Mechanisms of HbF Activation by Non-deletional HPFH

非缺失 HPFH 激活 HbF 的机制

• 批准号: 8854128
• 负责人: KENNETH R PETERSON
• 金额: $ 37.18万
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8854128
• 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; Elements; 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; Health; 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; 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; beta Globin; 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; targeted treatment; transgene expression

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.

描述（由申请人提供）：长期以来，人们一直认为了解人γ-至β-珠蛋白基因开关的分子机制在镰状细胞病（SCD）治疗中非常重要，因为大量证据表明，胎儿血红蛋白（HbF）增加可显著降低与该疾病相关的病理生理学。因此，了解如何在成人红细胞生成中重新激活γ-珠蛋白（HbF）将使SCD患者受益。本研究的目的是了解γ-珠蛋白基因沉默在成熟阶段的决定性红细胞生成。我们的临床目标是根据本研究的结果确定新的分子靶点，这些靶点可以在治疗上进行调节，以上调γ-珠蛋白合成，从而治疗SCD。胎儿血红蛋白（HPFH）点突变的非缺失遗传持续性可能是关于γ-珠蛋白基因抑制和激活机制的高度信息，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HPFH突变通过消除转录抑制因子复合物的募集来防止γ-珠蛋白基因表达沉默或维持γ-珠蛋白基因表达激活的假设通常位于γ-珠蛋白基因或基因座的更远基因间区域的阻遏体（“阻遏体”）组分，或替代地，通过产生允许γ-珠蛋白基因在与基因座控制区（LCR）相互作用方面部分胜过β-珠蛋白基因的有利染色质结构，或两者我们在人β-珠蛋白基因座酵母人工染色体（β-YAC）上引入了几个HPFH突变（包括-566、-195、-175和-117），获得了γ-珠蛋白非缺失型HPFH点突变和转基因小鼠。在具体目标1中，我们将使用小鼠模型和分子生物学/生物化学方法，包括转基因结构/表达研究、3C、组蛋白修饰、DNA酶I敏感性、共激活子/阻遏子募集、Bcl 11 A募集和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的蛋白质组学（PICh）和细胞培养中氨基酸稳定同位素标记（SILAC）结合质谱（MS）来研究这四种HPFH突变如何改变Aγ-珠蛋白启动子中的DNA结合蛋白复合物。在特定目标3中，我们将使用基于细胞的报告基因试验选择HPFH突变，在Aγ-珠蛋白基因启动子内产生并鉴定新的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，可以开发高度特异性的治疗方法，以增加HbF治疗SCD，而不会产生与广谱治疗相关的副作用。