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

在过去的几年中，一些出版物报道了人类基因组DNA中N6-甲基脱氧腺苷（m6 dA）的可变水平，并将其与对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m6 dA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（MTases）的有效性。因此，迫切需要在这一领域的新的，替代的研究方法。鉴于低水平的内源性m6 dA和缺乏无争议的真正的人N6-MTases导致大量的技术问题，我们开发了一种正交方法来研究m6 dA在人DNA中的潜在影响，通过在人细胞中表达充分表征的、高活性的细菌N6-MTases，然后分析全基因组引入的m6 dA的细胞效应。在这个过程之后，我们观察到在整体GANTC和GATC甲基化之后细胞增殖的减少。使用无催化活性的MT酶的对照以及不表达MT酶的细胞的培养确保了观察到的效应与引入的腺嘌呤-N6 DNA甲基化直接相关。我们确定了在GANTC背景下由m6 dA直接调控的几个基因。上调的基因显示H3 K27 me 3的m6 dA依赖性减少，表明PRC 2复合物被m6 dA抑制。m6 dA下调的基因显示JUN家族转录因子（TF）结合位点的富集。这些TF以降低的亲和力结合含有m6 dA的DNA，表明m6 dA可以减少JUN TF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，这些实验将在本项目中进行：1）研究在不同序列背景下和/或以更高水平引入人类细胞中的m6 dA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m6 dA应答的其他TF家族和染色质调节剂。3)触发m6 dA的生理效应后，其基因座特异性（而不是全基因组）交付m6 dA敏感位点从我们以前的工作使用表观基因组编辑。4)在m6 dA敏感位点采用表观基因组编辑来验证人N6-MTase候选物的功能并研究其功能。5)将m6 dA靶向于内源性m6 dA区域，并研究其生物学效应。这些新的实验方法开发和应用在这里是迫切需要在这个快速发展，但极具争议的领域。该项目的结果将为人类细胞中m6 dA的存在和生物学作用以及负责的N6-MTases提供新的线索。我们的数据将有助于巩固我们对m6 dA在人类DNA和细胞中的潜在作用的理解。

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.