Epigenetic regulation of stem cells and development by the DNA dioxygenase Tet2

摘要 Ten-Eleven易位(Tet1/2/3)家族是基因表达的表观遗传调节因子 对干细胞生物学和胚胎发育很重要。Tet酶是一种促进DNA的双加氧酶 5-甲基胞嘧啶(5mC)转化为5-羟甲基胞嘧啶(5hmC)的脱甲基化反应 衍生品。除了这种酶活性，Tet酶还可以结合染色质修饰复合体，以 以一种可能不依赖催化作用的方式调控基因。TET2是这个家族的关键成员。它是高度的 在胚胎干细胞(ESCs)中表达和控制干细胞所需的基因表达程序 世系规范。TET2在血液系统恶性肿瘤中也经常发生突变，并与 神经退行性疾病。虽然TET2的催化功能已经被很好地研究了，但它的非催化作用 仍然没有明确的定义。在这项建议中，我们试图确立催化依赖和 TET2在ESC基因调控和谱系承诺中的独立功能。我们假设TET2，in 除了通过DNA去甲基酶活性调节基因外，还可以在非催化作用下调节基因 通过将组蛋白修饰物招募到染色质来流行，这种双重基因调控模式对于 胚胎干细胞在神经和造血系中的适当分化。为了验证这一假设，我生成了 TET2催化突变体(Tet2m/m)和敲除(TET2-/-)ESCs，我将利用它们作为平台：(1)鉴定 TET2在胚胎干细胞中的催化和非催化直接靶基因的整合 TET2基因组占有率，(2)建立TET2介导的基因调控的激活和抑制机制 涉及与组蛋白修饰物OGT和HDAC2的相互作用，最后(3)定义了生物学意义 TET2的酶和非酶功能在胚胎干细胞分化和沿神经的谱系承诺中的作用 和造血血统。这些实验的发现将阐明新的表观遗传学机制 胚胎干细胞中涉及TET2催化和非催化功能的基因调控。它们将增强我们的 对干细胞生物学和发育的了解可能对血液系统恶性肿瘤有意义 TET2受到影响的地方。在米拉德·道拉蒂博士和伯妮斯·莫罗的共同指导下，我将 成功执行建议的研究和培训计划。这将使我能够为 表观遗传学和干细胞生物学领域，并发展必要的研究、专业和人际关系 成为独立的内科医生-科学家调查员的技能。

ABSTRACT The Ten-eleven translocation (Tet1/2/3) family of enzymes are epigenetic regulators of gene expression important for stem cell biology and embryonic development. Tet enzymes are dioxygenases that promote DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethycytosine (5hmC) and higher oxidized derivatives. In addition to this enzymatic activity, Tet enzymes can bind chromatin modifying complexes, to regulate genes in a presumably catalytic-independent manner. Tet2 is a key member of this family. It is highly expressed in embryonic stem cells (ESCs) and controls gene expression programs necessary for stem cell lineage specification. Tet2 is also frequently mutated in hematological malignancies and has been implicated in neurodegenerative diseases. While the catalytic functions of Tet2 have been well studied, its non-catalytic roles remain poorly defined. In this proposal, we seek to establish the significance of the catalytic dependent and independent functions of Tet2 in ESC gene regulation and lineage commitment. We hypothesize that Tet2, in addition to regulating genes through its DNA demethylase activity, can also modulate genes in a non-catalytic fashion by recruiting histone modifiers to the chromatin, and this dual mode of gene regulation is essential for proper ESC differentiation along the neural and hematopoietic lineages. To test this hypothesis, I have generated Tet2 catalytic mutant (Tet2m/m) and knock-out (Tet2–/–) ESCs, which I will use as a platform to: (1) identify the catalytic and non-catalytic direct target genes of Tet2 in ESCs by integrating changes in gene expression with Tet2 genomic occupancy, (2) establish Tet2-mediated activating and repressing mechanisms of gene regulation involving interactions with histone modifiers OGT and HDAC2, and finally (3) define the biological significance of Tet2 enzymatic and non-enzymatic functions in ESC differentiation and lineage commitment along the neural and hematopoietic lineages. Findings from these experiments will elucidate novel epigenetic mechanisms of gene regulation in ESCs involving Tet2 catalytic and non-catalytic functions. They will enhance our understanding of stem cell biology and development and can have implications in hematological malignancies where Tet2 is affected. Under the combined mentorship of Drs. Meelad Dawlaty and Bernice Morrow, I will successfully execute the proposed research and training plans. This will allow me to contribute greatly to the fields of epigenetics and stem cell biology and develop the necessary research, professional and interpersonal skills to become and independent physician-scientist investigator.