Mechanisms of HbF Activation by Non-deletional HPFH

描述（由申请人提供）：了解人类γ-到β-珠蛋白基因开关的分子机制在镰状细胞病（SCD）的治疗中一直被认为是重要的，因为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。因此，了解如何在成人红细胞生成中重新激活γ-珠蛋白（HbF）将有益于SCD患者。我们在这项研究的目的是了解γ-珠蛋白基因沉默在成人阶段的最终红细胞生成。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，这些靶点可以通过上调γ-珠蛋白的合成来治疗SCD。胎儿血红蛋白（HPFH）点突变的非缺失遗传持久性可能是γ-珠蛋白基因抑制和激活机制的重要信息，但迄今为止尚未在机制水平上进行广泛研究。该研究将验证以下假设：HPFH突变通过取消通常位于γ-珠蛋白基因或位点远端基因间区域的转录抑制复合物（“抑制体”）成分的募集，或通过创造一个有利的染色质结构，使γ-珠蛋白基因在与位点控制区（LCR）的相互作用中部分胜过β-珠蛋白基因，从而阻止沉默或维持γ-珠蛋白基因表达的激活。或两者兼而有之。在人β-珠蛋白位点酵母人工染色体（β-YAC）中引入了几种HPFH突变，包括-566、-195、-175和-117，a - γ-珠蛋白非缺失点突变，并产生了转基因小鼠。在Specific Aim 1中，我们将通过小鼠模型和分子生物学/生化方法研究- 566、-195、-175和-117 HPFH突变在体内发育过程中破坏γ-珠蛋白基因抑制和/或改变β-珠蛋白位点染色质结构域的机制，包括转基因结构/表达研究、3C、组蛋白修饰、DNase I敏感性、共激活物/抑制物募集、Bcl11A募集以及HPFH突变之间的协同作用。在特异性目标2中，我们将使用分离染色质片段的蛋白质组学（PICh）和细胞培养氨基酸的稳定同位素标记（SILAC）结合质谱（MS）来研究这四种HPFH突变如何改变a γ-球蛋白启动子中的dna结合蛋白复合物。在Specific Aim 3中，我们将在a γ-珠蛋白基因启动子中产生并鉴定新的HPFH突变，使用基于细胞的报告基因试验来选择HPFH突变，然后在β-YAC转基因小鼠中对这些突变进行表型表征，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，可以开发出高度特异性的治疗方法，以增加治疗SCD的HbF，而没有广谱治疗相关的副作用。

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms underlying the human γ- to β-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 γ-globin (HbF) in adult erythropoiesis will benefit SCD patients. Our goal in this study is to understand γ-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 γ-globin synthesis to treat SCD. Non-deletional hereditary persistence of fetal hemoglobin (HPFH) point mutations are likely to be highly informative regarding mechanisms of γ-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 γ-globin gene expression by abrogating recruitment of transcriptional repressor complex ("repressosome") components normally located at the γ-globin gene or more distal intergenic regions of the locus, or alternately, by creating a favorable chromatin structure that allows the γ-globin genes to partly outcompete the β-globin gene for interaction with the locus control region (LCR), or both. Several HPFH mutations have been introduced into our human β-globin locus yeast artificial chromosome (β-YAC) including the -566, -195, -175, and -117 , Aγ-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 γ-globin gene repression and/or alter the β-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 Aγ-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 Aγ-globin gene promoter using a cell-based reporter assay to select for HPFH mutations, followed by phenotypic characterization of these mutations in β-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.