Regulation of Pituitary Genes by the Transcription Factor, Pit-1, in the Domestic Turkey (A Turkey is NOT a Feathered Rat)
Weatherly, Kristy Lynn Jr.
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The transcription factor, Pit-1, is involved in the transcriptional regulation of the mammalian prolactin (Prl), growth hormone (GH) and thyroid stimulating hormone b-subunit genes (TSHb) as well as its own gene. The role of Pit-1 in avian species is unknown. Three turkey (t) Pit-1 isoforms have been identified that arise from alternative transcription initiation and alternative splicing. Splicing of exon 1 to an alternative acceptor splice site in exon 2 results in a 28 amino acid insertion in tPit-1β* relative to tPit-1*. Both isoforms initiate transcription at exon 1. A tPit-1 transcript unique to the turkey has been identified and arises following transcription initiation upstream of the alternative acceptor splice site in exon 2. Western blot analysis of pituitary extracts has revealed two isoforms of 37 and 40 kDa. The ability of Pit-1 to transactivate the Prl, GH, and Pit-1 promoters was determined with cotransfection assays. The tPrl, tGH, tPit-1 and rat (r) Prl promoters were cloned upstream of the luciferase gene in a reporter construct. Turkey Pit-1 isoforms and rPit-1 were expressed under the control of the Avian Sarcoma Virus Long Terminal Repeat (ASVLTR) promoter. Cotransfection analyses in mouse L cells indicate that tPit-1* activates the tPrl, tGH, tPit-1 and rPrl promoters 4.6-, 3.8-, 1.7-, and 29.0-fold, respectively. Similar results were observed when cotransfection assays were performed in a turkey pituitary-derived cell line and in primary turkey pituitary cells. These results indicate that tPit-1 is not a strong activator of the tPrl, tGH, or tPit-1 genes, whereas Pit-1 does activate these genes in mammals. A point mutation at amino acid position 176 (ser ⟹ leu) in the POU-homeodomain results in a mutant tPit-1 that shows decreased activity on all promoters tested. Turkey Pit-1* (ser-176) activates the rPrl promoter 14-fold lower than the wild type tPit-1* (leu-176).
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