Reinforcement of transcriptional silencing by a positive feedback between DNA methylation and non-coding transcription

Non-coding transcription is an important determinant of heterochromatin formation. In Arabidopsis thaliana a specialized RNA polymerase V (Pol V) transcribes pervasively and produces long non-coding RNA. This facilitates locus-specific establishment of RNA-directed DNA methylation (RdDM). Subsequent maintenance of RdDM is associated with elevated levels of Pol V transcription, which is consistent with a mutual reinforcement of DNA methylation and non-coding transcription. However, the impact of DNA methylation on Pol V transcription remained unresolved. We found that loss of DNA methylation leads to a strong reduction of Pol V transcription. This occurs when DNA methylation is lost in all sequence contexts, which may happen not only in mutants defective in RdDM but also in mutants lacking maintenance DNA methyltransferases. Our results support a model where RdDM is maintained by a mutual reinforcement of DNA methylation and Pol V transcription with a strong crosstalk with other silencing pathways. Overall design: RNA immunoprecipitation with an anti-NRPE1 antibody was performed on 5 samples, which include 3 different genotypes (cmt3:2replicates, drm2: 2replicates, met13)