Mechanisms of CMT3 activation and histone methylation in Arabidopsis thaliana

DNA methylation and histone methylation are modifications that allow the epigenetic control of gene expression. CHROMOMETHYLASE 3 (CMT3) is one of the DNA methyltransferases responsible for DNA methylation in the CHG context (where H is either A,T or C) in Arabidopsis thaliana. CMT3-mediated CHG methylation was shown to be dependent on histone methylation, in particular on the methylation of lysine 9 on the histone H3. At the same time, methylation of lysine 27 histone H3 was also implicated in CMT3 function. Despite these data, the exact mechanism of CMT3 activation and the correlation of CHG methylation with histone methylation were elusive. To gain a deeper understanding of CMT3 function, a multifaceted approach was taken. We have studied CMT3 both in vitro and in vivo and searched for histone methyltransferases that might influence CMT3 activity. We also profiled the localization of di-methylated lysine 9 histone H3 (H3K9me2) genome-wide and compared the data with whole-genome DNA methylation.;Our findings demonstrated that CMT3 activity is partially dependent on lysine 9 histone H3 methylation and not dependent on lysine 27 histone H3 methylation. Specifically, in vitro analysis of CMT3 methyltransferase activity revealed that CMT3 is dependent on the methylation of lysine 9 histone H3, when the DNA substrate is unmethylated. Interestingly, when the substrate is hemi-methylated DNA, CMT3 becomes activated by the histone H3 tail even in the absence of lysine 9 histone H3 methylation. Dissection of CMT3 function in vivo demonstrated that both BAH and chromo domains of CMT3 play an important role in the ability of CMT3 to propagate CHG methylation. We have also showed that H3K9me2 is correlated genome-wide with CMT3 dependent CHG methylation. In addition, we demonstrated that mutants in histone methyltransferases responsible for lysine 27 histone H3 mono-methylation do not affect DNA methylation and H3K9me2, suggesting that H3K27me1 acts independently of these epigenetic marks. Taken together, our findings provide a greater insight into how CMT3 becomes activated and recruited to the sites of action.