FACTORS REGULATING GAMMA-GLOBIN GENE VIA ITS CACCC BOX

通过 CACCC 盒调节伽马珠蛋白基因的因素

• 批准号: 6777960
• 负责人: JOYCE A. LLOYD
• 金额: $ 15万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-15 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6777960
• 关键词: RNA interference; affinity chromatography; binding proteins; cell line; chick embryo; chromatin immunoprecipitation; developmental genetics; electrospray ionization mass spectrometry; gel mobility shift assay; gene expression; genetic regulation; genetically modified animals; globin; laboratory mouse; matrix assisted laser desorption ionization; protein sequence; transcription factor; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): The CACCC element in the human gamma-globin gene promoter is essential for gamma- to beta-globin switching, a developmental process with clinical relevance. Two complementary approaches are described to identify the factor(s) that binds to the gamma-globin CACCC element and regulates the gene. The first aim is a functional candidate gene approach. The erythroid Kruppel-like factor (EKLF) regulates the beta-globin gene through its CACCC element. The gamma-globin CACCC-binding regulator is likely to be similar to EKLF. There are 21 C2H2 zinc finger proteins that form a subgroup with EKLF in phylogenetic analyses. Several of the mRNAs for these proteins are expressed in primary erythroid cells from the chick, and in human erythroid K562 and HEL cells. Knockout mice are available for KLF2, 4, 5 and 9. These mice will be bred with mice with the human beta-globin locus, and the effects of these KLFs on globin gene expression will be determined. RNA interference (RNAi) will be used in K562 and HEL cells to knockdown KLF3 and 8, which are expressed in erythroid cells and for which antibodies are available. For those proteins that affect gamma-globin gene expression in these assays, it will be determined whether they bind to the gamma- and/or beta-globin CACCC elements in vitro in gel mobility shift assays and in vivo in chromatin immunoprecipitation (CHIP) assays. In the second aim, gamma-globin CACCC binding factors will be identified and characterized in a non-biased approach. Nuclear extracts will be prepared from purified chicken red blood cells from adults and 9-dpc embryos. The chicken system is ideal because erythroid cells are nucleated and readily available, but if necessary an alternative approach with mouse fetal livers will be used. The nuclear extracts will be affinity-purified to enrich for gamma-globin CACCC binding proteins. Proteins that bind to an affinity column with a wild-type oligonucleotide, but not to a similar oligonucleotide with a CACCC box mutation, will be distinguished by 2-D gel electrophoresis. These proteins will be excised and subjected to MALDI-TOF and/or electrospray ionization tandem mass spectrometry. The human homolog for the chicken (or mouse) protein will be identified using protein sequence comparisons to databases. To determine the importance of the identified protein(s) in gamma-globin gene regulation, RNAi knock down, DNA binding, and ChIP assays will be performed.

描述（由申请人提供）：人 γ-珠蛋白基因启动子中的 CACCC 元件对于 γ- 至 β-珠蛋白转换至关重要，这是一个具有临床相关性的发育过程。描述了两种互补的方法来识别与伽玛球蛋白 CACCC 元件结合并调节该基因的因子。第一个目标是功能性候选基因方法。红系 Kruppel 样因子 (EKLF) 通过其 CACCC 元件调节 β-珠蛋白基因。 γ-珠蛋白 CACCC 结合调节因子可能与 EKLF 相似。在系统发育分析中，有 21 个 C2H2 锌指蛋白与 EKLF 形成一个亚组。这些蛋白质的一些 mRNA 在鸡的原代红系细胞以及人红系 K562 和 HEL 细胞中表达。可提供 KLF2、4、5 和 9 的基因敲除小鼠。这些小鼠将与具有人类 β-珠蛋白基因座的小鼠交配，并确定这些 KLF 对珠蛋白基因表达的影响。 RNA 干扰 (RNAi) 将用于 K562 和 HEL 细胞，以敲低 KLF3 和 8，这两种细胞在红细胞中表达，并且已有抗体可用。对于在这些测定中影响γ-珠蛋白基因表达的那些蛋白质，将在体外凝胶迁移率变动测定中和体内染色质免疫沉淀（CHIP）测定中确定它们是否与γ-和/或β-珠蛋白CACCC元件结合。在第二个目标中，将以无偏见的方法鉴定和表征 γ-珠蛋白 CACCC 结合因子。核提取物将从成人和 9-dpc 胚胎的纯化鸡红细胞中制备。鸡系统是理想的，因为红细胞有核并且容易获得，但如果有必要，将使用小鼠胎儿肝脏的替代方法。核提取物将进行亲和纯化以富集 γ-珠蛋白 CACCC 结合蛋白。与具有野生型寡核苷酸的亲和柱结合但不与具有 CACCC 盒突变的类似寡核苷酸结合的蛋白质将通过 2-D 凝胶电泳进行区分。这些蛋白质将被切除并进行 MALDI-TOF 和/或电喷雾电离串联质谱分析。鸡（或小鼠）蛋白质的人类同源物将通过与数据库的蛋白质序列比较来鉴定。为了确定已识别蛋白质在 γ-球蛋白基因调控中的重要性，将进行 RNAi 敲低、DNA 结合和 ChIP 测定。