From the Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Straße 2, D-35037 Marburg, Germany
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ABSTRACT |
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It has been reported that respiratory
epithelium-specific transcription is mediated by thyroid transcription
factor 1 and members of the HNF3/forkhead family of transcription
factors. Here, we show that the uteroglobin/Clara cell 10-kDa promoters from rabbit and man are regulated by HNF3 and HNF3
but not by HFH-4 and TTF-1. We have identified two HNF3-responsive elements in the
rabbit uteroglobin/CC10 promoter located around 95 and 130 base pairs
upstream of the transcriptional start site. Both elements contribute to
promoter activity in H441 cells expressing uteroglobin/CC10 and
HNF3
. Gene transfer experiments into Drosophila Schneider cells that lack many mammalian transcription factor homologs
revealed that HNF3
and HNF3
on their own cannot activate the
uteroglobin/CC10 promoter. However, HNF3
and HNF3
strongly enhanced Sp1-mediated promoter activation. Synergistic activation by
HNF3
and Sp1 was absolutely dependent on the integrity of two Sp1
sites located at around
65 and
230. We show further that multiple
activation domains of Sp1 are required for cooperativity with HNF3
.
These studies demonstrate that transcription from the rabbit
uteroglobin/CC10 promoter in lung epithelium is controlled by the
combinatorial action of the cell-specific factor HNF3
and the
ubiquitous factor Sp1.
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INTRODUCTION |
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The level and pattern of expression of a gene is determined mainly by the combinatorial action of transcription factors binding to distinct promoter and enhancer elements. In the last years, we have started to define DNA elements and transcription factors responsible for the expression of the rabbit uteroglobin/CC10 (Clara cell 10-kDa protein) gene, which is expressed in several ontogenetically unrelated epithelial tissues. Highest expression of the uteroglobin/CC10 gene is observed in endometrium and lung (1, 2). In endometrium, expression of the gene is restricted to the glandular and luminal epithelium (1), where it is induced by progesterone and estradiol (3). In lung, Clara cells lining the respiratory epithelium express constitutively uteroglobin/CC10 at high levels. In these cells, glucocorticoids modulate the uteroglobin/CC10 mRNA level only slightly (4, 5).
DNase I protection, promoter deletion, and linker-scanning analyses
revealed that at least six distinct regions contribute to the activity
of the promoter in the epithelial endometrium cell line Ishikawa and
and the lung cell line H441 (6, 7). Some of the transcription factors
acting through these elements have been identified in the last years.
Regions VI and II located 230 and 65 bp1 upstream of the
transcription start site are noncanonical binding sites for the closely
related transcription factors Sp1 and Sp3 (8). Region V spans at least
60 nucleotides between 208 and
148 and probably contains several
DNA elements that have not been characterized further. Regions III
(around
95) and IV (around
130) are characterized by two A/T-rich
nucleotide stretches. Region I contains a noncanonical TATA box motif
(TACA box) which is bound specifically by two factors, the TATA core
factor and the TATA palindrome factor (9). Both are different from the TATA box-binding protein. TATA palindrome factor has recently been
identified as the transcription factor Yin Yang 1 (YY1) (10).
Studies of other genes specifically expressed in lung epithelium (surfactant protein A, surfactant protein B, and murine CC10) provided evidence that thyroid transcription factor-1 (TTF-1), which is expressed in addition to the thyroid and the brain also in lung type II and Clara cells (11, 12), is a major player involved in the expression of genes in the respiratory epithelium (13-17). In addition, hepatocyte nuclear factor 3 (HNF3) family members and the HNF3/forkhead homologs (HFH), which are also expressed in lung epithelia (18, 19), are involved in expression of surfactant protein A (14) and surfactant protein B (13, 15, 18) genes as well as of murine CC10 genes (16, 17, 20-24).
In the present study, we analyzed various different human lung cell
lines for the expression of lung epithelium-specific marker genes. We
show that H441 cells express human uteroglobin/CC10 mRNA in
addition to several other lung epithelial cell-specific mRNAs. We
demonstrate that the rabbit uteroglobin/CC10 gene promoter responds to
the transcription factors HNF3 and HNF3
, but not to TTF-1 and
HFH-4. Promoter deletion and linker-scanning analysis identified two
functional elements for HNF3
/HNF3
around
130 and
95. The
factors present in H441 cells and acting through these elements are
HNF3
and Oct-1. Transfection studies into Drosophila
Schneider cells that lack many mammalian transcription factor homologs
revealed that HNF3
and HNF3
on their own cannot activate the
uteroglobin/CC10 promoter, but that both proteins strongly enhance
Sp1-mediated promoter activation. This study also demonstrates that
synergistic activation by HNF3
and Sp1 is absolutely dependent on
the integrity of the two Sp1 sites located at around
230 bp and
65
bp. In addition, we show that several activation domains of Sp1 are
required for cooperativity with HNF3
.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructions--
The construction of the wild-type
rabbit uteroglobin/CC10 promoter construct pUG(395)CATSV as well as
the linker-scanning mutants LS(
92/
99), LS(
127/
132),
LS(
64/
72), and LS(
222/
229) fused to the CAT gene have been
described (7). The double linker-scanning mutants DLS(
92/
127) and
DLS(
64/
222) were generated by polymerase chain reaction-based
mutagenesis using appropriate oligonucleotides. The human CC10
promoter-CAT construct (hCC10-CATSV) was obtained by replacing the
410-bp BamHI rabbit UG promoter fragment in pUG(
395)CATSV by a 405-bp HinfI fragment containing the human CC10
promoter sequences from
395 to +10 (25). An appropriate rat CC10
promoter-CAT construct (ratCC10-CATSV) was obtained by cloning a 350-bp
HpaII fragment containing rat CC10 sequences from
300 to
+60 (26) into the blunted BamHI site of pUG(
395)CATSV.
During the course of our experimental studies, we recloned the various
uteroglobin/CC10 promoters and linker-scanning mutants thereof as
HindIII-XhoI fragments into the luciferase
reporter vector pGAW, which is a self-made derivative of the pGL3 basic
vector (Promega). In pGAW the single BamHI site of pGL3 is
destroyed by blunt end religation and the polylinker of pGL3 is
replaced by a polylinker containing single recognition sequences for
the restriction enzymes PstI, EcoRI,
EcoRV, HindIII, BamHI,
BglII, XhoI, SmaI, NheI,
SacI, and KpnI. The luciferase reporter plasmids
were used for transfections in Drosophila Schneider cells
(SL2 cells).
Nuclear Extract Preparation and Electrophoretic Mobility Shift
Analysis (EMSA)--
Nuclear extracts of mammalian and transfected SL2
cells were prepared according to Andrews and Faller (30). The sequences of the oligonucleotides used for EMSAs are presented in the appropriate figures. After annealing of the single-stranded oligonucleotides the
double-stranded DNA was separated from unannealed strands by gel
electrophoresis on a native 12% polyacrylamide gel in Tris borate-EDTA
buffer. Double-stranded DNA was labeled by filling in the ends with
[-32P]dCTP and Klenow fragment (20 ng of DNA in 20 µl). Labeled DNA was separated from free [
-32P]dCTP
by filtration through ProbeQuant G-50 Micro Columns (Amersham Pharmacia
Biotech). EMSAs were performed by preincubating 1-3 µl of nuclear
extract with 1.5 µg of unspecific competitor poly(dI-dC) in a buffer
containing 10 mM HEPES (pH 7.9), 150 mM KCl, 1 mM dithiothreitol, 0.5 mM MgCl2,
0.1 mM EDTA, 8.5% glycerol for 10 min on ice.
Subsequently, 0.1 ng of labeled double-stranded oligonucleotide was
added to a final volume of 20 µl, and samples were incubated for
another 20 min on ice.
Cell Culture and Transfections-- Ishikawa cells were grown as monolayers in minimum essential medium containing 10% fetal calf serum. The human lung adenocarcinoma cell-line H441 was maintained in RPMI medium containing 4% fetal calf serum. SL2 cells (32) were grown in Schneider medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (insect cell qualified; Life Technologies, Inc.) at 25 °C. All media were supplemented with L-glutamine and antibiotics.
Ishikawa cells were transfected on 60-mm dishes at 50-70% confluence by the DEAE-dextran method as described (27) with 6-8 µg of reporter plasmid, 0.5-3 µg of expression vector and 2 µg of RSV-Luc or RSV-Northern Blot Analysis--
Total RNA from various cell lines
was provided by M. Kalff-Suske. For Northern blots, the RNA was
separated on 0.8% and 1.2% agarose gels containing 2.2 M
formaldehyde and blotted to nylon membranes. Prehybridization was
carried out in 5 × SSC, 5 × Denhardt's solution, 25 mM sodium phosphate, pH 6.4, 0.1% SDS, 250 µg/ml sonicated denaturated herring sperm DNA, 25 µg/ml poly(A), and 50%
formamide at 42 °C for 4 h. For hybridization, 10% dextran sulfate and appropriate 32P-labeled cDNA probes for SP
A, SP B, SP C, uteroglobin/CC10, TTF-1, HNF3, HNF3
, and HFH-4
(specific activity of 1 × 109 cpm/µg) were included
prior to overnight incubation. Control hybridizations of 18 S ribosomal
RNA have been performed as described (37) using a
32P-labeled single-stranded oligonucleotide probe that has
the sequence 5'-ACGGTATCTGATCGTCTTCGAACC-3'.
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RESULTS |
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Identification of Cell Lines Expressing Lung Epithelial Marker Genes-- To identify cell lines that express lung epithelium-specific marker genes, we performed Northern blot analysis of RNA from different human lung cell lines. As a negative control, we used RNA from Ishikawa cells, a cell line derived from an endometrium tumor (Fig. 1). Surfactant protein A and B (SP A and SP B) mRNAs were present exclusively in H441 cells whereas surfactant protein C mRNA was not detectable in any of the cell lines (data not shown). Human uteroglobin/CC10 mRNA was expressed in H441 cells, in addition to H60 and H69 cells (Fig. 1A). This finding was surprising inasmuch as it has been reported previously that H441 cells do not express the Clara cell marker protein CC10 gene (16, 38).
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Activation of the Uteroglobin/CC10 Promoters from Rabbit, Rat, and
Man by HNF3 and HNF3
--
Since H441 cells expressed
uteroglobin/CC10 as well as TTF-1 and HNF3
, we asked whether TTF-1
and members of the HNF3/forkhead family of transcription factors could
activate transcription from the uteroglobin/CC10 promoters of various
species. The promoters of the rat, human, and rabbit uteroglobin/CC10
genes were fused to the CAT gene and cotransfected along with CMV
promoter-driven expression constructs for TTF-1, HNF3
, HNF3
, and
HFH-4 into Ishikawa cells lacking these transcription factors (see Fig.
1B). TTF-1 enhanced transcription from the rat
uteroglobin/CC10 promoter up to 18-fold but had essentially no
influence on the homologous human and rabbit gene promoters (Fig.
2). In contrast, both HNF3
and HNF3
stimulated transcription from all three homologous uteroglobin/CC10 promoters up to 8-fold. Cotransfection of an HFH-4 expression plasmid
did not alter uteroglobin/CC10 promoter activities (Fig. 2). Thus, it
appears that TTF-1 can activate the uteroglobin/CC10 promoter only in
certain species. HNF3
and HNF3
, however, can stimulate
uteroglobin/CC10 promoters from all species tested. These results
suggest that the presence of HNF3
or HNF3
in respiratory epithelial cells is indispensable for the expression of
uteroglobin/CC10 genes in mammals.
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Identification of Rabbit Uteroglobin/CC10 Promoter Elements
Responsible for Activation by HNF3 and HNF3
--
To identify the
promoter elements in the rabbit uteroglobin/CC10 promoter responsible
for the activation by HNF3
and HNF3
, we cotransfected a series of
5'-promoter deletion-CAT constructs (7) along with the expression
constructs for HNF3
and HNF3
in Ishikawa cells (Fig.
3). Promoter deletion mutants with 5' end
points at
395,
220,
205,
177, and
159 were all activated by
HNF3
and HNF3
. Promoter mutants that contain only 96 or 35 nucleotides upstream of the transcription start site, however, were not
stimulated by these two transcription factors. Thus, the DNA elements
that are essential for stimulation by HNF3
and HNF3
lie within
159 bp and
96 bp upstream of the transcription start site.
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HNF3 and Oct-1 Are the Nuclear Factors Binding to the A/T-rich
Elements III and IV of the Uteroglobin/CC10 Gene in H441
Cells--
The experiments described so far were performed with
Ishikawa cells. In similar cotransfection experiments with H441 cells, we could not detect a significant activation of the rabbit
CC10/uteroglobin promoter by HNF3
and HNF3
. This observation is
likely due to the presence of high levels of endogenous HNF3
(Fig.
1; see below). However, transfection of the wild-type promoter and the
linker-scanning mutants LS(
92/
99), LS(
127/
132), and
DLS(
92/
127) revealed that both elements are necessary for full
promoter activity in H441 cells (Fig. 5).
Mutation of either the distal or proximal HNF3-responsive element (in
LS(
127/
132) and LS(
92/
99)) reduced promoter activity 2-3-fold.
Mutation of both elements (in DLS(
92/
127)) diminished promoter
activity at least 5-fold.
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HNF3 and HNF3
on Their Own Are Not Sufficient to Activate the
Uteroglobin/CC10 Promoter--
To further substantiate the conclusion
that HNF3
and HNF3
or Oct-1, but not TTF-1, contribute to the
level of transcription from the rabbit uteroglobin/CC10 promoter in
lung, we performed gene transfer experiments into the Drosophila
melanogaster Schneider cell line (SL2 cells) that lacks many
mammalian transcription factor activities (Fig.
7). For these experiments, luciferase constructs instead of CAT reporter constructs that we generated in the
course of our studies were used. The uteroglobin/CC10 promoter fused to
the luciferase gene was cotransfected along with plasmids expressing
Oct-1, TTF-1, HNF3
, or HNF3
, specifically designed for insect
cells (pPacOct-1, pPacTTF-1, pPacHNF3
, and pPacHNF3
). Cotransfection of pPacOct-1 along with the uteroglobin/CC10-luciferase gene led to a 2-3-fold increase of luciferase activity. As expected, TTF-1 did not activate the uteroglobin/CC10 promoter, confirming the
results of the cotransfection experiments obtained with mammalian cell
lines. Surprisingly, HNF3
as well as HNF3
did also not stimulate
transcription from the uteroglobin promoter (Fig. 7A). Control experiments showed, however, that all proteins were expressed in SL2 cells after transfection and that they specifically bind their
appropriate promoter elements in electrophoretic mobility shift assays
(Fig. 7B). This result suggested that HNF3
or HNF3
on
their own are not sufficient to activate the uteroglobin/CC10 promoter.
Additional transcription factors or coactivators not present in SL2
cells might be necessary for stimulation of the uteroglobin/CC10
promoter by HNF3
and HNF3
.
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HNF3 and HNF3
Strongly Enhance Sp1-mediated Activation of the
Rabbit Uteroglobin/CC10 Promoter--
Previously, we have shown that
Sp1 and Sp3 are also regulators of uteroglobin promoter activity (8).
Thus, we asked next whether HNF3
, HNF3
, or TTF-1 may stimulate
the uteroglobin/CC10 promoter in combination with these two ubiquitous
transcription factors. Sp1 on its own activated the uteroglobin
promoter in a dose-dependent manner (13-fold in Fig. 7).
Under these conditions, Sp3 also stimulated the uteroglobin/promoter to
a certain extent (2.5-fold). TTF-1 had no effect on Sp-mediated
activation, and Oct-1 in combination with Sp1 enhanced Sp1-mediated
activation 2-fold. Most significantly, cotransfection of HNF3
or
HNF3
along with Sp1 or Sp3 strongly increased uteroglobin/CC10
promoter activity (Fig. 7A). Thus, the presence of Sp1 or
Sp3 appears to be essential for uteroglobin/CC10 promoter stimulation
by HNF3 factor members. This conclusion was further substantiated by
experiments in which variable amounts of Sp1 and HNF3
expression
plasmids were transfected in the presence or absence of constant
amounts of HNF3
or Sp1, respectively (Fig.
8). Increasing amounts of transfected Sp1
expression plasmid activated the uteroglobin/CC10 promoter in a
dose-dependent manner (Fig. 8A), whereas HNF3
alone was inactive even at highest DNA levels (Fig. 8B).
HNF3
stimulated Sp1-mediated activation under all conditions.
Highest stimulation was obtained with 50 ng of Sp1-expression plasmid
and 100 ng of HNF3
expression plasmid (up to 12-fold over the value
obtained with Sp1 alone). Similar titration experiments were performed
also with TTF-1 or Oct-1 and Sp1. These experiments revealed that TTF-1
does not at all influence Sp1-mediated activation whereas Oct-1
enhanced Sp1-mediated activation 2-3-fold (data not shown). Thus,
Oct-1 and Sp1 act in an additive manner, whereas HNF3
and Sp1
strongly synergize with each other.
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Both Sp1-binding Sites Are Absolutely Necessary for
HNF3-mediated Activation of the Uteroglobin/CC10 Promoter--
The
uteroglobin/CC10 promoter contains two non-GC box binding sites for Sp1
located 65 and 230 bp upstream of the transcriptional start site (8).
We wanted to know how these sites contribute individually to HNF3
stimulation. Linker-scanning mutants in which either a single
(LS(
64/
72) and LS(
222/
229)) or both Sp1 binding sites
(DLS(
64/
222)) are mutated were cotransfected in the presence or
absence of HNF3
(Fig. 9). This series of experiments revealed that
enhancement of Sp1-mediated activation by HNF3
is dependent on the
integrity of both Sp1-binding sites of the uteroglobin/CC10 promoter.
Mutation of either the distal (LS(
222/
229)) or the proximal binding
site (LS(
64/
72)) reduced synergistic activation by HNF3
and Sp1
close to background levels (Fig. 9). Mutation of both Sp-binding sites
(LS(
64/
222)) abolished activation completely. Thus, it appears that
each Sp1-binding site flanking the HNF3-binding sites contributes
similarly to HNF3
-mediated enhancement of activation. The essential
contribution of the distal Sp1-binding site for HNF3
activation in
addition to the proximal Sp1 site indicates that Sp1 does not act
simply as a bridging factor between HNF3
and the basal transcription
machinery. Additional mechanisms must be at work in the combinatorial
activation of the rabbit uteroglobin/CC10 promoter by HNF3
and Sp1.
It should be noted that this result is compatible with experiments
performed previously with H441 cells (7). In H441 cells, the two
linker-scanning mutants LS(
222/
229) and LS(
64/
72) reduced
promoter activity 10- and 15-fold, respectively.
Multiple Activation Domains of Sp1 Are Required for Cooperativity
with HNF3--
Sp1 contains a zinc finger DNA binding domain and
four activation domains designated A, B, C, and D (28, 29). To identify those activation domains of Sp1 that are essential for cooperativity with HNF3
, we tested various Sp1 deletion derivatives (Fig.
10). All Sp1 mutants were poor
activators of the rabbit/CC10 promoter, indicating that all four
activation domains of Sp1 are involved in activation of the
uteroglobin/CC10 promoter. Surprisingly, the activity of none of the
mutants could be enhanced by HNF3
. This result suggests that several
activation domains of Sp1 including the glutamine-rich activation
domain A and B, the highly charged domain C, and the most C-terminal
activation domain D are involved in transcriptional synergy with
HNF3
.
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DISCUSSION |
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Human Uteroglobin/CC10 Is Expressed in Some Lung Epithelial Cell
Lines--
H441 cells have been widely used to study lung
epithelium-specific promoters from different species. This cell line
was derived from a human adenocarcinoma with morphological
characteristics of Clara cells lining the bronchiolar epithelium. Here,
we show for the first time that this cell line as well as H60 and H69 cells express the Clara cell-specific marker gene uteroglobin/CC10. This finding was surprising because, in two previous reports, human
CC10 could not be detected in H441 cells (24, 38). H441 cells express
also surfactant protein A and B mRNAs. However, it should be noted
that we estimate the expression levels of all three mRNAs in these
cells to be 3 orders of magnitude lower than in vivo. In
addition, H441 cells express the transcription factors TTF-1 and
HNF3, which are involved in regulation of lung epithelium-specific genes (for a review, see Ref. 41). Thus, this cell line reflects in
many aspects an in vivo situation.
Differential Activation of Uteroglobin/CC10 Promoters by TTF-1,
HNF3, and HNF3
--
An essential role of TTF-1 for the
activation of the rat and mouse uteroglobin/CC10 promoter regions,
which contain several binding sites for TTF-1, has been reported (16).
Our experiments confirmed this conclusion since we found a strong
activation of the rat uteroglobin/CC10 promoter by TTF-1. However,
neither the homologous rabbit nor the human uteroglobin/CC10 promoters
were activated by TTF-1 although purified TTF-1 specifically recognized two sites of the rabbit/uteroglobin promoter (around
85 and
1) in
DNase I protection experiments (data not shown). This result suggests
species-specific differences in the role of TTF-1 for uteroglobin/CC10
transcription in lung. However, we cannot exclude that potential TTF-1
binding sites located far upstream of the uteroglobin/CC10 5'-flanking
region could confer activation of the gene by TTF-1. In contrast to
TTF-1, both HNF3
and HNF3
, but not HFH-4 could activate all three
uteroglobin/CC10 promoters we have tested.
HNF3 Synergizes with Members of the Sp Family of Transcription
Factors--
We found that HNF3
and HNF3
on their own cannot
activate transcription from the uteroglobin/CC10 promoter in an insect
cell line that lacks many mammalian transcription factor activities. However, both factors, HNF3
and HNF3
, strongly enhanced
Sp1-mediated activation of the uteroglobin/CC10 promoter. Thus, it
appears that HNF3
and Sp1 act in a synergistic manner.
Interestingly, the surfactant protein B promoter also contains binding
sites for Sp1/Sp3 adjacent to an HNF3 binding site. Moreover, both
sites are necessary for lung specific activation of SP B gene
transcription (15). Thus, the combinatorial action of Sp family
transcription factors and the lung epithelial-specific transcription
factors HNF3
and HNF3
might be a common theme in lung epithelial
gene expression.
How Might HNF3 Cooperate with Sp1 Mechanistically?--
Several
mechanisms underlying the synergistic activation of HNF3
and Sp1
have to be considered. One possibility would be that HNF3
on its own
is transcriptionally inactive on the uteroglobin/CC10 promoter in the
absence, but active in the presence of Sp1. In such a model, Sp1 bound
to its proximal site could function as a bridging factor between
HNF3
and the basal transcriptional machinery. Since HNF3
does
have activation domains (45), a direct interaction of HNF3
and
components of the basal transcription machinery seems possible.
However, such a model would not explain why the distal Sp1 site is also
essential for synergistic activation by HNF3
and Sp1.
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ACKNOWLEDGEMENTS |
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We thank S. Meier for excellent technical assistance. We gratefully acknowledge J. Dennig, M. Kalff-Suske, A. Smid, and J. Klug for critically reading the manuscript. We thank M. Beato for stimulating discussions and providing research facilities.
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FOOTNOTES |
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* This work was supported by grants from the Bundesministerium für Bildung, Wissenschaft, Forschung, und Technologie and the Stiftung P. E. Kempkes.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-6421-286697;
Fax: 49-6421-285398; E-mail: suske{at}imt.uni-marburg.de.
1 The abbreviations used are: bp, base pair(s); LS, linker-scanning mutant; DLS, double linker-scanning mutant; LDL, low density lipoprotein; SREBP, sterol receptor element-binding protein; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; HFH, HNF3/forkhead homolog; RSV, Rous sarcoma virus; SP, surfactant protein.
2 C. Scheidereit, personal communication.
3 H. Braun and G. Suske, unpublished results.
4 R. H. Costa, personal communication.
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REFERENCES |
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