TRANSCRIPTIONAL REGULATION OF CRH GENE EXPRESSION

哺乳动物的应激反应在很大程度上是由 下丘脑-垂体-肾上腺（HPA）轴。关键的下丘脑释放 该轴中的因子是促肾上腺皮质激素释放激素（CRH）。CRH 刺激促肾上腺皮质激素（ACTH）的合成和分泌， 垂体前叶，反过来刺激生产和释放 肾上腺皮质中的糖皮质激素然后糖皮质激素介导 身体对压力的适应性反应。除了其在 在下丘脑，CRH在大脑的许多其他区域产生， 它似乎在协调适应性的功能， 应答因此，CRH生产和放行代表了关键控制水平 人类对外界刺激的生理反应能力， 被监管。 CRH分泌的调节已被广泛研究，但 负责激活或抑制表达的细胞机制 对CRH基因的了解仍然很少。这个项目的总体目标是 建议是定义参与的分子机制， 大鼠CRH基因的转录调控，特别关注 类固醇和第二信使调节途径。以往基因 转移实验表明，CRH基因受cAMP 和糖皮质激素。该基因的糖皮质激素调节是 特别有趣的是，因为CRH基因似乎与 在不同的细胞类型中由糖皮质激素调节。这本小说 糖皮质激素对CRH表达的调节将在 本建议特别集中于：1）本地化， 表征（通过定点诱变）阳性和/或 使用基因转移方法的阴性糖皮质激素反应元件 在培养的细胞系和原代培养物中;和2）鉴定 使用DNase I的纯化糖皮质激素受体的DNA结合位点 以及硫酸二甲酯保护和干扰测定。然而，在这方面， 转录因子通常相互作用以介导转录因子的独特调节。 任何特定的基因，因此糖皮质激素对CRH表达的调节 不能独立于其他相关的调节机制进行研究 CRH表达。因此，我们将继续本地化其他cisacting 参与调节大鼠CRH基因的控制元件， 描述了DNA-蛋白质相互作用的5'侧翼序列， 基因使用体外和体内生物化学方法。获得的知识 这些研究将大大增加我们对分子生物学的理解， 参与大鼠CRH基因转录控制的机制，以及 将使我们能够更好地了解这一重要的复杂的监管， 体内神经内分泌肽，

The mammalian stress response is mediated in large part by the hypothalamic-pituitary-adrenal (HPA) axis. The key hypothalamic releasing factor in this axis is corticotropin releasing hormone (CRH). CRH stimulates synthesis and secretion of adrenocorticotropin (ACTH) from the anterior pituitary which in turn stimulates the production and release of glucocorticoids from the adrenal cortex. Glucocorticoids then mediate the body's adaptive response to stress. In addition to its role in the hypothalamus, CRH is produced in many other regions of the brain and periphery where it appears to function in the coordination of adaptive responses. Thus, CRH production and release represent key control levels at which man's ability to respond physiologically to external stimuli can be regulated. The regulation of CRH secretion has been extensively studied, but the cellular mechanisms responsible for activating or repressing the expression of the CRH gene are still poorly understood. The overall goal of this proposal is to define the molecular mechanisms involved in the transcriptional regulation of the rat CRH gene, focusing specifically on the steroid and second messenger regulation pathways. Previous gene transfer experiments have shown that the CRH gene is regulated by both cAMP and glucocorticoids. The glucocorticoid regulation of this gene is especially intriguing, since the CRH gene appears to be differentially regulated by glucocorticoids in different cell types. This novel glucocorticoid regulation of CRH expression will be carefully examined in the present proposal by focusing specifically on: 1) the localization and characterization (by site-directed mutagenesis) of the positive and/or negative glucocorticoid responsive element(s) using gene transfer methods in cultured cell lines and primary cultures; and 2) the identification of DNA-binding sites for the purified glucocorticoid receptor using DNase I and dimethylsulfate protection and interference assays. However, transcription factors often interact to mediate the unique regulation of any specific gene, so the regulation of CRH expression by glucocorticoids cannot be studied independently of the other regulatory mechanisms involved in CRH expression. Therefore, we will continue to localize other cisacting control elements involved in regulation of the rat CRH gene and further characterize the DNAprotein interactions in the 5' flanking sequence of the gene using in vitro and in vivo biochemical methods. The knowledge gained from these studies will greatly increase our understanding of the molecular mechanisms involved in transcriptional control of the rat CRH gene, and will allow us to better understand the complex regulation of this important neuroendocrine peptide in vivo,