MECHANISM OF STEROL REGULATION OF LDL RECEPTOR GENE

甾醇对LDL受体基因的调控机制

• 批准号: 2228453
• 负责人: KAMAL D MEHTA
• 金额: $ 9.87万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2228453
• 关键词: CHO cells; DNA footprinting; HeLa cells; Saccharomyces cerevisiae; complementary DNA; cytokine; gene expression; gene induction /repression; genetic promoter element; genetic regulation; genetic transcription; interleukin 1; lac operon; low density lipoprotein; molecular cloning; polymerase chain reaction; sterols; transcription factor; tumor necrosis factor alpha

Every year in the United States over 550,000 deaths occur in more than five million Americans affected with atherosclerosis. The cause of atherosclerosis is not clear, however several risk factors are strongly related to its development. Of all known risk factors promoting atherosclerosis and heart disease, a high serum LDL-cholesterol level is the most important, and its significance in the pathogenesis of atherosclerotic coronary heart disease is well established. LDL receptors bind to the most atherogenic plasma protein LDL , and interestingly, the LDL receptor gene is negatively regulated at the transcriptional level by both the intracellular cholesterol obtained from plasma LDL and the cholesterol synthesized de novo. The goal of our research is to unravel the molecular mechanism of transcriptional regulation of the LDL receptor gene by sterols in human cells. The proposed research addresses a specific question concerning the mechanism of sterol regulation of the LDL receptor gene, by blending the biochemical and molecular biology techniques with yeast genetics. The proposed approach has become available only recently. All attempts to identify a protein involved in the sterol regulation of this receptor gene using the conventional in vitro methods have failed. Earlier, we have provided evidence for the in vivo role of the sterol regulatory element in controlling the LDL receptor gene expression by sterols. Now, protein-DNA interactions that occur in vivo in the promoter region of the LDL receptor gene in response to sterols will be investigated. Initial results using the in vivo footprinting technique are very encouraging, and the significance of these footprints in the sterol regulation of the LDL receptor gene will be established in future experiments. Furthermore, our investigation will provide an opportunity to isolate the cDNA of physiologically relevant crucial factor(s) involved in the sterol regulation of the LDL receptor gene by developing novel in vivo genetic screening systems in yeast. We will use human LDL receptor gene promoter placed upstream of two different reporter genes to screen for SREBP cDNA clones using yeast. The first system, Sp1-interactive selection system will screen for cDNA clones that causes enhancement of the LacZ gene expression in an SRE-1 dependent manner, and the second system, growth selection system, utilizes HIS3 reporter gene. Both systems provides a tight "ON-OFF" genetic selection and have proved useful in cloning the cDNAs of difficult transcription factors. Finally, experiments are also designed to understand the molecular mechanism of cytokines induction of the LDL receptor gene transcription, and its repression by sterols in human cells. These have been included as a result of new In vivo footprint observed in the human LDL receptor gene promoter. Interestingly, in vivo footprinting studies have identified a new region In the LDL receptor gene promoter which footprints only in the absence of sterols, and contain consensus sequences for two transcription factors binding sites (Ets and CREBP), whose activities are modulated by growth factors, cAMP and intracellular calcium levels. We are confident that this pioneering work will enable us to refine the techniques involved, and apply them successfully to other less well characterized but important sterol-responsive genes that have complex promoters.

在美国，每年有超过55万人死于 500万美国人受到动脉粥样硬化的影响。的起因 动脉粥样硬化尚不清楚，但有几个危险因素是强烈的。 与它的发展有关。在所有已知的风险因素中， 动脉粥样硬化和心脏病，高血清低密度脂蛋白-胆固醇水平是 最重要的，及其在发病机制中的意义 动脉粥样硬化性冠心病是公认的疾病。低密度脂蛋白受体 与最易致动脉粥样硬化的血浆蛋白低密度脂蛋白结合，有趣的是， 低密度脂蛋白受体基因在转录水平上受到负调控 从血浆低密度脂蛋白中获得的细胞内胆固醇和 胆固醇合成从头开始。 我们研究的目标是解开其分子机制。 甾醇对人低密度脂蛋白受体基因转录的调节 细胞。拟议的研究解决了一个具体的问题，即 甾醇调节低密度脂蛋白受体基因的机制 生物化学和分子生物学技术与酵母遗传学。这个 提议的方法直到最近才变得可行。 所有试图确定一种参与类固醇调节的蛋白质 这种受体基因使用传统的体外方法已经失败了。 早些时候，我们已经为类固醇在体内的作用提供了证据 低密度脂蛋白受体基因表达调控元件的研究 类固醇。现在，体内发生在启动子中的蛋白质-DNA相互作用 低密度脂蛋白受体基因对固醇的反应区域将是 调查过了。使用体内足迹技术的初步结果是 非常鼓舞人心，以及这些脚印在甾醇中的意义 低密度脂蛋白受体基因的调控将在未来建立 实验。此外，我们的调查将提供一个机会 分离与生理相关的关键因子(S)c DNA 体内开发新的类固醇对低密度脂蛋白受体基因的调节 酵母中的遗传筛选系统。我们将使用人类低密度脂蛋白受体基因 启动子位于两个不同报告基因的上游以进行筛选 用酵母菌克隆SREBP基因。第一个系统，Sp1-交互式选择 系统将筛选导致LacZ增强的cDNA克隆 以依赖SRE-1的方式进行基因表达，以及第二系统， 生长选择系统，利用HIS3报告基因。两个系统 提供紧凑的“开关”基因选择，并已被证明在 克隆困难转录因子的cDNA。最后， 实验也被设计用来理解分子机制 细胞因子对低密度脂蛋白受体基因转录的诱导作用 类固醇对人体细胞的抑制作用。这些已被包括在 在人低密度脂蛋白受体基因中观察到新的体内足迹的结果 推动者。有趣的是，活体足迹研究发现 低密度脂蛋白受体基因启动子中的新区域 不含甾醇，并包含两个转录的共同序列 因子结合位点(ETS和CREBP)，其活性受 生长因子、cAMP和细胞内钙水平。 我们相信，这项开创性的工作将使我们能够完善 所涉及的技术，并将其成功地应用于其他不太好的井 具有特征但重要的固醇反应基因，具有复杂的 推动者。