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. 1996 Jun 1;316(Pt 2):467–473. doi: 10.1042/bj3160467

The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene.

R F Toonen 1, S Gowan 1, C D Bingle 1
PMCID: PMC1217373  PMID: 8687389

Abstract

The mechanisms that direct expression of the Clara cell secretory protein (CCSP) gene to the bronchiolar epithelial cells of the lung remain to be elucidated. Previous studies have identified a number of proteins which bind to a functionally important region (Region 1) located -132 to -76 bp from the transcription start site in the rat CCSP gene. Subsequently we have shown that while Region 1 is an important positive regulator of CCSP gene expression, sequences 3' of this region (-75 to +38) are sufficient to confer tissue-specific expression of a reporter gene. In the present study we have used transient transfections with a deletion series of CCSP-CAT reporter plasmids (where CAT is chloramphenicol acetyltransferase) and gel mobility shift assays with a series of overlapping oligonucleotides covering the whole minimal promoter region to study protein-DNA interactions within this region. These studies have identified a conserved functional binding site for the lung and thyroid enriched homeodomain transcription factor TTF-1, located between positions -51 and -42 from the transcription start site. CCSP-CAT chimaeric reporters containing this region are specifically activated by TTF-1 in co-transfection assays, and nuclear extracts from cells which express TTF-1 bind to this region, as does in vitro translated rat TTF-1. Three additional conserved regions were identified, and in further gel mobility shift studies with an oligonucleotide spanning the conserved region immediately 5' to the TTF-1 site we identified a binding site for the ubiquitously expressed zinc-finger-containing proteins Sp1 and Sp3. These studies suggest that cell-type-restricted and ubiquitous nuclear proteins may play a combined role in the regulation of the CCSP gene within the bronchiolar epithelium by interacting with the minimal promoter region.

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Selected References

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  1. Bingle C. D., Gitlin J. D. Identification of hepatocyte nuclear factor-3 binding sites in the Clara cell secretory protein gene. Biochem J. 1993 Oct 1;295(Pt 1):227–232. doi: 10.1042/bj2950227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bingle C. D., Hackett B. P., Moxley M., Longmore W., Gitlin J. D. Role of hepatocyte nuclear factor-3 alpha and hepatocyte nuclear factor-3 beta in Clara cell secretory protein gene expression in the bronchiolar epithelium. Biochem J. 1995 May 15;308(Pt 1):197–202. doi: 10.1042/bj3080197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bohinski R. J., Di Lauro R., Whitsett J. A. The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis. Mol Cell Biol. 1994 Sep;14(9):5671–5681. doi: 10.1128/mcb.14.9.5671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bohinski R. J., Huffman J. A., Whitsett J. A., Lattier D. L. Cis-active elements controlling lung cell-specific expression of human pulmonary surfactant protein B gene. J Biol Chem. 1993 May 25;268(15):11160–11166. [PubMed] [Google Scholar]
  5. Bruno M. A., Bohinski R. J., Carter J. E., Foss K. A., Whitsett J. A. Structure and function of the mouse surfactant protein B gene. Am J Physiol. 1995 Mar;268(3 Pt 1):L381–L389. doi: 10.1152/ajplung.1995.268.3.L381. [DOI] [PubMed] [Google Scholar]
  6. Bruno M. D., Bohinski R. J., Huelsman K. M., Whitsett J. A., Korfhagen T. R. Lung cell-specific expression of the murine surfactant protein A (SP-A) gene is mediated by interactions between the SP-A promoter and thyroid transcription factor-1. J Biol Chem. 1995 Mar 24;270(12):6531–6536. doi: 10.1074/jbc.270.12.6531. [DOI] [PubMed] [Google Scholar]
  7. Damante G., Tell G., Formisano S., Fabbro D., Pellizzari L., Di Lauro R. Effect of salt concentration on TTF-1 HD binding to specific and non-specific DNA sequences. Biochem Biophys Res Commun. 1993 Dec 15;197(2):632–638. doi: 10.1006/bbrc.1993.2526. [DOI] [PubMed] [Google Scholar]
  8. De Felice M., Damante G., Zannini M., Francis-Lang H., Di Lauro R. Redundant domains contribute to the transcriptional activity of the thyroid transcription factor 1. J Biol Chem. 1995 Nov 3;270(44):26649–26656. doi: 10.1074/jbc.270.44.26649. [DOI] [PubMed] [Google Scholar]
  9. Dennig J., Hagen G., Beato M., Suske G. Members of the Sp transcription factor family control transcription from the uteroglobin promoter. J Biol Chem. 1995 May 26;270(21):12737–12744. doi: 10.1074/jbc.270.21.12737. [DOI] [PubMed] [Google Scholar]
  10. Fleming R. E., Gitlin J. D. Structural and functional analysis of the 5'-flanking region of the rat ceruloplasmin gene. J Biol Chem. 1992 Jan 5;267(1):479–486. [PubMed] [Google Scholar]
  11. Gstaiger M., Knoepfel L., Georgiev O., Schaffner W., Hovens C. M. A B-cell coactivator of octamer-binding transcription factors. Nature. 1995 Jan 26;373(6512):360–362. doi: 10.1038/373360a0. [DOI] [PubMed] [Google Scholar]
  12. Guazzi S., Price M., De Felice M., Damante G., Mattei M. G., Di Lauro R. Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J. 1990 Nov;9(11):3631–3639. doi: 10.1002/j.1460-2075.1990.tb07574.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hackett B. P., Gitlin J. D. Cell-specific expression of a Clara cell secretory protein-human growth hormone gene in the bronchiolar epithelium of transgenic mice. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9079–9083. doi: 10.1073/pnas.89.19.9079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hackett B. P., Shimizu N., Gitlin J. D. Clara cell secretory protein gene expression in bronchiolar epithelium. Am J Physiol. 1992 Apr;262(4 Pt 1):L399–L404. doi: 10.1152/ajplung.1992.262.4.L399. [DOI] [PubMed] [Google Scholar]
  15. Hagen G., Müller S., Beato M., Suske G. Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res. 1992 Nov 11;20(21):5519–5525. doi: 10.1093/nar/20.21.5519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hagen G., Müller S., Beato M., Suske G. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J. 1994 Aug 15;13(16):3843–3851. doi: 10.1002/j.1460-2075.1994.tb06695.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hagen G., Wolf M., Katyal S. L., Singh G., Beato M., Suske G. Tissue-specific expression, hormonal regulation and 5'-flanking gene region of the rat Clara cell 10 kDa protein: comparison to rabbit uteroglobin. Nucleic Acids Res. 1990 May 25;18(10):2939–2946. doi: 10.1093/nar/18.10.2939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ikeda K., Clark J. C., Shaw-White J. R., Stahlman M. T., Boutell C. J., Whitsett J. A. Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells. J Biol Chem. 1995 Apr 7;270(14):8108–8114. doi: 10.1074/jbc.270.14.8108. [DOI] [PubMed] [Google Scholar]
  19. Lazzaro D., Price M., de Felice M., Di Lauro R. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development. 1991 Dec;113(4):1093–1104. doi: 10.1242/dev.113.4.1093. [DOI] [PubMed] [Google Scholar]
  20. Margraf L. R., Finegold M. J., Stanley L. A., Major A., Hawkins H. K., DeMayo F. J. Cloning and tissue-specific expression of the cDNA for the mouse Clara cell 10 kD protein: comparison of endogenous expression to rabbit uteroglobin promoter-driven transgene expression. Am J Respir Cell Mol Biol. 1993 Sep;9(3):231–238. doi: 10.1165/ajrcmb/9.3.231. [DOI] [PubMed] [Google Scholar]
  21. Mizuno K., Gonzalez F. J., Kimura S. Thyroid-specific enhancer-binding protein (T/EBP): cDNA cloning, functional characterization, and structural identity with thyroid transcription factor TTF-1. Mol Cell Biol. 1991 Oct;11(10):4927–4933. doi: 10.1128/mcb.11.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nordlund-Möller L., Andersson O., Ahlgren R., Schilling J., Gillner M., Gustafsson J. A., Lund J. Cloning, structure, and expression of a rat binding protein for polychlorinated biphenyls. Homology to the hormonally regulated progesterone-binding protein uteroglobin. J Biol Chem. 1990 Jul 25;265(21):12690–12693. [PubMed] [Google Scholar]
  23. Ray M. K., Magdaleno S. W., Finegold M. J., DeMayo F. J. cis-acting elements involved in the regulation of mouse Clara cell-specific 10-kDa protein gene. In vitro and in vivo analysis. J Biol Chem. 1995 Feb 10;270(6):2689–2694. doi: 10.1074/jbc.270.6.2689. [DOI] [PubMed] [Google Scholar]
  24. Sawaya P. L., Stripp B. R., Whitsett J. A., Luse D. S. The lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families. Mol Cell Biol. 1993 Jul;13(7):3860–3871. doi: 10.1128/mcb.13.7.3860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schreiber E., Matthias P., Müller M. M., Schaffner W. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acids Res. 1989 Aug 11;17(15):6419–6419. doi: 10.1093/nar/17.15.6419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Singh G., Katyal S. L. An immunologic study of the secretory products of rat Clara cells. J Histochem Cytochem. 1984 Jan;32(1):49–54. doi: 10.1177/32.1.6418790. [DOI] [PubMed] [Google Scholar]
  27. Stripp B. R., Sawaya P. L., Luse D. S., Wikenheiser K. A., Wert S. E., Huffman J. A., Lattier D. L., Singh G., Katyal S. L., Whitsett J. A. cis-acting elements that confer lung epithelial cell expression of the CC10 gene. J Biol Chem. 1992 Jul 25;267(21):14703–14712. [PubMed] [Google Scholar]
  28. Strubin M., Newell J. W., Matthias P. OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins. Cell. 1995 Feb 10;80(3):497–506. doi: 10.1016/0092-8674(95)90500-6. [DOI] [PubMed] [Google Scholar]
  29. Suske G., Lorenz W., Klug J., Gazdar A. F., Beato M. Elements of the rabbit uteroglobin promoter mediating its transcription in epithelial cells from the endometrium and lung. Gene Expr. 1992;2(4):339–352. [PMC free article] [PubMed] [Google Scholar]
  30. Wikenheiser K. A., Wert S. E., Wispé J. R., Stahlman M., D'Amore-Bruno M., Singh G., Katyal S. L., Whitsett J. A. Distinct effects of oxygen on surfactant protein B expression in bronchiolar and alveolar epithelium. Am J Physiol. 1992 Jan;262(1 Pt 1):L32–L39. doi: 10.1152/ajplung.1992.262.1.L32. [DOI] [PubMed] [Google Scholar]
  31. Yu C. Y., Motamed K., Chen J., Bailey A. D., Shen C. K. The CACC box upstream of human embryonic epsilon globin gene binds Sp1 and is a functional promoter element in vitro and in vivo. J Biol Chem. 1991 May 15;266(14):8907–8915. [PubMed] [Google Scholar]

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