Gamma-globin gene silencing in human red cells

描述(由申请人提供)： 在地中海贫血和镰状细胞疾病中，症状的严重性可以通过增强成年红系细胞中胎儿伽马珠蛋白基因的表达而得到改善，在成人红细胞中，伽马基因通常是沉默的。这项应用旨在检验这样一种假设，即包括psi/β-珠蛋白基因增强子和启动子、Alu重复DNA以及β-珠蛋白启动子在内的α-和β-珠蛋白基因之间的基因间DNA通过与γ-启动子竞争结合存在于成人红系细胞中的有限数量的特定转录因子(TF)来参与伽马基因的沉默。这项研究将使用两个新的系统：从成人血液中获得的人类红系祖细胞CFU-E的体外培养和携带100 kb BAC DNA的转基因斑马鱼(TG斑马鱼)，其中伽马珠蛋白基因在红系分化和发育过程中经历沉默。具体目标是：(1)。鉴定调节γ-珠蛋白基因失活的因子，并通过i)微阵列分析识别在CFU-E和红细胞细胞中差异表达的已知的以及新的和未知的TF，通过i)微阵列分析来识别在CFU-E和红细胞细胞中差异表达的已知的以及新的和未知的TF，以确定这些TF在CFU-E细胞中过表达以确定它们对y基因表达的活性，iii)与CFU-E和红细胞细胞的核提取物进行凝胶迁移率变化分析，以研究TF与珠蛋白启动子和基因间DNA中的同源DNA基序的结合亲和力，以及iv)染色质免疫沉淀法(ChIP)，将这些转录因子在体内的变化映射到β-珠蛋白基因座上PolII或PolIII转录机制中。2)。转基因斑马鱼个体发育过程中调节人γ-珠蛋白基因失活的顺式DNA元件的鉴定。Cre-loxP介导的重组将在体内从整合的人BAC DNA中删除IL-DNA和珠蛋白启动子。这些缺失对伽马基因表达的影响，以及对转录因子在人类珠蛋白基因座上转录机制的组装的影响，将通过定量RT-PCR和芯片进行研究。建立和理解体外人类成年红细胞生成和转基因斑马鱼的伽马珠蛋白基因沉默模型将为测试旨在调节伽马珠蛋白的药物化合物提供新的靶点和新的系统。

DESCRIPTION (provided by applicant): In thalassemia and sickle cell disease the severity of symptoms can be ameliorated by enhanced expression of the fetal gamma-globin genes in adult erythroid cells in which the gamma-genes are normally silenced. This application proposes to test the hypothesis that the intergenic DNA between the Agamma- and delta-globin genes including the psi/beta-globin gene enhancer and promoter and the Alu repetitive DNAs as well as the beta-globin promoter participates in the silencing of gamma-genes by competing with the gamma-promoter for binding with specific transcription factors (TFs) present in limited amounts in adult erythroid cells. Two new systems will be used for the study: in vitro culture of human erythroid progenitor CFU-E cells obtained from adult blood and transgenic (Tg) zebrafish harboring 100 kb of BAC DNA spanning the entire human beta-globin gene locus, in which the gamma-globin genes undergo silencing during erythroid differentiation and development. The specific aims are: (1). Identification of TFs that regulate gamma-globin gene inactivation and assess their relative abundance during human erythropoiesis from CFU-E to erythroblast cells by i) microarray analysis to identify known and also new and unknown TFs differentially expressed in CFU-E and erythroblast cells, ii) over-expressing these TFs in CFU-E cells to determine their activities on y-gene expression, iii) gel mobility shift assays with nuclear extracts of CFU-E and erythroblast cells to study the binding affinities of the TFs to cognate DNA motifs in the globin promoters and the intergenic DNAs, and iv) Chromatin immunoprecipitation (CHIP) to map in vivo changes in the assembly of these TFs into pol II or pol III transcriptional machinery in the beta-globin gene locus. 2). Identification of cis-DNA elements that regulate inactivation of human gamma-globin genes during ontogeny in Tg zebrafish. Interginic DNA and globin promoters will be deleted in vivo from the integrated human BAC DNA by Cre-loxP mediated recombination. The effects of the deletions on gamma-gene expression and on the assembly of the TFs into transcriptional machinery at the human globin gene locus will be studied by quantitative RTPCR and CHIP. Establishing and understanding the in vitro human adult erythropoiesis and Tg zebrafish models of gamma-globin gene silencing will provide new targets and new systems to test pharmacological compounds designed to regulated gamma-globin.