Small RNAs Prevent Transcription-Coupled Loss of Histone H3 Lysine 9 Methylation in Arabidopsis thaliana

In eukaryotes, histone H3 lysine 9 methylation (H3K9me) mediates silencing of invasive sequences to prevent deleterious consequences including the expression of aberrant gene products and mobilization of transposons. In Arabidopsis thaliana, H3K9me maintained by SUVH histone methyltransferases (MTases) is associated with cytosine methylation (5meC) maintained by the CMT3 cytosine MTase. The SUVHs contain a 5meC binding domain and CMT3 contains an H3K9me binding domain, suggesting that the SUVH/CMT3 pathway involves an amplification loop between H3K9me and 5meC. However, at loci subject to read-through transcription, the stability of the H3K9me/5meC loop requires a mechanism to counteract transcription-coupled loss of H3K9me. Here we use the duplicated PAI genes, which stably maintain SUVH-dependent H3K9me and CMT3-dependent 5meC despite read-through transcription, to show that when PAI sRNAs are depleted by dicer ribonuclease mutations, PAI H3K9me and 5meC levels are reduced and remaining PAI 5meC is destabilized upon inbreeding. The dicer mutations confer weaker reductions in PAI 5meC levels but similar or stronger reductions in PAI H3K9me levels compared to a cmt3 mutation. This comparison indicates a connection between sRNAs and maintenance of H3K9me independent of CMT3 function. The dicer mutations reduce PAI H3K9me and 5meC levels through a distinct mechanism from the known role of dicer-dependent sRNAs in guiding the DRM2 cytosine MTase because the PAI genes maintain H3K9me and 5meC at levels similar to wild type in a drm2 mutant. Our results support a new role for sRNAs in plants to prevent transcription-coupled loss of H3K9me.