Mechanisms of HbF Activation by Non-deletional HPFH

非缺失 HPFH 激活 HbF 的机制

基本信息

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
项目类别：
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

项目摘要

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，人们认为了解人类 γ- 至 β- 珠蛋白基因转换的分子机制对于治疗镰状细胞病 (SCD) 非常重要，因为大量证据表明胎儿血红蛋白增加(HbF) 显着降低与这种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 γ-珠蛋白 (HbF) 将有益于 SCD 患者。这项研究的目的是了解成人阶段确定性红细胞生成过程中 γ 珠蛋白基因沉默的情况，我们的临床目标是根据本研究的结果确定新的分子靶点，这些靶点可以通过治疗性调节来上调 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性持久性可能对 γ-珠蛋白基因抑制和激活机制提供大量信息，但迄今为止尚未得到广泛研究。拟议的研究将检验 HPFH 突变通过消除通常位于 γ-珠蛋白基因或更远端基因间区域的转录抑制复合物（“抑制体”）成分的募集来防止 γ-珠蛋白基因表达沉默或维持激活的假设。或者，通过创建有利的染色质结构，使 γ-珠蛋白基因在与基因座控制区相互作用方面部分胜过 β-珠蛋白基因(LCR) 或两者均已引入我们的人类 β-珠蛋白位点酵母人工染色体 (β-YAC)，包括 -566、-195、-175 和 -117、Aγ-珠蛋白非缺失 HPFH。在具体目标1中，我们将研究-566、-195、-175和-117的机制。 HPFH 突变在体内发育过程中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、co-在具体目标 2 中，我们将研究这四种 HPFH 突变如何改变 DNA 结合蛋白复合物。在特定目标 3 中，我们将使用分离染色质片段的蛋白质组学 (PICh) 和细胞培养中的氨基酸稳定同位素标记 (SILAC) 结合质谱 (MS) 来生成 Aγ-珠蛋白启动子，并鉴定新的 HPFH 突变。 Aγ-珠蛋白基因启动子使用基于细胞的报告基因测定来选择 HPFH 突变，然后对 β-YAC 转基因小鼠中的这些突变进行表型表征并确定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。