REGULATION OF CHOLESTEROL 7 ALPHA HYDROXYLASE

最重要的途径，为催化剂和排泄的 胆固醇在哺乳动物中是胆汁酸的形成。不当 这一途径的调控具有广泛的意义，因为 胆固醇的消除是疾病的一个非常重要的因素， 如动脉粥样硬化、胆结石病和一些脂质沉积病。 该途径的起始和限速步骤是羟基化 胆固醇的7位，并由胆固醇7 α- 羟化酶这种酶受到反馈控制，由此高 胆汁酸水平通过肠肝 循环抑制其活动。胆固醇， 酶，施加阳性对照。这两项规定都发生在 它的基因的转录。本提案的总体目标是 了解胆固醇的分子机制， 胆汁酸介导的胆固醇70：-羟化酶调节 转录。 我们最近证明，胆汁酸和 胆固醇介导7 α-羟化酶转录调控 在转基因小鼠中的启动子活性，以及 介导调控位于启动子的前342 bp 地区 我们还定量地复制了胆汁酸， 胆固醇介导的7 α-羟化酶启动子调节 在已建立的组织培养系统中的活性。我们会用这块纸巾 在核苷酸水平定位DNA元件的培养系统 参与胆汁酸和胆固醇介导的 7 α-羟化酶转录。7 α- 将产生羟化酶启动子和异源启动子， 来描绘这些序列提供重要融合基因 我们的组织培养实验中的关键信息将在 转基因小鼠，以确认获得的数据。一旦DNA元素 介导胆汁酸和胆固醇调节7 α-羟化酶 转录是已知的，我们将建立一个蛋白质-DNA结合试验 其显示了7 α-羟化酶启动子 活性和结合特定的寡核苷酸，我们将使用作为 probes.该测定将用于纯化反式作用因子， 使用标准和亲和力结合DNA调节元件 色谱技术。这种净化将导致准备 抗体和DNA探针的研究所必需的分子 这一监管机制。这些研究将提供一个 更好地了解参与的分子机制， 胆汁酸合成的调节。

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.