Knockout of DDM1 in Physcomitrium patens disrupts DNA methylation with a minute effect on transposon regulation and development.

The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. In flowering plants, DDM1 mediates methylation in heterochromatin, which is targeted primarily by MET1 and CMT methylases and is necessary for silencing transposons and for proper development. DNA methylation mechanisms evolved throughout plant evolution, whereas the role of DDM1 in early terrestrial plants remains elusive. Here, we studied the function of DDM1 in the moss, Physcomitrium (Physcomitrella) patens, which has robust DNA methylation that suppresses transposons and is mediated by a MET1, a CMT, and a DNMT3 methylases. To elucidate the role of DDM1 in P. patens, we have generated a knockout mutant and found DNA methylation to be strongly disrupted at any of its sequence contexts. Symmetric CG and CHG sequences were affected stronger than asymmetric CHH sites. Furthermore, despite their separate targeting mechanisms, CG (MET) and CHG (CMT) methylation were similarly depleted by about 75%. CHH (DNMT3) methylation was overall reduced by about 25%, with an evident hyper-methylation activity within lowly-methylated euchromatic transposon sequences. Despite the strong hypomethylation effect, only a minute number of transposons were transcriptionally activated in Ppddm1. Finally, Ppddm1 was found to develop normally throughout the plant life cycle. These results demonstrate that DNA methylation is strongly dependent on DDM1 in a non-flowering plant; that DDM1 is required for plant-DNMT3 (CHH) methylases, though to a lower extent than for MET1 and CMT enzymes; and that distinct and separate methylation pathways (e.g. MET1-CG and CMT-CHG), can be equally regulated by the chromatin and that DDM1 plays a role in it. Finally, our data suggest that the biological significance of DDM1 in terms of transposon regulation and plant development, is species dependent.

DNA methylation mutants in Physcomitrella patens elucidate individual roles of CG and non-CG methylation in genome regulation.

Cytosine (DNA) methylation in plants regulates the expression of genes and transposons. While methylation in plant genomes occurs at CG, CHG, and CHH sequence contexts, the comparative roles of the individual methylation contexts remain elusive. Here, we present Physcomitrella patens as the second plant system, besides Arabidopsis thaliana, with viable mutants with an essentially complete loss of methylation in the CG and non-CG contexts. In contrast to A. thaliana, P. patens has more robust CHH methylation, similar CG and CHG methylation levels, and minimal cross-talk between CG and non-CG methylation, making it possible to study context-specific effects independently. Our data found CHH methylation to act in redundancy with symmetric methylation in silencing transposons and to regulate the expression of CG/CHG-depleted transposons. Specific elimination of CG methylation did not dysregulate transposons or genes. In contrast, exclusive removal of non-CG methylation massively up-regulated transposons and genes. In addition, comparing two exclusively but equally CG- or CHG-methylated genomes, we show that CHG methylation acts as a greater transcriptional regulator than CG methylation. These results disentangle the transcriptional roles of CG and non-CG, as well as symmetric and asymmetric methylation in a plant genome, and point to the crucial role of non-CG methylation in genome regulation.