A myriad of epigenetic modifications play critical roles in the genome and transcriptome functions of organisms on our planet. Historically, N6-methyladenine (6mA) has been considered the typical chemical modification for bacterial DNA, while 5-methylcytosine (5mC) is the dominant epigenetic modification in eukaryotes. However, recent intensive research has revealed a more complex scenario, with 6mA gaining importance in the regulation of gene expression in eukaryotes, acting on both DNA and RNA.
Within eukaryotic DNA, two major groups of species can be distinguished based on 6mA levels and patterns. The majority, including more complex eukaryotes, exhibit low levels of 6mA with predominance of asymmetric 6mA (where only one DNA strand is methylated). In contrast, most early diverging fungi (EDF), green algae, and ciliates show elevated levels characterized by symmetric 6mA (where both DNA strands are methylated). This suggests the existence of different types of N6 adenine methyltransferases (writers) involved in adenine methylation on DNA.
Here, we examine the methylomes of 62 species across most phyla of the EDF, revealing that reveal that, although the overall levels of 6mA are high, there are differences in 6mA levels and patterns between phylogenetic groups and among species within a group. In addition, we characterize the role of 6mA and its relationship with 5mC in regulating gene expression and responding to environmental and developmental cues, providing the first glimpse into the dynamic nature of fungal 6mA methylation under varying conditions. Furthermore, we find that lower levels of 5mC in asexual structures of the mucoralean Phycomyces blakesleeanus correlate with transposable element activation, a phenomenon reminiscent of what occurs in the mammalian germline.
Interestingly, we identify specific enzymatic machineries responsible for symmetric and asymmetric adenine methylation in Mucorales, an order of EDF. Asymmetric methylation is performed by MetB, likely acquired via horizontal gene transfer from bacteria. This protein is conserved in EDF but absent in other eukaryotes, suggesting convergent evolution for this function among species showing low but detectable levels of asymmetric 6mA. Deletion of the metB gene in the mucoralean Mucor lusitanicus alters gene expression, resulting in phenotypic changes such as reduced resistance to hydroxyurea, diminished virulence, and increased lipid accumulation, indicating a role in gene regulation.
Symmetric 6mA methylation occurs at the short palindromic sequence ApT, in contrast to asymmetric 6mA, and is concentrated in methylated adenine clusters (MACs) surrounding the transcription start sites of actively expressed genes, suggesting a role in transcriptional activation transcription. In EDF, symmetric 6mA is written by a protein complex similar to those found in ciliates and green algae. This complex comprises an MTA-70 methyltransferase (Mta1), an inactive DNA methyltransferase (Mta9), and the DNA-binding proteins P1 and P2, although M. lusitanicus has only P1. Deletion of genes encoding key complex proteins such as Mta1 or P1 results in altered gene expression, indicating the involvement of symmetric 6mA in gene regulation. Consequently, mutants show slow growth, reduced sporulation, susceptibility SDS stress, and upregulation of DNA repair genes.
In summary, our work reveals that both asymmetric and symmetric 6mA play pivotal roles in the regulation of gene expression in Mucorales, and probably in other EDF, opening new avenues for understanding the evolution of 6mA-dependent regulation in eukaryotes.
Writers for DNA adenine methylation in early diverging fungi and the cellular processes regulated by 6mA in M. lusitanicus