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

摘要 Tet 1/2/3（Ten-eleven translocation）酶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。泰特酶是双加氧酶， 通过将5-甲基胞嘧啶（5 mC）转化为5-羟甲基胞嘧啶（5 hmC）和更高的氧化 衍生物.除了这种酶活性，泰特酶可以结合染色质修饰复合物， 以一种可能不依赖催化剂的方式调节基因。Tet 2是这个家族的重要成员。极有 在胚胎干细胞（ESC）中表达，并控制干细胞所必需的基因表达程序， 谱系规范Tet 2也经常在血液恶性肿瘤中突变，并与肿瘤的发生有关。 神经退行性疾病虽然Tet 2的催化功能已经得到了很好的研究，但其非催化作用 定义不明确。在这个建议中，我们试图建立催化依赖性和 Tet 2在ESC基因调控和谱系定型中的独立功能。我们假设Tet 2，在 除了通过其DNA去甲基化酶活性调节基因外，还可以调节基因的非催化 通过招募组蛋白修饰剂到染色质中，这种基因调控的双重模式对于 ESC沿神经和造血谱系的适当分化沿着。为了验证这一假设，我生成了 Tet 2催化突变体（Tet 2 m/m）和敲除（Tet 2-/-）ESCs，我将使用其作为平台：（1）鉴定 通过整合基因表达的变化， Tet 2基因组占有率，（2）建立Tet 2介导的激活和抑制基因调控机制 涉及与组蛋白修饰剂OGT和HDAC 2的相互作用，最后（3）定义生物学意义 Tet 2在ESC分化和沿着神经细胞系的谱系定型中的酶和非酶功能 和造血系统这些实验的发现将阐明新的表观遗传机制， 涉及Tet 2催化和非催化功能的ESCs中的基因调控。他们将提高我们的 了解干细胞生物学和发育，并可能对血液恶性肿瘤产生影响 Tet 2受到影响。在米拉德·道拉蒂和伯尼采·莫罗博士的共同指导下，我将 成功执行拟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 成为独立的医学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.