Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin beta-subunit promoter.
The molecular determinants governing cell-specific expression of the thyrotropin (TSH) beta-subunit gene in pituitary thyrotropes are not well understood. The P1 region of the mouse TSHbeta promoter (-133 to -88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern and Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors. In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells. The complex demonstrated binding specificity to the P1 region DNA probe and could be disrupted by a GATA-2 antibody. When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interaction was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulated mouse TSHbeta promoter activity 8.5-fold, while each factor alone had a minimal effect. Mutations that abrogated this functional stimulatory effect mapped to the P1 region. Finally, we show that GATA-2 directly interacts with Pit-1 in solution. In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression.
Gordon
D F
DF
Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. david.gordon@uchsc.edu
Lewis
S R
SR
Haugen
B R
BR
James
R A
RA
McDermott
M T
MT
Wood
W M
WM
Ridgway
E C
EC
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D050989:GATA2 Transcription Factor; D005786:Gene Expression Regulation; D051379:Mice; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013972:Thyrotropin / Q000235:genetics*; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; D012333:RNA, Messenger; D013972:Thyrotropin; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C494712:Gata2 protein, mouse; C494185:Pou1f1 protein, mouse