A plant DNMT3 induces non-CG methylation and affects gene expression in human cells

CG methylation is the predominant form of DNA methylation across eukaryotes, whereas non-CG methylation in vertebrates is largely restricted to neurons and embryonic stem cells and is mediated by DNMT3 enzymes. In contrast, plant DNMT3 homologs catalyze extensive non-CG methylation across diverse tissues. To test whether a plant DNMT3 can establish non-CG methylation and influence transcription in human cells, we expressed Physcomitrium patens DNMT3b (PpDNMT3) in HEK293 cells, which lack this modification. PpDNMT3 induced robust, genome-wide non-CG methylation in transiently and stably transfected cells, in a manner dependent on an intact catalytic domain. PpDNMT3 expression was associated with altered gene expression that coincided with methylation changes. Genes showing combined CG and non-CG hypermethylation within promoters and gene bodies exhibited the strongest transcriptional repression. These results demonstrate that plant DNMT3 enzymes can reprogram human epigenomes and provide a versatile tool for modulating DNA methylation in cell types lacking endogenous non-CG methylation.