Biochemical Characteristics And Regulatory Mechanism Of A KMT4/Dot1-like Methyltransferase From Archaeon Sulfolobus Islandicus

In eukaryotic cells, histones are subjected to different kinds of post-translational modifications. These covalent modifications play pivotal roles in dynamic regulation of chromatin structure and gene expression. Histone lysine methylation is one of the most important epigenetic modifications and is widely distributed throughout all eukaryotes. Does epigenetic mechanism similar to eukaryotes also exist among prokaryotes? Until now our knowledge of origin and evolution of the epigenetic regulation pathway is still very limited.In eukaryotes, there are two distinct classes of histone lysine methyltransferase, the SET domain and Dotl families. Through bioinformatic methods including PSI-BLAST, we noticed that many archaeal lineages encode at least one putative KMT4/Dotl-like methyltransferase, designate as aKMT4/Dotl (archaeal lysine methyltransferase4). KMT4/Dotl-like genes are conserved in archaea domain, especially among Crenarchaeal branch, whereas SET domain MTases only exist in a few methanogenic Methanosarcina species (G61-SET). aKMT4only comprises the relatively conserved catalytic core of Dotl family methyltransferase, including AdoMet binding and the active sites, but it completely lacks the substrate recognition domain.We cloned and purified aKMT4from archaeal model organism Sulfolobus islandicus. Characteristics and regulation of enzyme activity of aKMT4were carried out mainly through biochemical approaches. Recombinant aKMT4can methylate a set of various proteins in vitro, which is consistent with the lack of substrate recognition domain of enzyme itself and the variegated methylation pattern of a number of proteins in Sulfolobus cells. All known substrates are nucleic acid binding proteins, including the chromatin proteins Su17d and Cren7, DNA replication proteins, Helicase SisMCM, RNA exosome components, and the ribosomal protein RPL11. These results implied that aKMT4might mainly participate in the regulation of chromatin structure and gene expression in archaea. Interestingly, aKMT4methylates the two forms of RNA exosome complex in different patterns, called Rrp4-exosome and Cs14-exosome. These results provided clues that aKMT4may play a role in regulation of mRNA recognizing, processing, and degradation by RNA exosome.aKMT4can monomethylate the chromatin proteins Su17d and Cren7at multiple lysine residues. MS/MS analysis indicated that in vitro methylation data of Cren7catalyzed by aKMT4is highly consistent with native Cren7methylation data. Cren7methylation was completely abolished only when all12lysine residues were changed, indicating that aKMT4is specific to lysine and has extremely low amino acid sequence specificity. Interestingly, when DNA was added to the methylation system, DNA significantly promoted methylation level of Sul7d but not of Cren7. Kinetic analysis of Sul7d methylation showed that aKMT4achieves the highest methylation activity on DNA-associated Sul7d. MS/MS data demonstrated that only the in vitro methylation pattern of DNA-associated Su17d correlates well with the methylated sites of native Su17d in vivo. These results further suggested that aKMT4catalyzes methylation of Sul7d in the chromatin context. All these results showed that aKMT4might be at least the major methyltransferase of archaeal chromatin proteins. Despite aKMT4has very low sequence bias, its behavior might be regulated by the local chromatin environment.aKMT4can self-methylate intra-molecularly. The automethylated enzyme shows relatively compromised activity. aKMT4-8A mutant with decreased automethylation shows a more than150%increase in methylation of substrates compared with aKMT4. In addition, automethylation is inhibited by suitable substrates in a concentration dependent manner. This suggested a new mechanism to regulate methyltransferase activity through automethylation.In a word, this research systematically analyzed biochemical characteristics of the first widespread methyltransferase aKMT4among archaea. This study not only provides direct experimental evidence for the prokaryotic origin model of eukaryotic histone modification enzymes, but also lays good foundation for further study of dynamic regulation of archaeal chromatin through methylation.