REGULATION OF ERYTHROPOIETIN GENE

描述：此申请寻求项目的竞争性续期 它关注于组织中促红细胞生成素基因的调节， 培养模型Hep 3B细胞。 在过去的五年里，研究人员 已经表明，通过促红细胞生成素的合成， 缺氧涉及稳态mRNA水平增加100倍， 主要是为了增强转录。 血红素蛋白是一种主要的介质 的诱导。 这个小组和其他人也暗示了两个DNA Epo结构基因侧翼的序列元件。 一个躺在 117 bp的最小启动子，第二个在40 bp的最小增强子3'端， 到多聚腺苷酸化位点。 两者必须相互作用， 增加转录。 每个位点含有六核苷酸共有序列 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 它可能与其他因素相互作用， 称为HIF-1。 该提案要求5年的支持，以继续，完善和扩展 这一初步进展。 第一个具体目标将重点关注 顺式作用DNA序列元件的继续表征， 与Epo基因相互作用的反式作用蛋白， 高效转录 这些是标准实验， 使用报告基因、推定的启动子和增强子元件，和 发现具有功能活性的元素的诱变形式。 的 第二个具体目标将探索核受体的潜在作用 在Epo基因调控中，特别关注HNF-4。 的理由 基因的关键侧翼区域 含有这个蛋白质家族的识别位点， 位点与蛋白质结合，至少如凝胶阻滞测定所定义。 第三个具体目标将利用双杂交系统克隆基因 在Hep-3B细胞中编码与HNF-4结合的蛋白质。 调查人员 假设HNF-4结合伴侣的鉴定应该阐明 在血红素蛋白传感器和血红素蛋白传感器之间的途径中的附加步骤， 直接与Epo基因结合的元件。 第四个具体目标将 继续尝试鉴定和表征血红素结合蛋白 介导氧气感应的初始步骤。 最后，具体 目的5将是一个探索的遗传缺陷的红细胞生成素 在四名已被确定为终身 红细胞增多和血浆Epo水平升高。 Epo mRNA产生 通过这些患者的细胞，和DNA序列分析， 将对这些个体的基因组DNA进行检测。

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.