Nelson S B, Lawson M A, Kelley C G, Mellon P L
Department of Reproductive Medicine, University of California, San Diego, La Jolla 92093-0674, USA.
Mol Endocrinol. 2000 Sep;14(9):1509-22. doi: 10.1210/mend.14.9.0521.
Neuroendocrine control of the reproductive cascade is mediated by GnRH, which in mammals is produced by a subset of neurons scattered throughout the hypothalamus and forebrain. Utilizing a cultured cell model of GnRH neurons (GT1-7 cells), two regulatory regions in the rat GnRH 5' flanking DNA were identified as essential for cell-type specificity: a 300-bp enhancer and a 173-bp conserved proximal promoter. Using transient transfections to compare expression in GT1-7 cells to a non-GnRH-expressing cell type (NIH 3T3), we show that the GnRH enhancer and the proximal promoter each play roles in conferring this specificity. Deletion of footprint 2 (FP2; -26 to -76) from the promoter when coupled to the GnRH enhancer diminishes reporter activity in GT1-7 cells more strongly than in NIH 3T3 cells. Furthermore, deletion of FP2 from the promoter when coupled to the heterologous Rous sarcoma virus 5'-long terminal repeat promoter abolishes the difference in reporter activity between GT1-7 and NIH 3T3 cells, suggesting that FP2 of the GnRH promoter is necessary for cell-specific expression. In addition, FP2 alone is sufficient to confer cell-specific expression and can interact with the GnRH enhancer to augment reporter gene expression specifically in GT1-7 cells. Finally, a 31-bp sequence from within FP2 (-63 to -33) synergistically activates transcription when coupled with the GnRH enhancer in GT1-7 cells but not in NIH 3T3 cells. Thus, this 31-bp region contains elements necessary for interaction between the GnRH enhancer and promoter. We show that two of five protein complexes that bind to the -63 to -33 region are GT1-7 cell specific, and both of them appear to be homeodomain proteins. The identification of a cell-specific element in the GnRH proximal promoter significantly advances our understanding of the transcriptional basis for neuron-specific GnRH gene expression.
生殖级联反应的神经内分泌控制由促性腺激素释放激素（GnRH）介导，在哺乳动物中，GnRH由散布于下丘脑和前脑的一部分神经元产生。利用GnRH神经元的培养细胞模型（GT1-7细胞），大鼠GnRH 5'侧翼DNA中的两个调控区域被确定为细胞类型特异性所必需：一个300bp的增强子和一个173bp的保守近端启动子。通过瞬时转染比较GT1-7细胞与非GnRH表达细胞类型（NIH 3T3）中的表达，我们发现GnRH增强子和近端启动子在赋予这种特异性方面均发挥作用。当与GnRH增强子偶联时，从启动子中缺失足迹2（FP2；-26至-76）在GT1-7细胞中比在NIH 3T3细胞中更强烈地降低报告基因活性。此外，当与异源劳斯肉瘤病毒5'-长末端重复启动子偶联时，从启动子中缺失FP2消除了GT1-7和NIH 3T3细胞之间报告基因活性的差异，这表明GnRH启动子的FP2对于细胞特异性表达是必需的。此外，单独的FP2足以赋予细胞特异性表达，并且可以与GnRH增强子相互作用以特异性增强GT1-7细胞中的报告基因表达。最后，FP2内的一个31bp序列（-63至-33）与GnRH增强子偶联时在GT1-7细胞中协同激活转录，但在NIH 3T3细胞中则不然。因此，这个31bp区域包含GnRH增强子和启动子之间相互作用所需的元件。我们表明，与-63至-33区域结合的五个蛋白质复合物中的两个是GT1-7细胞特异性的，并且它们两者似乎都是同源域蛋白。GnRH近端启动子中细胞特异性元件的鉴定显著推进了我们对神经元特异性GnRH基因表达转录基础的理解。
