CELL SPECIFIC GNRH EXPRESSION

促性腺激素释放激素（GnRH）的细胞特异性表达 对哺乳动物生殖系统的协调调节至关重要 系统 GnRH神经元数量相对较少， 地理位置使得GnRH的研究变得困难。 人类GnRH基因具有 被克隆，它的转录起始位点定位在 下丘脑和胎盘。在这项提议中， 人促性腺激素释放激素（hGnRH）基因的5'侧翼区对细胞增殖的重要性 将表征特定表达。 转基因小鼠 使用基因构建体产生，所述基因构建体由多个缺失的 hGnRH启动子与荧光素酶报告基因（LUC）融合，产生了一个 GnRH细胞特异性元件的初步定位。 额外 转基因实验将证明这种元素是否足以 GnRH细胞特异性表达。GnRH细胞特异性元件还将 使用GnRH表达神经元细胞系在体外作图。 两高 相关细胞系NLT和Gn 11广泛表达GnRH mRNA， 不同的级别可用于此目的。 DNAse ⅰ足迹 将用于确定hGnRH启动子的哪些序列是 与来自表达GnRH的细胞提取物的核蛋白结合。 到 识别与功能重要的顺式作用结合的核蛋白 元素，凝胶位移分析将使用针对 候选蛋白质。脑2（Brn-2）mRNA已在一个 GnRH细胞系（NLT）表达高量GnRH mRNA，但在 另一个表达少量GnRH mRNA的细胞系（Gn 11），表明 它可以作为GnRH基因的细胞特异性激活剂。 Brn-2和 将其它POU蛋白表达载体转染到Gn 11细胞中 确定它们是否可以通过其细胞特异性激活GnRH启动子， 元素 最后，GnRH神经元中POU蛋白的存在将 可以使用原位杂交研究来确定。因此，顺式作用 负责控制GnRH的元件和转录因子 下丘脑中的基因表达将被分离和表征。 这些研究将提供重要的分子见解控制 并提出了转录因子， 在生殖轴的某些病理状态中有缺陷的。

The cell-specific expression of gonadotropin-releasing hormone (GnRH) is essential for the coordinate regulation of the mammalian reproductive system. The relatively small numbers of GnRH neurons and their diffuse locations has made the study of GnRH difficult. The human GnRH gene has been cloned and it's transcriptional start site mapped in the hypothalamus and placenta. In this proposal, DNA sequences within the 5' flanking region of the human GnRH (hGnRH) gene important for cell- specific expression will be characterized. Trasgenic mice have been generated using gene constructs consisting of various deletions of the hGnRH promoter fused to the luciferase reporter gene (LUC), yielded a preliminary localization of a GnRH cell-specific element. Additional transgenic experiments will prove whether this element is sufficient for GnRH cell-specific expression. The GnRH cell-specific element will also be mapped in vitro using GnRH-expressing neuronal cell lines. Two high related cell lines NLT and Gn11, which express GnRH mRNA at widely different levels, are available for this purpose. DNAse I footprinting will be used to determine which sequences of the hGnRH promoter are bound by nuclear proteins from GnRH-expressing cellular extracts. To identity nuclear proteins bound to functionally important cis-acting elements, gel-shift analyses will be performed with antibodies directed at candidate proteins. Brain 2(Brn-2) mRNA has been identified in one GnRH cell line (NLT) expressing high amount of GnRH mRNA but not in another cell line (Gn11) expressing low amounts of GnRH mRNA, suggesting it may serve as a cell-specific activator of the GnRH gene. Brn-2 and other POU protein expression vectors will be transfected into Gn11 cells determine if they can activate the GnRH promoter via its cell-specific element. Finally, the presence of POU proteins in the GnRH neuron will be determined using in situ hybridization studies. Thus, cis-acting elements and transcriptional factors responsible for the control of GnRH gene expression in the hypothalamus will be isolated and characterized. These studies will provide important molecular insights into the control of human reproduction and suggest transcription factors which might be defective in certain pathological conditions of the reproductive axis.