FACTORS REGULATING GAMMA-GLOBIN GENE VIA ITS CACCC BOX

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-珠蛋白至β-珠蛋白的转换（一种具有临床相关性的发育过程）至关重要。描述了两种互补的方法来鉴定与γ-珠蛋白CACCC元件结合并调节基因的因子。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元件调节β-珠蛋白基因。γ-珠蛋白CACCC结合调节剂可能与EKLF相似。在系统发育分析中，有21个C2 H2锌指蛋白与EKLF形成一个亚群。这些蛋白质的几种mRNA在来自鸡的原代红系细胞以及人红系K562和HEL细胞中表达。敲除小鼠可用于KLF 2、4、5和9。这些小鼠将与具有人β-珠蛋白基因座的小鼠交配，并将确定这些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KLF 3和8，它们在红系细胞中表达，并且可获得抗体。对于在这些试验中影响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试验和体内染色质免疫沉淀（CHIP）试验中确定它们是否与γ-和/或β-珠蛋白CACCC元件结合。在第二个目标中，γ-珠蛋白CACCC结合因子将被确定和表征在一个无偏见的方法。将从成年鸡和9-dpc胚胎的纯化鸡红细胞制备核提取物。鸡系统是理想的，因为红系细胞是有核的并且容易获得，但是如果必要的话，将使用小鼠胎肝的替代方法。将对核提取物进行亲和纯化以富集γ-珠蛋白CACCC结合蛋白。通过2-D凝胶电泳区分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。将这些蛋白质切除并进行MALDI-TOF和/或电喷雾电离串联质谱。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。为了确定已鉴定蛋白在γ-珠蛋白基因调控中的重要性，将进行RNAi敲除、DNA结合和ChIP试验。

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.