bioRxiv2023
Arabidopsis thaliana has two nucleolus organizer regions (NORs), consisting of long tandem arrays of 45S rRNA genes on chromosomes 2 (NOR2) and 4 (NOR4). The rRNA gene subtypes mapping to NOR2 are mostly silenced during development, whereas most of those mapping to NOR4 are active. Cytosine methylation of promoters plays a significant role in rRNA gene silencing, but recent findings demonstrate that gene body methylation also play a key role in gene silencing. Previous studies have demonstrated that CG methylation plays a role in rRNA gene silencing. However, it remains to be determined whether Chromomethylase 2 (CMT2)-mediated, RdDM-independent CHH methylation plays any role in rRNA gene silencing. To find out the relative importance of cytosine methylation in each of the sequence contexts (CG, CHG, and CHH, where H=A, T, or C), we studied multiple DNA methylation-deficient Arabidopsis mutants. Primarily, we show that the mutants that displayed higher losses of CG or CHH methylation at rDNA loci, display higher degrees of NOR2 rRNA gene silencing disruption. Using whole genome bisulfite sequencing data, we show that the loss of RdDM-independent CMT2, not the loss of RdDM-dependent DRM2, erases most of the CHH methylation at rDNA loci, while retaining CG methylation levels comparable to those of wild-type Col-0, demonstrating the sufficiency of CHH methylation loss for releasing rRNA gene silencing. Analysis of Arabidopsis and tomato NOR sequences revealed that in 45S rRNA gene sequences, cytosines mostly occur in the CHH context, followed by CG and the lowest being in CHG contexts. The predominance of CHH sites is even more pronounced when the rRNA gene promoter and spacer promoter(s) are considered (0-7% CG, 14-25% CHG, and 79-80% CHH), suggesting a mechanism to explain the stronger disruption of NOR2 gene silencing in mutants that display higher losses of CHH methylation. Our data also reveal a role for gene body methylation in rRNA gene silencing. We show that NOR2 genes are relatively hypermethylated compared to NOR4, consistent with findings from recent studies. By contrast, human rRNA genes have more CG sites than CHH and CHG, correlating with the evolutionary loss of CMTs in mammals. CMTs are plant-specific, which primarily methylate CHG and CHH sites. Our data define a critical role for CMT2-mediated RdDM-independent CHH methylation, in combination with MET1-mediated CG methylation, in rRNA gene silencing, which is entirely mediated by CG methylation in mammals.