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

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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 (< 15 sec), is destabilized bu 170 nM 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. In summary, the current studies show that TSH.beta.-producing cells contain distinct nuclear factor(s), which are absent in the TSH.beta.-nonproducing cells and which bind regions necessary for optimal TSH.beta. gnee expression, suggesting that such factor(s) may be critical in mediating the thyrotroph-specific expression of the TSH.beta. gene.