Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter.
Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.
Susa
Takao
T
Laboratory of Molecular Biology and Gene Regulation, Division of Life Science, Graduate School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Kanagawa 214-8571, Japan. tsusa@isc.meiji.ac.jp
Nakayama
Michie
M
Kitahara
Kousuke
K
Kimoto
Fuyuko
F
Kato
Takako
T
Kato
Yukio
Y
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D037201:Follicle Stimulating Hormone, beta Subunit / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012097:Repressor Proteins; D013552:Swine; D014162:Transfection
D037201:Follicle Stimulating Hormone, beta Subunit; C489733:Hesx1 protein, mouse; D018398:Homeodomain Proteins; C102134:Prophet of Pit-1 protein; D012097:Repressor Proteins; D004247:DNA; D003850:Deoxyribonuclease I