The study investigates the role of CHROMOMETHYLASE3 (CMT3) in maintaining CpXpG methylation, a type of epigenetic silencing in Arabidopsis. CMT3 is a novel cytosine methyltransferase homolog that was identified through a genetic screen for suppressors of a hypermethylated clark kent mutant. These mutants, which are associated with dense hypermethylation at noncanonical cytosines (CpXpG and asymmetric sites), exhibit decreased CpXpG methylation and reactivated expression of endogenous retrotransposon sequences. The cmt3 mutants display wild-type morphology but show significant loss of CpXpG methylation, particularly at the SUP gene and other sequences throughout the genome. Bisulfite genomic sequencing and Southern blot analysis confirmed the loss of CpXpG methylation in cmt3 mutants, while CpG methylation remained largely unaffected. Additionally, cmt3 mutants showed differential reactivation of endogenous retrotransposons, suggesting that different loci may depend on either CpXpG or CpG methylation for gene silencing. The findings indicate that CMT3 is specifically required for maintaining CpXpG methylation, highlighting its distinct role from the DNMT1/MET1 class of methyltransferases.The study investigates the role of CHROMOMETHYLASE3 (CMT3) in maintaining CpXpG methylation, a type of epigenetic silencing in Arabidopsis. CMT3 is a novel cytosine methyltransferase homolog that was identified through a genetic screen for suppressors of a hypermethylated clark kent mutant. These mutants, which are associated with dense hypermethylation at noncanonical cytosines (CpXpG and asymmetric sites), exhibit decreased CpXpG methylation and reactivated expression of endogenous retrotransposon sequences. The cmt3 mutants display wild-type morphology but show significant loss of CpXpG methylation, particularly at the SUP gene and other sequences throughout the genome. Bisulfite genomic sequencing and Southern blot analysis confirmed the loss of CpXpG methylation in cmt3 mutants, while CpG methylation remained largely unaffected. Additionally, cmt3 mutants showed differential reactivation of endogenous retrotransposons, suggesting that different loci may depend on either CpXpG or CpG methylation for gene silencing. The findings indicate that CMT3 is specifically required for maintaining CpXpG methylation, highlighting its distinct role from the DNMT1/MET1 class of methyltransferases.