Epigenetic regulation of neural stem cell biology by Tet DNA dioxygenases

摘要 10 - 11易位酶家族（TET 1/2/3）是基因表达的表观遗传调节因子 在神经干细胞（NSC）和哺乳动物神经系统发育过程中高度表达。泰特 酶是双加氧酶，其通过将5-甲基胞嘧啶转化为 (5mC)5-羟甲基胞嘧啶（5 hmC）和更高级的氧化衍生物。除了作为一个 5 hmC是一个去甲基化中间体，可以作为一个稳定的表观遗传标记，高度富集和动态 在发育中的神经系统。泰特酶和5 hmC失调与人类 神经发育综合征、智力残疾、颅面畸形和神经变性。 这些观察结果表明泰特酶在神经系统发育中的关键作用，并导致 他们在神经干细胞生物学中的作用。然而，泰特酶在神经干细胞中的功能和 神经发育仍然知之甚少。我们实验室的初步数据表明，泰特三重- 来源于胚胎干细胞的敲除NSC（T123-/-）在自我更新、多能性 和神经发育基因的表达。因此，我们假设泰特酶具有重要的 在表观遗传调控基因表达程序中的功能，对NSC的维持和多能性至关重要 和胚胎神经发育中的作用。为了验证这一假设，我们获得了胚胎前脑NSC细胞系， 含有Tet 1/2/3的floxed等位基因和从该基因表达的他莫昔芬诱导型Cre重组酶转基因， 组成型Rosa 26基因座（T123 F/F; +/R26-CreER）用于所有三种泰特基因的条件性组合缺失。我们 我还建立了一个Tet 1/2/3三重floxed小鼠群体，表达他莫昔芬诱导的Cre重组酶 在神经特异性巢蛋白启动子（T123 F/F; +/Nestin-CreERT 2）控制下的转基因。使用这些模型， 我们将（1）确定泰特酶在神经干细胞的维持和多能性中的作用，（2）建立 胚胎神经发育中TET的需求，以及（3）鉴定TET介导的表观遗传和 神经干细胞的转录调控机制。这些研究的结果将确定新的作用， NSC生物学和神经发育中的泰特酶，提供了对TET调节异常如何促进 人类神经发育障碍，并确定新的治疗目标。在以下机构的共同指导下， Drs. Meelad Dawlaty和Jean Hébert，我将成功地执行拟议的研究和培训计划。这 研究计划将进一步我的神经干细胞生物学的表观遗传调控的知识，并促进 我的科学和专业发展，使我具备成为一名医生的必要技能- 科学家

ABSTRACT The ten-eleven translocation family (TET1/2/3) of enzymes are epigenetic regulators of gene expression that are highly expressed in neural stem cells (NSCs) and during mammalian nervous system development. TET enzymes are dioxygenases that promote active and passive DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethycytosine (5hmC) and higher-order oxidized derivatives. In addition to its role as a demethylation intermediate, 5hmC can function as a stable epigenetic mark and is highly enriched and dynamic in the developing nervous system. TET enzymes and 5hmC dysregulation have been implicated in human neurodevelopmental syndromes, intellectual disability, craniofacial abnormalities, and neurodegeneration. These observations suggest a critical role for TET enzymes in the developing nervous system and has led to interest in their roles in the biology of NSCs. However, the functions of TET enzymes in NSCs and neurodevelopment remain poorly understood. Preliminary data from our lab demonstrates that Tet triple- knockout NSCs (T123–/–) derived from embryonic stem cells exhibit severe defects in self-renewal, multipotency, and expression of neurodevelopmental genes. We therefore hypothesize that TET enzymes have essential functions in epigenetic regulation of gene expression programs critical for NSC maintenance and multipotency and in embryonic neurodevelopment. To test this hypothesis, we have derived embryonic forebrain NSC lines containing floxed alleles of Tet1/2/3 and a tamoxifen-inducible Cre recombinase transgene expressed from the constitutive Rosa26 locus (T123F/F; +/R26-CreER) for conditional, combined deletion of all three Tet genes. We have also established a colony of Tet1/2/3 triple-floxed mice expressing a tamoxifen-inducible Cre recombinase transgene under control of the neural-specific Nestin promoter (T123F/F; +/Nestin-CreERT2). Using these models, we will (1) define the role of TET enzymes in the maintenance and multipotency of NSCs , (2) establish the requirement of TETs in embryonic neurodevelopment, and (3) identify TET-mediated epigenetic and transcriptional regulatory mechanisms in NSCs. Findings from these studies will define novel roles played by TET enzymes in NSC biology and neurodevelopment, provide insights into how dysregulation of TETs contribute to human neurodevelopmental disorders, and identify novel targets for therapy. Under the joint mentorship of Drs. Meelad Dawlaty and Jean Hébert, I will successfully execute the proposed research and training plan. This research program will further my knowledge of the epigenetic regulation of neural stem cell biology and facilitate my scientific and professional development by equipping me with the necessary skills to become a physician- scientist.