Study Of DDM1 And RNA-binding Protein DDM3 On Regulation Of Transcriptional Read-through In Arabidopsis

The strict transcriptional regulation mechanism is important to gene expression.The efficient transcription termination is required to prevent transcriptional read-through(TR T)that may cause transcriptional interference at neighboring genes.However,the exact TRT mechanism remains poorly understood.3'UTR plays important roles in transcription,especially during the termination stage.In this study,we mutagenized the WT transgenic seeds with EMS that carry 35S-LUC transgene,and screened for mutants in which the 35S-LUC was activated.We find that nucleosome remodeler DDM1(Decrease in DNA methylation 1)and RNA-binding protein DDM3(Decrease in DNA methylation 3)are key components of this regulatory machinery.DDM1 is an important chromatin remodeling protein of the Snf2 family in Arabidopsis.DDM1 alters nucleosome composition and placement,and its mutation has been reported to cause a profound loss of methylation in the regions of TEs and genes.In this study,we found DDM1 was required for preventing TRT of 35S-LUC and endogenous MULE-CYP40.By whole-genome strand-specific RNA sequencing,we identified and confirmed 43 endogenous TRT sites between genes,TEs or gene and TE in the ddml-11 mutant.TRT mainly occurred at heterochromatin regions.Analysis of DNA methylation in these regions revealed that TRT occurred frequently at the intergenic regions with a higher methylation level in wild type comparing to the regions that TRT did not occur.Our results suggest that the intergenic DNA methylation may involve in preventing aberrant gene TRT or producing new gene during evolution.We also discovered a new RNA-binding protein DDM3 which contains three RNA-binding KH domains.DDM3 is located in nucleus and expressed in pollen.We found the DDM3 mutation caused TRT of 35S-LUC and endogenous genes and TEs.Through genome-wide RNA sequencing,we found that there were 719 genes and 634 TEs up-regulated in ddm3-1 mutant.According to whole-genome bisulfate sequencing data,DNA methylation(CG,CHG and CHH)level was reduced in ddm3-1 mutant.The effects of DDM3 mutation on DNA methylation were similar to those of mutation in DDM1.The RIP-seq results suggest that DDM3 can bind to the specific RNA,and in the corresponding DNA region,the methylation level was significantly reduced in ddm3 mutant.Furthermore,DDM3 interacted with DNA methyltransferase DRM1.In conclusion,we demonstrate that DDM1 and DDM3 are required for preventing aberrant gene TRT.DNA methylation level in the intergenic regions is an important signature for this process.We propose that DDM3 regulates DNA methylation by binding the RNAs and recruiting DRM1 and other DNA methyltransferases to the corresponding DNA location.Moreover,this mechanism is not dependent on RdDM pathway.Our studies contribute to further understanding of DNA methylation mechanism and increase the knowledge towards regulation of transcriptional read-through in plants.