REGULATION OF ERYTHROPOIETIN GENE

促红细胞生成素基因的调控

• 批准号: 2141655
• 负责人: H. Franklin Bunn
• 金额: $ 47.44万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2141655
• 关键词: DNA binding protein; erythropoiesis; erythropoietin; gel mobility shift assay; genetic enhancer element; genetic promoter element; genetic regulation; genetic transcription; heme; hybrid cells; hypoxia; messenger RNA; molecular site; nucleic acid sequence; receptor binding; reporter genes; tissue /cell culture; transcription factor

DESCRIPTION: This application seeks competitive renewal of a project that has focused upon regulation of the erythropoietin gene in a tissue culture model Hep3B cells. During the past 5 years, the Investigators have shown that the induction of erythropoietin by a synthesis by hypoxia involves a 100 fold increase in steady state mRNA levels, due largely to enhanced transcription. A heme protein is a primary mediator of the induction. This group and others have also implicated two DNA sequence elements flanking the structural gene for Epo. One lies in a 117 bp minimal promoter, and the second in a 40 bp minimal enhancer 3' to the polyadenylation site. Both must interact in order to mediate increased transcription. Each site contains hexanucleotide consensus sequences that are involved in the binding of the nuclear receptor family (eg thyroid receptor) of transcription factors. Preliminary data included in this application suggests that the orphan receptor HNF-4 is likely to be involved, and that it may interact with another factor called HIF-1. This proposal requests 5-year's support to continue, refine, and extend this preliminary progress. The first Specific Aim will focus on continued characterization of the cis-acting DNA sequence elements and the transacting proteins that interact with the Epo gene to promote efficient transcription. These are standard experiments involving the use of reporter genes, putative promoter and enhancer elements, and mutagenized versions of elements found to have functional activity. The second Specific Aim will explore the potential role of nuclear receptors in Epo gene regulation, with a particular focus on HNF-4. The rationale for these studies is that the critical flanking regions of the genes contain recognition sites for this family of proteins, and that these sites bind to proteins, at least as defined by gel retardation assays. The third Specific Aim will utilize the two hybrid system to clone genes encoding proteins in Hep-3B cells that bind to HNF-4. The Investigators postulate that identification of HNF-4 binding partners should elucidate additional steps in the pathway between the heme protein sensor, and the elements binding directly to the Epo gene. The fourth Specific Aim will continue attempts to identify and characterize the heme-binding protein that mediates the initial steps in oxygen sensing. Finally, Specific Aim 5 will be an exploration of genetic defects in erythropoietin regulation in four patients who have been identified to have lifelong erythrocytosis and elevated levels of plasma Epo. Epo mRNA production by cells derived from these patients, and DNA sequence analysis of genomic DNA from these individuals will be performed.

描述：本应用程序寻求竞争性续订项目 这重点是组织中红细胞生成素基因的调节 培养模型HEP3B细胞。 在过去的5年中，调查人员 已经表明，通过 缺氧涉及稳态mRNA水平100倍，应育 主要是增强的转录。 血红素蛋白是主要介体 归纳。 这个组和其他人也牵涉到两个DNA EPO结构基因的序列元件。 一个人在 117 bp最小启动子，第二个BP中的第二个最小增强剂3' 到多腺苷酸位点。 两者必须相互作用才能调解 转录增加。 每个站点都包含六核苷酸共识 与核受体结合有关的序列 转录因子的家族（例如甲状腺受体）。 初步数据 本应用程序中包括 可能参与，并且可能与另一个因素互动 称为HIF-1。 该提案要求5年的支持继续，完善和扩展 这个初步的进步。 第一个具体目标将重点放在 顺式作用DNA序列元件的持续表征和 与EPO基因相互作用以促进的交易蛋白 有效的转录。 这些是涉及的标准实验 使用记者基因，推定的启动子和增强子元素，以及 发现具有功能活性的元素的诱变版本。 这 第二个特定目标将探索核受体的潜在作用 在EPO基因调节中，特别关注HNF-4。 理由 对于这些研究，基因的关键侧翼区域 包含该蛋白质家族的识别站点，这些 位点与蛋白质结合，至少由凝胶延迟测定定义。 第三个特定目的将利用两个混合系统来克隆基因 编码与HNF-4结合的HEP-3B细胞中的蛋白质。 调查人员 假设HNF-4结合伙伴的识别应阐明 血红素蛋白传感器和途径的其他步骤 直接与EPO基因结合的元素。 第四个特定目标将 继续尝试识别和表征血红素结合蛋白 这介导了氧气传感的初始步骤。 最后，具体 AIM 5将探索促红细胞生成素的遗​​传缺陷 四个被确定患有终身的患者的调节 红细胞增多和血浆EPO水平升高。 EPO mRNA产生 通过源自这些患者的细胞，以及DNA序列分析 将进行这些个体的基因组DNA。