Abstract
Gene expression is largely regulated by DNA methylation, transcription factor (TF), and microRNA (miRNA) before, during, and after transcription, respectively. Although the evolutionary effects of TF/miRNA regulations have been widely studied, evolutionary analysis of simultaneously accounting for DNA methylation, TF, and miRNA regulations and whether promoter methylation and gene body (coding regions) methylation have different effects on the rate of gene evolution remain uninvestigated. Here, we compared human-macaque and human-mouse protein evolutionary rates against experimentally determined single base-resolution DNA methylation data, revealing that promoter methylation level is positively correlated with protein evolutionary rates but negatively correlated with TF/miRNA regulations, whereas the opposite was observed for gene body methylation level. Our results showed that the relative importance of these regulatory factors in determining the rate of mammalian protein evolution is as follows: Promoter methylation ≈ miRNA regulation > gene body methylation > TF regulation, and further indicated that promoter methylation and miRNA regulation have a significant dependent effect on protein evolutionary rates. Although the mechanisms underlying cooperation between DNA methylation and TFs/miRNAs in gene regulation remain unclear, our study helps to not only illuminate the impact of these regulatory factors on mammalian protein evolution but also their intricate interaction within gene regulatory networks.