WGBS of Arabidopsis thaliana rll6 mutant

To gain deeper insights into the mechanisms of DNA demethylation pathway, we conducted a genetic screen for proteins that are involved in preventing epigenetic silencing, and then the ones, which are also implicated in DNA demethylation pathway, are used for further studies. Eventually, a mutant with low luciferase luminescence (low LUC luminescence) was recovered, and named reduced LUC luminescence 6-1 (rll6-1). Map-based cloning revealed thatrll6-1 mutation is located in bacterial artificial chromosome (BAC) clone F5I10 on chromosome 4, and there are a total of 10 candidate genes were found within such a region. Analysis of genome-wide methylation patterns of rll6-1 mutant showed that rll6-1 mutation led to 3863 hyper-DMRs throughout the five Arabidopsis chromosomes, and elevated DNA methylation level of 2×35S promoter, which was similar to that found in ros1 mutant. Further analysis demonstrated that there are 1456 common hyper-DMRs shared by rll6-1 and ros1-7, which account for 2407 and 5642 hyper-DMRs, respectively, suggesting both proteins act together in a synergistic manner to remove DNA methylation. Further investigations demonstrated that rll6-1 mutation does not affect the expression of the four genes of the DNA glycosylase/lyase family. Thus, our results demonstrated that RLL6 not only participated in transcriptional anti-silencing, but is also involved in DNA demethylation pathway. Overall design: BS-seq of rll6-1;Col-LUC;ros1-7.