated with increas- ing amounts of DNase I (0.1, 0.5, 1.25, 2.5, 5.0, and 50.0 units). Numbers on the left margin indicate the migration of DNA size standards, in kb. C, cell-type-specific hypersensitivity in the promoter proximal region of CFTR. Southern blot of the same DNA used in R, but digested with EcoRI. The probe used was the same as in R. Sites hypersensitive to DNase I in T84 chromatin are indicated by the arrowheads at the right margin. Size standards are marked as in B.

tional regulatory element in the interval reported. In fact, in our experiments the reference construct appears to be slightly less active than a larger clone containing 635 bp of 5‘-flanking sequence.

DISCUSSION

In uiuo expression of the gene encoding CFTR is limited primarily to specific cells in epithelial tissues (8-10). The mechanisms underlying this strict cellular control of CFTR gene expression are complex, presumably mediated through the combined action of several distinct regulatory processes. In this report we present evidence which suggests that CFTR transcription can be modulated both indirectly, through mod- ifications in the physical context or accessibility of the gene, and directly, through the interaction of promoter sequences and cell-specific trans-acting factors. In addition, we describe a novel alternatively initiated CFTR mRNA isoform, which may indicate that RNA processing contributes to the regulation of CFTR expression.

There are several aspects of CFTR transcription which are atypical of tissue-specific genes. The CFTR 5”flanking region superficially resembles sequences associated with constitutive, “housekeeping” genes but clearly possesses the capability to direct transcription in a tissue-specific, regulated manner. The promoter region is C + G-rich, and inspection of the

-3.2 +/-n . . . . . . . . I ,I / . . . . -

-.635 -0 s.253

0 HeLa El Pand

0 400 800 1200 1600 2000

Promoter Activity

FIG. 7. Promoter activity in the 5”flanking region of the CFTR gene. Deletion analysis of the 5”flanking region of the CFTR gene. A series of 5’ deletions of the CFTR gene 5”flanking region were fused a t position +I11 to the gene encoding firefly luciferase. The deletion construct terminating a t -83 bp was assembled using PCR as described under “Materials and Methods”; the other constructs terminate at restriction sites EcoRI ( R ) , AccI ( A ) , PmlI (PI , Eco47III ( E ) , and BssHII ( R ) , as indicated in the upper schematic diagram. pA3luc is the promoterless luciferase plasmid vector. The reporter gene fusions were introduced into the cell lines T84, HeLa, and PANC-1 for transient expression assays. Luciferase activity was measured and normalized for transfection efficiency by comparison to a cotransfected internal control as described under “Materials and Methods.” Promoter activity is defined as the ratio of luciferase activity to CAT protein concentration. Each value represents the mean & the standard error of the mean of a t least three independent transfection experiments.

I 4 -

3 -

2 -

1 ”“

0 I I I I

P A ~ I U C -3.7 -3.2 -2.5 -.635 -.253 -.083

Promoter Constructs FIG. 8. Relative promoter activity in the cell line CaCo2.

Relative promoter activity of 5’ deletion constructs in the cell line CaCo2. Normalized promoter activity values for the series of 5’ deletion constructs were obtained as described under “Materials and Methods.” The data are expressed here as promoter activity relative to the activity of the deletion construct terminating a t position -253 of CFTR. Each value shown represents the mean & the standard error of the mean of a t least three independent transfection experiments.

genomic sequence adjacent to the transcription start sites fails to reveal either TATA or CCAAT sequence motifs. Direct assessment of steady state CFTR mRNA abundance in a panel of cell lines indicates a broad quantitative range in vitro, consistent with the assertion that CFTR gene expression is

CFTR Promoter 15919

under tissue-specific transcriptional control. Even in cells which express CFTR, however, the transcript is present in relatively low abundance (11, 12), and maximal output of CFTR reporter constructs is only about 0.5-2% of the activity of positive control plasmids driven by the Rous sarcoma virus promoter/enhancer region (20).3 A number of independent assays demonstrate multiple but discrete transcription start sites which vary among cell lines expressing CFTR. The pattern of start site usage correlates with mRNA abundance, suggesting regulatory influence at the level of start site selec- tion. The multiple levels of control imposed upon the intrin- sically weak CFTR promoter imply that cellular constraints tightly limit CFTR gene expression.

The identification of CFTR mRNA isoforms differentially spliced at the 5' end introduces the possibility of an alternative regulatory pathway for control of CFTR gene expression. The exon -la initiated transcripts described in the current report bypass the canonical translation initiation codon of CFTR, located in exon 1. If translated, the -la isoforms would encode a protein with an amino terminus distinct from the exon 1 initiated polypeptide. Inclusion of exon la, which is closed in all reading frames, precludes translational read- through from exon -la. The exon -la/2 splice isoform, however, maintains an open reading frame which would allow complete translation of a full-length CFTR polypeptide, but exon -la does not contain an AUG initiation codon in this reading frame. The closest downstream candidate AUG is in exon 4. This codon lies in very poor sequence context for initiation and is adjacent to several better Kozak consensus matches out of phase from the CFTR open reading frame. Since additional exons upstream of -la are unlikely, translation of the -la/2 isoform must initiate at a non-AUG codon if it occurs at all. The efficiency of translation initiation from non-AUG codons is generally very poor in eukaryotes (38), but has been documented to occur in several systems (39, and references therein). In one recent example, a novel protein isoform of the murine p-retinoic acid receptor was found to be generated by CUG-initiated translation of an alternatively spliced transcript which excluded the gene's canonical AUG translation initiation codon (40). Perhaps CFTR utilizes a similar mechanism: exon -la contains an in-frame CUG codon at position -670 in the context of a relatively favorable match to the Kozak consensus (5/10 including the invariant purine at -3).

The physiological significance of transcriptional initiation in CFTR exon -la is unclear. Exon -la containing transcripts could be the manifestation of a negative regulatory mechanism, acting through the generation of non-productive, untranslatable mRNAs. Alternatively, - la transcripts may encode distinct isoforms of the CFTR protein, perhaps with novel functional properties. Exon l a is closed in all reading frames, and thus its role in CFTR expression remains partic- ularly obscure.

Using a series of reporter constructs transfected into several cell lines, we investigated CFTR promoter activity. While the proximal promoter region may confer tissue specificity to a large extent, upstream elements clearly contribute to the overall quantitative level of CFTR transcription. In both T84 and HeLa cells promoter strength declines incrementally with progressive sequence deletions, indicating that transcriptional control may depend upon the coordinate activity of multiple and individually weak cis- and trans-acting factors. Consist- ent with this model, we have found the CFTR promoter region to be densely bound by a complex population of sequence- specific nuclear protein^.^ Although the individual contributions of each of these DNA-protein interactions may prove to

be unresolvable, other systems have been described in which multiple weak effectors can form a combinatorial array capable of directing transcription with a high degree of specificity (41, 42). The gradual decline of promoter strength in T84 cells and the failure to unmask expression in HeLa cells with progressively smaller deletion constructs suggests that appropriate quantitative modulation of CFTR transcription is not dictated by dominant positive or negative 5' sequence elements. In experiments conducted specifically to assay for transcriptional silencer activity, we were unable to reproduce the results of Chou and co-workers (19), who reported a negative transcriptional regulatory element between positions -356 and -288 in the CFTR promoter. Our data are also inconsistent with a study by Yoshimura and co-workers (20) in which the authors assert that sequences 5' to position -53 do not augment basal promoter activity in the colon epithelial cell line HT-29. Explanations for these disparate results may include differences in cell lines, reporter construct design, and assay sensitivity.

In addition to direct modulation by transcriptional regulatory elements, CFTR gene expression may be subject to the influence of indirect mechanisms such as differential methylation in the promoter region and tissue-specific modifica- tions of chromatin structure. In the mammalian genome, inactive chromatin is usually methylated at CpG dinucleotides. Through a poorly understood mechanism, methylation can convert HTF islands such as the one found in the CFTR promoter region into high affinity substrates for factors which bind methylated DNA, resulting in long term transcriptional inactivation (37,43). At the CFTR locus, there appears to be a general correlation between level of expression and the degree of methylation detected in the promoter region. The CFTR promoter region is heavily methylated in the low level expressors HeLa and B3.1.0, but is hypomethylated in the high level expressor T84. In the cell line PANC-1, however, this simple pattern breaks down: although the promoter region is hypomethylated, CFTR mRNA is present at very low levels. This apparent discordance may indicate that absence of methylation in the CFTR promoter region could be a necessary predisposing condition but is not sufficient to influence CFTR expression without the participation of other specific factors. In the case of CFTR, the methylation status of the promoter region could reflect differences between an inactive promoter (B3.1.0, HeLa) and a potentially active promoter (PANC-l), rather than providing a direct index of transcriptional activity. Clearly, however, a much more com- prehensive survey of cell types would be required to exclude the alternative hypothesis that methylation in this region may not significantly affect CFTR promoter activity.

Regulated gene transcription is dependent upon specific protein-DNA interactions, and thus differential accessibility of gene sequences in chromatin represents a preemptive control point in the modulation of gene activity. Nuclease hypersensitive sites are believed to represent regions which are conformationally exposed in uiuo, allowing trans-acting factors enhanced access to important cis-acting regulatory sequences. Less distinct regions of uniformly heightened sensitivity to nuclease digestion may extend for many kilobases and generally define the locus of an active or potentially active gene (44, 45). The upstream domain boundary distin- guishing bulk chromatin from the active or potentially active chromatin associated with the CFTR gene lies within a 3.3- kb AccI genomic DNA fragment with end points located from 3.2 to 6.5 kb 5' to the transcription start sites. Except as noted, we found no evidence for general nuclease sensitivity or hypersensitivity in a 26-kb region encompassing the tran-

15920 CFTR Promoter

scription start sites. It is therefore unlikely that any &-acting regulatory elements relevant to CFTR gene expression would map 5' to this region. Proximally, five distinct sites hypersensitive to DNase I appear in the context of T84 nuclei. Con- sistent with a role in maintaining high level expression of CFTR, these sites were not detected in cell lines expressing low CFTR message levels (B 3.1.0, HeLa, PANC-1). A pre- vious study (12) has noted the presence of hypersensitive sites at approximately -3000, -1600, -900, and -200 bp relative to the transcription start site in chromatin isolated from HT29, a cell line comparable to T84 in tissue origin and CFTR expression levels. Although the locations of the HT29 and T84 sites do not appear to correlate precisely, the fact that chromatin hypersensitivity in the CFTR promoter region has only been observed in cells highly positive for CFTR is strongly suggestive of an association with transcriptional activity. All five sites are clustered within an interval of about 1.5 kb encompassing exons -la, la, and 1, with one site mapping in intron 1 approximately 100 bp downstream of the 3' end of exon 1, a location suggestive of enhancer activity.

Functional assessment of the hypersensitive sites indicates that these sequences do not act as strong positive cis elements in transient transfection assays. Elimination of the upstream hypersensitive sites results in only slight incremental loss of promoter activity; a construct lacking all five of the hypersensitive sites is still more active in T84 than in HeLa cells. Similarly, preliminary experiments fail to reveal transcriptional activation capability in constructs placing the intron 1-hypersensitive site DNA in cis to a reporter gene driven by either a heterologous promoter or 3.7 kb of the CFTR promoter region (data not shown). While these results suggest that the sites do not exert major direct effects on transcription, a significant or even primary role in the context of in vivo chromatin cannot be excluded on the basis of transient expression experiments. Precedent from other systems has established that sequences required for control of gene expression in uiuo may not necessarily manifest cis-acting potential in transient transfection assays (46-49). Integration into chromatin may be an important prerequisite for proper func- tioning of hypersensitive site clusters such as the &globin locus activating region (10, 50-52), and thus it is conceivable that the hypersensitive sites detected in the CFTR promoter region may behave in a similar manner, exerting regulatory influence only in the context of intact chromatin.

The discordance between promoter activity measurements and observed CFTR mRNA levels in PANC-1 cells empha- sizes the limitations of interpreting transient transfection data. Clearly the CFTR promoter, at least in the form tested in the transfection studies, is not alone sufficient for recapi- tulating the cell type-specific expression pattern observed for the endogenous transcript. The differences in apparent promoter activity in PANC-1, T84, and HeLa cells may reflect the influence of variables other than the ability of the promoter to support transcription. The behavior of CFTR promoter constructs in PANC-1 cells suggests that the trans- acting factors necessary for CFTR expression are present in this cell line. The possibility that the endogenous CFTR gene in PANC-1 cells contain disabling promoter mutations has not yet been tested. The structural constraints imposed by in uiuo chromatin conformation could contribute to or even dictate CFTR expression levels in this cell line. I t is possible that appropriate control of CFTR transcription initiation in PANC-1 cells may require regulatory elements not contained in our reporter constructs, or that the elevated template copy number introduced during transient transfection could titer out low abundance negative regulatory DNA-binding proteins

normally responsible for repression of CFTR transcription. PANC-1 cells, unmethylated in the promoter region but not manifesting the chromatin hypersensitivity associated with high level transcription, could represent a cellular environ- ment poised for expression of CFTR but lacking some critical in vivo signal or stimulus, an impediment overcome or masked by the artificial conditions imposed in a transient expression assay. Alternatively, regulated expression of CFTR in PANC- 1 cells may involve mechanisms other than control of transcription initiation, such as modulations in mRNA stability, transcriptional attenuation, or RNA processing.

The complexity of CFTR gene regulation attests to the extensive degree of cellular control dedicated to limiting expression of this important protein. In addition to proximal promoter region sequences, recapitulation of appropriate transcriptional control will likely require incorporation of factors which influence the physical context of the CFTR gene. Building upon the initial characterization of CFTR transcriptional control elements, more complex questions of in uiuo modulation can now be addressed in transgenic ani- mals expressing CFTR promoter fusion genes.

Acknowledgments-We are grateful for the gifts of a c-myc probe from Craig B. Thompson, and luciferase reporter plasmids from William M. Wood. We acknowledge the contributions of Theresa V. Strong to the exon - la work. For helpful advice, constructive criti- cism, and stimulating discussions, we thank Todd A. Gray and Deborah Gumucio.

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