Epigenetic regulation of neural stem cell biology by Tet DNA dioxygenases

摘要 十一十一易位家族(TET1/2/3)是基因表达的表观遗传调节因子 在神经干细胞(NSCs)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，通过转化5-甲基胞嘧啶来促进主动和被动的DNA去甲基化 (5mC)转化为5-羟甲基胞嘧啶(5HmC)和更高阶氧化衍生物。除了它作为一个 去甲基化中间体5hmC可以作为一个稳定的表观遗传标记，具有高度的丰度和动态化 在发育中的神经系统中。Tet酶和5hmC失调与人类 神经发育综合征、智力残疾、颅面畸形和神经退行性变。 这些观察表明Tet酶在发育中的神经系统中起着关键作用，并导致了 对它们在神经干细胞生物学中的作用感兴趣。然而，Tet酶在神经干细胞和 人们对神经发育仍然知之甚少。我们实验室的初步数据表明，Tet是三倍体 来源于胚胎干细胞的基因敲除神经干细胞(T123-/-)在自我更新、多潜能、 和神经发育基因的表达。因此，我们假设Tet酶具有重要的 对神经干细胞维持和多能性至关重要的基因表达程序的表观遗传调控功能 以及胚胎神经发育。为了验证这一假设，我们获得了胚胎前脑神经干细胞系。 含有Tet1/2/3的花等位基因和三苯氧胺诱导的Cre重组酶转基因 构成ROSA26基因座(T123F/F；/R26-Creer)，用于条件性、联合缺失所有三个Tet基因。我们 我还建立了一组Tet1/2/3三花小鼠，表达他莫昔芬诱导的Cre重组酶 在神经特异性Nestin启动子控制下的转基因(T123F/F；/Nestin-CreERT2)。使用这些模型， 我们将(1)确定Tet酶在神经干细胞维持和多能性中的作用，(2)建立 Tet在胚胎神经发育中的需求，以及(3)鉴定Tet介导的表观遗传学和 神经干细胞中的转录调控机制。这些研究的发现将定义 神经干细胞生物学和神经发育中的TET酶，提供了对TET调节失调如何起作用的见解 人类神经发育障碍，并确定新的治疗靶点。在…的共同指导下 Meelad Dawlaty博士和Jean HéBert博士，我将成功执行拟议的研究和培训计划。这 研究计划将加深我对神经干细胞生物学表观遗传调控的了解，并促进 我的科学和职业发展，使我具备成为医生所需的技能- 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.