MCM10 Mediates RNA-directed DNA Methylation In Arabidopsis

Gene silencing plays roles in many aspects like gene expression regulation,transposon expression and jumping repression,and genome stability maintaining.DNA methylation is one of the most important epigenetic modifications which is involved in gene silencing.RdDM(RNA-directed DNA methylation)has been found so far as the only pathway which can mediate the de novo DNA methylation in Arabidopsis.Meanwhile,it can maintain CHH methylation.DRM2 is a DNA methyltransferase in RdDM.Though many progresses on RdDM mechanism have been achieved by now,there are still some problems needing to be solved.L119 was a transgenic line which carried a T-DNA containing two silencing reporter genes,Pro35S::NPTII and ProRD29A::LUC.Mutagenized by EMS,mutants that can release the Pro35S::NPTII were selected.These mutants grew well on MS media supplemented kanamycin.mcm10 was one of the mutants identified by map-based cloning according to this phenotype.Further investigation found that the expression of ProRD29A::LUC was released in mcm10 too.The CHH DNA methylation level at ProRD29A::LUC but not Pro35S::NPTII was decreased in mcm10 by bisulfite DNA methylation sequencing.The whole genome DNA methylation analysis revealed that DNA methylation at a large scale of RdDM targets was decreased in mcm10 and 70%of mcm10 hypomethylation DMRs were overlapped with that of drm2.The expression of RdDM targets were derepressed in mcm10.The expression of ROS2 was down regulated in RdDM mutants.In according with other RdDM mutants,the expression of ROS1 was reduced in mcm10.All of these results demonstrated that MCM10 participated in RdDM pathway.RdDM can be divided into two phases,a upstream of Pol ?-mediated siRNA production phase and a downstream of Pol ?-mediated DNA methylation phase.Previous study showed that disruption of either phase could lead to decrease of siRNAs,but the affected siRNA cluster types were not exactly the same.According to this,the two phases specific 24 nt-siRNA was detected by Northern-blot and analyzed by whole genome small RNA sequencing.Both of the two results showed that MCM10 affected downstream but not upstream 24 nt-siRNA accumulation,which suggested that MCM10 acted at downstream of RdDM.The Pol V transcripts were not reduced in detected by strand-specific RT-PCR.This result suggested that MCM10 did not affect Pol V transcription and might have a function downstream of Pol V transcription.Further analysis showed that MCM10 interacted with DRM2 by in vitro and in vivo assays.The current model proposes that DRM2 functions at the Pol V transcription fork,where the double-stranded DNA is unwound.And there was a report that plant DRM can only catalyze dsDNA,but not single stranded DNA.Therefore,we speculated that whether MCM10 could anneal the two strands at Pol ?transcription fork,which would facilitate DRM2 to catalyze DNA methylation.In vitro tests on MCM10 DNA strand annealing activity showed that MCM10 possessed a strong annealing activity and its C-terminal fragment was important to its activity.Besides,the annealing activity ofMCM10 could facilitate plant DRM to catalyze DNA methylation tested by a DNA methyltransferase assay.In summary,we propose that MCM10 facilitates DRM2-catalyzed DNA methylation by annealing the two DNA strands at Pol V transcription fork.The work here discovers a new component of RdDM,MCM10,and the further investigation on its function mechanism improves the current RdDM model.The finding of the DNA strand annealing activity of MCM10 could be of great values to researches on studying MCM10 functions in other species.