Identification of thyrotroph-specific factors and cis-acting sequences of the murine thyrotropin beta subunit gene.
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)
Alexander
L M
LM
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Gordon
D F
DF
Wood
W M
WM
Kao
M Y
MY
Ridgway
E C
EC
Gutierrez-Hartmann
A
A
D000818:Animals; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000032:analysis; D003850:Deoxyribonuclease I; D004560:Electricity; D051379:Mice; D010903:Pituitary Gland, Anterior / Q000032:analysis / Q000166:cytology; D010911:Pituitary Neoplasms / Q000032:analysis; D012045:Regulatory Sequences, Nucleic Acid; D013972:Thyrotropin / Q000235:genetics*; D014157:Transcription Factors / Q000032:analysis*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D013972:Thyrotropin; D003850:Deoxyribonuclease I