Evolutionary persistence of DNA methylation for millions of years after ancient loss of ade novomethyltransferase

SUMMARYCytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles, yet has been lost many times in diverse eukaryotic lineages. In the yeastCryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase, Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificityin vitroandin vivoand utilizes similarin vivocofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for ade novomethylase, DnmtX, between 50-150 MYA. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns inC. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 MY through a process analogous to Darwinian evolution of the genome.