CMT3-mediated <i>ONSEN</i> activation is regulated by H3K9 methylation.

(A) Genome browser view of H3K9me2 abundance at ONSEN in Col-0 and the SUVH4, SUVH5, and SUVH6 loss-of-function mutant (suvh456). (B) Genome browser view of CHH methylation at ONSEN2 in Col-0, cmt3-11, and suvh456. (C) Metaplots showing average CHH methylation of 8 ONSEN copies. -2kb indicates the upstream 2000 bp of the start site, and 2kb indicates the downstream of 2000 bp of the end site. (D) Boxplots showing CWA (CAA and CTA) and non-CWA CHH methylation level in Col-0, cmt3-11, and suvh456 of the open reading frame (ORF) of all eight copies of ONSEN. P-values are from Wilcoxon test. (E) Relative ONSEN transcript level in the corresponding mutants under non-stress (NS) and heat stress (HS). The ONSEN transcripts were normalized to 18S rRNA. All bars represent mean + SD from three biological replicates. Student’s t-test. *P ONSEN in Col-0,cmt3, and cmt2 under NS and HS. H3K9me2 ChIP samples were first normalized to parallel H3 ChIP, and then to the respective spike-in human chromatin. ACT7 served as a negative control. TA3 was used as a positive control. All bars represent mean + SEM from three biological replicates with all technical replicates shown. Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001. ns indicates not significant.