Functional Analysis of Surfactant Protein B (SP-B) Promoter
1997; Elsevier BV; Volume: 272; Issue: 5 Linguagem: Inglês
10.1074/jbc.272.5.3083
ISSN1083-351X
AutoresRamgopal K. Margana, Vijayakumar Boggaram,
Tópico(s)Epigenetics and DNA Methylation
ResumoSurfactant 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 3ä (HNF-3ä) 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-3ä interact cooperatively with SP-B promoter to activate gene transcription. 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 3ä (HNF-3ä) 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-3ä interact cooperatively with SP-B promoter to activate gene transcription. INTRODUCTIONSurfactant protein B (SP-B), 1The abbreviations used are: SPsurfactant proteinCATchloramphenicol acetyltransferaseTTF-1thyroid transcription factor 1HNF-3hepatocyte nuclear factor-3PCRpolymerase chain reaction. a hydrophobic protein of pulmonary surfactant, is essential for maintenance of biophysical properties and physiological function of surfactant (1Weaver T.E. Whittsett J.A. Biochem. J. 1991; 273: 249-264Crossref PubMed Scopus (360) Google Scholar). The critical role of SP-B in surfactant function is suggested by its deficiency in newborns with congenital alveolar proteinosis (2Nogee L.M. Demello D.E. Dehner L.P. Colten H.R. N. Engl. J. Med. 1993; 328: 406-410Crossref PubMed Scopus (530) Google Scholar). 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 (3Clark J.C. Wert S.E. Bachurski C.J. Stahlman M.T. Stripp B.R. Weaver T.E. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7794-7798Crossref PubMed Scopus (554) Google Scholar). 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 (4Phelps D.S. Floros J. Am. Rev. Respir. Dis. 1988; 137: 939-942Crossref PubMed Scopus (130) Google Scholar, 5Wohlford-Lenane C.L. Snyder J.M. Am. J. Respir. Cell. Mol. Biol. 1992; 7: 335-343Crossref PubMed Scopus (62) Google Scholar). SP-B mRNA is increased by glucocorticoids and agents that increase intracellular cyclic AMP (6Whitsett J.A. Weaver T.E. Clark J.C. Sawtell N. Glasser S.W. Korfhagen T.R. Hull W.M. J. Biol. Chem. 1987; 262: 15618-15623Abstract Full Text PDF PubMed Google Scholar, 7Liley H.G. White R.T. Warr R.G. Benson B.J. Hawgood S. Ballard P.L. J. Clin. Invest. 1989; 83: 1191-1197Crossref PubMed Scopus (143) Google Scholar, 8Floros J. Gross I. Nichols K.V. Veletza S.V. Dynia D. Lu H. Wilson C.M. Peterec S. Am. J. Respir. Cell Mol. Biol. 1991; 4: 449-454Crossref PubMed Scopus (32) Google Scholar, 9Xu J. Possmayer F. Biochim. Biophys. Acta. 1993; 1169: 146-155Crossref PubMed Scopus (7) Google Scholar, 10Margana R.K. Boggaram V. Am. J. Physiol. 1995; 268PubMed Google Scholar).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 (11Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2184) Google Scholar, 12Goodrich J.A. Cutler G. Tjian R. Cell. 1996; 84: 825-830Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). 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 (13Margana R.K. Boggaram V. Am. J. Physiol. 1996; 270: 601-612PubMed Google Scholar). We also determined that the SP-B minimal promoter contained a lung cell/tissue-specific enhancer (13Margana R.K. Boggaram V. Am. J. Physiol. 1996; 270: 601-612PubMed Google Scholar). 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 (13Margana R.K. Boggaram V. Am. J. Physiol. 1996; 270: 601-612PubMed Google Scholar). TTF-1 and hepatocyte nuclear factor 3ä (HNF-3ä) have been shown recently to play important roles in human SP-B promoter activity in NCI-H441 cells (14Bohinski R.J. Di Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar, 15Clevidence D.E. Overdier D.G. Peterson R.S. Porcella A. Ye H. Eric Paulson K. Costa R.H. Dev. Biol. 1994; 166: 195-209Crossref PubMed Scopus (116) Google Scholar). 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 (16Lazzaro D. Price M. De Felice M. Di Lauro R. Development (Camb.). 1991; 113: 1093-1104Crossref PubMed Google Scholar, 17Monaghan A.P. Kaestner K.H. Grau E. Schutz G. Development (Camb.). 1993; 119: 567-578Crossref PubMed Google Scholar). 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-3ä 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 (18Boggaram V. Margana R.K. FASEB J. 1996; 10Google Scholar).DISCUSSIONSP-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 (4Phelps D.S. Floros J. Am. Rev. Respir. Dis. 1988; 137: 939-942Crossref PubMed Scopus (130) Google Scholar, 5Wohlford-Lenane C.L. Snyder J.M. Am. J. Respir. Cell. Mol. Biol. 1992; 7: 335-343Crossref PubMed Scopus (62) Google Scholar). 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 (14Bohinski R.J. Di Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar, 15Clevidence D.E. Overdier D.G. Peterson R.S. Porcella A. Ye H. Eric Paulson K. Costa R.H. Dev. Biol. 1994; 166: 195-209Crossref PubMed Scopus (116) Google Scholar). During lung development HNF-3ä and TTF-1 proteins are expressed at the onset of lung morphogenesis (16Lazzaro D. Price M. De Felice M. Di Lauro R. Development (Camb.). 1991; 113: 1093-1104Crossref PubMed Google Scholar, 17Monaghan A.P. Kaestner K.H. Grau E. Schutz G. Development (Camb.). 1993; 119: 567-578Crossref PubMed Google Scholar) at which time expression of SP-B mRNA is not detected, and in fully developed lung the expression of HNF3ä, TTF-1, and SP-B co-localize to cells of distal epithelium. These data suggest that TTF-1 and HNF-3ä 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 (13Margana R.K. Boggaram V. Am. J. Physiol. 1996; 270: 601-612PubMed Google Scholar). Human SP-B promoter is transactivated by HNF-3ä/HFH-8 proteins in HepG2 cells (15Clevidence D.E. Overdier D.G. Peterson R.S. Porcella A. Ye H. Eric Paulson K. Costa R.H. Dev. Biol. 1994; 166: 195-209Crossref PubMed Scopus (116) Google Scholar), and human (14Bohinski R.J. Di Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar) and rabbit (13Margana R.K. Boggaram V. Am. J. Physiol. 1996; 270: 601-612PubMed Google Scholar) 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-3ä, 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 (26Pilot-Matias T.J. Kister S.E. Lawrence Fox J. Kropp K. Glasser S.W. Whitsett J.A. DNA (N. Y.). 1989; 8: 75-86Crossref PubMed Scopus (110) Google Scholar) and mouse (34Bruno M.A. Bohinski R.J. Carter J.C. Foss K.A. Whitsett J.A. Am. J. Physiol. 1995; 268: 381-389PubMed Google Scholar) 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 (35Kingsley C. Winoto A. Mol. Cell. Biol. 1992; 12: 4251-4261Crossref PubMed Scopus (487) Google Scholar, 36Hagen G. Muller S. Beato M. Suske G. Nucleic Acids Res. 1992; 20: 5519-5525Crossref PubMed Scopus (522) Google Scholar, 37Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22: 4914-4921Crossref PubMed Scopus (202) Google Scholar). 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 (37Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22: 4914-4921Crossref PubMed Scopus (202) Google Scholar, 38Hagen G. Denning J. Preiß A. Beato M. Suske G. J. Biol. Chem. 1995; 270: 24989-24994Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) 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 (35Kingsley C. Winoto A. Mol. Cell. Biol. 1992; 12: 4251-4261Crossref PubMed Scopus (487) Google Scholar, 36Hagen G. Muller S. Beato M. Suske G. Nucleic Acids Res. 1992; 20: 5519-5525Crossref PubMed Scopus (522) Google Scholar, 37Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22: 4914-4921Crossref PubMed Scopus (202) Google Scholar) with similar binding affinities, the differential expression of Sp1 in various cell types, as well as developmental regulation of Sp1 (39Saffer J.D. Jackson S.P. Annarella M.B. Mol. Cell. Biol. 1991; 11: 2189-2199Crossref PubMed Scopus (483) Google Scholar) support a regulatory role for Sp1. In the lung notable expression of Sp1 was detected in alveolar epithelial cells (39Saffer J.D. Jackson S.P. Annarella M.B. Mol. Cell. Biol. 1991; 11: 2189-2199Crossref PubMed Scopus (483) Google Scholar), and Sp1 mRNA levels increased during postnatal development of mice.Our data suggest that besides TTF-1 and HNF-3ä, 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 (40Leggett R.W. Armstrong S.A. Barry D. Mueller C.R. J. Biol. Chem. 1995; 270: 25879-25884Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). 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 (41Toonen R.F.G. Gowan S. Bingle C.D. Biochem. J. 1996; 316: 467-473Crossref PubMed Scopus (60) Google Scholar) 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 (32Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar). Studies have also suggested that ETS proteins play important roles in the control of growth and differentiation (32Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar). Specifically ETS 1 expression increases during fetal development, and high levels of expression are found in fetal lung (42Kola I. Brookes S. Green A.R. Garber R. Tymms M. Papas T.S. Seth A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7588-7592Crossref PubMed Scopus (217) Google Scholar). 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 (43Bruno M.D. Bohinski R.J. Huelsman K.M. Whitsett J.A. Korfhagen T.R. J. Biol. Chem. 1995; 270: 6531-6536Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), SP-B, SP-C, as well as Clara cell-specific protein (14Bohinski R.J. Di Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar) 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 (44Francis-Lang H. Price M. Polycarpou-Schwarz M. Di Lauro R. Mol. Cell. Biol. 1992; 12: 576-588Crossref PubMed Scopus (208) Google Scholar). 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 (45Zannini M. Francis-lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (271) Google Scholar).Functional analysis of 5′-flanking regions has shown that human (27Bohinski R.J. Huffman J.A. Whitsett J.A. Lattier D.L. J. Biol. Chem. 1993; 268: 11160-11166Abstract Full Text PDF PubMed Google Scholar, 46Venkatesh V.C. Planer B.C. Schwartz M. Vanderbilt J.N. Tyler White R. Ballard P.L. Am. J. Physiol. 1995; 268: 674-682PubMed Google Scholar) and rabbit (13Margana R.K. Boggaram V. Am. J. Physiol. 1996; 270: 601-612PubMed Google Scholar) 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-3ä, 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-3ä 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. INTRODUCTIONSurfactant protein B (SP-B), 1The abbreviations used are: SPsurfactant proteinCATchloramphenicol acetyltransferaseTTF-1thyroid transcription factor 1HNF-3hepatocyte nuclear factor-3PCRpolymerase chain reaction. a hydrophobic protein of pulmonary surfactant, is essential for maintenance of biophysical properties and physiological function of surfactant (1Weaver T.E. Whittsett J.A. Biochem. J. 1991; 273: 249-264Crossref PubMed Scopus (360) Google Scholar). The critical role of SP-B in surfactant function is suggested by its deficiency in newborns with congenital alveolar proteinosis (2Nogee L.M. Demello D.E. Dehner L.P. Colten H.R. N. Engl. J. Med. 1993; 328: 406-410Crossref PubMed Scopus (530) Google Scholar). 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 (3Clark J.C. Wert S.E. Bachurski C.J. Stahlman M.T. Stripp B.R. Weaver T.E. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7794-7798Crossref PubMed Scopus (554) Google Scholar). 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 (4Phelps D.S. Floros J. Am. Rev. Respir. Dis. 1988; 137: 939-942Crossref PubMed Scopus (130) Google Scholar, 5Wohlford-Lenane C.L. Snyder J.M. Am. J. Respir. Cell. Mol. Biol. 1992; 7: 335-343Crossref PubMed Scopus (62) Google Scholar). SP-B mRNA is increased by glucocorticoids and agents that increase intracellular cyclic AMP (6Whitsett J.A. Weaver T.E. Clark J.C. Sawtell N. Glasser S.W. Korfhagen T.R. Hull W.M. J. Biol. Chem. 1987; 262: 15618-15623Abstract Full Text PDF PubMed Google Scholar, 7Liley H.G. White R.T. Warr R.G. Benson B.J. Hawgood S. Ballard P.L. J. Clin. Invest. 1989; 83: 1191-1197Crossref PubMed Scopus (143) Google Scholar, 8Floros J. Gross I. Nichols K.V. Veletza S.V. Dynia D. Lu H. Wilson C.M. Peterec S. Am. J. Respir. Cell Mol. Biol. 1991; 4: 449-454Crossref PubMed Scopus (32) Google Scholar, 9Xu J. Possmayer F. Biochim. Biophys. Acta. 1993; 1169: 146-155Crossref PubMed Scopus (7) Google Scholar, 10Margana R.K. Boggaram V. Am. J. Physiol. 1995; 268PubMed Google Scholar).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 (11Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2184) Google Scholar, 12Goodrich J.A. Cutler G. Tjian R. Cell. 1996; 84: 825-830Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). 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 (13Margana R.K. Boggaram V. Am. J. Physiol. 1996; 270: 601-612PubMed Google Scholar). We also determined that the SP-B minimal promoter contained a lung cell/tissue-specific enhancer (13Margana R.K. Boggaram V. Am. J. Physiol. 1996; 270: 601-612PubMed Google Scholar). 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 (13Margana R.K. Boggaram V. Am. J. Physiol. 1996; 270: 601-612PubMed Google Scholar). TTF-1 and hepatocyte nuclear factor 3ä (HNF-3ä) have been shown recently to play important roles in human SP-B promoter activity in NCI-H441 cells (14Bohinski R.J. Di Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar, 15Clevidence D.E. Overdier D.G. Peterson R.S. Porcella A. Ye H. Eric Paulson K. Costa R.H. Dev. Biol. 1994; 166: 195-209Crossref PubMed Scopus (116) Google Scholar). 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 (16Lazzaro D. Price M. De Felice M. Di Lauro R. Development (Camb.). 1991; 113: 1093-1104Crossref PubMed Google Scholar, 17Monaghan A.P. Kaestner K.H. Grau E. Schutz G. Development (Camb.). 1993; 119: 567-578Crossref PubMed Google Scholar). 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-3ä 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 (18Boggaram V. Margana R.K. FASEB J. 1996; 10Google Scholar).
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