Protein Factors in Thyrotropic Tumor Nuclear Extracts Bind to a Region of the Mouse Thyrotropin β-Subunit Promoter Essential for Expression in Thyrotropes

Abstract

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, TSH 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-I 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 areas from -271 or -201 to -77 were used, suggesting combinatorial cis interactions between these regions of the TSH beta promoter. DNase-I 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.

A Soluble Transcription Factor, Oct-1, Is Also Found in the Insoluble Nuclear Matrix and Possesses Silencing Activity in Its Alanine-Rich Domain

“…Interestingly, however, silencing has not been reported to occur in mouse and rat TSH␤ gene regulation. It was previously shown that upstream regions of the mouse and rat TSH␤ genes that roughly correspond to the hTSH␤ silencer element activated heterologous promoters such as those of Rous sarcoma virus or thymidine kinase (TK) in transient-transfection assays with TtT-97 or pituitary cells (34,48,69), and they therefore were labeled enhancers. Despite the sequence homology between the mouse and rat genes, the localizations of such enhancers did not coincide.…”

“…Despite the sequence homology between the mouse and rat genes, the localizations of such enhancers did not coincide. Furthermore, the murine element (Ϫ271/Ϫ117) that conferred activation to the Rous sarcoma virus promoter failed to show any enhancing activity in the context of the homologous promoter in TtT-97 cells (69). When tested on the homologous promoter, however, the proximal Ϫ133/Ϫ100 region was shown to be important for basal promoter activity and Pit-1-dependent activation (17,31).…”

TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper proteins.

“…However, the analysis of genetic dwarf mice by Li et al 11990) has implicated the pituitary-specific transcription factor Pit-1 (Bodner et al 1988;Ingraham et al 1988) in the maintenance of the thyrotroph phenotype even though Pit-1 is expressed in the pituitary after the initiation of TSH~ expression ). In addition, several thyrotroph-specific footprints within the TSHf~ promoter have been observed by using nuclear extracts from cells obtained from the thyrotroph TtT-97 transplantable tumor, which expresses both the ~-subunit and B-subunit of TSH (Wood et al 1990).…”

Pit-1 and GATA-2 Interact and Functionally Cooperate to Activate the Thyrotropin β-Subunit Promoter

“…Within this broad area, four DNase I protected regions have been identified using nuclear extracts from TSH␤ expressing mouse TtT-97 thyrotropic tumor cells (7)(8)(9). These have been termed D1 (Ϫ253 to Ϫ222), D2 (Ϫ196 to Ϫ176), P1 (Ϫ133 to Ϫ88), and P2 (Ϫ86 to Ϫ64).…”