Susa Takao, Nakayama Michie, Kitahara Kousuke, Kimoto Fuyuko, Kato Takako, Kato Yukio
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.
Biochem Biophys Res Commun. 2007 Jun 8;357(3):712-7. doi: 10.1016/j.bbrc.2007.03.197. Epub 2007 Apr 9.
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.
同源结构域抑制因子Hesx1/Rpx在垂体器官发生起始阶段Rathke囊的形成过程中发挥关键作用，并抑制Pit-1基因的Prop-1依赖性表达，而Pit-1基因可促进Pit-1依赖性激素产生细胞的分化。最近，我们发现了Prop-1的一种新功能，即它通过Fd2区域（-852/-746）激活猪促卵泡激素β亚基（FSHβ）基因。本研究旨在确定Hesx1是否在FSHβ基因的Prop-1依赖性激活中发挥作用。我们进行了猪FSHβ启动子-985/+10的瞬时转染实验、Fd2区域的电泳迁移率变动分析（EMSA）和DNase I足迹分析。在GH3细胞中的转染实验表明，单独的Hesx1表达不会改变启动子活性，但与Prop-1共表达会抑制FSHβ启动子的Prop-1依赖性激活。对于缺失-745/-104区域的突变报告载体也得到了类似结果，这表明Fd2区域是Hesx1以及Prop-1的靶位点。使用重组Hesx1和Prop-1蛋白进行的EMSA和DNase I足迹分析表明，Hesx1和Prop-1确实以不同方式结合富含AT的区域。这些结果表明，Hesx1在垂体发育早期阻断FSHβ基因的晚期表达，随后Prop-1出现并激活该基因。
