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Laboratory of Molecular Biology and Gene Regulation, Department of Life Science, School of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan

(Requests for offprints should be addressed to T Sato; Email: yukato{at}isc.meiji.ac.jp)

Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone ß subunit (FSHß) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone subunit (GSU), and luteinizing hormone ß subunit (LHß). A series of deletion mutants of the porcine GSU (up to –1059 bp) and LHß (up to –1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine GSU and LHß promoters by Prop-1, which was found to activate the GSU promoter of –1059/+12 bp up to 11.7-fold but not the LHß promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, –1038/–1026, –942/–928, –495/–479, –338/–326, –153/–146, and –131/–124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of GSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for GSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of GSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHß gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of GSU and FSHß gene expressions.