New approaches to study the biological role of 6-methyladenine in human DNA

研究人类 DNA 中 6-甲基腺嘌呤生物学作用的新方法

• 批准号: 527466121
• 负责人: Professor Dr. Albert Jeltsch
• 金额: --
• 依托单位: Abteilung für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/527466121?language=en
• 关键词: New; approaches; study; biological; role

During the last years, several publications have reported variable levels of N6-methyldeoxyadenosine (m6dA) in human genomic DNA and connected it with effects on cell physiology and in human diseases. However, other publications raised doubts on these findings including the existence and levels of m6dA in human cells, its way of incorporation, potential biological effects, and the validity of proposed human N6-methyltransferases (MTases). Hence, there is an urgent demand for novel, alternative research approaches in this field. Given that low levels of endogenous m6dA and the lack of non-controversial bona fide human N6-MTases cause massive technical problems, we developed an orthogonal approach to study the potential effects of m6dA in human DNA by expressing well-characterized, highly active bacterial N6-MTases in human cells followed by the analysis of the cellular effects of the genome-wide introduced m6dA. Following this procedure, we observed reductions in cell proliferation after global GANTC and GATC methylation. Controls using catalytically inactive MTases as well as cultivation of the cells without expression of the MTase ensured that the observed effects were directly related to the introduced adenine-N6 DNA methylation. We identified several genes that are directly regulated by m6dA in a GANTC context. Upregulated genes showed m6dA-dependent reduction of H3K27me3 suggesting that the PRC2 complex is inhibited by m6dA. Genes downregulated by m6dA showed enrichment of JUN family transcription factor (TF) binding sites. These TFs bind m6dA containing DNA with reduced affinity suggesting that m6dA can reduce the recruitment of JUN TFs to target genes. Based on these important initial discoveries and proof of concept of our experimental approach, several interesting follow-up experiments appear, which will be approached in this project: 1) Investigation of the effect of m6dA introduced in human cells in different sequence contexts and/or at higher levels on cell proliferation and gene expression. 2) Identification of additional TF families and chromatin regulators that respond to m6dA in different sequence contexts. 3) Triggering of physiological effects of m6dA after its locus-specific (instead of genome-wide) delivery at m6dA sensitive sites taken from our previous work using epigenome editing. 4) Employing epigenome editing at m6dA sensitive sites to validate the function of human N6-MTase candidates and study their function. 5) Targeted m6dA at endogenous m6dA containing regions and study its biological effects. These new experimental approaches developed and applied here are urgently needed in this rapidly developing, but highly controversial, field. The results of this project will shed new light on the existence and biological role of m6dA in human cells and the responsible N6-MTases. Our data will help to consolidate our understanding of the potential role of m6dA in human DNA and cells.

在过去的几年里，一些文献报道了人类基因组DNA中不同水平的N6-甲基脱氧腺苷(M6dA)，并将其与细胞生理学和人类疾病的影响联系起来。然而，其他出版物对这些发现提出了质疑，包括人类细胞中m6dA的存在和水平、其掺入方式、潜在的生物学效应以及拟议的人类N6-甲基转移酶(MTase)的有效性。因此，在这一领域迫切需要新颖的、可替代的研究方法。鉴于低水平的内源性m6dA和缺乏无争议的真正的人类N6-MTase造成了大量的技术问题，我们开发了一种正交方法来研究m6dA在人类DNA中的潜在影响，方法是在人类细胞中表达特征良好、高活性的细菌N6-MTase，然后分析全基因组引入的m6dA的细胞效应。遵循这一程序，我们观察到全球GANTC和GATC甲基化后细胞增殖减少。对照组使用催化失活的MTase，以及培养不表达MTase的细胞，确保观察到的效果与引入的腺嘌呤-N6 DNA甲基化直接相关。我们在GANTC背景下确定了几个直接受m6dA调控的基因。上调的基因显示依赖于m6dA的H3K27me3的减少，这表明m6dA抑制了PRC2复合体。M6dA下调的基因表现为Jun家族转录因子(Tf)结合位点的丰富。这些转录因子与含有DNA的m6dA结合亲和力降低，提示m6dA可以减少Jun转录因子向靶基因的募集。基于这些重要的初步发现和我们实验方法的概念证明，出现了几个有趣的后续实验，本项目将探讨这些实验：1)研究m6dA在不同序列背景下和/或更高水平引入人类细胞对细胞增殖和基因表达的影响。2)鉴定在不同序列背景下对m6dA有反应的其他TF家族和染色质调节子。3)m6dA在m6dA敏感部位的位点特异性(而不是全基因组)传递后，触发其生理效应。4)在m6dA敏感部位进行表观基因组编辑，验证人N6-MTase候选基因的功能，并研究其功能。5)将m6dA靶向内源性m6dA所在区域，研究其生物学效应。这些在这里开发和应用的新的实验方法在这个快速发展但极具争议性的领域是迫切需要的。该项目的结果将为人类细胞中m6dA的存在和生物学作用以及相关的N6-MTase提供新的线索。我们的数据将有助于巩固我们对m6dA在人类DNA和细胞中的潜在作用的理解。