REGULATION OF CHOLESTEROL 7 ALPHA HYDROXYLASE

分解代谢和排泄的最重要途径 哺乳动物体内的胆固醇是胆汁酸的形成。不适当 对这一途径的调控具有广泛的影响，因为 胆固醇的消除是导致这种疾病的一个非常重要的因素 如动脉粥样硬化、胆石症和一些脂肪堆积症。 该途径中的起始和限速步骤是羟化。 胆固醇在7位，由胆固醇7α催化- 羟基酶。这种酶受到反馈控制，因此高 胆汁酸通过肠-肝回到肝脏的水平 循环抑制了它的活动。胆固醇，人体的底物 酶，发挥积极的控制作用。这两项法规都发生在 其基因的转录。这项提案的总体目标是 为了了解胆固醇的分子机制--和 胆汁酸对胆固醇70：羟基酶的调节作用 抄写。我们最近证明了胆汁酸和 胆固醇介导7α-羟基酶的转录调控 转基因小鼠的启动子活性，以及DNA元件(S) 介导调控位于启动子的第一个342bp 区域。我们还定量复制了胆汁酸--和 胆固醇对7α-羟基酶启动子的调节作用 在已建立的组织培养系统中的活动。我们将用这张纸巾 在核苷酸水平上定位DNA元件的培养系统(S) 参与胆汁酸和胆固醇介导的 7α-羟基酶转录。7α-的缺失突变体 将创建羟基酶启动子和异源启动子，并 测试来描述这些序列。重要的融合基因提供了 我们的组织培养实验中的关键信息将在 转基因小鼠以证实所获得的数据。一旦这些DNA元素 7α-羟基酶对胆汁酸和胆固醇的调节作用 转录是已知的，我们将建立一种蛋白质-DNA结合分析 这表明7α-羟基酶启动子之间存在精确相关性 活性和与特定寡核苷酸的结合，我们将用作 探测器。这个测试将被用来提纯 使用标准和亲和力与DNA调控元件结合 层析技术。这种提纯将导致准备 对分子研究至关重要的抗体和DNA探针 该法规所涉及的机制。这些研究应该提供一个 更好地理解与细胞周期相关的分子机制 胆汁酸合成的调控。

The most important pathway for the catabolism and excretion of cholesterol in mammals is the formation of bile acids. Improper regulation of this pathway has widespread implications because the rate of elimination of cholesterol is a very important factor in diseases such as atherosclerosis, gallstone disease, and some lipid storage diseases. The initial and rate limiting step in this pathway is the hydroxylation of cholesterol at position 7 and is catalyzed by cholesterol 7alpha- hydroxylase. This enzyme is subject to a feedback control, whereby high levels of bile acids returning to the liver via the enterohepatic circulation suppress its activity. Cholesterol, the substrate of the enzyme, exerts a positive control. Both regulations occur at the level of the transcription of its gene. The overall goal of this proposal is to understand the molecular mechanisms involved in the cholesterol- and bile acid-mediated regulation of cholesterol 70:-hydroxylase transcription. We have recently demonstrated that bile acids and cholesterol mediate transcriptional regulation of 7alpha-hydroxylase promoter activity in transgenic mice, and that the DNA element(s) that mediates that regulation is located in the first 342 bp of the promoter region. We have also quantitatively reproduced the bile acid- and cholesterol-mediated regulation of the 7alpha-hydroxylase promoter activity in an established tissue culture system. We will use this tissue culture system to localize, at the nucleotide level, the DNA element(s) involved in the bile acid- and cholesterol-mediated 'regulation of 7alpha-hydroxylase transcription. Deletion mutants of the 7alpha- hydroxylase promoter and heterologous promoters will be created and tested to delineate those sequences. Important fusion genes that provide critical information in our tissue culture experiments will be tested in transgenic mice to confirm the obtained data. Once the DNA elements that mediate the bile acid and cholesterol regulation of 7alpha-hydroxylase transcription are known, we will establish a protein-DNA binding assay that show a precise correlation between 7alpha-hydroxylase promoter activity and binding to specific oligonucleotides that we will use as probes. This assay will be used to purify the transacting factors that bind to the DNA regulatory elements using standard and affinity chromatography techniques. This purification will lead to the preparation of antibodies and DNA probes essential for the study of the molecular mechanisms involved in that regulation. These studies should provide a better understanding of the molecular mechanisms involved in the regulation of bile acid synthesis.