CHOLESTEROL 7ALPHA-HYDROXYLASE IN TRANSGENIC MICE

胆固醇代谢和排泄的最重要途径 是胆汁酸的形成。 监管不当， 途径具有广泛的影响，因为消除的速度 胆固醇是诸如动脉粥样硬化的疾病中非常重要的因素， 胆结石疾病和一些脂质沉积疾病。 初始和速率 该途径的限制步骤是胆固醇的羟基化， 位置7，并被胆固醇7 α-羟化酶催化， 微粒体细胞色素P450。这种酶受到反馈控制， 由此高水平的胆汁酸通过肝脏返回肝脏， 肝肠循环抑制其活性，胆固醇， 酶的底物，发挥阳性对照。 这两项规定都发生在 在RNA积累的水平上，推测并通过与其他类似的方法， 参与胆固醇稳态的基因，在 转录其基因。 本提案的总体目标是 了解胆固醇和胆汁相关的分子机制 酸介导的胆固醇7 α-羟化酶表达的调节。 我们 克隆了胆固醇7 α-羟化酶基因，其特征是 转录起始位点，并测序了其5 '-侧翼的1.6kb序列。 地区 通过转染实验，我们已经证明， 片段含有促进其表达所必需的DNA元件。 以肝脏特异性的方式转录。 然而，我们无法 显示胆固醇或胆汁酸依赖性基因调控 由7 α-羟化酶的5 ′-侧翼区介导的转录 基因 因为这种监管似乎需要复杂的相互作用， 整个器官系统，我们将开发出具有融合基因的转基因小鼠， 含有7 α-羟化酶启动子的基因。 一旦我们获得了 启动子构建体在转基因小鼠中表现出调节表达，我们 将使用体内和体外相结合的方法来识别和 描述与胆固醇和胆汁酸有关的DNA元素- 介导的7 α-羟化酶表达调节。 我们将通过 修剪7 α-羟化酶基因的5 ′侧翼区， 产生缺失突变体， 转录系统，然后将它们引入小鼠。 蛋白质/DNA结合研究将使我们能够定位启动子 最有可能包含监管要素的区域。 这些区域将 通过诱变技术进行突变， 上述体内和体外方法。 这些研究将提供 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 catalized by cholesterol 7 alpha-hydroxylase, a microsomal cytochrome P450. 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, and cholesterol, the substract of the enzyme, exerts a positive control. Both regulations occur at the level of RNA accumulation, presumably and by analogy with other genes involved in cholesterol homeostasis, 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 7alpha-hydroxylase expression. We have cloned the cholesterol 7alpha-hydroxylase gene, characterized its transcriptional initiation site, and sequenced 1.6 kb of its 5'-flanking region. By transfection experiments we have demonstrated that this fragment contains the DNA elements necessary for the promotion of its transcription in a liver-specific manner. However, we have been unable to demonstrate either cholesterol or bile acid-dependent regulation of gene transcription mediated by the 5'-flanking region of the 7alpha-hydroxylase gene. Because that regulation seems to require the complex interaction of whole organ systems, we will develop transgenic mice that harbor fusion genes containing the 7alpha-hydroxylase promoter. Once we obtain a promoter construct that shows regulated expression in transgenic mice, we will use combined in vivo and in vitro approaches to identify and characterize the DNA elements involved in cholesterol and bile acid- mediated regulation of 7alpha-hydroxylase expression. We will do this by trimming down the 5'-flanking region of the 7alpha-hydroxylase gene, creating deletion mutants that will be analyzed in an in vitro transcription system, prior to introducing them into mice. Protein/DNA-binding studies will allow us to localize the promoter region(s) most likely to contain regulatory elements. These region(s) will be mutated by oligonucleotide-mutagenesis techniques and studied by the in vivo and in vitro approaches mentioned above. These studies should provide a better understanding of the molecular mechanisms involved in the regulation of bile acid synthesis.