FACTORS REGULATING GAMMA-GLOBIN GENE VIA ITS CACCC BOX

描述(由申请人提供)：人类伽马-珠蛋白基因启动子中的CACCC元件对于从伽马-珠蛋白到β-珠蛋白的转换是必不可少的，这是一个具有临床意义的发育过程。描述了两种互补的方法来确定与伽马珠蛋白CACCC元件结合并调节该基因的因子(S)。第一个目标是功能候选基因方法。红系Kruppel样因子(EKLF)通过其CACCC元件调控β-珠蛋白基因。γ-珠蛋白CACCC结合调节因子可能类似于EKLF。在系统发育分析中，有21个C2H2锌指蛋白与EKLF形成一个亚组。这些蛋白的几个mRNAs在鸡的原代红系细胞以及人红系K562和HEL细胞中表达。KLF2、KLF4、KLF5和KLF9基因敲除小鼠是可用的。这些小鼠将与具有人类β-珠蛋白基因的小鼠杂交，这些KLF对珠蛋白基因表达的影响将被确定。将在K562和HEL细胞中使用RNA干扰(RNAi)来敲除KLF3和8，这两个基因在红系细胞中表达，并对其有抗体可用。对于那些在这些检测中影响伽马珠蛋白基因表达的蛋白质，将在凝胶迁移率变化分析中确定它们是否在体外与伽马和/或β-珠蛋白CACCC元件结合，在染色质免疫沉淀(ChIP)分析中确定它们是否与体内结合。在第二个目标中，将以一种无偏见的方法识别和表征伽马珠蛋白CACCC结合因子。核提取物将从纯化的成年鸡红细胞和9-DPC胚胎中制备。鸡肉系统是理想的，因为红系细胞是有核的，很容易获得，但如果有必要，将使用另一种方法处理小鼠胚胎肝脏。核提取液将进行亲和纯化，以丰富γ-珠蛋白CACCC结合蛋白。与带有野生型寡核苷酸的亲和柱结合的蛋白质，而不是与具有CACCC盒突变的类似寡核苷酸结合的蛋白质，将通过二维凝胶电泳法进行区分。这些蛋白质将被切除，并接受MALDI-TOF和/或电喷雾电离串联质谱仪。人类与鸡(或鼠)蛋白的同源物将通过与数据库的蛋白质序列比较来识别。为了确定确定的蛋白质(S)在伽马珠蛋白基因调控中的重要性，将进行RNA干扰、DNA结合和芯片分析。

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.