5-Hydroxymethylcytosine and Globin Gene Switching

描述（由申请方提供）：胎儿血红蛋白（HbF）水平升高可减轻镰状细胞病和β地中海贫血相关症状的严重程度，并提高患者的生活质量和寿命。全面了解珠蛋白基因表达的发育调节机制（珠蛋白基因转换）对于开发新的治疗方法以增加HbF非常重要。来自多个实验室的超过30年的实验证据强烈支持DNA甲基化在成年人中γ-珠蛋白基因的沉默表达中起重要作用的假设。在我们实验室的狒狒模型中率先使用DNA甲基转移酶的药理学抑制剂来增加HbF水平，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。利用狒狒模型，我们实验室还进行了新的和重要的观察，表明γ-珠蛋白基因启动子在胎肝红细胞生成过程中以进行性方式去甲基化。在胎肝红细胞生成过程中，γ-珠蛋白启动子的DNA去甲基化机制仍不清楚。我们的初步数据显示，增加水平的5-羟甲基胞嘧啶（5-hmC），5-甲基胞嘧啶（5-mC）的一种新的修饰最近发现在真核细胞中，由泰特双加氧酶家族催化，与γ-珠蛋白启动子在红细胞表达水平升高的γ-珠蛋白。5-hmC被认为是DNA去甲基化的被动和主动机制中的关键中间体，因此我们的初步数据强烈表明，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。本研究将探讨胎儿红系分化过程中γ-珠蛋白基因的DNA去甲基化是通过泰特介导的5-hmC通路完成的这一假说。验证这一假设将确定一个重要的机制（S）在正常发育胎儿阶段特异性激活γ-珠蛋白表达。我们设想，详细了解胎儿肝脏红细胞生成过程中γ-珠蛋白基因去甲基化的正常生理机制，将促进针对该机制的新策略的开发，以实现DNA去甲基化和γ-珠蛋白基因的高水平激活，从而增加患者的HbF。

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.