The role of DNMT3A in gene regulation and stem cell expansion

项目摘要 DNA甲基化是一种表观遗传修饰，在调节干细胞中起关键作用， 发展和许多疾病。在癌症中观察到的异常DNA甲基化 二十多年来，许多研究都集中在启动子超甲基化上， 肿瘤抑制基因此外，DNMT 3A，一种从头DNA甲基转移酶， 在一系列血液恶性肿瘤中经常发生突变。我们的实验室已经证明， Dnmt 3a缺失损害造血干细胞（HSC）分化，同时在造血干细胞中扩增HSC数量。 这表明DNMT 3A可能在肿瘤发生和干细胞调节中起作用。 然而，白血病患者样本的DNA甲基化谱显示DNA甲基化相关 基因组中的基因表达很差，突出了我们对特定基因表达的有限理解。 DNA甲基化的功能使用最常见的DNMT 3A的鼠模型的最新研究 血液恶性肿瘤中的突变DNMT 3AR 882，证明DNMT 3AR 882协同 与FLT 3-ESTITD和NPM 1c突变，有助于白血病转化。然而，我们的 了解哪些剩余的DNMT 3A突变导致白血病发生以及 它们对癌症形成的贡献仍然缺乏。因此， 拟议的研究是了解DNMT 3A如何影响癌症的基因调控，以及如何 DNMT 3A突变体易使干细胞扩增。在目标1中，我建立了一个新的DNA表观基因组， 编辑工具（dCas 9-DASSunTag-DASDNMT 3A系统），以研究DNA之间的因果关系 甲基化和基因表达。通过对全基因组范围的泛癌症分析， 确定了发生在峡谷基因组体区域的DNA超甲基化（广泛的， 甲基化不足的区域），并激活相应的基因表达。在目标2中，使用 Dnmt 3a突变小鼠模型，我将阐明一个Dnmt 3a突变体在引发中的作用 干细胞扩增这项研究的结果将揭示异常DNA 癌症中的甲基化和DNMT 3A-DNA相关恶性肿瘤的分子机制。之甚少 已知表观遗传修饰剂的突变如何影响癌症的3D基因组结构。 因此，在目标3中，我计划利用我的博士后研究来了解表观遗传修饰剂如何 塑造癌症的基因组景观及其潜在机制。该培训计划是 为我量身定制了一个基础科学研究的全面教育，这将是非常有用的 在实现我的长期职业目标，成为一名独立的癌症研究人员。

PROJECT SUMMARY DNA methylation is an epigenetic modification that plays a key role in regulating stem cells, development and many diseases. Abnormal DNA methylation has been observed in cancer for more than two decades, with many investigations focusing on promoter hypermethylation, which silences tumor suppressor genes. Additionally, DNMT3A, one of de novo DNA methyltransferases, is frequently mutated in a spectrum of hematological malignancies. Our lab has demonstrated that Dnmt3a loss impairs hematopoietic stem cell (HSC) differentiation, while expanding HSC numbers in bone marrow, suggesting DNMT3A may have a role in tumorigenesis and stem cell regulation. However, DNA methylation profiling of leukemia patient samples shows DNA methylation correlates poorly with gene expression across the genome, highlighting our limited understanding of the specific functions of DNA methylation. Recent studies using a murine model of the most frequent DNMT3A mutation in hematological malignancies, DNMT3AR882, demonstrated that DNMT3AR882 cooperates with FLT3-­ITD and NPM1c mutations to contribute to leukemic transformation. Nevertheless, our knowledge of which remaining DNMT3A mutations lead to leukemogenesis and the mechanisms by which they contribute to cancer formation remains lacking. Therefore, the long-­term goal of the proposed research is to understand how DNMT3A affects gene regulation in cancer, and how DNMT3A mutants predispose stem cell expansion. In Aim 1, I established a novel DNA epigenome editing tool (dCas9-­SunTag-­DNMT3A system) to investigate the causal relationship between DNA methylation and gene expression. Using pan-­cancer analysis of genome-­wide profiles, we have identified DNA hypermethylation occurring in the gene-­body regions of canyons (broad and undermethylated regions) with activation of corresponding gene expression. In Aim 2, using a Dnmt3a mutant murine model I developed, I will elucidate the role of one Dnmt3a mutant in priming stem cell expansion. The findings from this proposed research will shed the light on abnormal DNA methylation in cancer and molecular mechanisms of DNMT3A-­associated malignancies. Little is known about how mutations in epigenetic modifiers affect the 3D genomic structure in cancer. Therefore, in Aim 3, I plan to use my postdoctoral studies to understand how epigenetic modifiers shape the genomic landscape in cancer and their underlying mechanism. This training program is tailored to give me a comprehensive education in basic science research that will be extremely useful in achieving my long-­term career goal of becoming an independent cancer researcher.