OXYGEN SENSING AND ERYTHROPOIETIN GENE REGULATION

本提案的目的是了解 作为人类Epo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DNA序列和核蛋白的特征， 反应 Hep 3B细胞系已显示出显著增加Epo 生产缺氧。 使用荧光素酶报告系统， 在Hep 3B细胞中的转染研究中，我们已经定位了缺氧- Epo基因3 ′端24个碱基对（bp）部分的诱导型增强子 侧翼序列 为了进一步确定 对于这种反应，将使用以下方法进行转染研究： 该24 bp片段的截短和点突变衍生物。 点 根据结果设计待测突变片段 获得的截短突变体和同源性的存在下， 具有已知转录因子结合位点的增强子序列。 在 为了识别和表征DNA-蛋白质相互作用， 将进行缺氧响应元件、凝胶迁移率变化研究 用24 bp的片段来探测来自常氧环境的核蛋白提取物， 以及来自产生Epo的其它细胞系的缺氧Hep 3B细胞（Hep G2 和RC-3）和不产生Epo的对照细胞系。 的 蛋白结合的特异性将通过凝胶迁移分析来确定 使用未标记的正常和突变的24 bp增强子片段作为 竞争对手 为了定义DNA-蛋白质相互作用， 特异于缺氧，核提取物将用于脱氧核糖核酸酶 我对这个基因区域的足迹研究。 由于在体外 足迹研究只能近似于生活的实际情况 细胞，还将进行体内足迹研究，以表征 Hep 3B中Epo基因这一部分的DNA-蛋白质相互作用 细胞在常氧和缺氧条件下。 为了分离基因 编码调节Epo基因对缺氧反应的蛋白质， 以缺氧Hep 3B细胞mRNA为模板构建表达cDNA文库 并将用寡核苷酸探针进行筛选， 网站的因素，直接这种反应。 分离与 这些调节因素的特征将导致更好的 了解氧调节基因的机制 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.