Function Of Rice Chromatin Remodeling Factor DDM1 In Regulation Of DNA Methylation And Gene Expression

The major genetic information is encoded by DNA,which wrapped around the histone octamers and is packaged into chromatin.The ovalent modifications of histone and DNA constitute an additional layer of information that influences gene expression.DNA cytosine methylation is mainly enriched in transposable elements(TEs)and repetitive sequences.The rice gemome contains about 40% repetitive sequence and TEs and many small TEs that are located near genes in euchromatin regions,suggesting that DNA methylation in the rice genome may have more important function in regulating gene expression and plant development.In this work,I studied the precise DNA methylation profiles and characteristics in rice seedling tissues,the function of rice chromatin remodeling factor DDM1 in regulating DNA methylation and gene expression as well as plant development.Rice has two genes homologous to Arabidopsis DDM1.Two genes display high level of identity in protein sequence and the coding sequence.The single mutants osddm1 a and osddm1 b show no obvious phenotypes diversity,although slight DNA methylation decrease was decteted.The osddm1a/1b double mutant obtained by genetic crosses produced many severe developmental abnormalities including extremely reduced plant height and complete sterility.We generated single-nucleotide resolution maps of cytosine methylation in seedling leaf tissues of wild type and osddm1 a,osddm1b and osddm1a/1b mutants by bisulfite sequencing(BS-seq),the DNA methylation of osddm1 a and osddm1 b reduced by 9.7% and 16.5% respectively,thus the osddm1a/1b double mutant reduced 54% DNA methylation,CG,CHG and CHH methylation reduced by44.9%,73.5% and 49.1% respectively.We observed that methylation at CG and CHG sites were enriched in heterochromatic regions,whereas CHH methylation was located essentially in euchromatic regions in the rice genome.In wild type,CG methylation was at similar levels between the 5’ and 3’ regions and gene body(except the ends of the body),while non-CG methylation is much higher in the 5’ and 3’ regions than the body in non-TEgenes,The osddm1a/1b double mutation reduced methylation at all contexts in non-TE genes.In osddm1a/1b mutant,CG and CHG methylation in TEs and TEGs were grestly decreased.So OsDDM1 is mainly required for CG and CHG methylation of TEs and TEGs.Rice TEs and TE-related genes show high CG and CHG methylation,only small TEs(less than 1kb)show relatively high CHH methylation.DRM2 is a CHH methyltransferase in RdDM pathway.We showed that rice DRM2 was necessary for CHH methylation at genic regions of euchromatin.Comparison of CHH methylation losses between osddm1a/1b and osdrm2 mutants revealed that most loci with CHH loss were overlapped.These data suggest that OsDDM1 and OsDRM2 may cooperate for euchromatin CHH methylation in the rice genome.To study mutation effects of osddm1a/1b and osdrm2 in gene expression,we analyzed the transcriptomes of the same tissues by RNA-seq.Data analysis revealed 1453up-regulated and 141 down-regulated genes in osddm1a/1b compared to wild type(>4folds,p-value <0.001).In osddm1a/1b,about 60%(859/1453)of the up-regulated genes were TEGs,among which 80.3%(687/859)were hypo-CHG DMGs and about 34%(200/594)of the up-regulated protein-coding genes were hypo-CHG DMGs.Moreover,the up-regulated protein-coding genes were more enriched for H3K9me2 than unaffected genes,so OsDDM1 repress the expression of TEGs and heterochromatic non-TE genes by CHG methylation.In osddm1a/1b,we observed obvious gain of CHH methylation in centromere and loss of CHH methylation in euchromatin.To test whether CHH methylation change correlated with sRNA,we analyzed sRNA in osddm1a/1b by sRNA-seq.The level of 24 nt siRNA reduced on euchromatin and increased on heterochromatin in osddm1a/1b.The results indicate that the change of 24 nt siRNA show positive correlation with change CHH methylation.The increased CHH methylation in osddm1a/1b was lost methylation in osddm1a/1b/drm2,suggesting that the CHH methylation gained in osddm1a/1b were de nove and maintained by OsDRM2.The results indicate that rice OsDDM1 can maintain CG,CHG methylation and repress CHH methylation in heterchromatin.Rice OsDDM1 cooperates with OsDRM2 for CHH methylation in euchromatic region.Therefore,in addition to mediate high levels of CG ad CHG methylation of the genome,rice OsDDM1 appears to play an important role in gene regulation.