Globin Gene Switching: Molecular Control

描述(申请人提供)：这项建议的总体目标是研究人类血红蛋白转换的机制，描绘LCR中HSS的功能，并开发一种新的方法来研究人类血红蛋白转换的分子和细胞控制。在SA1中，我们将测试这一假设，即人类珠蛋白基因的切换是由发育时钟类型的机制控制的，该机制通过沿着贝塔珠蛋白基因座进行的一系列与时间相关的染色质修饰来操作：a)表现出与体内相似的时间过程的伽马到贝塔开关的人胎儿红系×MEL细胞杂交将被用来确定(通过高通量技术确定DNaseI敏感性、DNA甲基化和染色质组蛋白修饰)假定的发育时钟切换的位置。B)将使用BetaYAC×MEL杂交种来实验验证发育时钟的位置。C)影响假定时钟序列的人类突变将被用来测试YACxMEL或淋巴x GM-979杂交细胞模型中对开关的影响。在SA2中，我们将通过对不是由于贝塔基因序列突变引起的贝塔地中海贫血的研究来确定LCR调节元件的体内功能。将搜索106例现有病例，以寻找HSS核心元素的点突变或缺失。淋巴杂交将确定这些突变是否是β基因的顺式突变，以及是否与βmRNA缺乏症有关。YAC/MEL杂交车将用于验证地中海贫血的影响并研究其机制。我们还将测试这一假设，即某些β地中海贫血的表型是由于LCR突变导致该调节元件与下游伽马珠蛋白基因之间不稳定的相互作用。在SA3中，我们将开发一种新的方法用于体外研究血红蛋白开关的分子控制，该方法将基于人ES细胞与MEL细胞的融合，2)将允许在类似于体内人血红蛋白开关的时间框架内对epsilon到Gamma和Gamma到Beta开关的纵向分析，以及3)通过在人ES细胞中引入所需的调控元件的突变来极大地增强开关的分子分析，并测试它们对人ES细胞×MEL细胞杂交的开关的影响。

DESCRIPTION (provided by applicant): The overall goals of this proposal are to investigate the mechanisms of human hemoglobin switching, delineate the function of HSs of the LCR and develop a new approach for the study of the molecular and cellular control of human hemoglobin switching. In SA1, we will test the hypothesis that the human globin gene switching is controlled by a developmental clock-type of mechanism operating through a series of time-related chromatin modifications along the beta globin locus: a) human fetal erythroid x MEL cell hybrids which display a gamma to beta switch with a time course similar to that in vivo will be used to identify (with high throughput technologies determining DNAse I sensitivity, DNA methylation and chromatin histone modifications) the location of the postulated developmental clock of switching. b) betaYAC x MEL hybrids will be used to experimentally verify the location of the developmental clock. c) Human mutations affecting sequences of the postulated clock will be used to test effects on switching in the YACxMEL or lymphoid x GM-979 hybrid cell models. In SA2 we will determine the in vivo function of regulatory elements of the LCR with studies of beta thalassemias not due to mutations of beta gene sequences. 106 cases already available will be searched for point mutations or deletions of core elements of the HSs. Lymphoid x MEL hybrids will determine whether these mutations are in cis to the beta gene and are associated with beta mRNA deficiency. YAC/MEL hybrids will be used to verify the thalassemia effect and to investigate mechanisms. We will also test the hypothesis that the phenotype of certain delta beta thalassemias is due to LCR mutations causing unstable interactions between this regulatory element and the downstream gamma globin genes. In SA3 we will develop a new approach for the ex vivo investigation of the molecular control of hemoglobin switching which: 1) will be based on fusion of human ES cells with MEL cells, 2) will allow the longitudinal analysis of epsilon to gamma and gamma to beta switch in a time frame similar to that of the in vivo human hemoglobin switching and 3) will greatly enhance the molecular analysis of switching through introduction of desired mutations of regulatory elements in human ES cells and testing their effects on switching of human ES cells x MEL cell hybrids.