Mechanisms and functions of N6-methyladenosine (m6A) in cancer

总结 基因表达调节的一个重要机制是动态调节，并且可能是可逆的， mRNA中的核苷酸修饰。这些修饰可以对mRNA稳定性具有显著影响， 翻译和mRNA代谢的其他方面。我们在这一领域发挥了奠基作用， 用于N6-甲基腺苷（m6A）的全转录组作图的技术。我们的绘图研究提供了 首次证明m6A可以被动态调节，并可能赋予mRNA新的功能。我们 最近发现急性髓性白血病（AML）细胞显示出升高的胃L3和胃L14水平， 作为m6A形成甲基转移酶的异二聚体。我们发现，m6A促进自我更新， 在AML和CD34+干细胞中，m6A的耗竭触发分化程序。因此，m6a具有关键性 在特定发育阶段的造血分化中起作用，并且这一过程在发育过程中被解除管制。 急性髓细胞白血病因此，在全转录组水平上精确表征m6A的这些阶段特异性模式 对于了解m6A如何影响发育过渡至关重要。开发新的方法来映射m6A， 与造血和AML相关的罕见细胞群将有助于揭示这种表观转录组学 修饰对于AML中观察到的分化的调节和失调是至关重要的，并且可能对其他 癌的此外，m6A的作用在很大程度上被认为反映了特定"阅读器"蛋白的作用， 其结合mRNA中的m6A以影响其在细胞中的命运。YTHDC1是核心的主要读者， YTHDF家族在细胞质中，其包括三个几乎相同的旁系同源物，并且其可能具有 冗余功能为了显著提高我们对m6A在AML中的作用的理解， 该建议的具体目的是：（1）以细胞类型特异性方式可视化和绘制mRNA中的m6A。 在这里，我们描述了以细胞类型特异性方式检测和映射m6A的方法的发展 及其在理解造血和AML中的m6A动力学中的应用。(2)定义函数 在正常血细胞和AML中对m6A读数器YTHDC1的要求。基于全基因组的 屏幕和我们的初步数据，YTHDC1是一个强有力的候选人，为读者可能调解的主要方面 m6A在AML中的作用。在此，我们评估YTHDC1在正常和恶性肿瘤中的功能作用。 使用人脐带血细胞、AML细胞系和原发性AML患者进行造血。(3)确定 YTHDF胞质m6A阅读器对mRNA命运的作用和调节。YTHDF蛋白似乎 是胞质溶胶中m6A mRNA的主要调节因子。我们将确定YTHDF蛋白是如何调节的， 介导m6A-mRNA去稳定作用，如果YTHDF蛋白影响细胞分化， 在癌细胞系和AML中的增殖。总的来说，我们的项目将开发新的使能技术， 研究癌症中的m6A，并测试m6A阅读器促进癌症进展的机制。

SUMMARY An important mechanism of gene expression regulation is dynamically regulated, and possibly reversible, nucleotide modifications in mRNA. These modifications can have marked effects on mRNA stability, translation, and other aspects of mRNA metabolism. We had a founding role in this field by developing the technology for transcriptome-wide mapping of N6-methyladenosine (m6A). Our mapping study provided the first evidence that m6A could be dynamically regulated, and potentially impart new functions in mRNA. We recently showed that acute myeloid leukemia (AML) cells exhibit elevated levels of METTL3 and METTL14, the heterodimer that acts as the m6A-forming methyltransferase. We found that m6A promotes self-renewal in AML and in CD34+ stem cells, and depletion of m6A triggers a differentiation program. Thus, m6A has critical roles in hematopoietic differentiation at specific stages of development, and this process is deregulated in AML. Therefore, precise characterization of these stage-specific patterns of m6A at a transcriptome-wide level is critical to understand how m6A affects developmental transitions. Developing new methods to map m6A in the rare cell populations relevant to hematopoiesis and AML would help to reveal how this epitranscriptomic modification is critical for the regulation and deregulation of differentiation seen in AML, and possibly other cancers. Additionally, the effects of m6A are largely thought to reflect the actions of specific “reader” proteins, which bind m6A in mRNA to affect its fate in cells. The major readers are YTHDC1 in the nucleus, and the YTHDF family in the cytoplasm, which comprise three nearly identical paralogs, and which may have redundant functions. In order to significantly advance our understanding of the role of m6A in AML, the specific aims of this proposal are: (1) To visualize and map m6A in mRNA in a cell-type specific manner. Here we describe the development of methods for detecting and mapping m6A in a cell type-specific manner and their application to understand m6A dynamics in hematopoiesis and AML. (2) To define the functional requirement for the m6A reader YTHDC1 in normal blood cells and in AML. Based on a genome-wide screen and our preliminary data, YTHDC1 is a strong candidate for the reader that may mediate major aspects of the effect of m6A in AML. Here we assess the functional role for YTHDC1 in both normal and malignant hematopoiesis using human cord blood cells, AML cell lines and primary AML patients. (3) To determine the roles and regulation of the YTHDF cytosolic m6A readers on mRNA fate. The YTHDF proteins appear to be the major regulators of m6A mRNAs in the cytosol. We will determine how YTHDF proteins are regulated to mediate their m6A-mRNA destabilizing effects and if YTHDF proteins influence cellular differentiation and proliferation in cancer cell lines and in AML. Overall, our project will develop new enabling technologies for studying m6A in cancer and test mechanisms by which the m6A readers contribute to cancer progression.