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Departments of Medicine, University of Colorado Health Sciences Center Denver, Colorado 80262
Departments of Biochemistry, Biophysics, and Genetics, University of Colorado Health Sciences Center Denver, Colorado 80262
Division of Clinical Endocrinology, Medizinische Hochschule Hannover D-3000 Hannover, Germany
Address requests for reprints to: Dr. William M. Wood, University of Colorado Health Sciences Center, Box B-151, 4200 East Ninth Avenue, Denver, Colorado 80262.
Abstract
The β-subunit gene of TSH is specifically expressed in thyrotrope cells of the anterior pituitary gland. To define the particular TSH β-subunit gene sequences responsible for tissue-specific expression, TSHβ promoter fragments were assessed for promoter activity by gene transfer into TSH-expressing thyrotropic tumor cells (TtT-97). Previous studies have shown that the murine TSHβ gene promoter was more efficiently used in TtT-97 cells compared to other pituitary-derived cells or nonpituitary fibroblasts and that a 191-basepair DNA sequence of the 5' flanking region between –271 and –80 was sufficient for maximal promoter activity in thyrotropes. Further deletional analysis within this region has localized the area responsible for expression in thyrotropes to a 37-basepair region between –117 and –80 up-stream of the major transcriptional initiation site. DNase-l protection assays demonstrated that this functionally defined 5' flanking area, in addition to the adjacent sequences immediately up-stream and down-stream, interacts with protein factors present in nuclear extracts from TtT-97 tumor cells. When fused to a heterologous promoter, fragments derived from the region between –271 and –80 exhibited cell-specific activity, although this was not conferred solely by the TSHβ promoter fragment from –117 to –80. Heterologous promoter activity was further stimulated when fragments containing the areas from –271 or –201 to –77 were used, suggesting combinatorial cis interactions between these regions of the TSHβ promoter. DNase-l protection studies suggest that there are multiple protein-binding domains in the mouse TSHβ 5' flanking sequence. Only the more proximal domains, which encompass important promoter elements, appear to be required for efficient expression in thyrotropes, whereas other more up-stream sites of protein interaction may be involved in regulatory aspects of TSHβ gene expression.
FOOTNOTES
This work was supported in part by NIH Grants DK-36843 and CA-47411 (to E.C.R.), NIH Grant DK-37667 (to A.G.-H.), and NIH Grant NRSA-08358 (to L.M.A.).
* Supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Oc 17/1–1).
Pew Scholar in the Biomedical Sciences.
Received for publication July 16, 1990. Revision received September 5, 1990. Accepted for publication September 18, 1990.
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