Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene

Ramgopal Margana, Kiflu Berhane, M. Nurul Alam, and Vijayakumar Boggaram

Department of Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas 75708-3154


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted manner in alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. In NCI-H441 cells, a lung cell line with characteristics of Clara cells, a minimal promoter region comprising -236 to +39 nucleotides supports high-level expression of chloramphenicol acetyltransferase reporter activity. In the present investigation, we characterized the upstream promoter region, -236 to -140 nucleotides, that is essential for promoter activity. Deletion mapping identified two segments, -236 to -170 and -170 to -140 nucleotides, that are important for promoter activity. Mutational analysis and gel mobility shift experiments identified thyroid transcription factor-1, Sp1, and Sp3 as important trans-acting factors that bind to sequences in the upstream promoter region. Our data suggest that SP-B promoter activity is dependent on interactions between factors bound to upstream and downstream regions of the promoter.

lung; gene regulation; respiratory distress syndrome; transcription factors; surfactant protein B; thyroid transcription factor-1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SURFACTANT, A LIPOPROTEIN COMPLEX synthesized and secreted by alveolar type II epithelial cells, maintains the integrity of the alveoli during respiration by reducing surface tension at the alveolar air-tissue interface (14). Surfactant also plays important roles in host defense against certain bacteria and viruses through its actions to enhance phagocytosis by alveolar macrophages (32). Deficiency of surfactant is associated with newborn respiratory distress syndrome (2), the leading cause of neonatal morbidity and mortality in developed countries. Reduced levels and/or activity of surfactant may contribute to respiratory failure in adult respiratory distress syndrome (16). Surfactant protein (SP) B, a 9-kDa hydrophobic protein, is essential for the maintenance of biophysical properties and physiological function of surfactant. A number of studies have shown that SP-B promotes the adsorption and spreading of phospholipids (25) and stabilizes the phospholipid monolayer formed on the alveolar surface (10). Deficiency of SP-B due to a frame shift mutation in the coding region is associated with fatal respiratory failure in infants with congenital alveolar proteinosis (22). Targeted disruption of SP-B gene causes respiratory failure in newborn mice, further supporting the important role of SP-B in lung function (8).

Margana and Boggaram (19) previously found that a promoter region containing the -236 to +39 nucleotides of the rabbit SP-B gene is necessary and sufficient for high-level expression of chloramphenicol acetyltransferase (CAT) reporter gene in NCI-H441 cells, a human pulmonary adenocarcinoma cell line with characteristics of bronchiolar epithelial cells (Clara cells). Further deletion of the 5' region to the -140 nucleotide resulted in ~80% reduction in promoter activity (19), suggesting that the -236 to -140 region is required for full expression of SP-B promoter activity. The minimal promoter, -236 to +39 nucleotides, supported high-level CAT expression in a lung cell type-specific manner in cell cultures, suggesting that it contained a cell- and/or tissue-specific enhancer (19). A study by Margana and Boggaram (20) also identified binding sites for Sp1, Sp3, thyroid transcription factor (T TF)-1, and hepatocyte nuclear factor (HNF)-3 transcription factors within the -140 nucleotide of the minimal SP-B promoter that appeared to function in a cooperative or combinatorial manner to maintain SP-B promoter activity. These data suggested that the upstream promoter region, -236 to -140 nucleotides, contains functionally important cis-acting DNA elements for which the identity remains to be determined.

The objective of our investigation was to identify and characterize functionally important cis-acting DNA elements in the upstream region (-236 to -140 nucleotides) of the SP-B minimal promoter. Deletion mapping of the upstream promoter region identified two distinct regions, -236 to -170 and -170 to -140 nucleotides, that are important for SP-B promoter activity. Mutational analysis and gel mobility shift analysis of NCI-H441 nuclear proteins interacting with the putative transcription factor binding sites identified functionally important binding sites for TTF-1, Sp1, and Sp3 factors in the SP-B upstream promoter region.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nuclear extract preparation. Nuclear extracts from cells were prepared according to the method described by Schreiber et al. (28). Typically, cells from a confluent 75-cm2 flask were used for preparation of the extract. The buffer composition of the final nuclear extract was 20 mM HEPES, pH 7.9, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothrietol, 1 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml each of leupeptin and aprotinin, and 0.5 mg/ml of benzamidine. Nuclear extracts were divided into aliquots, which were placed into chilled tubes, rapidly frozen in liquid nitrogen, and stored at -80°C. The protein concentration of nuclear extract was determined by the method of Bradford (5) with Bio-Rad protein assay reagent.

Plasmid constructions and site-directed mutagenesis. SP-B fragments -218 to +39 and -170 to +39 were amplified by PCR with oligonucleotide sense primers 5'-CCCAGGC<UNL>CTG<B>CA</B>G</UNL> GA- GTCGG-3' and 5'-ACCAGCC<UNL><B>G</B>AA<B>T</B>TC</UNL>AGGGGAG-3' that correspond to -225 to -206 and -176 to -157 nucleotide sequences within SP-B promoter region and an antisense primer, 5'-GCAGCCACGG<UNL>C<B>T</B>GCAG</UNL>GTGTGACTTGGCCGTGACG-3', that corresponds to +14 to +48 sequence of SP-B coding region. The primers contained mutations (in boldface) that generated Pst I and EcoR I sites (underlined) for cloning purposes. The amplified fragments were ligated upstream of CAT gene in the vector pSKCATDelta S (1). pSKCATDelta S containing the SP-B promoter fragment -236 to +39 nucleotides served as the template for site-directed mutagenesis by PCR according to the method of Nelson and Long (21) as described (20). Oligonucleotides that served as primers (sense) to introduce mutations into the transcription factor binding sites in the SP-B promoter are shown in Table 1. The mutated DNA fragments were inserted upstream of the CAT gene in pSKCATDelta S. The presence of introduced mutations in the insert DNA was established by DNA sequencing.

                              
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Table 1.   SP-B promoter oligonucleotides used as primers in PCR-based mutagenesis

Electrophoretic mobility shift assays. Synthetic oligonucleotides were annealed by heating 10 µM sense and antisense oligonucleotides in 10 mM Tris · HCl 7.5, 10 mM MgCl2, and 50 mM NaCl at 95°C for 5 min and then allowing them to cool to room temperature over a period of 1 h. The concentration of the double-stranded oligonucleotides was determined by measuring absorbance at 260 nm (50 µg/ml = 1.0 A260 unit). Double-stranded oligonucleotides were end labeled with [gamma -32P]ATP and T4 polynucleotide kinase. Electrophoretic mobility shift assays (EMSAs) were performed by incubating 0.5-1.0 ng (3-10 × 104 counts/min) of the oligonucleotide probe (Table 2) 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 dithiothrietol, 1 mM EDTA, and 0.5 or 1 µg of poly(dI-dC) as nonspecific competitor] for 20 min at 30°C. Nuclear proteins were preincubated for 20 min at 30°C in binding buffer before addition of the labeled probe. Competition experiments were performed by addition of indicated molar excess of cold wild-type or mutant oligonucleotides along with addition of the labeled probe. In antibody supershift assays, nuclear extracts were preincubated with 1.0 µg of preimmune IgG or polyclonal antibodies to transcription factors for 1 h or overnight at 4°C before incubation with the oligonucleotide probe. Four microliters of 6× loading buffer [0.25% bromphenol blue, 0.25% xylene cyanol, and 15% Ficoll (type 400) in H2O] were added to the incubation mixtures before they were loaded onto a 6% nondenaturing polyacrylamide gel containing 0.5× Tris-borate-EDTA. Electrophoresis was performed at constant current (30-35 mA) for 1.5-2 h with 0.5× Tris-borate-EDTA as running buffer. Gels were vacuum-dried and exposed to autoradiographic film. Polyclonal antibodies to human Sp1, Sp2, Sp3, and Sp4 and consensus and mutant Sp1 oligonucleotides were purchased from Santa Cruz Biotechnology. Polyclonal antisera to the NH2-terminal portion of rat thyroid-specific enhancer-binding protein (TTF-1) was kindly supplied by Dr. Shioko Kimura (National Cancer Institute, Bethesda, MD) and polyclonal antibodies to nuclear factor (NF)-1 (alpha -CTF1) were kindly supplied by Dr. Naoko Tanese (New York University Medical Center, New York, NY).

                              
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Table 2.   WT and MT double-stranded SP-B promoter oligonucleotides used in gel mobility shift assays

Cell culture and transfections. NCI-H441, a human pulmonary adenocarcinoma cell line with characteristics of Clara cells that expresses SP-B endogenously (23), was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C in a humidified atmosphere of 5% CO2 and air. Cells were purchased from American Type Culture Collection (Manassas, VA).

Plasmids were amplified in Escherichia coli Top10F' strain and purified by anion-exchange chromatography (QIAGEN, Valencia, CA), according to the manufacturer's protocol. The quality of the plasmid DNA was analyzed by agarose gel electrophoresis and ethidium bromide staining. Plasmid DNA preparations routinely contained supercoiled DNA as the major DNA species. DNA concentration was determined by measuring absorbance at 260 nm. At least two independent preparations of plasmids were used for transfection. pCDNA3.1 (Invitrogen, Carlsbad, CA), a beta -galactosidase expression vector, served as an internal control for normalization of transfection efficiency. Plasmid DNAs were transiently transfected into cells by liposome-mediated DNA transfer with LipofectAMINE (GIBCO BRL, Life Technologies, Gaithersburg, MD) as described previously (19). SP-B-CAT DNA (4.0 µg) and pCDNA3.1 (0.5 µg) were used to transfect cells plated on a 60-mm dish. Generally, CAT activity was determined after 48 h of incubation after DNA transfection.

CAT and beta -galactosidase assays. CAT activity of cell extracts was determined by the liquid scintillation counting assay (30) with [14C]chloramphenicol and n-butyryl coenzyme A as described previously (19). beta -Galactosidase activity was determined by chemiluminescent assay with Galacto-Light Plus (TROPIX, Bedford, MA) substrate according to the recommended protocol. Cell extracts were heat treated at 60°C for 10 min or 48°C for 50 min to inactivate endogenous acetylase and beta -galactosidase activities before incubation with substrates. CAT activities of cell extracts were normalized to cotransfected beta -galactosidase activity to correct for variations in transfection efficiency.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Deletion analysis of SP-B minimal promoter. A previous study by Margana and Boggaram (19) showed that deletion of the 5' region of SP-B minimal promoter from -236 to -140 nucleotides resulted in >80% loss of promoter activity. This suggested that the promoter region extending from -236 to -140 nucleotides is required for full expression of SP-B promoter activity and may contain functionally important cis-DNA elements. To further map sequences in the upstream promoter region that are important for promoter activity, we generated SP-B-CAT promoter constructs that contained deletions at the 5'-end and determined promoter activity after transient transfection into NCI-H441 cells. Results (Fig. 1) showed that deletion of the 5' region of the minimal promoter to -170 and -140 nucleotides caused significant reduction in SP-B promoter activity, indicating that the upstream promoter regions, -236 to -170 and -170 to -140 nucleotides, contained important cis-DNA elements. To further assess the importance of these regions in SP-B promoter activity, we analyzed the enhancer activities of segments of SP-B minimal promoter (Fig. 2). SP-B fragments lacking the TATA sequence, -236 to -27, -236 to -140, and -140 to -27 nucleotides, were generated by PCR and ligated upstream of the SV40 promoter in the vector pSKSVCAT (1), and CAT expression was determined after transient transfection into NCI-H441 cells. Results showed that SP-B promoter fragments -236 to -140 and -140 to -27 increased CAT expression twofold compared with CAT expression by SV40 promoter alone (Fig. 2). The enhancing activity of the -236 to -27 nucleotide region (~4-fold) was equivalent to the combined activities of the -236 to -140 and -140 to -27 nucleotide regions (Fig. 2). These data further suggested that the -236 to -140 and -140 to -27 nucleotide regions of SP-B minimal promoter contributed toward enhancer activity and that contributions of both regions are required for expression of SP-B promoter function. Consistent with the results of the deletion mapping studies, the -236 to -140 nucleotide region displayed significant enhancing activity.


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Fig. 1.   Deletion analysis of minimal surfactant protein (SP) B promoter. The 5' region of SP-B minimal promoter (-236 to +39 nucleotides) was deleted by PCR, and deleted fragments were inserted upstream of chloramphenicol acetyltransferase (CAT) gene in vector pSKCATDelta S. Plasmids carrying promoter deletions were transiently transfected into NCI-H441 cells, and after 48 h of incubation, CAT activity of cell extracts was determined. CAT activities were normalized to cotransfected beta -galactosidase activity to correct for variations in transfection efficiencies. Data are means ± SE of 4 independent experiments. * Significantly different from -236 nucleotide promoter construct: P < 0.05 vs. -218 construct; P < 0.01 vs. -170 and -135 constructs. Two-tailed P values were derived from 1-sample t-test.



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Fig. 2.   Enhancer activity of SP-B promoter fragments. SP-B promoter fragments lacking TATA sequence were generated by PCR and inserted upstream of SV40 promoter in plasmid pSKSVCAT. CAT activity of enhancer fragments was determined by transient transfection into NCI-H441 cells. CAT activities were normalized to cotransfected beta -galactosidase activity to correct for variations in transfection efficiencies. Data are means ± SE of 4 independent experiments. * Significantly different from pSVCAT, 2-tailed P < 0.01 by 1-sample t-test.

Mutational analysis of SP-B upstream promoter region. Deletion mapping experiments and analysis of enhancing activity showed that the SP-B upstream promoter region, -236 to -140 nucleotides, is essential for promoter function. We searched the upstream promoter region for the presence of putative transcription factor binding sites using a transcription factor database (MacDNASIS, Hitachi Software, San Francisco, CA) and by comparison with the consensus binding sequences for the vertebrate-encoded transcription factors (12). SP-B upstream promoter region contained putative binding sites for Ets, activator protein-2, Sp1, NF-1 and TTF-1 transcription factors (Table 3). We investigated the functional importance of the transcription factor binding sites by mutational analysis (Table 1). Nucleotides in the binding sites were altered by substitution mutagenesis, and the effect of mutations on CAT expression was determined after transient transfection. Sp1 sites were mutated by GG to TT substitution in the 5' portion of the binding site. Mutation of these nucleotides has been shown to drastically reduce the binding of Sp1 to its binding site (15). TTF-1 binding site was mutated by substituting nucleotides in the 3' portion of the binding sequence. A previous study by Margana and Boggaram (20) showed that alteration of nucleotides in the 3' portion of the TTF-1 binding sequences at -112 and -102 nucleotides of SP-B promoter significantly reduced binding of TTF-1, leading to reduction of promoter activity. Ets binding site was mutated by GAA to CAG substitution. Similar mutations caused a significant reduction in the promoter activity and binding of Ets immunoreactive protein to Ets response element in the human HER2 gene (29). In all cases, mutations of binding sites caused significant reduction in binding of transcription factors as evaluated by EMSA. Results (Fig. 3) showed that mutations of Ets (-229 and -192), activator protein-2 (-225), NF-1 (-181), and Sp1 (-155) nucleotide sites did not have any significant effect on SP-B promoter activity, whereas mutations of TTF-1 (-187), Sp1 (-162), or Ets (-149) nucleotides caused an ~50% decrease in promoter activity.

                              
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Table 3.   Putative cis-DNA elements in SP-B upstream promoter



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Fig. 3.   Mutational analysis of upstream region of minimal SP-B promoter. Putative transcription factor binding sites (1-8) were altered by mutagenesis (left) as described in MATERIALS AND METHODS. CAT activity of promoter plasmids containing mutations was determined by transient transfection into NCI-H441 cells (right). CAT activities were normalized to cotransfected beta -galactosidase activity to correct for variations in transfection efficiencies. CAT activities of mutated promoter constructs are expressed relative to activity of wild-type (WT) SP-B promoter. X, mutations in transcription factor binding sites in SP-B promoter map; arrows, transcription start site; AP-2, activator protein-2; TTF-1, thyroid transcription factor-1; NF-1, nuclear factor-1. Top, map of minimal SP-B promoter. Nos., nucleotide positions in promoter. Data are means ± SE of 3 or more independent experiments.

Identification of transcription factors binding to cis-DNA elements in the upstream promoter region. Mutational analysis of putative transcription factor binding sites in the upstream promoter region identified that TTF-1 (-187), Sp1 (-162), and Ets (-149) binding sites serve important roles in SP-B promoter function. We performed EMSA using NCI-H441 nuclear extracts to identify proteins binding to the cis-DNA elements. EMSA with SP-B promoter oligonucleotide containing the TTF-1 binding site (-187) showed the formation of two protein-DNA complexes (Fig. 4). Formation of these complexes was abolished in the presence of molar excess of wild-type but not of mutant oligonucleotide. Furthermore, excess SP-B promoter oligonucleotide containing TTF-1 binding sites at -112 and -102 nucleotides but not mutant oligonucleotide abolished formation of the complex. These data indicated that the formation of a protein-DNA complex is the result of binding of TTF-1. Incubation of nuclear extracts with a polyclonal antibody to TTF-1 resulted in a further retardation of the DNA-protein complex, demonstrating the identity of the binding protein as TTF-1.


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Fig. 4.   Electrophoretic mobility shift assay (EMSA) of NCI-H441 nuclear proteins binding to TTF-1 site at -187 site in SP-B promoter. Binding of nuclear proteins was analyzed as described in MATERIALS AND METHODS by incubating 32P-labeled double-stranded SP-B promoter oligonucleotide (-194 to -179 nucleotides) with 5 µg of NCI-H441 nuclear proteins in presence of 200-fold molar excess of unlabeled WT or mutant (Mt) oligonucleotides as competitors (lanes 3-6), polyclonal antibodies to TTF-1 (lanes 7 and 9), or nonimmune IgG (lane 8). Sequences of oligonucleotides are in Table 2. Lane 1, free oligonucleotide probe; lane 2, probe incubated with nuclear proteins; lane 3, WT TTF-1 oligonucleotide (-194 to -179); lane 4, WT TTF-1 oligonucleotide (-118 to -90); lane 5, mutant TTF-1 oligonucleotide (-194 to -179); lane 6, mutant TTF-1 oligonucleotide (-118 to -90). TTF-1 oligonucleotide corresponding to nucleotides -118 to -90 of SP-B promoter contains TTF-1 sites at -112 and -102. Sequences of WT and Mt TTF-1 oligonucleotides (-118 to -90) have been described (19). Arrows, mobilities of protein-DNA and antibody-protein-DNA complexes; -, absence; +, presence. EMSA gels represent 1 of 3 independent experiments.

EMSA using a SP-B promoter oligonucleotide containing the Sp1 site (-162) and H441 nuclear extract showed the formation of two protein-DNA complexes. The formation of these complexes was abolished in the presence of excess wild-type but not of mutant oligonucleotide. Similarly, excess Sp1 consensus oligonucleotide but not mutant oligonucleotide inhibited the formation of the complex. To identify proteins binding to the Sp1 element, the effects of antibodies to various members of the Sp1 family of proteins on the mobility of the protein-DNA complex were investigated. Nuclear proteins were reacted with antibodies before incubation with the labeled oligonucleotide, and the protein-DNA complexes were analyzed by EMSA. Results showed that Sp1 and Sp3 antibodies reacted with protein-DNA complexes to further retard their mobility (Fig. 5), whereas Sp2 and Sp4 antibodies had no effect on the mobility of the protein-DNA complex (data not shown). Specifically, complex 2 was abolished by the Sp3 antibody, resulting in the formation of a supershifted complex. These data suggested that a major component of complex 2 must be Sp3. These results demonstrated the identity of proteins binding to the Sp1 DNA element as Sp1 and Sp3 proteins. In a previous study, Margana and Boggaram (20) found that the protein-DNA complexes formed by Sp1 elements in the SP-B promoter were supershifted by Sp1 and Sp3 antibodies but not by Sp2 and Sp4 antibodies.


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Fig. 5.   EMSA of NCI-H441 nuclear proteins binding to Sp1 site at -162 in SP-B promoter. Binding of nuclear proteins was analyzed as described in MATERIALS AND METHODS by incubating 32P-labeled double-stranded SP-B promoter oligonucleotide (-171 to -151) with 5 µg of NCI-H441 nuclear proteins in presence of 200-fold molar excess of unlabeled WT or Mt oligonucleotides as competitors (lanes 3-6) or polyclonal antibodies of Sp1 (lanes 7 and 9), Sp3 (lane 10), or nonimmune IgG (lane 8). Sequences of oligonucleotides are shown in Table 2. Lane 1, free oligonucleotide probe; lane 2, probe incubated with nuclear proteins; lane 3, WT Sp1 oligonucleotide (-171 to -151); lane 4, Sp1 consensus oligonucleotide; lane 5, mutant Sp1 oligonucleotide (-171 to -151); lane 6, mutant Sp1 oligonucleotide. Arrows, mobilities of protein-DNA and antibody-protein-DNA complexes. EMSA gels represent 1 to 3 independent experiments.

EMSA using the SP-B promoter oligonucleotide containing the Ets site (-149) produced a minor band that was not supershifted by Ets antibodies (data not shown). The Ets site is overlapped by a sequence that has similarities to NF-kappa B binding element, and the DNA binding activity of the NF-kappa B element was strongly induced by treatment with tumor necrosis factor-alpha (25 ng/ml) (data not shown). The identity of the protein(s) present in untreated cells that interacts with the Ets/NF-kappa B site remains to be investigated.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SP-B mRNA is expressed in a highly cell-restricted manner by alveolar type II and bronchiolar (Clara) epithelial cells of the lung (24, 34) and is developmentally induced (18). Human (4, 33) and rabbit (19) SP-B promoter fragments containing the -218 to +41 and -236 to +39 nucleotides support high-level expression of CAT activity in NCI-H441 cells. SP-B promoter serves as a target for TTF-1 and HNF-3/forkhead transcription factors that bind and activate SP-B promoter function (3, 9, 20). Margana and Boggaram (19) previously found, by deletion mapping, that a region of rabbit SP-B gene comprising the -236 to +39 nucleotides is necessary and sufficient for high-level promoter activity in NCI-H441 cells. Deletion of the 5' region of the rabbit SP-B minimal promoter to -140 nucleotides reduced promoter activity by nearly 80% (19). A previous study by Margana and Boggaram (20) showed that Sp1, Sp3, TTF-1, and HNF-3 transcription factors bind to sequence motifs within the -140 nucleotide to activate the promoter. These data suggested that the upstream region of the rabbit SP-B promoter, -236 to -140 nucleotides, is essential for promoter function and contains functionally important cis-DNA elements.

Identification of cis-DNA elements and interacting transcription factors important for SP-B promoter activity is essential for the understanding of mechanisms underlying cell type-specific and developmental regulation of SP-B gene transcription. In the present study, we mapped the SP-B upstream promoter region for the identification of functionally important regions and identified cis-DNA elements and interacting transcription factors important for SP-B promoter activity. Deletion of 5'-flanking DNA of SP-B minimal promoter to the -170 and -140 nucleotides reduced promoter activity by 40 and 80%, respectively, indicating that the -236 to -170 and -170 to -140 nucleotide regions are essential for promoter activity. A previous study (19) showed that a region derived from the SP-B minimal promoter, -236 to -39 nucleotides, increased CAT expression from SV40 minimal promoter in a lung cell-specific manner in cell cultures. We further examined SP-B sequences -236 to -140 and -140 to -27 for enhancing activity. Both regions increased CAT expression from SV40 minimal promoter by a similar magnitude, indicating that they contained enhancing activities. These data indicated that the upstream and proximal regions of the promoter are required for the full expression of the activity of the SP-B enhancer.

The SP-B upstream promoter region, -236 to -140 nucleotides, is essential for promoter activity and contains significant enhancing activity, indicating that it contains functionally important cis-DNA elements. We analyzed the functional importance of putative cis-DNA elements by mutational analysis. cis-DNA elements were mutated to prevent binding of transcription factors, and the effects of the mutations on the promoter activity were determined. Mutational analysis of the SP-B upstream promoter region revealed the presence of three functionally important DNA elements with similarities to TTF-1, Sp1, and Ets/NF-kappa B binding motifs. Mutation of TTF-1 (-187 to -182 nucleotides), Sp1 (-162 to -154 nucleotides), or Ets/NF-kappa B (-149 to -146 nucleotides) elements reduced promoter activity by ~50%. Margana and Boggaram's previous study (20) demonstrated that sequence elements with similarities to Sp1 and Ets motifs at -207 and -162 nucleotides did not have functional roles in SP-B promoter activity. The results of mutational analysis are in agreement with the deletion analysis of the SP-B upstream region. Deletion of 5' region of SP-B promoter to the -170 nucleotide removes the TTF-1 binding sequence and consequently reduces promoter activity by ~50%. Similarly, further deletion to the -140 nucleotide removes Sp1 and Ets/NF-kappa B sequences and further decreases promoter activity by ~50%.

EMSA using TTF-1 antibodies resulted in further retardation of the protein-DNA complex identifying TTF-1 as the major protein factor binding to the TTF-1 element at the -187 nucleotide. In a previous study, Margana and Boggaram (20) identified that TTF-1 binding sites at -112 and -102 nucleotides serve important roles in SP-B promoter function. TTF-1, a homeodomain-containing transcription factor, plays important roles in the regulation of thyroid-specific gene expression (13). Apart from its expression in the thyroid and certain regions of the fetal brain, TTF-1 is also expressed in the lung. TTF-1 is selectively expressed in alveolar type II cells and subsets of bronchiolar epithelial cells in the lung and functions as a transcriptional activator of SP-A (6), SP-B, and SP-C and Clara cell secretory protein (CCSP) genes (3).

Of the two putative Sp1 elements, the element at the -162 nucleotide was found to be important for promoter activity and binding Sp1 and Sp3 transcription factors. Margana and Boggaram (20) found previously that Sp1 and Sp3 proteins bind to functional Sp1 elements at -130 and -35 nucleotides in the SP-B promoter. Because all members of the Sp family of transcription factors display overlapping binding specificity and affinities, it is likely that lack of binding of Sp2 and Sp4 proteins to the Sp1 element is due to the absence of the proteins in NCI-H441 cells rather than to their inability to recognize the binding sequence. The Sp1 element (-162 site) contained an overlapping sequence with similarities to the Ets binding motif. The previous study (20) showed that mutation of the Ets motif at the -162 site had no effect on promoter activity, and proteins that bound to the Ets element were not recognized by Ets antibodies.

The identity of proteins binding to the putative Ets element (-149 nucleotide) is not clear. The Ets element overlapped with a NF-kappa B binding site and reacted weakly with nuclear proteins from untreated cells. The protein-DNA complex was not recognized by Ets antibodies. It remains to be investigated whether the Rel family of proteins that bind to the Ets/NF-kappa B element in nuclear extracts from tumor necrosis factor-alpha -treated cells plays an important role in SP-B promoter function.

Results of the present study and a previous study by Margana and Boggaram (20) have shown that the SP-B minimal promoter contains functional binding sites for Sp1, Sp3, TTF-1, and HNF-3 transcription factors (Fig. 6). The previous study (20) also indicated that combinatorial or cooperative interactions between transcription factors are necessary for promoter function. During fetal lung development, TTF-1 and HNF-3 are expressed before differentiation of alveolar type II cells and expression of SP-B mRNA (36), suggesting that additional factors may be required for activation of SP-B gene transcription. Our data indicate that Sp1 and Sp3 serve as additional factors essential for the activation of SP-B gene expression. The present and a previous study (20) have demonstrated the importance of Sp1 and Sp3 transcription factors in the function of SP-B promoter. Sp1 and Sp3, which belong to a family of transcription factors that recognize GC boxes and contain zinc finger motifs, also serve as important transcriptional activators for other genes that are expressed in the respiratory epithelium, such as 10-kDa Clara cell secretory protein (31), uteroglobin (11), ClC-2 chloride channel (7), and the alveolar type I cell gene T1alpha (26).


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Fig. 6.   Schematic representation of minimal SP-B promoter (-236 to +39 nucleotides) showing positions of TATA element and binding sites (nos. at bottom) for Sp1/Sp3, TTF-1, and hepatocyte nuclear factor (HNF)-3 factors. Functional characterization of Sp1 (-130 and -35), TTF-1 (-112 and -102), and HNF-3 (-88) binding sites has been described previously (19).

TTF-1 and Sp1 serve as targets for phosphorylation by cAMP-dependent protein kinase and could potentially mediate the actions of hormones and other regulatory agents on SP gene expression in the developing lung. cAMP-dependent protein kinase A phosphorylation of TTF-1 has been implicated in the activation of SP-B (35) and SP-A (17) gene expression in lung epithelial cells. Sp1 has been shown to undergo phosphorylation by cAMP-dependent protein kinase A in HL60 leukemia cells (27). Whether phosphorylation of Sp1 by cAMP or other hormone-activated protein kinases controls SP-B gene expression in pulmonary epithelial cells is not known.

Our studies have identified multiple binding sites for TTF-1, Sp1, and Sp3 transcription factors in the SP-B promoter (Fig. 6). The clustered organization of the transcription factor binding sites in the promoter and their close proximity to the TATA element may promote cooperative interactions among the factors and facilitate stabilization of the transcriptional complex that may be required for activation of SP-B gene transcription. Our studies have also revealed that the SP-B promoter is a target for multiple transcription factors, some of which are expressed more selectively (such as TTF-1 and HNF-3) than others (such as Sp1 and Sp3). Interactions between multiple transcription factors and the general transcriptional complex on the SP-B promoter could give rise to the formation a stereospecific transcription complex that is essential for cell-type specific activation of SP-B gene transcription.


    ACKNOWLEDGEMENTS

This research was supported by National Heart, Lung, and Blood Institute Grant HL-48048.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: V. Boggaram, Dept. of Molecular Biology, The Univ. of Texas Health Science Center at Tyler, 11937, US Highway 271, Tyler, TX 75708-3154 (E-mail: vijay{at}uthct.edu).

Received 18 August 1999; accepted in final form 5 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alam, J., N. Yu, S. Irias, J. L. Cooj, and E. Vig. Reduced chloramphenicol acetyltransferase activity observed with vectors containing an upstream SphI recognition sequence. Biotechniques 10: 423-425, 1991.

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