Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone alpha subunit gene expression.
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 beta subunit (FSHbeta) 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 alpha subunit (alphaGSU), and luteinizing hormone beta subunit (LHbeta). A series of deletion mutants of the porcine alphaGSU (up to -1059 bp) and LHbeta (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 alphaGSU and LHbeta promoters by Prop-1, which was found to activate the alphaGSU promoter of -1059/+12 bp up to 11.7-fold but not the LHbeta 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 alphaGSU, 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 alphaGSU 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 alphaGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHbeta 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 alphaGSU and FSHbeta gene expressions.
Sato
Takanobu
T
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. yukato@isc.meiji.ac.jp
Kitahara
Kousuke
K
Susa
Takao
T
Kato
Takako
T
Kato
Yukio
Y
D024761:AT-Hook Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism / Q000502:physiology*; D037101:Luteinizing Hormone, beta Subunit / Q000235:genetics; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; D018398:Homeodomain Proteins; D037101:Luteinizing Hormone, beta Subunit; C102134:Prophet of Pit-1 protein