REGULATION OF ERYTHROPOIETIN GENE

描述：此应用程序寻求项目的竞争性续订 专注于组织中促红细胞生成素基因的调节 培养模型：Hep3B细胞。在过去的5年里，调查人员 已经表明，通过一种合成方法诱导促红细胞生成素 低氧涉及稳态mRNA水平的100倍增加，由于 很大程度上归因于增强转录。一种血红素蛋白是一种主要的媒介。 归纳法的一部分。该组织和其他人还牵涉到两个DNA 促红细胞生成素结构基因两侧的序列元件。一个躺在一个 最小启动子117个碱基，第二个启动子为40个碱基最小增强子3‘ 到多聚腺苷酸化部位。两者都必须相互作用才能进行调解 增加了转录。每个位点都包含六核苷酸共识 参与核受体结合的序列 转录因子家族(如甲状腺受体)。初步数据 包括在本申请中表明孤儿受体HNF-4是 很可能参与其中，而且可能与另一个因素相互作用 称为HIF-1。 该提案需要5年的支持才能继续、完善和扩展 这一初步进展。第一个具体目标将集中在 继续表征顺式作用的DNA序列元件和 与EPO基因相互作用以促进 高效的转录。这些都是标准的实验，涉及 使用报告基因、假定的启动子和增强子元件，以及 被发现具有功能活性的元素的诱变版本。这个 第二个特定目标将探索核受体的潜在作用 在促红细胞生成素基因调控方面，特别关注HNF-4。其基本原理是 对于这些研究来说，基因的关键侧翼区域 包含这个蛋白质家族的识别位点，这些 至少根据凝胶滞留分析的定义，结合部位可以与蛋白质结合。 第三个具体目标是利用这两个杂交系统来克隆基因。 编码Hep-3B细胞中与HNF-4结合的蛋白质。调查人员 假设HNF-4结合伙伴的鉴定应该澄清 在血红素蛋白感受器和 与EPO基因直接结合的元件。第四个具体目标是 继续尝试鉴定和表征血红素结合蛋白 这是氧气感应的最初步骤。最后，具体 目标5将探索促红细胞生成素的遗传缺陷 对四名已被确认为终身患者的患者的管理 红细胞增多和血浆促红细胞生成素水平升高。促红细胞生成素基因的产生 从这些患者中提取的细胞，并进行DNA序列分析 这些个体的基因组DNA将被执行。

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.