Domains of Pit-1 required for transcriptional synergy with GATA-2 on the TSH beta gene.
Previous studies showed that Pit-1 functionally cooperates with GATA-2 to stimulate transcription of the TSH beta gene. Pit-1 and GATA-2 are uniquely coexpressed in pituitary thyrotropes and activate transcription by binding to a composite promoter element. To define the domains of Pit-1 important for functional cooperativity with GATA-2, we cotransfected a set of Pit-1 deletions with an mTSH beta-luciferase reporter. Plasmids were titrated to express equivalent amounts of protein. A mutant containing a deletion of the hinge region between the POU and homeodomains retained the ability to fully synergize with GATA-2. In contrast, mutants containing deletions of amino acids 2-80 or 72-125 demonstrated 56 or 34% of the synergy found with the full-length protein, suggesting that these regions contributed to cooperativity. Mutants with deletions of the POU-specific or homeodomain further reduced the effect signifying the requirement for DNA binding. GST interaction studies demonstrated that only the homeodomain of Pit-1 interacted with GATA-2. Finally, several mutations between the Pit-1 and GATA-2 sites on the TSH beta promoter reduced binding for each factor and greatly reduced ternary complex formation. Thus multiple domains of Pit-1 are required for full synergy with GATA-2 and sequences between the two binding sites contribute to co-occupancy with both factors on the proximal TSH beta promoter.
Gordon
David F
DF
Department of Medicine, University of Colorado Health Sciences Center, Box B151, 4200 E Ninth Avenue, Denver, CO 80262, USA. david.gordon@uchsc.edu
Woodmansee
Whitney W
WW
Black
Jennifer N
JN
Dowding
Janet M
JM
Bendrick-Peart
Jamie
J
Wood
William M
WM
Ridgway
E Chester
EC
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004357:Drug Synergism; D050989:GATA2 Transcription Factor; D051379:Mice; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D017384:Sequence Deletion; D037322:Thyrotropin, beta Subunit / Q000235:genetics*; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014162:Transfection
D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; D037322:Thyrotropin, beta Subunit; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C494712:Gata2 protein, mouse; C494185:Pou1f1 protein, mouse