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病中，症状的严重程度可以通过增强成人红系细胞中胎儿γ-珠蛋白基因的表达来改善，其中γ-基因通常是沉默的。本申请提出检验以下假设：包括psi/β-珠蛋白基因增强子和启动子和Alu重复DNA以及β-珠蛋白启动子的Δ γ-和δ-珠蛋白基因之间的基因间DNA通过与γ-启动子竞争结合以有限量存在于成体红细胞中的特异性转录因子（TF）而参与γ-基因的沉默。两个新的系统将用于研究：在体外培养的人红系祖细胞CFU-E细胞从成人血液和转基因（Tg）斑马鱼窝藏100 kb的BAC DNA跨越整个人类β-珠蛋白基因座，其中γ-珠蛋白基因在红系分化和发育过程中进行沉默。具体目标是：（1）.鉴定调节γ-珠蛋白基因失活的TF并评估其在从CFU-E到成红细胞的人红细胞生成期间的相对丰度，通过i）微阵列分析以鉴定在CFU-E和成红细胞中差异表达的已知的以及新的和未知的TF，ii）在CFU-E细胞中过表达这些TF以确定其对γ-基因表达的活性，iii）用CFU-E和成红细胞的核提取物进行凝胶迁移率变动测定，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，和iv）染色质免疫沉淀（CHIP），以在β-珠蛋白基因座中绘制这些TF组装成pol II或pol III转录机器的体内变化。2）。在Tg斑马鱼个体发育过程中调节人类γ-珠蛋白基因失活的顺式DNA元件的鉴定。通过Cre-loxP介导的重组，将整合的人BAC DNA中的内含子DNA和珠蛋白启动子在体内缺失。将通过定量RTPCR和CHIP研究缺失对γ-基因表达和对TF组装成人珠蛋白基因座处的转录机器的影响。建立和了解γ-珠蛋白基因沉默的体外人成红细胞生成和Tg斑马鱼模型将为检测设计用于调节γ-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.