Identification of Thyrotroph-Specific Factors andCis-Acting Sequences of the Murine Thyrotropinβ Subunit Gene

Abstract

Pituitary thyrotroph cells specialize in the synthesis of TSH, and thus represent a model to study cell-specific gene expression. We have used the murine TSH beta (mTSH beta) gene promoter and TSH-producing and nonproducing transplantable tumors derived from murine thyrotroph cells, referred to as TtT-97 and MGH 101A, respectively, to identify nuclear factors which selectively interact with the mTSH beta gene. DNase I protection analyses demonstrate that factors present in TtT-97 nuclear extracts bind with high affinity to five separate sites in the TSH beta promoter region, denoted as distal D1 (-253 to -227) and proximal, P1 (-76 to -68), P2 (-106 to -98), P3 (-126 to -112), and P4 (-142 to -131) footprints. By contrast, non-TSH beta expressing thyrotroph cell nuclear extracts and L-cell nonpituitary cell extracts did not appear to footprint the D1 site; whereas the nonpituitary nuclear extracts revealed minimal DNase I protection in the P1-P4 regions. These data show that the distal D1 site is thyrotroph specific and contains a 6 base pair direct repeat sequence (5'-AGATAT-3'). Factor occupancy of the D1 site is protein dependent, occurs rapidly (less than 15 sec), is destabilized by 170 mM KCl, and results in an associated DNase I hypersensitive region. A double-stranded oligonucleotide spanning the D1 footprint competes only the distal factor binding region. Transfection of plasmid constructs containing progressive 5'-deletions of the mTSH beta promoter linked to the reporter gene luciferase into primary TtT-97 cells demonstrate a marked decrease in activity between the regions -270 and -79, which contains the D1 region.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple Dna Elements Determine Basal and Thyroid Hormone Regulated Expression of the Human Glycoprotein Α Subunit Gene in Pituitary Cells

“…The con¬ trasting patterns of basal expression of the human a promoter in pituitary and choriocarcinoma cells suggest that pituitary cell-specific trans-acting factors may exert both stimulatory and inhibitory actions on 5' flanking a elements in such pituitary cells. This hypothesis is supported by description of thyrotroph-specific factors which bind to regions of the murine TSH-ß gene required for optimal TSH-ß expression (Alexander, Gordon, Wood et al 1989) and of multiple overlapping protein-binding sites in human a 5'flanking DNA (Jameson et al, 1988), includ¬ ing those shown to be important functionally in the present studies.…”

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).…”

Interaction of basal positive and negative transcription elements controls repression of the proximal rat prolactin promoter in nonpituitary cells.

“…1). The trimerized poly(A) stops upstream of the polylinker and luciferase reporter gene in pA3Luc result in a promoterless reporter vector with negligible activity in gene transfer studies, and thus the activity of any promoter fragment cloned into the polylinker can be assayed with increased sensitivity owing to the lack of significant contribution from vector readthrough (3,4,42,68). The orientation of the inserted promoter fragment and documentation of specific rPRL promoter mutations in either the pA3Luc-or the pGEM4-based vectors were verified by DNA sequencing using a luciferase oligonucleotide primer as previously reported (3,4,68).…”

“…Following electroporation, cells were maintained in culture for 8 to 24 h, depending on the optimal time of luciferase expression for each cell line (30). Cells were then harvested and lysates were measured for luciferase activity as previously reported (3,12,30,31,68 (39,40,53,57). While significant reduction in promoter activity could be due to removal of a critical positive transcription element, such as FPIII, the almost complete loss of promoter activity with the -127 deletion suggested to us that this 5' deletion endpoint unmasked a strong repressor element.…”