Leclerc Gilles M, Boockfor Fredric R
Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Avenue, Charleston, 29425, USA.
Mol Cell Endocrinol. 2005 Dec 21;245(1-2):86-92. doi: 10.1016/j.mce.2005.10.026. Epub 2005 Dec 7.
Recent evidence from our laboratory demonstrated that the OCT1 protein was necessary for GnRH gene promoter pulse activity through its interaction with a specific OCT1 binding site (OCT1BS-a, -1,774/-1,781). In light of the importance of this POU homeoprotein in pulsatile function, we focused on two other highly conserved OCT1 sites within this region, OCT1BS-b (-1,694/-1,701, previously AT-b), and OCT1BS-c (-1,569/-1,562). Mutagenesis of these sites revealed that alteration of OCT1BS-c, but not OCT1BS-b, virtually abolished gene expression pulses in GT1-7 cells. EMSAs confirmed that OCT1 can bind to both sites. Taken together, our findings demonstrate clearly that more than one Oct1 binding site is necessary for GnRH promoter pulses. Moreover, the lack of an influence observed with OCT1BS-b on pulse activity indicates that OCT1 action is not general to all OCT1 sites, but specific to certain octamer sequences in the NSE region of the GnRH promoter.
我们实验室最近的证据表明，OCT1蛋白通过与特定的OCT1结合位点（OCT1BS-a，-1,774 / -1,781）相互作用，对GnRH基因启动子脉冲活性是必需的。鉴于这种POU同源蛋白在脉冲功能中的重要性，我们聚焦于该区域内另外两个高度保守的OCT1位点，即OCT1BS-b（-1,694 / -1,701，先前称为AT-b）和OCT1BS-c（-1,569 / -1,562）。对这些位点的诱变显示，OCT1BS-c的改变而非OCT1BS-b的改变几乎消除了GT1-7细胞中的基因表达脉冲。电泳迁移率变动分析证实OCT1可以与这两个位点结合。综上所述，我们的研究结果清楚地表明，GnRH启动子脉冲需要不止一个Oct1结合位点。此外，观察到OCT1BS-b对脉冲活性缺乏影响，表明OCT1的作用并非对所有OCT1位点都相同，而是对GnRH启动子NSE区域中的某些八聚体序列具有特异性。
