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

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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.