TRANS-ACTING FACTOR(S) CONTROLLING GLOBIN GENE EXPRESSION IN K562 CELLS & MICE

K562 细胞中控制珠蛋白基因表达的反式作用因子

• 批准号: 3854693
• 负责人: A N SCHECHTER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3854693
• 关键词: DNA binding protein; DNA footprinting; HeLa cells; gel electrophoresis; gene expression; genetic manipulation; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; globin; hemoglobin; human genetic material tag; human tissue; laboratory mouse; mutant; neoplastic cell culture for noncancer research; nucleic acid sequence; transcription factor

K562 is an erythroleukemic cell line used as a model for the study of the control of human globin gene expression. These cells do not support transcription of the beta-globin gene but do express transcripts of epsilon- and gamma-globin genes at very high levels when exposed to a number of inducing agents. Results from this and other laboratories suggest that the control of this pattern of expression is mediated by the presence and/or absence of trans-acting factors which exert their action on sequences corresponding to the promoters of these genes. We have previously reported the presence of a transcriptional control element with properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin gene. We also showed that this silencer has stronger inhibitory activity in HeLa cells than K562 human erythroleukemia cells. Using deletion mutants and synthetic oligonucleotides in transient expression assays, DNA sequences responsible for this effect have been further delimited to 44 nucleotides located between -294 and -251 bp. Gel electrophoresis mobility shift assays and DNasel footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. The protein present in K562 cells, but not in HeLa cells, that interacts specifically with this silencer binds to the same sequence recognized by the yeast binding protein ABF1. Possible mechanisms by which these proteins may regulate epsilon- globin gene transcription in erythroid and non-erythroid cells are discussed. We have constructed several mice using transgenic techniques that express both the human alpha-globin and beta-S-globin polypeptides at levels of 15- 20% of endogenous mouse hemoglobin. Data on erythrocyte functional properties now being studied include morphological analysis oxygen affinity curves, hematological parameters and rheology. Further cross breeding studies should result in a line of transgenic animals manifesting the sickle cell phenotype.

K562是一种红白血病细胞系，用作研究白血病细胞的模型。 人珠蛋白基因表达的调控。 这些细胞不支持 β-珠蛋白基因的转录，但不表达 当暴露于一种 诱导剂的数量。 这个实验室和其他实验室的结果 这表明，这种表达模式的控制是介导的， 存在和/或不存在反式作用因子， 对应于这些基因的启动子的序列。 我们以前曾报道存在一个转录控制 从-392到-177 bp具有沉默器特性的元件 相对于人ε-珠蛋白基因的帽位点。 我们还展示 该沉默子对HeLa细胞的抑制活性强于K562 人红白血病细胞 使用缺失突变体和合成的 瞬时表达试验中的寡核苷酸， 对于这种效应，已经进一步限定为44个核苷酸， 在-294和-251 bp之间。 凝胶电泳迁移率变动分析和 DNA酶1足迹分析表明，这些负调控 序列被存在于细胞核中的蛋白质以不同的方式识别。 从HeLa和K562细胞获得的提取物。 K562中存在的蛋白质 细胞，但不是在HeLa细胞，这是相互作用，特别是与此 沉默子与酵母结合蛋白识别的相同序列结合 ABF1。 这些蛋白质可能通过哪些机制来调节蛋白质- 红系和非红系细胞中的珠蛋白基因转录是 讨论 我们用转基因技术构建了几只小鼠， 人α-珠蛋白和β-S-珠蛋白多肽在15- 20 μ g/ml的水平下， 20%的内源性小鼠血红蛋白。 红细胞功能数据 目前正在研究的性质包括形态分析氧亲和力 曲线、血液学参数和流变学。 进一步杂交育种 研究应该会产生一系列转基因动物， 镰状细胞表型