Báo cáo khoa học: Evaluation of potential regulatory elements identi®ed as DNase I hypersensitive sites in the CFTR gene doc

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:
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 .
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.
In the present studies we have evaluated a number of the
intragenic DHS to de®ne which might be involved in speci®c
tissues or regulatory pathways and which might merely
re¯ect structural motifs within the CFTR gene. Further
in vivo evaluation is warran ted to fully understand the

CFTR regulatory mechanisms.
ACKNOWLEDGEMENTS
We thanks Dr Gabriel Capella for the NP31 cell line and Drs Nathalie
Mouchel, David Smith and Sytse Henstra for contributions. This work
was funded b y the Cystic Fibrosis Trust and Vaincre La Muco visci-
dose. D. U. is funded by the Danish Research Foundation. A. W. was
in receipt o f a Wellcome T rust vacation scholarship.
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