Targeting DNA Methylation and the Cancer Epigenome

项目摘要 我们的实验室在1980年发现DNA甲基化(表观遗传修饰)影响基因表达 和细胞分化。高通量测序和癌症基因组的意外结果 Atlas和其他项目表明，癌症中的许多突变是在修改表观基因组的基因中发生的。这 证实了我们的长期假设，即异常的表观遗传过程是人类 治疗癌症并提供新的治疗机会。了解表观基因组在癌症中是如何改变的 需要一种我们在过去五年中制定的综合方法。我们希望确定 DNA甲基化、核小体定位和组蛋白等关键特征的机制 修改是相互影响的。然后我们将确定DNA甲基化抑制剂(DNMTi)是如何发挥作用的。 这笔赠款旨在使用我们强大的新Nome-seq技术来了解 DNA甲基化和核小体定位；使用敲击法和其他方法检查 改变染色质重塑和组蛋白修饰酶对整个表观基因组的影响； 并了解表观基因组如何控制内源性逆转录病毒(ERV)。我们将研究如何 在人类癌症中，表观基因组发生了变化，不仅表现在基因启动子区域的变化，也表现在 增强子和绝缘子区域以及基因本身。要解决的主要问题包括1)为什么 染色质修饰物中有如此多的突变，这些突变对细胞的结构有什么影响 表观基因组？2)激活癌症/睾丸基因表达的功能后果是什么 通过5-氮杂核苷？3)哪些双链RNA被5-氮杂核苷激活？这些双链RNA是如何被激活的 与细胞反应有关？4)我们能设计出可能增加细胞反应的表观遗传药物组合吗 5-氮杂核苷治疗的疗效如何？以及5)冷冻-EM能否帮助可视化相关的复合体 染色质的结构和功能？我们还将研究Tet酶的作用，Tet酶可以氧化5-甲基-4-氨基丁酸。 甲基胞嘧啶为5-羟甲基胞嘧啶，需要维生素C作为辅因子，G9a和G9a酶 SETDB1，它甲基化组蛋白H3K9。增加ERV表达的组合将是 优先，因为最近的数据强烈表明ERV的表达可能与细胞变化有关，并且 很有可能对癌症的临床结果产生影响。癌症患者往往缺乏维生素C，这意味着 补充剂可显著提高Tet活性和患者反应。我们的方法不仅仅是设计成 从整体上理解表观基因组，并设计出策略，以增加患者对 药物，也许通过定义未来合理的药物组合，特别是利用DNMTI的成功。 该项目应具有迅速的机械和翻译影响，因为DNMTI的是FDA批准的或 目前正在进行癌症治疗的试验。

Project Summary Our laboratory discovered in 1980 that methylation of DNA (epigenetic modification) affected gene expression and cell differentiation. High-throughput sequencing and the unexpected outcomes from The Cancer Genome Atlas and other projects show that many mutations in cancer are in genes that modify the epigenome. This has validated our long-term hypothesis that abnormal epigenetic processes are major contributors to human cancer and offer novel therapeutic opportunities. Understanding how the epigenome is changed in cancer requires an integrated approach, which we have developed over the last five years. We wish to determine the mechanisms by which key features such as DNA methylation, nucleosome positioning, and histone modifications influence each other. We then will determine how DNA methylation inhibitors (DNMTi’s) work. This grant is designed to use our powerful new NOMe-seq technology to understand the relationship between DNA methylation and nucleosomal positioning; to use knock-down and other approaches to examine the effects of altering both chromatin-remodeling and histone-modifying enzymes on the epigenome as a whole; and to understand how the epigenome controls endogenous retroviruses (ERVs). We will study how the epigenome is altered in human cancer, characterizing changes not only in gene promoter regions but also in enhancer and insulator regions and in genes themselves. Major questions to be addressed include 1) Why are there so many mutations in chromatin modifiers, and what are the effects of these mutations on the structure of the epigenome? 2) What are the functional consequences of activating the expression of cancer/testis genes by 5-azanucleoside? 3) What double-stranded RNAs are activated by 5-azanucleosides and how do these relate to cellular responses? 4) Can we design combinations of epigenetic drugs which might increase the effectiveness of 5-azanucleoside treatment? and 5) Can cryo-EM help to visualize complexes relevant to chromatin structure and functions? We will also study the roles of TET enzymes, which oxidize 5- methylcytosine to 5-hydroxymethylcytosine and require vitamin C as a cofactor, and the enzymes G9A and SETDB1, which methylate histone protein H3K9. Combinations that increase the expression of ERVs will be prioritized, because recent data strongly suggests that ERV expression may be linked to cellular changes, and quite possibly to clinical outcomes in cancer. Cancer patients are often deficient in vitamin C, implying that supplementation may markedly increase TET activity and patient response. Our approach is designed not only to understand the epigenome holistically but also to devise strategies which will increase patients' responses to drugs, perhaps by defining future rational drug combinations, in particular making use of DNMTi’s. Success of this project should have rapid mechanistic and translational impact, as DNMTi’s are FDA-approved or are currently in trials for cancer treatment.