5-Hydroxymethylcytosine and Globin Gene Switching

描述(由申请人提供)：增加胎儿血红蛋白(HBF)水平可以减轻与镰状细胞病和β-地中海贫血相关的症状的严重程度，并提高患者的生活质量和寿命。全面了解珠蛋白基因表达(珠蛋白基因转换)的发育调节机制(S)对于开发增加HbF的新疗法非常重要。来自多个实验室的30多年的实验证据有力地支持了这样的假设，即DNA甲基化在抑制成年人伽马珠蛋白基因的表达方面发挥了基础性作用。使用DNA甲基转移酶的药理抑制剂来提高HBF水平在我们实验室的狒狒模型中是首创的，并导致了一系列临床试验，证明了这些药物在患者中的有效性。本实验室还利用恒河猴模型进行了一项新颖而重要的观察，发现在胎肝红细胞生成过程中，γ-珠蛋白基因启动子以进行性的方式去甲基化。在胎肝红细胞生成过程中，γ-珠蛋白启动子DNA去甲基化的机制尚不清楚。我们的初步数据显示，5-羟甲基胞嘧啶(5-HMC)是最近在真核细胞中发现的由Tet双加氧酶家族催化的一种新的5-甲基胞嘧啶(5-MC)修饰物，其水平的升高与红系细胞中伽马珠蛋白启动子的表达水平升高有关。5-HMC被认为是DNA去甲基化被动和主动机制中的关键中间体，因此我们的初步数据有力地表明，5-HMC参与了胎肝红细胞生成过程中γ-珠蛋白基因去甲基化的机制。这项建议将探讨这样一种假设，即胎儿红系分化过程中伽玛珠蛋白基因的DNA去甲基化是通过Tet介导的涉及5-HMC的途径完成的。这一假说的验证将定义一个关键的机制(S)，在正常发育的胎儿阶段特异性激活伽马珠蛋白的表达。我们预计，对胎肝红细胞生成过程中伽玛珠蛋白基因去甲基化的正常生理机制的详细了解将促进针对这一机制的新策略的开发，以实现DNA去甲基化和伽马珠蛋白基因的高水平激活，以增加患者的HBF。 公共卫生相关性：升高的胎儿血红蛋白水平可减轻镰状细胞病和β-地中海贫血的严重程度，并延长患者的寿命。对调节γ-珠蛋白基因表达的机制的完整描述在未来增加HBF的药物和分子疗法的设计中将是重要的。DNA甲基化是调节γ-珠蛋白基因表达的关键因素。这项建议中描述的实验试图了解5-羟甲基胞嘧啶在胎儿肝脏红细胞生成过程中负责γ-珠蛋白启动子脱甲基化的机制(S)中的作用。我们的长期目标是了解珠蛋白基因表达的发育调节机制，以此作为开发镰状细胞病和β地中海贫血治疗方法的手段。我们期望我们的研究将促进针对正常的生理机制的新策略的开发，这些机制负责伽玛珠蛋白基因DNA去甲基化，以实现患者胎儿血红蛋白水平的升高。

DESCRIPTION (provided by applicant): Increased fetal hemoglobin (HbF) levels lessen the severity of symptoms associated with sickle cell disease and beta-thalassemia and increase the quality of life and life span of patients. A complete understanding of the mechanism(s) responsible for developmental regulation of globin gene expression (globin gene switching) is important to develop new therapies to increase HbF. Over 30 years of experimental evidence from multiple laboratories strongly supports the hypothesis that DNA methylation plays a fundamental role in silencing expression of the gamma-globin gene in the adults. The use of pharmacological inhibitors of DNA methyltransferase to increase HbF levels was pioneered in the baboon model in our laboratory and led to a series of clinical trials that demonstrated the effectiveness of these drugs in patients. Using the baboon model, our laboratory also made the novel and important observation showing that the gamma-globin gene promoter was demethylated in a progressive manner during fetal liver erythropoiesis. The mechanism responsible for DNA demethylation of the gamma-globin promoter during fetal liver erythropoiesis remains unknown. Our preliminary data shows that increased levels of 5-hydroxymethylcytosine (5-hmC), a novel modification of 5-methylcytosine (5-mC) recently found in eukaryotic cells that is catalyzed by the TET dioxygenase family, are associated with the gamma-globin promoter in erythroid cells expressing elevated levels of gamma-globin. 5-hmC has been proposed to be a key intermediate in both passive and active mechanisms of DNA demethylation and therefore our preliminary data strongly suggests that 5-hmC is involved in the mechanism responsible for demethylation of the gamma-globin gene during fetal liver erythropoiesis. This proposal will investigate the hypothesis that DNA demethylation of the gamma-globin gene during fetal erythroid differentiation is accomplished through a TET- mediated pathway involving 5-hmC. Validation of this hypothesis will define a crucial mechanism (s) in normal developmental fetal stage-specific activation of gamma-globin expression. We envision that detailed knowledge of the normal, physiological mechanism responsible for gamma-globin gene demethylation during fetal liver erythropoiesis will foster the development of new strategies targeting this mechanism to achieve DNA demethylation and high level activation of the gamma-globin gene to increase HbF in patients. PUBLIC HEALTH RELEVANCE: Elevated fetal hemoglobin levels lessen the severity of sickle cell disease and beta-thalassemia and increase the lifespan of patients. A complete description of the mechanism responsible for the regulation of gamma-globin gene expression would be important in the design of future pharmacological and molecular therapies to increase HbF. DNA methylation is a key factor in the regulation of gamma-globin gene expression. The experiments described in this proposal seek to understand the role of 5-hydroxymethylcytosine in the mechanism(s) responsible for DNA demethylation of the gamma-globin promoter during fetal liver erythropoiesis. It is our long term goal to understand the mechanism responsible for developmental regulation of the globin gene expression as a means to developing therapies for sickle cell disease and beta-thalassemia. We expect that our studies will foster the development of new strategies targeting the normal, physiological mechanism responsible for gamma-globin gene DNA demethylation to achieve increased fetal hemoglobin levels in patients.