Mechanisms of HbF Activation by Non-deletional HPFH

描述(由申请人提供)：由于大量证据表明，胎儿血红蛋白(HBF)的增加显著降低了与该疾病相关的病理生理学，因此，了解人类球蛋白基因转换的潜在分子机制在治疗镰状细胞病(SCD)中一直被认为是重要的。因此，了解如何在成人红细胞生成中重新激活β-珠蛋白(HBF)将使SCD患者受益。我们在这项研究中的目标是了解？-珠蛋白基因在成虫阶段的沉默 绝对的红血球生成。我们的临床目标是根据这项研究的结果确定新的分子靶点，这些靶点可以通过治疗来调节β-珠蛋白的合成，从而治疗SCD。胎儿血红蛋白非缺失性遗传性持久性(HPFH)点突变可能对β-珠蛋白基因抑制和激活的机制有很高的信息量，但到目前为止还没有在机制水平上进行广泛的研究。这项拟议的研究将检验这样一种假设，即HPFH突变通过取消通常位于β-珠蛋白基因或基因座更远的基因间隔区的转录抑制复合体(“抑制体”)组件的招募，或者通过创造有利的染色质结构，允许-珠蛋白基因在与基因座控制区(LCR)的相互作用中部分地竞争-珠蛋白基因，从而防止-珠蛋白基因的沉默或维持-珠蛋白基因的激活。我们已将-566、-195、-175和-117等几个HPFH突变引入到我们的人珠蛋白基因座酵母人工染色体(YAC)中，并产生了转基因小鼠。在具体目标1中，我们将使用小鼠模型和分子生物学/生化方法，包括转基因结构/表达研究、3C、组蛋白修饰、DNase I敏感性、共激活/抑制因子招募、BCL11A招募和HPFH突变之间的协同作用，研究-566、-195、-175和-117 HPFH突变在体内发育过程中扰乱-珠蛋白基因抑制和/或改变-珠蛋白基因染色质区域的机制。在特定的目标2中，我们将使用分离的染色质片段的蛋白质组学(PICH)和细胞培养中的氨基酸稳定同位素标记(SILAC)结合质谱仪(MS)来研究这四个HPFH突变是如何改变A？珠蛋白启动子上的DNA结合蛋白复合体的。在具体目标3中，我们将使用基于细胞的报告分析来选择HPFH突变，并在A？珠蛋白基因启动子内产生和鉴定新的HPFH突变，然后在？-YAC转基因小鼠中对这些突变进行表型鉴定，并测定这些位点的DNA结合活性。我们从这些研究中获得的知识将揭示新的治疗靶点，针对这些靶点，可以开发高度特异性的治疗方法，以增加SCD治疗的HBF，而不会出现与广谱治疗相关的副作用。

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms underlying the human ?- to ¿-globin gene switch has long been recognized as important in the treatment of sickle cell disease (SCD), since a wealth of evidence has demonstrated that increased fetal hemoglobin (HbF) significantly decreases the pathophysiology associated with this disease. Thus, knowledge of how to reactivate ?-globin (HbF) in adult erythropoiesis will benefit SCD patients. Our goal in this study is to understand ?-globin gene silencing during the adult stage of definitive erythropoiesis. Our clinical goal is to identify new molecular targets based on the outcome of this study that can be modulated therapeutically for up-regulation of ?-globin synthesis to treat SCD. Non-deletional hereditary persistence of fetal hemoglobin (HPFH) point mutations are likely to be highly informative regarding mechanisms of ?-globin gene repression and activation, but to date have not been studied extensively at the mechanistic level. The proposed study will test the hypothesis that HPFH mutations prevent silencing or maintain activation of ?-globin gene expression by abrogating recruitment of transcriptional repressor complex ("repressosome") components normally located at the ?-globin gene or more distal intergenic regions of the locus, or alternately, by creating a favorable chromatin structure that allows the ?-globin genes to partly outcompete the ¿-globin gene for interaction with the locus control region (LCR), or both. Several HPFH mutations have been introduced into our human ¿-globin locus yeast artificial chromosome (¿-YAC) including the -566, -195, -175, and -117 ,?-globin non-deletional HPFH point mutations and transgenic mice have been produced. In Specific Aim 1, we will study the mechanisms by which the - 566, -195, -175, and -117 HPFH mutations disrupt ?-globin gene repression and/or alter the ?-globin locus chromatin domain during development in vivo using murine models and molecular biology/biochemical approaches including transgene structure/expression studies, 3C, histone modification, DNase I sensitivity, co-activator/repressor recruitment, Bcl11A recruitment, and synergy between the HPFH mutations. In Specific Aim 2 we will examine how these four HPFH mutations alter DNA-binding protein complexes in the A?-globin promoter using proteomics of isolated chromatin segments (PICh) and Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) coupled with mass spectrometry (MS). In Specific Aim 3, we will generate and identify novel HPFH mutations within the A?-globin gene promoter using a cell-based reporter assay to select for HPFH mutations, followed by phenotypic characterization of these mutations in ¿-YAC transgenic mice and determination of the DNA-binding activity at these sites. The knowledge we gain from these studies will reveal novel therapeutic targets for which highly-specific treatments may be developed to increase HbF for the treatment of SCD without the side-effects associated with broad-spectrum therapies.