Establishing and Interpreting Abnormal DNA Methylation in Cancer

项目摘要/摘要 哺乳动物的从头DNA甲基转移酶DNMT3A对大脑的出生后发育是必不可少的， 控制身体大小和调节造血。该基因的突变通常出现在年龄- 与不确定潜能相关的克隆性造血，是某些白血病的驱动因素 重要的是，我们要了解这种酶如何在活细胞中发挥作用。所有人类癌症都含有DNA 然而，DNMT3A的甲基化异常在实体肿瘤中相对罕见，这表明 酶调节可能是这些变化的原因。尽管我们非常详细地知道DNMT3A 甲基化裸露的DNA，我们几乎不知道这是如何发生在核小体的背景下， 染色质的基本构件。本应用程序的重点是使用新的见解来解决这个问题 最近在实验室里开发的。我们将集中讨论截断的异构体DNMT3A2的作用 在胚胎发育过程中表达，但在大多数实体癌中过度表达。我们的新型低温电磁结构 展示了这种酶如何与辅助蛋白DNMT3B3通过酸性结合到核小体上 补丁--一个完全出乎意料的发现。我们将使用生化、低温EM、细胞和 小鼠的研究是为了更准确地了解DNA甲基化是如何在染色质的背景下工作的。 在小鼠模型中，Dnmt3a2，而不是更广泛研究的更长的异构体Dnmt3a1，已经被敲打 OUT将用于检验遗传和化学物质永生化和致癌过程中的因果关系。 癌症的小鼠模型。这种全面的方法将有助于我们理解这一基本的 这一过程在正常细胞和转化细胞中起作用。 长期以来，关于DNA甲基化标记在细胞中如何解释的研究一直局限于 完全和完全未甲基化的CpG二联体状态。由于R35筹资机制的灵活性， 我们偶然发现CpG半甲基化可以刺激或抑制CTCF的结合 这取决于哪条双链是甲基化的。我们将调查半甲基化在癌症中的患病率 细胞，并研究其对其他转录因子结合和染色质结构的影响。差动 结合可能在癌细胞的不对称细胞分裂中发挥作用。我们希望回答三个关键问题： 1)DNMT3A2的过度表达是否有助于癌症的发生？2)DNMT3A2的从头甲基化是如何发生在 核小体背景？3)半甲基化的潜在生物学作用是什么？

Project Summary/Abstract The mammalian de novo DNA methyltransferase, DNMT3A is essential for postnatal development of the brain, control of body size and for the regulation of hematopoiesis. Mutations in the gene are commonly found in age- associated clonal hematopoiesis of indeterminant potential and are drivers for certain leukemias making it important that we understand how the enzyme functions in living cells. All human cancers contain DNA methylation anomalies, however mutations in DNMT3A are relatively rare in solid tumors, suggesting that altered enzyme regulation might be responsible for these changes. Although we know in exquisite detail how DNMT3A methylates naked DNA, we know virtually nothing about how this occurs in the context of nucleosomes, the fundamental building blocks of chromatin. The focus of this application is to address this issue using new insights recently developed in the lab. We will concentrate on the role of a truncated isoform, DNMT3A2, normally expressed during embryonic development but overexpressed in most solid cancers. Our novel cryo-EM structure shows how this enzyme partners with an accessory protein DNMT3B3 to bind to nucleosomes through the acidic patch – a totally unexpected discovery. We will follow up on this work using biochemical, cryo-EM, cellular and mouse studies to gain a more precise understanding of how DNA methylation works in the context of chromatin. A mouse model in which Dnmt3a2, but not the more widely studied longer isoform, Dnmt3a1, has been knocked out will be used to test for causality in the process of immortalization and carcinogenesis in genetic and chemical mouse models of cancer. This comprehensive approach will help in our understanding of how this fundamental process works in normal and transformed cells. Studies on how DNA methylation marks are interpreted in cells has long been confined to comparison between fully and completely unmethylated CpG dyad states. Because of the flexibility in the R35 funding mechanism, we serendipitously discovered that CpG hemimethylation can either stimulate or inhibit binding by CTCF depending on which duplex strand is methylated. We will investigate the prevalence of hemimethylation in cancer cells and investigate its effects on binding of other transcription factors and chromatin structure. Differential binding may play a role in asymmetric cell divisions in cancer cells. We hope to answer three critical questions: 1) Does DNMT3A2 overexpression contribute to carcinogenesis? 2) How does de novo methylation occur in a nucleosomal context? 3) What are the potential biological roles for hemimethylation?