MECHANISM OF STEROL REGULATION OF LDL RECEPTOR GENE

甾醇对LDL受体基因的调控机制

• 批准号: 2771365
• 负责人: KAMAL D MEHTA
• 金额: $ 11.06万
• 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 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2771365
• 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; low density lipoprotein receptor; 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55万人死亡， 五百万美国人患有动脉粥样硬化事业 动脉粥样硬化尚不清楚，但几个危险因素是强烈的， 与其发展有关。所有已知的危险因素 动脉粥样硬化和心脏病，高血清LDL-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。LDL受体 与最易致动脉粥样硬化的血浆蛋白LDL结合，有趣的是， LDL受体基因在转录水平上受到 从血浆LDL获得的细胞内胆固醇和 胆固醇从头合成。 我们研究的目标是揭示 甾醇对人LDL受体基因转录的调控 细胞拟议的研究涉及一个具体问题， 胆固醇调节LDL受体基因的机制，通过混合 生物化学和分子生物学技术与酵母遗传学。的 所提议的方法只是最近才可用。 所有试图鉴定参与固醇调节的蛋白质的尝试， 这种受体基因使用常规的体外方法都失败了。 早些时候，我们已经提供了甾醇在体内作用的证据， 调节元件在控制低密度脂蛋白受体基因表达， 甾醇类。现在，在体内启动子中发生的蛋白质-DNA相互作用 低密度脂蛋白受体基因的区域对固醇的反应将是 研究了使用体内足迹技术的初步结果是 非常令人鼓舞，这些足迹在甾醇中的意义 LDL受体基因的调控将在未来建立 实验此外，我们的调查将提供机会， 分离参与以下过程的生理相关关键因子的cDNA： 通过开发新的体内LDL受体基因的甾醇调控， 酵母中的遗传筛选系统。我们将使用人LDL受体基因 启动子置于两个不同的报告基因的上游，以筛选 使用酵母的SREBP cDNA克隆。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SRE-1依赖性方式的基因表达，以及第二系统， 生长选择系统，利用HIS 3报告基因。 两个系统 提供了一种紧密的“开-关”遗传选择， 克隆了难转录因子的cDNA。 最后， 实验还旨在了解分子机制， 细胞因子对低密度脂蛋白受体基因转录的诱导及其作用 在人体细胞中被固醇抑制。 这些已被列为一个 在人LDL受体基因中观察到的新体内足迹的结果 启动子 有趣的是，体内足迹研究已经确定了一个 LDL受体基因启动子中的一个新区域， 没有甾醇，并含有两个转录的共有序列 因子结合位点（Ets和CREBP），其活性由 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 特征性但重要的甾醇反应基因，具有复杂的 发起人。