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

K562 细胞中控制珠蛋白基因表达的反式作用因子

• 批准号: 3875730
• 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/3875730
• 关键词: DNA binding protein; DNA footprinting; gel electrophoresis; gene expression; genetic manipulation; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; globin; human tissue; 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. Sequence specific DNA binding protein acting on cis-regulatory elements have been hypothesized to be key elements in eukaryotic gene transcription, and even though considerable progress has been made in their isolation, only one DNA binding proteins with affinity for the human globin gene promoters have been identified. We have defined several positive and negative regulatory regions 5' to the epsilon-globin promoter, and detected binding of proteins to these regions. 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 e-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 silencer binding protein ABF1. Possible mechanisms by which these proteins may regulate epsilon-globin gene transcription in erythroid and non-erythroid cells are discussed.

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表明 这种表达模式的控制是由 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。序列特异性 作用于顺式调节元件的DNA结合蛋白已经被发现， 假设是真核基因转录的关键元件，甚至 尽管在分离它们方面已经取得了相当大的进展，但只有一个DNA 对人珠蛋白基因启动子具有亲和力的结合蛋白具有 被识别。我们已经定义了几个积极和消极的监管 ε-珠蛋白启动子的5 '区，并检测到蛋白质的结合 到这些地区。 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e-珠蛋白基因的帽位点。我们还证明了 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 沉默子与酵母沉默子识别的相同序列结合 结合蛋白ABF1。这些蛋白质可能 调节红系和非红系中ε-珠蛋白基因转录 细胞进行了讨论。