OXYGEN SENSING AND ERYTHROPOIETIN GENE REGULATION

当前提案的目标是了解这些机制 人促红细胞生成素基因表达增加的基础 缺氧。这些研究的具体目标是确定和 描述其背后的DNA序列和核蛋白 回应。Hep 3B细胞株被证明能显著增加EPO 在低氧条件下生产。使用荧光素酶报告系统 在Hep 3B细胞中的转染研究，我们已经定位了一种缺氧- 促红细胞生成素基因3‘端24个碱基对的可诱导增强子 侧翼序列。为了进一步定义负责的序列 对于这种反应，将使用以下方法进行转染性研究 该24个碱基片段的截短和点突变衍生。点 将根据结果设计待测试的突变片段 用截断突变体获得的，并根据在 具有已知转录因子结合位点的增强子序列。在……里面 为了识别和表征DNA与蛋白质之间的相互作用 将进行低氧反应元件、凝胶迁移率变化研究 利用24bp片段探测常氧大鼠核蛋白提取液 以及低氧的Hep 3B细胞，来自其他产生EPO的细胞系(Hep G2 和RC-3)和不产生EPO的对照细胞系。这个 蛋白质结合的特异性将通过凝胶移位分析来确定。 使用未标记的正常和突变的24bp增强子片段作为 竞争对手。为了定义DNA-蛋白质相互作用 针对缺氧，核抽提物将用于脱氧核糖核酸酶 我对该基因的这一区域进行了研究。因为在体外 足迹研究只能接近生活中的实际情况 细胞，体内足迹研究也将进行，以表征 Hep-3B中EPO基因这一部分的DNA-蛋白质相互作用 常氧和低氧条件下的细胞。为了分离基因 编码调节EPO基因对低氧反应的蛋白质，以及 低氧条件下Hep 3B细胞基因表达文库的构建 并将用含有结合的寡核苷酸探针进行筛选 网站寻找指导这一反应的因素。隔离和 对这些监管因素进行表征将导致更好的 对氧调节基因潜在机制的认识 表情。

The objective of the current proposal is to understand the mechanisms underlying the increased expression of the human Epo gene in response to hypoxia. The specific goal of these studies is to identify and characterize the DNA sequences and nuclear proteins underlying this response. The Hep 3B cell line has been shown to markedly increase Epo production with hypoxia. Using a luciferase reporter system in transfection studies in Hep 3B cells, we have localized a hypoxia- inducible enhancer to a 24 base pair (bp) portion of the Epo gene 3' flanking sequence. In order to further define the sequences responsible for this response, transfection studies will be performed using truncation and point mutation derivatives of this 24 bp fragment. Point mutation fragments to be tested will be designed based on results obtained with truncation mutants and on the presence of homology in the enhancer sequence with known transcription factor binding sites. In order to identify and characterize DNA-protein interactions in the hypoxia-responsive element, gel mobility shift studies will be performed using the 24 bp fragment to probe nuclear protein extracts from normoxic and hypoxic Hep 3B cells, from other cell lines that produce Epo (Hep G2 and RC-3) and from control cell lines which do not make Epo. The specificity of protein binding will be determined by gel shift assays using unlabeled normal and mutant 24 bp enhancer fragments as competitors. In order to define DNA-protein interactions that are specific to hypoxia, nuclear extracts will be used in Deoxyribonuclease I footprint studies of this region of the gene. Since in vitro footprinting studies can only approximate the actual situation in living cells, in vivo footprint studies will also be performed to characterize DNA-protein interactions in this portion of the Epo gene within Hep 3B cells under normoxic and hypoxic conditions. In order to isolate genes encoding the proteins that regulate Epo gene response to hypoxia, an expression cDNA library will be constructed from hypoxic Hep 3B cell mRNA and will be screened with oligonucleotide probes that contain binding sites for the factors that direct this response. Isolation and characterization of these regulatory factors would lead to a better understanding of the mechanisms underlying oxygen-regulated gene expression.