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Volume 270, Number 12, Issue of March 24, 1995 pp. 6531-6536
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (*)

(Received for publication, December 2, 1994; and in revised form, January 13, 1995)

Michael D. Bruno ,  Robert J. Bohinski ,  Karen M. Huelsman ,  Jeffrey A. Whitsett ,  Thomas R. Korfhagen (§)

From the Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039

ABSTRACT

Cis-acting elements determining lung epithelial cell-selective transcription of the murine surfactant protein A (SP-A) gene were identified between nucleotide positions -255 and -57. This region of the murine SP-A gene contained nucleotide sequences consistent with thyroid transcription factor-1 (TTF-1) binding motifs. An SP-A-CAT plasmid containing the TTF-1 binding sites was transcriptionally active in mouse lung epithelial (MLE-15) cells but not in HeLa, 3T3, or H441 cells. However, transcription of the SP-A-CAT construct was activated after cotransfection of HeLa cells with a vector expressing recombinant TTF-1, pCMV-TTF-1. Recombinant TTF-1 homeodomain protein bound to four distinct binding sites located between nucleotides -231 to -168. Proteins in nuclear extracts of MLE-15 cells bound TTF-1 binding sites and were supershifted by TTF-1 antibody. Mutations of three of the TTF-1 binding sites in this region reduced expression of the SP-A-CAT construct in transfected MLE-15 cells and reduced transactivation in HeLa cells. TTF-1 interacts with complex protein/DNA binding sites located in the 5`-flanking region of the murine SP-A gene enhancing lung epithelial cell-specific expression in vitro.

FOOTNOTES

*
This work was supported by Grant HL28623, Center for Gene Therapy Grant HL51832, and the Cystic Fibrosis Foundation. Portions of this work were presented at the American Thoracic Society Meeting, May, 1994, Boston, MA and the Eighth Annual Cystic Fibrosis Conference, October, 1994, Orlando, FL. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Children's Hospital Medical Center, Division of Pulmonary Biology, TCHRF, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-559-8920; Fax: 513-559-7868.

(^1)
The abbreviations used are: SP-A, surfactant protein A; TTF-1, thyroid transcription factor-1; CAT, chloramphenicol acetyltransferase; HNF, hepatic nuclear factor; EMSA, electrophoretic mobility shift assay.

(^2)
Ikeda, K., Clark, J. C., Stahlman, M. T., Boutell, C. J., Whitsett, J. A.(1995) J. Biol. Chem., in press.

ACKNOWLEDGEMENTS

We thank Dr. Roberto Di Lauro for the anti-TTF-1 antibody and TTF-1 homeodomain protein and Ann Maher for excellent secretarial assistance.

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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