Mechanisms of HbF Activation by Non-deletional HPFH

非缺失 HPFH 激活 HbF 的机制

• 批准号: 8721479
• 负责人: KENNETH R PETERSON
• 金额: $ 37万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8721479
• 关键词: 3-Dimensional; Adult; Adverse effects; Affect; African American; Amino Acids; Binding; Binding Sites; Biochemical; Biological Assay; Bone Marrow Cells; Cell Culture Techniques; Cells; Chromatin; Chromatin Structure; Chromosomes, Artificial, Yeast; Clinical; Complex; Coupled; DNA Binding; DNA-Binding Proteins; Data; Deoxyribonuclease I; Development; Disease; Distal; EMSA; Environment; Erythropoiesis; Ethylnitrosourea; Fetal Hemoglobin; Fluorescence-Activated Cell Sorting; Functional disorder; Gene Activation; Gene Expression; Gene Silencing; Genes; Globin; Goals; Greek; Green Fluorescent Proteins; Growth; Hemoglobin F Disease; Hereditary Disease; Human; Hypersensitivity; Intercistronic Region; Knowledge; Label; Lead; Light; Locus Control Region; Mass Spectrum Analysis; Modeling; Modification; Molecular; Molecular Biology; Molecular Conformation; Molecular Target; Mus; Mutagenesis; Mutation; Mutation Spectra; Nature; Nucleotides; Outcome Study; Patients; Phenotype; Point Mutation; Population; Process; Promoter Regions; Protein Binding; Proteins; Proteomics; Regulatory Element; Reporter; Reporting; Repression; Research; Resolution; Sickle Cell Anemia; Site; Stable Isotope Labeling; Staging; Stem cells; Structure; Switch Genes; Testing; Therapeutic; Transcription Repressor/Corepressor; Transgenes; Transgenic Mice; Up-Regulation; base; effective therapy; embryo/fetus; fetal; gene interaction; gene repression; hematopoietic tissue; histone modification; in vivo; melting; mutant; new therapeutic target; novel; prevent; promoter; protein complex; public health relevance; transgene expression

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.

描述（由申请人提供）：理解人类 α- 至 β-珠蛋白基因转换的分子机制长期以来被认为在镰状细胞病 (SCD) 的治疗中非常重要，因为大量证据表明胎儿血红蛋白 (HbF) 的增加可显着降低与该疾病相关的病理生理学。因此，了解如何重新激活成人红细胞生成中的β-珠蛋白（HbF）将使 SCD 患者受益。我们这项研究的目标是了解成年阶段的β-珠蛋白基因沉默。 确定的红细胞生成。我们的临床目标是根据本研究的结果确定新的分子靶点，可以通过治疗性调节来上调 β-珠蛋白合成来治疗 SCD。胎儿血红蛋白（HPFH）点突变的非缺失遗传性持久性可能对β-珠蛋白基因抑制和激活机制提供大量信息，但迄今为止尚未在机制水平上进行广泛研究。拟议的研究将测试以下假设：HPFH 突变通过消除通常位于 β-珠蛋白基因或更远端基因间区域的转录阻遏复合物（“阻抑体”）成分的募集来防止 β-珠蛋白基因表达沉默或维持激活，或者通过创建有利的染色质结构，允许 β-珠蛋白基因部分地表达 胜过 ¿-珠蛋白基因与位点控制区 (LCR) 的相互作用，或两者兼而有之。几种HPFH突变已被引入我们的人类Ф-珠蛋白位点酵母人工染色体(Ф-YAC)中，包括-566、-195、-175和-117，?-珠蛋白非缺失HPFH点突变，并已产生转基因小鼠。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）研究 - 566、-195、-175 和 -117 HPFH 突变在体内发育过程中破坏 β-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 敏感性、共激活子/阻遏子招募、Bcl11A 招募以及 HPFH 突变之间的协同作用。在具体目标 2 中，我们将使用分离染色质片段 (PICh) 的蛋白质组学和细胞培养中氨基酸稳定同位素标记 (SILAC) 以及质谱 (MS) 来研究这四种 HPFH 突变如何改变 Aβ-珠蛋白启动子中的 DNA 结合蛋白复合物。在具体目标 3 中，我们将使用基于细胞的报告基因测定来生成和鉴定 Aβ-珠蛋白基因启动子内的新 HPFH 突变，以选择 HPFH 突变，然后对 ¿-YAC 转基因小鼠中的这些突变进行表型表征，并确定这些位点的 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，可以开发高度特异性的治疗方法来增加 HbF 来治疗 SCD，而不会产生与广谱疗法相关的副作用。