(Received for publication, May 25, 1996, and in revised form, October 16, 1996)
From the Department of Molecular Biology, The University of Texas Health Science Center at Tyler, Tyler, Texas 75710
Surfactant protein B (SP-B) is essential for
maintenance of biophysical properties and physiological function of
pulmonary surfactant. SP-B mRNA expression is restricted to
alveolar type II epithelial cells and bronchiolar epithelial cells
(Clara cells) of adult lung. We previously (Margana, R. K., and
Boggaram, V. (1996) Am. J. Physiol. 270, L601-L612)
found that a minimal promoter region (236 to +39) of rabbit SP-B gene
is sufficient for high level expression of chloramphenicol
acetyltransferase reporter gene in NCI-H441 cells, a cell line with
characteristics of Clara cells. In the present study we used mutational
analysis, electrophoretic mobility shift assays, and DNase I
footprinting to identify cis-DNA regulatory elements and
trans-acting protein factors required for lung
cell-specific expression of SP-B gene. We found that in addition to
thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor
3
(HNF-3
) binding sites, two spatially separate DNA sequences
that bind Sp1 and Sp3 factors are necessary for the maintenance of SP-B
promoter activity. Mutation of any one of the transcription factor
binding sites caused a significant reduction in SP-B promoter activity
suggesting that Sp1, Sp3, and TTF-1 and HNF-3
interact cooperatively
with SP-B promoter to activate gene transcription.
Surfactant protein B (SP-B),1 a hydrophobic protein of pulmonary surfactant, is essential for maintenance of biophysical properties and physiological function of surfactant (1). The critical role of SP-B in surfactant function is suggested by its deficiency in newborns with congenital alveolar proteinosis (2). Infants diagnosed with alveolar proteinosis die of respiratory failure despite maximal medical assistance. Targeted disruption of SP-B gene causes abnormalities of surfactant metabolism and respiratory failure in newborn mice, further supporting the important role of SP-B in lung function (3). SP-B mRNA is developmentally induced and in adult lung SP-B mRNA is expressed in a highly cell type-specific manner in alveolar type II cells and bronchiolar epithelial (Clara) cells (4, 5). SP-B mRNA is increased by glucocorticoids and agents that increase intracellular cyclic AMP (6-10).
Transcription initiation plays a key role in the control of gene expression during terminal differentiation of cell types. Activation of cell/tissue-specific gene transcription is dependent on interactions between transcription factors (activators and repressors), some of which are expressed widely and others are restricted in their distribution, and the general transcriptional machinery (11, 12). How interactions between various transcription factors result in cell/tissue-specific activation of gene transcription is not yet well understood.
We previously isolated and sequenced rabbit SP-B gene and determined
that a minimal SP-B promoter region spanning 236 to +39 nucleotides
is sufficient for high level expression of CAT reporter gene in a
cell-specific manner in NCI-H441 cells, a human pulmonary
adenocarcinoma cell line with characteristics of Clara cells (13). We
also determined that the SP-B minimal promoter contained a lung
cell/tissue-specific enhancer (13). SP-B promoter activity was enhanced
in HeLa cells by co-expression of thyroid transcription factor 1 (TTF-1), suggesting that it contained sequence element(s) for TTF-1
binding (13). TTF-1 and hepatocyte nuclear factor 3
(HNF-3
) have
been shown recently to play important roles in human SP-B promoter
activity in NCI-H441 cells (14, 15). TTF-1 and HNF-3 are also expressed
in tissues other than lung, and during lung development TTF-1 and HNF-3
are expressed before differentiation of alveolar type II cells and
expression of SP-B mRNA (16, 17). These observations suggest that
TTF-1 and HNF-3 are not sufficient for cell type-specific activation of
SP-B gene transcription and that additional factors might be required
for activation of SP-B gene transcription.
The objective of our investigation was to identify
cis-acting DNA elements and interacting protein factors
necessary for lung cell-specific expression of rabbit SP-B gene. We
used mutational analysis, electrophoretic mobility shift assays, and
DNase I footprinting to identify DNA sequence elements and interacting
protein factors important for the functional activity of SP-B promoter.
We found that in addition to TTF-1 and HNF-3 binding sites, two
spatially separate DNA sequences that bind Sp1 and Sp3 factors play
critical roles in maintaining functional activity of SP-B promoter.
Mutation of any one of these sites resulted in a significant reduction in SP-B promoter activity, suggesting that combined or cooperative interactions of Sp1, Sp3, and TTF-1 and HNF-3 transcription factors with SP-B promoter is necessary for activation of gene transcription. Some of the findings reported in the present study have been presented in preliminary form (18).
Nuclear extracts from NCI-H441
cells and other cells were prepared according to the method described
by Ausubel et al. (19). Typically cells from 10 confluent
75-cm2 flasks were used for preparation of nuclear
extracts. Nuclear extracts were aliquoted into chilled tubes and
rapidly frozen in liquid nitrogen and stored at 80 °C. The protein
concentration of nuclear extract was determined by Bio-Rad protein
assay (20).
DNase I footprinting reactions were
performed as described by Lakin (21) with modifications. The sense and
antisense strands of SP-B fragment 236 to +39 were labeled as
follows: pSKCAT
S (22) containing SP-B fragment from
236 to +39 was
linearized with BamHI or HindIII, and the DNA was
dephosphorylated. Dephosphorylated DNA was digested with
PstI or BamHI to release the SP-B fragment, and
the fragment was end-labeled using [
-32P]ATP and T4
polynucleotide kinase. For footprinting 0.5-1 ng of labeled DNA probe
(2.5-5.0 × 104 cpm) was incubated with H441 or HepG2
nuclear proteins or 1 footprinting unit (50 ng) of purified human
recombinant Sp1 protein (Promega) for 20 min at 30 °C in 20 µl of
binding buffer that contained 13 mM HEPES, pH 7.9, 13%
glycerol, 80 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 67 µg of bovine
serum albumin, and 0.2 µg of poly(dI-dC) as nonspecific competitor.
Following incubation the reaction mixture was treated with 3 µl of
DNase I (Promega) (3 µl of DNase I (1 unit/µl) was diluted to 50 µl in 5 mM CaCl2 and 10 mM
MgCl2) for 30 s at room temperature. DNase I reaction was terminated by addition of buffer containing 8.5 mM
EDTA, 8.5 µg/ml proteinase K, 85 µg/ml tRNA, and 0.07% SDS
followed by incubation at 37 °C for 20 min and then at 68 °C for
2 min. The samples were phenol/CHCl3-extracted, and DNA was
ethanol-precipitated. DNA was dissolved in 10 µl of denaturing
loading solution (90% formamide, 10 mM EDTA, pH 8.0, 0.01% bromphenol blue, and 0.01% xylene cyanol) and analyzed on a 6%
denaturing polyacrylamide sequencing gel.
pSKCATS containing SP-B promoter fragment
236 to
+39 or
730 to +39 served as the template for site-directed
mutagenesis by polymerase chain reaction (PCR) according to the method
described by Nelson and Long (23). Briefly, the method uses four
synthetic oligonucleotide primers. One oligonucleotide contained the
desired mutation(s), and the other three oligonucleotides were designed to allow selective amplification of the mutated sequence by PCR. Oligonucleotides that served as primers (sense) to introduce mutations into the DNA binding sites of transcription factors are shown in Table
I.
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In all reactions the upstream primer (246 sense) was
5
-CTGTTCAGAAGGATCCAGGAACCCAGGCCTG-3
and the downstream
hybrid primer (+29 antisense) was
5
-ATTAACCCTCACTAAAGGGAGCAGCCACGGCTGCAGGTGT. The
downstream primer contained a single mutation at nucleotide +37 that
generated a PstI site in the amplified DNA. The sequences of
BamHI and PstI sites in the oligonucleotide
primers are shown in italics. The 5
20-nucleotide unique sequence of
the downstream hybrid primer is shown in bold.
All reactions (100 µl) were performed in a Perkin-Elmer gene amp PCR
system 2400 using 1-2 fmol of template DNA, 0.3 µM
amounts of primers and AmpliTaq DNA polymerase. Unless stated otherwise the reaction conditions included denaturation at 94 °C for 5 min followed by a 30-cycle amplification that consisted of denaturation at
94 °C for 30 s, annealing at 55 °C for 30 s, and
extension at 72 °C for 30 s. Amplification was followed by a
final extension at 72 °C for 7 min. The method consisted of four
separate reaction steps. In step 1 DNA was amplified using pSKCATS
containing SP-B promoter fragment
730 to +39 as template and
mutagenic and hybrid primers. The amplified DNA was purified by agarose
gel electrophoresis and used as the primer for step 2. The step 2 reaction contained approximately 0.6 pmol of the product of step 1 and
pSKCAT
S containing SP-B promoter fragment
730 to +39 as template.
Amplification was carried out as a single cycle that consisted of
denaturation at 94 °C for 5 min, annealing at 37 °C for 2 min,
and extension at 72 °C for 10 min. In step 3, the upstream sense
primer and the oligonucleotide primer identical to the 5
segment of
the hybrid primer, 5
-ATTAACCCTCACTAAAGGGA-3
, were added, and 30-cycle DNA amplification was performed.
Nucleotides in the HNF-3 sequence motif were replaced by PCR
mutagenesis in two steps; mutant DNA obtained using mutagenic primer
(i) was used as the template with mutagenic primer (ii) to obtain SP-B
promoter fragment in which all of the nucleotides in the HNF-3 binding
site had been mutated. SP-B promoter fragment containing mutations in
the Myc-Max binding motif at +30 was obtained by PCR amplification
using pSKCATS containing SP-B promoter fragment
730 to +39 as the
template and upstream primer (
246, sense), 5
-CTGTTCAGAAGGATCCAGGAACCCAGGCCTG-3
and downstream primer
(+50, antisense),
5
-CTTGCCTGCAGCCACGGCGG
GACTTGGCCGT-3
. The DNA binding site is underlined, and the mutated nucleotides are
shown in bold italics. BamHI and PstI sites are
shown in italics. In all cases the final amplified DNA was
phenol/CHCl3 extracted and digested with BamHI
and PstI. The digested DNA was purified by agarose gel
electrophoresis and inserted upstream of CAT gene in pSKCAT
S. The
sequence of insert DNA was determined to verify that it contained the
desired mutations.
Double-stranded
synthetic oligonucleotides were end-labeled using
[-32P]ATP and T4 polynucleotide kinase.
Electrophoretic mobility shift assays were performed by incubating 0.5 or 1.0 ng (104 to 2 × 104 cpm) of the
oligonucleotide probe (Table II) with 5 µg of nuclear protein in 20 µl of binding buffer (13 mM HEPES, pH 7.9, 13% glycerol, 80 mM KCl, 5 mM
MgCl2, 1 mM dithiothreitol, 1 mM
EDTA, 67 µg of bovine serum albumin and 0.2 µg of poly(dI-dC) as
nonspecific competitor) for 20 min at 30 °C. In some experiments
nuclear proteins were preincubated in binding buffer prior to addition
of the labeled probe. Competition experiments were performed by
addition of indicated molar excess of cold wild type or mutant
oligonucleotides prior to addition of the labeled probe. In case of
antibody supershift assays, nuclear extracts were preincubated with
preimmune IgG or polyclonal antibodies to transcription factors for
1 h or overnight at 4 °C prior to incubation with
oligonucleotide probe. Polyclonal antibodies to human Sp1, Sp2, Sp3,
and Sp4 were purchased from Santa Cruz Biotechnology. Polyclonal
antibodies to forkhead domains of mouse HNF-3
, HNF-3
, and
HNF-3
were kindly supplied by Drs. Gunther Schutz and Wolfgang
Schmid, German Cancer Research Center, Heidelberg, Germany. Polyclonal
antisera to the N-terminal portion of rat T/EBP (TTF-1) was kindly
supplied by Dr. Shioko Kimura, National Cancer Institute. After
incubation the DNA-protein complexes were resolved by electrophoresis
on a 4% nondenaturing polyacrylamide gel containing 0.5 × TBE
(0.045 M Tris borate and 0.001 M EDTA) using
0.5 × TBE as running buffer. Electrophoresis was performed at
constant current (30-35 mA) for 1.5-2 h. Gels were vaccum-dried and
exposed to autoradiographic film.
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Cell lines were maintained in culture medium supplemented with 10% fetal bovine serum, penicillin (100 units), streptomycin (100 µg), and amphotericin B (0.25 µg) at 37 °C in a humidified atmosphere of 5% CO2 and air. NCI-H441 (24), a human pulmonary adenocarcinoma cell line with characteristics of Clara cells which expresses SP-B endogenously, was maintained in RPMI 1640 medium. HeLa, a human cervical cancer cell line, was maintained in Dulbecco's modified Eagle's medium.
Plasmid DNAs were transiently transfected into cells by
liposome-mediated DNA transfer using LipofectAMINE (Life Technologies, Inc.) as described previously (13). At least two independent preparations of plasmids were used for transfection. pCH110 (Pharmacia Biotech Inc.), a -galactosidase expression vector served as an internal control for normalization of transfection efficiency.
CAT activity of cell
extracts was determined by the liquid scintillation counting assay (25)
using [14C]chloramphenicol and n-butyryl
coenzyme as described previously (13). -Galactosidase activity was
determined by chemiluminescent assay using Galacto-Light Plus (Tropix,
Bedford, MA) substrate according to the recommended protocol. CAT
activities were normalized for transfection efficiency based on
-galactosidase activity.
Proximal promoter regions of rabbit (236
to +39) (13) and human (
218 to +41) (26) SP-B genes display high
degree of sequence conservation and support high level expression of
CAT reporter gene in NCI-H441 cells (13, 27). These observations suggest that SP-B proximal promoter regions contain binding sites for
transcription factors necessary for expression in NCI-H441 cells. DNA
sequence alignment of rabbit and human SP-B promoter regions (13)
showed that rabbit SP-B promoter contains sequence motifs that are
nearly identical to TTF-1 and HNF-3 motifs identified in human SP-B
promoter. As in the case of human SP-B promoter (14), the TTF-1 binding
site in rabbit SP-B promoter has limited identity to the consensus
binding sequence for TTF-1 (GNNCACTCAAG) (28). The TTF-1 binding site
consisted of two closely juxtaposed elements with the sequence CTGGAG
and CTCCAG that resemble the 3
segment of consensus TTF-1 binding
site. The putative HNF-3 binding site is characterized by the sequence,
5
-TGTTTG-3
, that occurs in the regulatory elements of a wide variety
of liver-specific genes (29).
We searched rabbit SP-B proximal promoter (236 to +39) for the
presence of other putative transcription factor binding sites using a
transcription factor data base (MacDNASIS 3.0) and by comparison
with the consensus binding sequences for vertebrate-encoded transcription factors (30). Results showed that the SP-B promoter, besides containing TTF-1 and HNF-3 sites, also contained putative binding sites for Sp1, ETS, and Myc-Max transcription factors (Fig.
1).
DNase I Footprinting Reveals Multiple Interactions between NCI-H441 Nuclear Proteins and SP-B Promoter
SP-B proximal promoter
contained putative binding sites for several transcription factors. To
map out regions of SP-B promoter that bind transcription factors, we
analyzed the interactions of nuclear proteins present in NCI-H441 cells
with SP-B promoter by DNase I footprinting assay. In DNA samples
complexed with NCI-H441 nuclear proteins, several protected regions
were observed (Fig. 2), suggesting that multiple
proteins present in nuclear extracts of NCI-H441 cells interact with
SP-B promoter. The protected regions of SP-B promoter included binding
sites for Sp1, ETS, TTF-1, HNF-3, and Myc-Max transcription factors
(Fig. 1).
Sp1, TTF-1, and HNF-3 Factors Are Critical for SP-B Promoter Activity
We investigated the functional importance of putative
binding sites for Sp1 (207,
130,
35), ETS (
161,
51), TTF-1
(
112,
102), HNF-3 (
88) and Myc-Max (
115, +30) transcription
factors by mutational analysis (Fig. 3).
Sp1 binding sites were mutated by GG to TT substitution in the 5
portion of the binding site. Mutation of these nucleotides drastically
reduces binding of Sp1 to its binding site (31). Results showed that
mutation of Sp1 binding site at
207 did not significantly alter SP-B
promoter activity, whereas mutation of binding site at
130 or
35
caused 80% or greater reduction in SP-B promoter activity. TTF-1
binding sites were mutated by substituting nucleotides in DNA elements
that closely resemble the 3
portion of consensus TTF-1 binding
sequence. Mutation of TTF-1 sites at
112 and
102 caused
approximately 70% reduction in SP-B promoter activity and 60%
reduction in trans-activation of promoter by TTF-1 in HeLa cells.
Mutation of the HNF-3 binding sequence by substituting all six
nucleotides resulted in nearly 70% reduction in SP-B promoter
activity.
SP-B promoter contains several purine-rich sequence elements that are
similar to the sequence 5-GGAA/T-3
that is known to bind the Ets
family of transcription factors (32). Electrophoretic mobility shift
assays with SP-B promoter oligonucleotides showed that among various
Ets binding sites, the ones located at
162 and
51 displayed strong
binding to NCI-H441 nuclear proteins. We therefore analyzed the
functional importance of Ets binding sites at
162 and
51 in SP-B
promoter activity by mutational analysis. Mutation of the ETS binding
site at
162 did not alter SP-B promoter activity, whereas mutation of
the ETS site at
51 caused approximately 30% increase in promoter
activity. Mutation of both the ETS sites caused approximately 30%
increase in SP-B promoter activity. Two sequence elements that closely
resemble the consensus binding motif for Myc-Max transcription factors (33) are located at
115 and +30. Mutation of the Myc-Max binding site
at
115 resulted in approximately 40% reduction in SP-B promoter activity, whereas mutation of the site at +30 had no significant effect
on promoter activity.
To verify if Sp1, TTF-1, HNF-3, and ETS factors are
components of the binding activity in nuclear extracts, we analyzed
binding of factors present in nuclear extracts by electrophoretic
mobility shift assays. Results showed that SP-B promoter
oligonucleotide that contained Sp1 binding site at 207 did not form
any complex, indicating absence of interaction of nuclear proteins
(data not shown). However, SP-B promoter oligonucleotides that included binding sites for Sp1 at
130 or
35 formed two complexes that were
competed by excess amounts of unlabeled wild type oligonucleotide and
by an oligonucleotide containing a consensus Sp1 binding site (Figs.
4A and 5A).
However, an oligonucleotide that contained a mutant Sp1 site failed to
prevent formation of complex, suggesting the identity of binding
factors as Sp1 or Sp1-like proteins (Fig. 4A).
Electrophoretic mobility shift assays in the presence of monospecific
antibodies to different members of the Sp1 family of proteins revealed
the identity of proteins binding to Sp1 sites as Sp1 and Sp3 (Figs.
4B and 5B). Results also showed that the lower
mobility complex (complex I) was completely supershifted by Sp3
antibody, indicating that complex I results from binding of Sp3. We
further confirmed binding of Sp1 to SP-B promoter by DNase I
footprinting assays with purified recombinant Sp1. Results demonstrated
that purified Sp1 binds to Sp1 binding sites at
130 and
35 in SP-B
promoter (Fig. 6).
In electrophoretic mobility shift assays, the SP-B promoter
oligonucleotide containing the TTF-1 binding site formed two major complexes (Fig. 7). Formation of these complexes was
significantly reduced in the presence of excess wild type
oligonucleotide but not in the presence of excess mutant
oligonucleotide (Fig. 7). In the presence of monospecific antibodies to
TTF-1 the higher mobility complex (complex II) was specifically
supershifted, indicating the identity of the protein species present in
complex II as TTF-1 or TTF-1-related protein (Fig. 7). The lower
mobility complex (complex I) was not supershifted in the presence of
antibodies to TTF-1, indicating that complex I arises from interaction
with protein(s) unrelated to TTF-1.
Electrophoretic mobility shift analysis with the SP-B promoter
oligonucleotide containing the HNF-3 binding site demonstrated formation of two complexes (Fig. 8). Formation of these
complexes was significantly reduced in the presence of excess wild type oligonucleotide. In the presence of excess mutant oligonucleotide formation of complex I was not reduced but formation of complex II was
reduced, suggesting that complex I likely results from interaction with
HNF-3 (Fig. 8). In the presence of monospecific antibodies to different
members of the HNF-3 family of transcription factors, formation of a
supershifted complex was observed only in the presence of antibodies to
HNF-3 (Fig. 8). Furthermore in the presence of HNF-3
antibodies
complex I was specifically supershifted, indicating that complex I
arises from interactions with HNF-3
or a related protein. The
identity of proteins present in complex II remains to be
determined.
Electrophoretic mobility shift assays with SP-B promoter oligonucleotides containing binding sites for the ETS family of transcription factors showed formation of three distinct complexes that were competed by excess wild type oligonucleotide but not by a mutant oligonucleotide (data not shown). The complexes were not recognized by polyclonal ETS antibody (Santa Cruz Biotechnology) capable of cross-reacting with ETS family proteins.
SP-B mRNA is induced during fetal lung development, and in
adult lung its expression is restricted to alveolar epithelial (type
II) cells and bronchiolar epithelial (Clara) cells (4, 5). Molecular
mechanisms that mediate cell type-specific activation of SP-B gene
transcription are not well understood. Recent studies have shown that
TTF-1 and HNF-3/forkhead proteins play important roles in the
functional activity of human SP-B promoter (14, 15). During lung
development HNF-3 and TTF-1 proteins are expressed at the onset of
lung morphogenesis (16, 17) at which time expression of SP-B mRNA
is not detected, and in fully developed lung the expression of HNF3
,
TTF-1, and SP-B co-localize to cells of distal epithelium. These data
suggest that TTF-1 and HNF-3
are not sufficient for cell-specific
activation of SP-B promoter and that additional factors are required
for activation of the promoter.
DNA footprinting analysis showed that multiple proteins present in
nuclear extracts of NCI-H441 cells bind to the SP-B promoter and that
protected regions contain binding sites for Sp1, ETS, Myc-Max, TTF-1,
and HNF-3 transcription factors. TTF-1 and HNF-3 binding sites are
nearly identical in rabbit, human, and mouse SP-B promoters and so is
their placement relative to the TATAA element (13). Human SP-B promoter
is transactivated by HNF-3/HFH-8 proteins in HepG2 cells (15), and
human (14) and rabbit (13) SP-B promoters are transactivated by TTF-1
in HeLa cells, further supporting functional roles for TTF-1 and HNF-3
binding sites in rabbit SP-B promoter. We further assessed the
functional roles of TTF-1 and HNF-3 binding sites in SP-B promoter
activity by mutational analysis. Mutation of TTF-1 and HNF-3 binding
sites significantly reduced SP-B promoter activity as did activation of
mutant SP-B promoter by TTF-1, demonstrating the importance of these
sites in the functional activity of SP-B promoter. Electrophoretic mobility shift analysis demonstrated that TTF-1 and HNF-3 binding sites, in addition to binding TTF-1 and HNF-3
, also bind other nuclear proteins whose identities remain to be determined.
Putative Sp1 binding sites are located at 207,
130, and
35 in
SP-B proximal promoter. We determined the functional importance of
these elements in SP-B promoter activity by mutational analysis. Mutation of the site at
207 had no significant effect on SP-B promoter activity, but mutations of the site at
130 or
35 caused a
significant reduction in promoter activity. These data demonstrated that Sp1 elements at
130 and
35 play equally important roles in the
functional activity of SP-B promoter. Gel mobility shift and DNase I
footprinting experiments indicated the identities of proteins
interacting with these sites as Sp1 and Sp3.
The occurrence and role of Sp1 elements in the promoter functions of
human and mouse SP-B genes have not been investigated. Examination of
human (26) and mouse (34) SP-B proximal promoter sequences reveals that
the human and mouse SP-B promoters contain putative Sp1 binding sites
at 36 and
42. The putative Sp1 binding sequences in human and mouse
promoters, 5
-GCCCGCCCA-3
and 5
-TCCAGCCCC-3
, display a high degree
of similarity to Sp1 element at
35 in rabbit SP-B promoter. The high
degree of conservation of sequence as well as similar placements of Sp1
binding sites in rabbit, human, and mouse SP-B promoters undescores the
importance of Sp1 binding element in the functional activity of SP-B
promoter. Sp1 binding site at
130 in rabbit SP-B promoter appears to
be unique to rabbit SP-B gene, since a similar sequence element could
not be found in human and mouse SP-B promoters.
Sp1 binds to GC boxes present in promoters of a wide variety of genes and modulates promoter activity. To date three Sp1-related proteins, Sp2, Sp3, and Sp4, have been described (35-37). Similar to Sp1, all three Sp1-related proteins are expressed widely and contain zinc finger structures and glutamine- and serine/threonine-rich amino acid stretches. The DNA binding domains of Sp1, Sp3, and Sp4 proteins are highly conserved and display similar binding affinity to GC boxes. Of the different members of Sp1 family proteins, Sp3 (37, 38) was found to act as a potent repressor of basal and Tat-mediated activation of the human immunodeficiency virus promoter. Although Sp1 is expressed ubiquitously, several lines of evidence suggest a regulatory role for Sp1. The recent identification of several Sp1-related proteins (35-37) with similar binding affinities, the differential expression of Sp1 in various cell types, as well as developmental regulation of Sp1 (39) support a regulatory role for Sp1. In the lung notable expression of Sp1 was detected in alveolar epithelial cells (39), and Sp1 mRNA levels increased during postnatal development of mice.
Our data suggest that besides TTF-1 and HNF-3, Sp1 and Sp3 serve as
important regulators of SP-B gene expression. The critical role of Sp1
and Sp3 binding sites in the functional activity of SP-B promoter
suggests that cell type-specific and developmental induction of SP-B
gene expression is dependent on expression of Sp1 and Sp3 proteins/or
activity. Sp1 is modified by phosphorylation, and modification by
phosphorylation modulates binding of Sp1 to its target sites (40). The
role of cell type-specific and developmental control of Sp1 and Sp3
expression, and the putative role of phosphorylation of Sp1 in the
regulation of SP-B gene expression in fetal lung, remain to be
investigated. Since Sp3 has the potential to function as a
transcriptional repressor, developmental and cell type-specific regulation of SP-B gene expression might be maintained by a dynamic positive and negative regulation exerted by Sp1 and Sp3. The putative role of Sp1-related transcription factors in the control of other lung-specific genes, particularly other surfactant protein genes, is
not known. The recent identification of binding sites for Sp1 and Sp3
proteins in the minimal promoter of rat Clara cell-specific protein
(41) suggests that Sp1-related transcription factors might serve as
important regulators of lung-specific gene expression.
Recent studies have suggested that ETS proteins can interact with other
transcription factors to modulate promoter activity (32). Studies have
also suggested that ETS proteins play important roles in the control of
growth and differentiation (32). Specifically ETS 1 expression
increases during fetal development, and high levels of expression are
found in fetal lung (42). The role of ETS 1 in control of lung growth
and differentiation is not known. Rabbit SP-B promoter contains a
number of putative binding sites for ETS proteins. Results of
mutational analysis of ETS binding sites showed that the element at
51 can function as a weak suppressor of SP-B promoter activity.
Proteins that bound to ETS sites were not recognized by an antibody
capable of cross-reacting with members of ETS family transcription
factors, suggesting that the proteins are either unrelated to ETS
proteins or that they represent new members of the ETS family of
transcription factors. Further investigations are needed to define the
role of the ETS binding site at
51 and of other putative ETS sites in
the control of SP-B promoter activity.
SP-B promoter contained putative Myc-Max binding sites at 115 and
+30. Mutation of the site at +30 did not alter SP-B promoter activity,
but mutation of site at
115 reduced SP-B promoter activity by nearly
40%. The site at
115 overlaps with TTF-1 binding site; whether
reduction in SP-B promoter activity as a result of mutation in the
Myc-Max element is due to interference with binding of TTF-1 remains to
be investigated.
TTF-1 activates surfactant protein (SP)-A (43), SP-B, SP-C, as well as Clara cell-specific protein (14) promoters, suggesting that it plays an important role in the control of lung-specific gene expression. TTF-1 has also been shown to be a key regulator of thyroid-specific gene expression (44). Our studies of the control of SP-B promoter activity has demonstrated that significant differences exist between TTF-1-regulated control of gene expression in thyroid and lung. Whereas lung-specific expression of SP-B is controlled by combined interactions of multiple transcription factors with the promoter, thyroid-specific expression of thyroglobulin and thyroid peroxidase genes is dependent on mutually exclusive interactions of TTF-1 and Pax-8 factors (45).
Functional analysis of 5-flanking regions has shown that human (27,
46) and rabbit (13) SP-B proximal promoter regions comprising
nucleotides
218 to +436 and
236 to +39 are sufficient for high
level expression of the CAT reporter gene in NCI-H441 cells, but
further deletion of 5
DNA to
130 nucleotides significantly reduces
CAT expression. Functionally important transcription factor binding
sites thus far identified in human and rabbit SP-B promoter regions,
namely TTF-1, HNF-3
, and Sp1 and Sp3 sites, are located within
130
nucleotides, suggesting that factors binding to upstream sequences are
necessary for activation of the promoter.
In summary our studies have identified Sp1 and Sp3 transcription
factors as important regulators of SP-B promoter activity and that
combined or cooperative effects of Sp1, Sp3, and TTF-1 and HNF-3
proteins on SP-B promoter is required for activation of promoter.
Further characterization of regulatory DNA elements and interacting
proteins of SP-B promoter region
236 to
136 will aid in
understanding mechanisms that mediate lung cell-specific activation of
SP-B gene transcription.
We thank Dr. Shioko Kimura, National Cancer Institute, for providing pCMV4-T/EBP-1 and antibodies to rat T/EBP and Drs. Gunther Schutz and Wolfgang Schmid German Cancer Research Center, Heidelberg, Germany for providing antibodies to HNF-3 proteins.