REGULATION OF ERYTHROPOIETIN PRODUCTION

红细胞生成调控中一个主要的未解之谜是 是缺氧引发增加的 促红细胞生成素（Epo）。 在胎儿中，肝脏是Epo的主要部位 合成，而出生后生产转移到肾脏。 我们有 显示人肝癌细胞系Hep 3B产生高水平的Epo 缺氧或钴处理后的mRNA和蛋白质 氯化物，一种先前显示在体内增加Epo产生的试剂。 核径流和放线菌素追踪实验将在 为了确定Epo mRNA的调节是否仅在 转录水平。 Epo的顺式作用调控元件 将研究基因的瞬时和稳定表达 实验使用Hep 3B和对照细胞系转染非- Epo基因的编码区连接到报告基因（人生长 激素）。 生长激素的生产后的挑战， 缺氧或钴将在用 全面的建设。 DNA酶敏感位点的定位 将提供关于基因的独立信息， 监管要素。 这项建议的另一个主要目的是 更好地了解氧传感器的生化性质。 已经设计了实验来测试传感器是 一种血红素蛋白，其构象取决于 卟啉环以及它是否与氧结合。 反式作用 调节Epo基因的因素将通过比较 从对照细胞制备的核提取物与 钴或缺氧。 将进行凝胶阻滞和足迹分析。 用于鉴定显示缺氧的Epo DNA区域， 钴特异性蛋白结合。 这些发现将与 上述横切实验的结果。 绵延的 似乎起调节元件作用的DNA将附着在 琼脂糖凝胶，用于亲和纯化特异性反式作用DNA结合 proteins. 特别注意将集中在含血红素的蛋白质。 Hep 3B细胞系对缺氧和钴的Epo反应是 与体内观察到的结果相当。 因此，它是一种理想的模型 系统，以获得对分子电路的连贯理解， 从缺氧信号到Epo基因表达增强。

One of the major unanswered questions in the regulation of erythropoiesis is the mechanism by which hypoxia triggers increased production of erythropoietin (Epo). In the fetus, the liver is the primary site of Epo synthesis whereas after birth production shifts to the kidney. We have shown that the human hepatoma cell line Hep3B produces high levels of Epo mRNA and protein following exposure to hypoxia or treatment with cobalt chloride, an agent previously shown to increase Epo production in vivo. Nuclear run-off and actinomycin chase experiments will be performed in order to ascertain whether the regulation of Epo mRNA is solely at the transcriptional level. The cis-acting regulatory elements of the Epo gene will be investigated in both transient and stable expression experiments using Hep3B and control cell lines transfected with non- coding regions of the Epo gene linked to a reporter gene (human growth hormone). The production of growth hormone following challenge with hypoxia or cobalt will be measured following transfection with a comprehensive set of constructions. Mapping of the DNAse sensitive sites of the EPO gene will provide independent information on the gene's regulatory elements. An additional major aim of this proposal is to better understand the biochemical nature of the oxygen sensor. Experiments have been designed to test the hypothesis that the sensor is a heme protein whose conformation depends on the metal atom within the porphyrin ring and whether it is bound to oxygen. The trans-acting factors that regulate the Epo gene will be investigated by comparison of nuclear extracts prepared from control cells versus those challenges with cobalt or hypoxia. Gel retardation and footprinting analyses will be employed to identify those regions of Epo DNA that display hypoxia- and cobalt-specific protein binding. These findings will be correlated with the results of the transection experiments described above. Stretches of DNA that appear to function as regulatory elements will be attached to Sepharose and used to affinity purify specific trans-acting DNA binding proteins. Special attention will focus on heme-containing proteins. The Epo response of the Hep3B cell line to hypoxia and cobalt is comparable to that observed in vivo. Therefore it is an ideal model system to gain a coherent understanding of the molecular circuit leading from the hypoxia signal to enhanced expression of the Epo gene.