A thyrotrope-specific variant of Pit-1 transactivates the thyrotropin beta promoter.
Thyrotropin (TSH) beta is a subunit of TSH, the expression of which is limited to the thyrotrope cells of the anterior pituitary gland. Tissue-specific expression of the mouse TSH beta gene is conferred by sequences between -270 and -80 of the 5'-flanking region. We have investigated tissue-specific expression of the TSH beta promoter in two thyrotrope-derived cell types: 1) TtT-97 thyrotropic tumors, which express the endogenous TSH beta gene, and 2) an alpha-TSH cell line, which was generated from a thyrotropic tumor that has lost the ability to express the TSH beta gene. The pituitary-specific transcription factor Pit-1 is present in thyrotropes and interacts with three cis-acting elements in the functionally important region of the TSH beta promoter. Pit-1 protein is present in TtT-97 tumor cells but is absent from alpha-TSH cells. Reintroduction of Pit-1 into alpha-TSH cells by transient transfection does not restore TSH beta promoter activity. We have identified an alternately spliced variant of Pit-1, called Pit-1T, the mRNA and protein expression of which is limited to thyrotrope-derived cells. Pit-1T contains a 14-amino acid insert in the transactivation domain due to an alternate 3' splice acceptor site. Transiently transfected Pit-1T increases TSH beta promoter activity in TtT-97 thyrotropic tumor cells, whereas additional Pit-1 has no effect. The alpha-TSH cell line, which lacks all Pit-1 proteins, requires both isoforms in order to stimulate TSH beta promoter activity. These data suggest that Pit-1T is a thyrotrope-specific splice variant of Pit-1 that is required for TSH beta promoter stimulation; furthermore, both Pit-1 and Pit-1T are required for TSH beta promoter activity in thyrotrope cells.
Haugen
B R
BR
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Wood
W M
WM
Gordon
D F
DF
Ridgway
E C
EC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002478:Cells, Cultured; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013972:Thyrotropin / Q000235:genetics*; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D012260:Ribonucleases; D013972:Thyrotropin; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C494185:Pou1f1 protein, mouse