(Received for publication, June 15, 1995; and in revised form, September 11, 1995)
From the
The cystic fibrosis transmembrane conductance regulator (CFTR)
gene in man is controlled by a tightly regulated and weak promoter. The
architecture of the CFTR promoter suggests regulatory characteristics
that are consistent with the absence of a TATA-like sequence, including
the ability to initiate RNA transcription at numerous positions.
Detailed investigation of the most proximal region of the human CFTR
gene promoter through deletion and mutational analysis reveals that
expression is contingent on the conservation of the inverted CCAAT
sequence. Basal expression of CFTR transcription and cAMP-mediated
transcriptional regulation require the presence of an imperfect and
inverted CCAAT element recognized as 5`-AATTGGAAGCAAAT-3`, located
between 132 and 119 nucleotides upstream of the translational start
site. RNA isolated from a transfected pancreatic cell line carrying
integrated wild-type and mutant CFTR-directed transgenes was used to
map the 5` termini of the transgenic transcripts. Analysis of the
transcript termini by ribonuclease protection analysis reflects the
direct association of the conserved inverted CCAAT sequence in
promoting transcript initiation. Because of the requirement for the
inverted CCAAT sequence for promoting transcription of CFTR, the
involvement of CCAAT-binding factors is suspected in the regulation of
CFTR gene transcription. To test this, we used electrophoretic mobility
shift assays to demonstrate that the majority of the binding to the
inverted CCAAT element, between -135 and -116, was easily
competed for by binding to cognate nucleotide sequences for
CCAAT-enhancer binding protein (C/EBP). An antibody specific for the
C/EBP-related protein, C/EBP, detected C/EBP
as part of a
nuclear protein complex bound to the inverted CCAAT sequence of the
CFTR gene. Also, the detection of specific activating transcription
factor/cyclic-AMP response element binding protein antigens by antibody
supershift analysis of nuclear complexes suggest that species of this
family of transcription factors could be involved in the formation of
complexes with C/EBP
within the CFTR gene inverted CCAAT-like
element. These studies raise the possibility of interactions between
individual members of the C/EBP and activating transcription
factor/cyclic-AMP response element binding protein families potentially
contribute to the tight transcriptional control rendered by the CFTR
gene promoter.
The gene responsible for cystic fibrosis encodes the cystic
fibrosis transmembrane conductance regulator (CFTR) ()gene
product(1, 2) . The expression of CFTR is
predominantly confined to specific epithelial cell types and is
ordinarily expressed in low levels. The low levels and cell
type-specific expression of CFTR appears to be dictated, primarily, by
genomic sequences 5` upstream of the translational start of CFTR, which
correspond to functional promoter sequences(3, 4) .
The requirements for active and cell type-specific CFTR transcription
relies presumably within a narrow band of nucleotide sequences,
proximal to the multiple transcript initiations(5) . The levels
of CFTR transcripts in individual cell types appear to be concomitant
with the ability of certain epithelial cell types, which variably
modulate expression of CFTR in response to 1) levels of
cAMP(6) , 2) the stimulation of protein kinase A and C
activities(7, 8, 9) , and 3) phorbol esters (10) likely through signaling pathways or mechanisms that
converge ultimately on gene transcription (for review, see (11) ). The mechanisms that modulate CFTR gene expression
through extracellular and intracellular signals may implicate
activities analogous to that of both the C/EBP and ATF/CREB families of
transcription factors(12) .
The C/EBP-related family of nuclear transcription factors represent a class of proteins characterized by the ability to bind the CCAAT nucleotide consensus sequence and confer either transcriptional activation or repression on target genes(13, 14, 15, 16) . All members in the C/EBP class of transcription factors contain a basic region and a leucine zipper (bZIP) domain, which correspond to both DNA binding (17, 18) and protein dimerization potentials(19) . The C/EBP family of nuclear proteins are also members of a larger superfamily of transcription factors also characterized by the bZIP motif that includes the ATF/CREB family of transcription factors(18) . The ATF/CREB family has been characterized based on the recognition of several proteins to the cAMP-response element (CRE) nucleotide consensus(20, 21, 22, 23, 24) . It has been previously demonstrated that individual C/EBP proteins characterized thus far can homodimerize or heterodimerize within the C/EBP family of bZIP domain proteins to initiate a specific transcriptional effect (15, 24) and could elicit transcriptional stimulation or repression in response to cAMP(25) . It is now believed that the diverse transcriptional regulation of genes containing the recognition sites of either C/EBP and ATF/CREB may include the heterodimerization between different members in each of the C/EBP and ATF/CREB families(26) . This mechanism may be utilized to respond to complex signals and transcriptional cues through single sequence elements including a response to cAMP, despite the absence of active CRE, AP1, and AP2 consensus nucleotide sequences (27, 28) within the gene.
In this report we focused on the regulation of CFTR gene
transcription mediated by the region encompassing only 135 nucleotides
5` upstream of the translational start site in pancreatic carcinoma,
PANC1, cells. Through deletion analysis of the proximal region of the
CFTR gene 5`-flanking sequences, we have identified a single cis-acting element. This cis-acting element is
significant to the expression of CFTR gene transcription and represents
the most proximal inverted and an imperfect CCAAT consensus essential
for any detectable transcription. Analysis of the transcript termini
provide evidence for the direct association of the conserved inverted
CCAAT sequence in promoting and positioning transcript initiation.
Thus, the core CCAAT element of the CFTR gene 5`-flanking region may
reflect a weak initiator nucleotide consensus necessary for the
generation of the multiple transcripts for CFTR gene expression.
Studies to identify DNA-protein interactions were confirmed by both
electrophoretic mobility shift and DNase protection assays to determine
the extent of protein binding to the CFTR inverted CCAAT consensus
region. Characterization of DNA-protein interactions between the CFTR
inverted CCAAT sequence and nuclear proteins from PANC1 cells now
provide evidence for the interaction with one member of the C/EBP
family of transcription factors, C/EBP (15, 16) and also referred to as
NFIL-6
(29) . In addition, electrophoretic mobility shift
assays also indicate the presence of CREB1 and ATF1 in the protein
complexes bound to the CFTR inverted CCAAT element. Thus, these results
suggest that the mechanism regulating CFTR gene transcription in PANC1
cells may include the trans-acting potentials of both the
C/EBP and ATF/CREB families of transcription factors on a single
inverted CCAAT element. Indication that the transcription factor
C/EBP
(NFIL-6
) is associated with a pancreatic cell nuclear
complex bound to the CFTR inverted CCAAT element may also implicate
regulation mediated by interleukin-6(29) . Hence, the complex
nature of CFTR gene transcription in specific cells may require
multiple interactions between bZIP families of transcription factors to
direct the highly complex nature of transcriptional regulation
demonstrated by the CFTR gene.
Plasmid construct
ptk(pL)GH (
)was used as a heterologous
promoter to insert fragments of the CFTR 5` genomic region containing
the wild-type and mutant inverted CCAAT elements. Cloned fragments,
corresponding to the CFTR wild-type and mutant gene promoters pCF-197wt
and pCF-197mut served as DNA templates for the generation of PCR
fragments. Synthetic oligonucleotides,
5`-ccaagcttgggcagtgaaggcgggggaaagagcaa-3` and
5`-ccaagcttgggctcaaccctttttctctgacctg-3`, were used to generate a
114-bp fragment spanning from -198 to -24 of CFTR promoter
sequences, containing HindIII restriction adaptors, using
Vent(TM) polymerase (New England Biolabs, Inc.) according to the
manufacturer's directions. These fragments were, subsequently,
cloned into a HindIII site of the heterologous
ptk(pL
)GH thymidine kinase promoter vector, and
appropriate plasmid clones were verified by restriction analysis and
sequenced by the Sequenase kit (U. S. Biochemical Corp.) The plasmids
p[CF+ccaat]tk/hGH, p[CF-ccaat]tk/hGH, and
p[CFc
t]tk/hGH contain, respectively, the wild-type
inverted CCAAT box element of the CFTR promoter in opposing
orientations independently and the mutant inverted CCAAT element of the
CFTR gene promoter.
Stable
co-transfections of pancreatic carcinoma cells were carried out using
DOTAP, identically, as described above with the constructs,
pCF-197wt/hGH or pCF-197/hGH or the control plasmid pØGH
and the selectable marker, pMC1neoA (Stratagene, Inc.) at a molar ratio
of 15:1 (reporter gene/selectable marker gene) in PANC1 cells. These
co-transfections were carried in the same manner as above with the
exception that cells were plated on 60-mm plates at a lower density of
approximately 5
10
cells/plate. Following
transfection, the culture medium containing the DOTAP/DNA mixture was
replaced by the selection medium containing 400 µg/ml
Geneticin® (G418) (Life Technologies, Inc.). Stable cell
populations were established after 14 days in selectable medium.
Genomic DNAs were isolated from individual clonal cell populations, and
the integration of the transgenes along with the copy numbers were then
confirmed by Southern dot hybridization analysis using probes spanning
the bacterial sequences of the transgenes. Monoclonal populations of
stably transfected cells pCF-197wt/hGHpac, pCF-197
/hGHpac, or
pØGHpac were then expanded further in 60-mm plates and
maintained in selectable medium containing G418. 20 confluent plates of
each stably transfected cell line containing either the pCF-197/hGH,
pCF-197
/hGH, or pØGH transgene construct were used for the
preparation of cytoplasmic RNA (33) .
DNase protection assays were performed using
nuclear extracts prepared from untreated and 8-Br-cAMP-treated cells on
the 127-bp fragment homologous to the region of the CFTR promoter
between -198 and -71 upstream of the translation start
site. A DNA fragment was generated with the primers
5`-gggcagtgaaggcgggggaaagagc-3` and 5`-ctgggtgcctgccgctcaaccctt-3`. The
5` oligonucleotide 5`-gggcagtgaaggcgggggaaaga-3` was labeled by using
[-
P]ATP and T4 polynucleotide kinase.
Addition of the [
-
P]ATP-labeled
oligonucleotide (
5 pmol) to the PCR was used to generate a
P-labeled DNA fragment. The DNA fragment was excised and
purified from a 5% polyacrylamide gel. DNase protection experiments
were carried out as described previously(38) . To confirm the
location of protection from DNase hydrolysis, corresponding
Maxam-Gilbert (G + A) reactions were conducted on the
P-labeled DNA fragment.
Fig. 1shows that the major elements of transcriptional control of CFTR gene promoter function in the pancreatic cell line are confined to sequences approximately 135 nucleotides from the translational start site. In control cultures (hatched bars) not induced by 8-Br-cAMP, the promoter sequence extending 912 nucleotides from the open reading frame produced significant levels of hGH expression, approximately 5-fold greater than control cells transfected with reporter gene constructs lacking all CFTR gene sequences (pØGH). Subsequent deletion from the 5` end up to 135 nucleotides from the translational start of CFTR diminished basal transcription activity only by 20%; however, deletion of a further 19 bp (pCF-116/hGH) reduced transcriptional activity to a level 50% lower than that of the negative control, pØGH, which lacked all CFTR nucleotide sequences. This reduction of promoter activity in pCF-116/hGH to a point below the clearly detectable level of hGH expression pØGH likely indicates that cryptic promoter sites exist within the pØGH plasmid backbone or the hGH gene, and that the activity of these sites is suppressed by sequences within the proximal 116 bp of the CFTR promoter. Consistent with this interpretation is the finding that successive deletion of sequences downstream of -116 restored hGH expression to the background levels expressed by pØGH (data not shown); however, we did not perform experiments to substantiate the presence of DNA element corresponding to transcriptional repression.
Figure 1:
Functional analysis of the
proximal region of the CFTR gene 5`-flanking sequences. Several
fragments of the CFTR 5`-flanking sequences were generated by PCR and
ligated to the hGH gene, as shown as the schematic diagram. Plasmid
constructs were transiently transfected into the cell line PANC1, which
was either left untreated or treated with 8-Br-cAMP, and hGH reporter
gene expression levels were determined as described under
``Materials and Methods.'' The expression of hGH levels were
evaluated and normalized to the the level of constitutive expression of
chloramphenicol acetyltransferase generated by the internal control
plasmid pRSVcat. Values represented in the graph are shown as the mean
percent plus the standard error of hGH expression relative to the level
of constitutive expression generated by the plasmid pCMV/hGH, indicated
at the top of the graph. Numbers refer to
the number of nucleotides relative to the translational start site
consensus for CFTR when fused to the hGH reporter gene in each of the
plasmid constructs represented. Mutation to the wild-type inverted
CCAAT sequence (5`-ggaattggaagcaaatt-3`) is denoted as the nucleotide
sequence ggaat[]gaagcaaatt. The plasmid hGH
construct pØGH is the parental vector, lacking 5`-flanking
sequences of the CFTR gene, included in this experiment. The # refers to the number of data points generated for each of the
constructs shown.
In addition to supporting basal transcriptional activity, the same CFTR promoter constructs also demonstrate cAMP-stimulated expression of the hGH gene (Fig. 1, black bars). Essentially the same level of activity in the presence of 8-Br-cAMP, representing 3-4-fold stimulation over untreated controls, was seen in cells transfected with promoter constructs extending 135 or more bp upstream of the translational start site. Deletion of sequences upstream -116 bp reduced activity to below that of the pØGH control construct, abolishing the effect of 8-Br-cAMP.
In
addition to mediating basal expression of CFTR gene transcription, the
inverted CCAAT element was strictly required for cAMP-mediated
induction of transcriptional activity. Moreover, the absence of any
demonstrable cAMP-mediated activation of CFTR gene transcription via
existing sequences homologous to both CRE and AP1 suggest that this
inverted CCAAT element is, alone, sufficient for cAMP responsiveness
within the CFTR gene (data not shown). Recent data have indicated that
the induction of CFTR gene expression was mediated by cAMP likely
acting through putative cis elements of the CFTR gene
promoter(6) . The best studied transcriptional response by cAMP
was identified through the analysis of the conventional CRE nucleotide
consensus(20) . Despite the presence of both CRE and AP1
consensus sites upstream in the CFTR gene 5`-flanking
region(3, 4) , these sequences appeared to lack
substantial cis-acting potential within the context of the
CFTR gene(5, 41) . To map the nucleotide sequences
responsible for the observed induction of CFTR by cAMP, deletion
constructs (Fig. 1) were examined for their ability to support
cAMP-mediated induction of CFTR gene transcription. Constructs
pCF-912/hGH, pCF-492/hGH, pCF-262/hGH, pCF-197/hGH, and pCF-135/hGH
each averaged a stimulation of approximately 3-fold, upon induction
with the cAMP analog, 8-Br-cAMP (Fig. 1). This activation was
detected following a 12-h treatment of cells with 8-Br-cAMP. A 19-bp
deletion downstream of the -135 nucleotide position abolished
cAMP induction as indicated by construct pCF-116/hGH (Fig. 1).
Mutation to the inverted CCAAT sequence demonstrates the strict
requirement of the conserved CCAAT sequence to mediate the induction by
8-Br-cAMP on hGH expression. Construct pCF-197/hGH contains a
three-nucleotide deletion in the inverted CCAAT sequence to C-T.
The loss in cAMP-mediated regulation directly corresponds to the
deletion of three nucleotides in the inverted CCAAT element. In
addition, the absence of any CRE consensus indicates the level of
cAMP-inducible transcription of CFTR is mediated by the inverted CCAAT
element, alone. The cAMP-mediated induction of hGH expression directed
by the CFTR gene promoter from construct pCF-197/hGH requires the
conservation of the inverted CCAAT element consensus in the CFTR gene
promoter. This result is consistent with other examples of cAMP
induction mediated by the CCAAT element, including the recent
examination of the G-protein
subunit gene
promoter(42) . Thus, the results indicate here that the
potential role of the inverted CCAAT element in the CFTR gene is to
mediate both the basal transcription of CFTR and induction by cAMP.
Figure 2:
Proximal element of the human CFTR gene
directs cAMP-mediated transcription through the inverted CCAAT element
in a heterologous gene. Plasmid constructs containing wild-type and
mutant inverted CCAAT sequence fragment of the human CFTR gene
5`-flanking nucleotide sequences were ligated to a thymidine kinase
gene promoter linked to the human growth hormone gene (see
``Materials and Methods''). Transient transfections were
performed on the PANC1 (top graph) and HeLa S3 (bottom
graph) cell lines with the constructs containing the inverted
CCAAT element of the CFTR gene in the sense orientation
(p[CF+ccaat]tk/hGH), antisense orientation
(p[CF-ccaat]tk/hGH), and with the mutated CCAAT consensus,
p[CFct]tk/hGH, followed by treatment of cells with
8-Br-cAMP for 24 h. The levels of human growth hormone were determined
in the medium as described under ``Material and Methods'' and
were quantitated relative to the levels of hGH generated by the
pCMV/hGH as described in the legend to Fig. 1. Levels of hGH
expression were normalized for transfection efficiency by the
comparison to a internal control plasmid
pRSV/cat.
Figure 3: The inverted CCAAT sequence of the CFTR gene directs multiple but discrete transcript initiations. Ribonuclease protection was performed on stably transfected cells carrying the pCF-197wt/hGHpac and pCF-197mut/hGHpac transgenes, containing the CFTR promoter-human growth hormone fusion constructs as described under ``Materials and Methods.'' Radiolabeled antisense riboprobes of 422 and 419 nucleotides in length corresponding to the wild-type and mutant CFTR transgene sequences, respectively, were generated by Sp6 RNA polymerase. A 425-nucleotide riboprobe, spanning the hGH gene sequences and pUC12 sequences of the parental vector pØGH, was used as a negative control to correct for background signals generated from the stably transfected cell line carrying the pØGHpac gene. 50 µg of cytoplasmic RNA from stably transfected cells or yeast tRNA were allowed to anneal to their respective antisense riboprobes overnight. Hybridized RNA strands were hydrolyzed by ribonuclease, and the protected RNA fragments were resolved on a 6% polyacrylamide sequencing gel. The size of the protected transcripts and position of the transcript termini were mapped using a DNA sequence ladder in adjacent lanes (not shown). The schematic depicted above the representative autoradiograph indicates position of transcript termini relative to the region spanning the CFTR promoter between -154 and -48 upstream relative to the translational start site of the CFTR gene. The nucleotide sequence on top of the schematic is that of the wild-type CFTR gene. The line above the sequence refers to the inverted CCAAT sequence; the line below the sequence denotes the weak CAAT consensus found in the CFTR gene(3) . Closed triangles indicate the positions of the 5` termini of the major transcripts, relative to the CFTR promoter, apparent from the wild-type transgene pCF-197wt/hGHpac. The bent arrows indicate the presence and direction of the transcripts detected within the 5`-flanking region of the CFTR gene evaluated in this assay. Down arrows refer to the position of transcript 5` termini emanating from minor transcriptional start sites.
Figure 4:
Competition analysis of PANC1 cell nuclear
proteins bound to the CFTR gene inverted CCAAT element. Panel
A, EMSA was performed using a double-stranded oligonucleotide
corresponding to sequences of the CFTR-inverted CCAAT element
5`-tggggggaattggaagccaaatgacat-3`. The oligonucleotide was synthesized
and labeled at the 5` end by incubating with
[-
P]ATP and T4 polynucleotide kinase.
Nuclear extract preparation, binding reactions to the radiolabeled
oligonucleotide, and electrophoresis were performed as described under
``Materials and Methods.'' 2 µg of nuclear extract and
unlabeled oligonucleotide DNA, identical to the
P-labeled
oligonucleotide sequence probe and used as competitor, was introduced
into the binding reaction in increasing amounts prior to the addition
of the radiolabeled oligonucleotide. Values indicate the molar excess (x-fold) used relative to the amount of
P-labeled
oligonucleotide introduced into the reaction. Panel B,
competing unlabeled oligonucleotides, as indicated above the figure,
were introduced into the binding reactions prior to the introduction of
the
P-labeled inverted CCAAT oligonucleotide sequence as
described above. A 10 or 100-fold excess (relative to the input of
P-labeled oligonucleotide) of competing oligonucleotides
were added to each of the reactions. The competitor oligonucleotides
used at 10-fold excess were the wild-type CFTR-inverted CCAAT element,
the C/EBP binding consensus of the c-fos gene promoter, the
C/EBP binding consensus of the albumin CCAAT box sequence, and the
symmetrical dyad consensus for optimal binding by C/EBP proteins. The
mutant CFTR-inverted CCAAT sequence unlabeled oligonucleotide was added
at a 100-fold excess in the binding reactions (see ``Materials and
Methods'').
To compare DNA binding affinities between cAMP-induced and basal levels of transcription, directed by the proximal 5` end of the human CFTR gene, nuclear extracts prepared from PANC1 cells treated with 8-Br-cAMP and untreated cells were used for DNase I protection assays. The footprint visualized in Fig. 5is indicative of protection of the CFTR gene promoter, is positioned between -146 and -90 upstream of the open reading frame of CFTR, and presents a large region protected from deoxyribonuclease hydrolysis (Fig. 5). Interestingly, this footprint is primarily a characteristic of the extract derived from the untreated control and not from nuclear extract isolated from cells treated with 8-Br-cAMP. The footprint demonstrated in nuclear extracts from untreated PANC1 cells generates extensive binding, overlapping the inverted CCAAT element of the human CFTR gene. Comparatively, DNA binding affinity is slightly detectable in PANC1 cells stimulated with 8-Br-cAMP (Fig. 5) in accord with the stimulation of reporter gene transcription by cAMP as shown in Fig. 1and Fig. 2. This result is consistent with a DNA-protein complex constitutively bound to CCAAT box sequences previously shown to contribute to the weak and basal levels of human gp91-hox gene transcription through the displacement of more potent transcriptional activators(44) . This model accounts for the relative absence of change in the detection of multiple DNA-protein complexes by EMSA from various cell types despite dramatic changes in the level of gene transcription mediated by the CCAAT box(44) .
Figure 5:
Comparative analysis of DNase I protection
of the inverted CCAAT element with nuclear extracts treated with
8-Br-cAMP. Genomic fragment of the CFTR gene encompassing nucleotides
between -196 and -74 nucleotides upstream of the CFTR gene
open reading frame for translation was used for the DNase protection
experiment. Preparation of the P-labeled DNA probe and
nuclear extract from PANC1 cells were performed as described under
``Materials and Methods.'' Nuclear extracts prepared from
PANC1 cells treated with 1 mM 8-Br-cAMP and untreated PANC1
cells were incubated with the
P-labeled DNA fragment. The
binding reactions were then subjected to hydrolysis with
deoxyribonuclease I and fractionated on a 8% nucleotide sequencing gel. Numbers above the lanes indicate the amount of protein from
the nuclear extract added to the reaction prior to partial
deoxyribonuclease I hydrolysis. Protein amounts (100 µg) remained
constant in each lane by the addition of bovine serum albumin. The bracket outline indicates the region of protection from DNase
I hydrolysis by nuclear extract. The footprint was mapped by comparison
of Maxam and Gilbert sequence reactions performed on the identical
P-labeled DNA fragment (not shown) and indicated as
numerical nucleotide position relative to the translational start site.
The position of the inverted CCAAT element of the CFTR gene promoter is
indicated by the solid box and nucleotide sequence adjacent to
the figure.
Figure 6:
C/EBP is detected in the nuclear
protein complex bound to the human CFTR gene promoter inverted CCAAT
element. EMSA experiments shown were carried out as described under
``Materials and Methods.'' Panel A, EMSA using the
oligonucleotide containing the inverted CCAAT element of the human CFTR
gene as a probe (see legend to Fig. 4). Nuclear extracts (10
µg) prepared from untreated and 8-Br-cAMP-treated PANC1 cells were
added along with the radiolabeled probe followed by the addition of 1
µg of antiserum directed against C/EBP
or rabbit preimmune
serum in the lanes indicated. Competitor oligonucleotide DNA
representing the typical dyad consensus for C/EBP binding was
introduced into the binding reactions in the lanes indicated at a
25-fold molar excess relative to the amount of
P-labeled
probe used. Arrows (A and B`), depict the
bandshifts of DNA-protein complexes. A indicates the position
of the prominent DNA-protein complex generated from the PANC1 cell
nuclear extract. B` indicates the position of the
DNA-protein-antibody supershift complex detected in the autoradiograph. Panel B, an EMSA experiment using nuclear extracts prepared
from PANC1 cells and an oligonucleotide probe carrying a prototypical
C/EBP
consensus site from the human hemopexin gene A
site(37) . 1 µg of C/EBP
-specific antibody or
preimmune serum was added to the binding reactions where indicated. Arrows, depict the bandshifts of DNA-protein
complexes.
Figure 7:
Identification of ATF/CREB proteins within
nuclear extracts bound to the CFTR gene promoter inverted CCAAT
element. PANC1 nuclear extracts (10 µg) were bound to the P-labeled oligonucleotide (see legend to Fig. 4).
Antibodies directed against CREB1, CREB2, ATF1, ATF2, and ATF3 (Santa
Cruz Biotechnology, Inc.) were added to the binding reaction, as shown
in the lanes indicated, following the binding by nuclear
extracts to the radiolabeled oligonucleotide. A serum control (shown in
the last lane) used as a polyclonal antibody against the RelB
protein (Santa Cruz Biotechnology, Inc.). Arrows indicate
position of complexes formed in the absence of
antibody.
The expression of CFTR is controlled by different regulatory
mechanisms(6, 7, 9, 45, 46, 47, 48) ,
suggesting that an array of signals could be required to maintain the
activity of the CFTR cAMP-mediated Cl channel.
Intracellular signaling by cAMP is a major component in the modulation
of CFTR activity(49) . With regard to the processes that
modulate the function of CFTR through cyclic AMP-mediated activation of
Cl
channels, a complementary model has included the
stimulatory effects mediated by cAMP on CFTR gene
transcription(6) , therefore providing further provocation of
epithelial cells to direct the expression of sufficient levels of CFTR
through signaling pathways mediated by cAMP. In this report, the CFTR
gene 5`-flanking region serves as a model promoter to examine the role
of nucleotide sequences proximal to both transcriptional and
translational start sites in the complex regulation of CFTR gene
transcription. The expression of CFTR gene transcription is consistent
with the characteristically weak promoter that utilizes multiple
initiation sites for transcription(3, 4) . Through
deletion and mutagenesis of the 5`-flanking sequences of the CFTR gene,
we demonstrate that the basal transcription of CFTR in PANC1 is
conserved by using sequences extending only 135 nucleotides upstream of
the open reading frame for translation. Experiments were performed and
indicate the role of an inverted CCAAT sequence in directing both basal
and cAMP-inducible regulation of CFTR. The presence of the cis-element containing both an inverted and imperfect repeat
of the canonical CCAAT sequence of ATTGGAAGCAAAT within the deleted
19-bp element indicates a possible relationship between the inverted
CCAAT element and transcriptional regulatory function. Analysis of the
inverted CCAAT element, proximal to many transcript initiations
directed by the CFTR gene(3, 4, 5) ,
demonstrates that conservation of the inverted CCAAT consensus is
essential for maintaining basal levels of reporter gene activity in a
transient expression assay. Based on this characterization of the CFTR
gene promoter, mutation of nucleotides within the inverted CCAAT
sequence virtually abolished the expression of reporter gene activity.
Although these studies cannot account for cell type-specific
differences in the expression of CFTR gene transcription, this provides
some fundamental understanding of the nucleotide requirements for
directing CFTR gene transcription.
Despite the absence of nucleotide
consensus for CRE, AP1, or AP2 activity within the context our promoter
constructs, we have evaluated reporter gene levels of transfected PANC1
cells stimulated with cAMP. The cis-acting element, which
apparently directs basal transcription, can also account for the
induction of CFTR gene transcription by cAMP in these studies. These
unexpected results from our transfection studies performed in this
report indicate that cAMP-mediated regulation resides within the
nucleotide sequences also directing basal levels of transcription. To
address the logical flaw in interpreting the cAMP-mediated regulation
conferred by the same nucleotide sequences directing basal expression
of CFTR gene transcription, we had constructed heterologous gene
promoter constructs using the CFTR gene 5`-flanking element fused to
the herpes simplex virus thymidine kinase promoter. Our results suggest
that the same sequence elements promoting basal CFTR gene transcription
confer cAMP-mediated regulation through the context of a heterologous
gene. Although models that demonstrate the ability to direct
cAMP-mediated transcription in the absence of conventional
cAMP-responsive regulatory elements are relatively few, some recent
investigations implicate the activity of the CCAAT element in directing
both basal and cAMP-mediated transcription(50) . This was also
previously demonstrated with the human G-protein gene
promoter(42) . It has been more recently described that the
regulation of the human tryptophan hydroxylase gene transcription
requires an inverted CCAAT motif for maintaining basal expression and
cAMP-mediated transcription (51) . The absence of a CRE, AP1,
or AP2 consensus sequence within the region of the CFTR promoter that
we have examined here reflects some similarities to these
unconventional genetic models for cAMP-mediated regulation. The
kinetics of cAMP-mediated transcription in these models (42, 51) bears some similarities to the cAMP induction
of CFTR gene expression (6) in relationship to the time
required for stimulation to occur (>6 h). This is in contrast to
stimulation mediated by cAMP through a conventional cis-acting
CRE, such as that previously demonstrated with the somatostatin
gene(52) , requiring only minutes for transcriptional
stimulation. This would suggest that transcription mediated by cAMP can
occur through several different mechanisms, accounting for differences
in the kinetics of gene transcription mediated by cAMP. The delayed
response to intracellular increases of cAMP to the activation of gene
transcription appears to fall into a category of genes possibly
requiring de novo protein synthesis(51) . This is in
contrast to the rapid transcriptional response to intracellular levels
of cAMP mediated by the CRE not requiring new protein
synthesis(53) . We indicate cAMP-responsive transcription
within the CFTR gene promoter is likely due to the inverted CCAAT
sequence. This result is indicated from co-transfection studies despite
the absence of more conventional sequences responsive to cAMP such as
the CRE(52) , AP1(54) , and AP2 (55) sites
within the nucleotide elements studied in this report. Interestingly,
the inverted CCAAT element of the CFTR gene was able to stimulate both
basal levels as well as cAMP-mediated transcription within the context
of a heterologous gene promoter. Although the placement of such cis-acting sequence elements of the CFTR gene within the
context of a heterologous gene promoter may demonstrate a misleading
effect with regard to actual CFTR promoter function, here we
demonstrate the ability of the inverted CCAAT element to only confer
cAMP-responsive transcriptional control.
To address the relevance of
the inverted CCAAT sequence in directing the transcriptional machinery
to the CFTR gene, stably transfected cells were obtained using two
independent CFTR promoter transgene constructs. RNA isolated from the
transgenic cells, containing wild-type and mutant inverted CCAAT
sequence fused to reporter sequences, were analyzed by ribonuclease
protection mapping. Results from the ribonuclease protection assay
indicate the direct association of the conserved inverted CCAAT
sequence with the positioning and pattern of the transcription start
sites, using antisense probes specific for each of the constructs
tested shown in Fig. 3, thus providing evidence for the
involvement of an inverted CCAAT nucleotide consensus in directing
basal transcription. Although many examples have alluded to the role of
CCAAT sequences in directing RNA polymerase II-dependent
transcription(40) , there are no apparent studies that
demonstrate a direct role for a CCAAT consensus nucleotide sequence as
a component of the basal transcription apparatus. One explanation for
the lack of evidence is due to the absence of example promoters and
cell types that could represent a model environment demonstrating both
basal and inducible transcription involving a CCAAT nucleotide
consensus, exclusively. Another explanation may involve multiple and
complex levels of regulation directed at specific CCAAT elements,
making the evaluation between basal and inducible regulation difficult
to interpret. Due to the relatively weak promoter function of the CFTR
gene, this may well represent a novel feature of transcriptional
regulation characteristic of the CFTR gene. In addition, specific Plabeled RNA antisense probes were created to span
specifically between the CFTR promoter and the human growth hormone
gene. Therefore, the qualitative evaluation of the protected fragments
was restricted to transcripts generated only from the CFTR gene
promoter fragment from the window of nucleotides depicted in Fig. 3. A study by Koh et al.(5) indicated
that the presence of an additional putative exon upstream of the CFTR
promoter, referred to as exon 1a, may utilize alternative or cryptic
starts in transcription. Although this may represent an additional
level of regulation for CFTR gene expression, we have no indication of
this yet in the cell type we have studied in this report (data not
shown). Through the context of the promoter sequences examined, we
indicate the possible significance of the inverted CCAAT sequence in
basal regulation of CFTR gene transcription. This would indicate that
the conservation of the inverted CCAAT sequence is associated with the
transcriptional start site selection and represents a sequence
requirement for basal transcription of the CFTR gene.
Studies to compare the affinity of DNA binding overlapping the inverted CCAAT sequence between cAMP-mediated and basal transcription indicate extensive protection of DNase hydrolysis in untreated cells when compared with cAMPstimulated cells. Contrary to our own expectations, extensive protection of the nucleotide sequences overlapping the inverted CCAAT element of the human CFTR gene was more characteristic of basal expression and not of cAMP-stimulated expression in the PANC1 cell type. Although EMSA experiments performed here do not yet account for some form of displacement of transcriptional activation through binding to the the inverted CCAAT element, one explanation may be due to the sensitivity of the DNase protection assay to detect subtle changes in DNA binding affinities. Arguably, the constitutive binding of a repressor to the CCAAT element, which may act to displace transcriptional activation as shown previously(44) , could function in tandem with other nuclear protein(s) to allow some access of more potent trans-activators to the CCAAT sequence. Thus, this mechanism may allow more or less activation of CFTR gene transcription through the competition between positive and negative effectors of CFTR gene transcription. The relief of repressor activity by other factors may represent a plausible mechanism for activation of CFTR gene transcription, but this hypothesis has not yet been experimentally tested.
Gel shift and competition analysis of the
CCAAT element indicate that in fact CCAAT binding protein(s) are
responsible as least in part for the interaction with the CFTR
gene-inverted CCAAT nucleotide consensus. Interestingly, competition
with nucleotide consensus elements corresponding to both the albumin
and c-fos gene CCAAT sequences(56, 36) ,
respectively, readily competed for the CFTR gene-inverted CCAAT
element, suggesting that specific CCAAT enhancer binding proteins may
target the CFTR gene promoter. A further attempt to characterize such
interaction by specific gene products was performed by antibody
supershift analysis to immunologically characterize the C/EBP species
from PANC1 cell nuclear protein bound to the inverted CCAAT element of
the CFTR gene. Results of experiments shown in Fig. 6indicate
the presence of C/EBP in nuclear complexes bound to the
CFTR-inverted CCAAT sequence. Due to the involvement of C/EBP
(NF-IL6
) in a response to cytokine-mediated inflammation by
interleukin-6(57, 37) , we cautiously speculate the
involvement of interleukin-6 in a signaling pathway in regulating CFTR
gene transcription.
Immunological detection of CREB1 and ATF1 associated with protein complexes bound to the inverted CCAAT element of the CFTR gene promoter (Fig. 7) may indicate another level of regulation associated with cAMP-responsive transcription factors despite the absence of a CRE nucleotide consensus within the sequences examined in this report. Although the absence of a CRE certainly may not preclude CREB or ATF proteins from targeting promoters devoid of such cis-acting elements, a recent example by Vallejo et al.(58) demonstrates that regulation of the somatostatin gene requires protein-protein interaction between both C/EBP and ATF/CREB factors to elicit a response by cAMP through the somatostatin gene CRE. Inversely, C/EBP proteins have been shown to bind, specifically, to the phosphoenolpyruvate carboxykinase gene CRE with high affinity to promote cAMP-mediated transcriptional activation(59) . With regard to the CFTR gene, this paradox may be accounted for by a reversed mechanism, suggesting the inverted CCAAT element as the cis-acting target directing multiple protein interactions between members of the C/EBP and ATF/CREB families of transcription factors. In addition, studies by Roesler et al.(60) demonstrate C/EBP as an effector of cAMP-mediated transcription through the combined interactions with liver-specific transcription factors to modulate the cAMP regulation of phosphoenolpyruvate carboxykinase. The models described here may represent complex features analogous to CFTR gene regulation. Therefore, through the identification of trans-acting factors responsible for directing CFTR gene transcription, we can begin to elucidate the mechanisms regulating CFTR gene expression that may help us better understand the pathology associated with cystic fibrosis.