5-Hydroxymethylcytosine and Globin Gene Switching

描述（由申请人提供）：增加胎儿血红蛋白（HbF）水平可减轻镰状细胞病和地中海贫血相关症状的严重程度，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控机制（珠蛋白基因开关）对于开发新的治疗方法来增加HbF非常重要。来自多个实验室的超过30年的实验证据有力地支持了DNA甲基化在成人中沉默γ -珠蛋白基因表达中起基本作用的假设。使用DNA甲基转移酶的药理学抑制剂来增加HbF水平是在我们实验室的狒狒模型中首创的，并导致了一系列的临床试验，证明了这些药物对患者的有效性。利用狒狒模型，我们的实验室还进行了新颖而重要的观察，表明γ -珠蛋白基因启动子在胎儿肝脏红细胞生成过程中以渐进的方式去甲基化。胎儿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。我们的初步数据表明，最近在真核细胞中发现的5-羟甲基胞嘧啶（5-hmC）的一种新修饰，由TET双加氧酶家族催化，其水平升高与红细胞中表达升高的γ -珠蛋白启动子有关。5-hmC被认为是DNA去甲基化被动和主动机制的关键中间体，因此我们的初步数据强烈表明，5-hmC参与了胎儿肝脏红细胞生成过程中γ -珠蛋白基因去甲基化的机制。本研究将探讨胎儿红细胞分化过程中γ -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。这一假设的验证将确定正常发育胎儿阶段特异性γ -珠蛋白表达激活的关键机制。我们设想，对胎儿肝红细胞生成过程中γ -珠蛋白基因去甲基化的正常生理机制的详细了解，将促进针对该机制的新策略的发展，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.