PROTEIN FACTORS IN THYROTROPIC TUMOR NUCLEAR EXTRACTS BIND TO A REGION OF THE MOUSE THYROTROPIN BETA-SUBUNIT PROMOTER ESSENTIAL FOR EXPRESSION IN THYROTROPES

The beta-subunit gene of TSH is specifically expressed in thyrotrope cells of the anterior pituitary gland. To define the particular TSH-beta-subunit gene sequences responsible for tissue-specific expression, THS-beta 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 TSH-beta 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-1 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 TSH-beta promoter fragment from -117 to -80. Heterologous promoter activity was further stimulated when fragments containing the area from -271 or -201 to -77 were used, suggesting combinatorial cis interactions between these regions of the TSH-beta promoter. DNase-l protection studies suggest that there are multiple protein-binding domains in the mouse TSH-beta 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 TSH-beta gene expression. (Molecular Endocrinology 4: 1897-1904, 1990)