While N6-methyldeoxyadenine (6mA) modification has been linked to fundamental regulatory processes in prokaryotes, its prevalence and functional implications in eukaryotes are controversial. Here, we report 6mA-Sniper to quantify 6mA sites in eukaryotes at single-nucleotide resolution. With 6mA-Sniper, we delineated an accurate 6mA profile in C. elegans with 2,034 sites, significantly enriched on sequences of [GC]GAG motif. Twenty-six of 39 6mA events with MnlI restriction endonuclease sites were experimentally verified, demonstrating the feasibility of this method. Notably, the enrichment of these 6mA sites on a specific sequence motif, their within-population conservation and the combinatorial patterns, and the selective constrains on them jointly support an active model for the shaping of the profile by some undiscovered methyltransferases. We then report the dominant contribution of one new methyltransferase, METL-9, in shaping the base level and the stress-dependent changes of 6mA profile in C. elegans, in that the levels of 6mAs at single-nucleotide resolution are significantly decreased in strains with the removal of METL-9 (METL-9 KO-OP50), while generally increased after P. aeruginosa infection. Accordingly, we identified 998 stress-dependent 6mA sites emerged specifically after the infection, enriched in stimulus response genes generally upregulated after the infection. The upregulation of these genes is likely regulated through a mutual exclusive crosstalk between 6mA and H3K27me3 modification, as supported by their cooccurrence, and the increasing of H3K27me3 at regions marked by decreased 6mA levels in METL-9 KO-OP50 strains. Notably, in different C. elegans strains, the cross-strain genetic variants removing 6mA sites are associated with the decreased expression of their host genes, and the removal of two randomly-selected 6mA events with genome editing directly decreased the expression of their host genes. We thus highlight 6mA regulation as a previously-neglected regulator of eukaryote transcriptomes in stress response.

N6-Methyldeoxyadenine (6mA) has been rediscovered as a DNA modification with potential biological function in metazoans. However, the physiological function and regulatory mechanisms regarding the establishment, maintenance and removal of 6mA in eukaryotes are still poorly understood. Here we show that genomic 6mA levels change in response to pathogenic infection in Caenorhabditis elegans (C. elegans). We further identify METL-9 as the methyltransferase that catalyzes DNA 6mA modifications upon pathogen infection. Deficiency of METL-9 impairs the induction of innate immune response genes and renders the animals more susceptible to pathogen infection. Interestingly, METL-9 functions through both 6mA-dependent and -independent mechanisms to transcriptionally regulate innate immunity. Our findings reveal that 6mA is a functional DNA modification in immunomodulation in C. elegans.

Animals respond to mitochondrial perturbation by activating the mitochondrial unfolded protein response (UPR mt) to induce the transcription of mitochondrial stress response genes. In C. elegans, activation of UPR mt allows the animals to maintain organismal homeostasis, activate the innate immune response and promote lifespan extension. Here we show that splicing factors such as PRP-19 are required for the induction of UPR mt in C. elegans. PRP-19 also modulates mitochondrial perturbation-induced innate immune response and lifespan extension. Knockdown of PRP-19 in mammalian cells suppresses UPR mt activation and disrupts the mitochondrial network. These findings reveal an evolutionarily conserved mechanism that maintains mitochondrial homeostasis and controls innate immunity and lifespan through splicing factors.