Study Of Epigenetic Mechanism On Rice DNA Adenine And Cytosine Methylation

Rice is not only an important cereal crop but also a model plant for studies of functional genomics and epigenomics.Epigenomes play vital roles in gene expression reprogramming during plant development and responses to environmental cues.As a cereal plant,rice has specific epigenomic features(such as DNA cytosine methylation landscape)and epigenetic regulations due to its distinct genomic structures.In addition,recent results have shown that DNA adenine methylation is also an important epigenomic mark in many eukaryotic organisms.However,the extent of DNA adenine methylation and its chromatin function in plants remain unknown.Here,we studied rice genomic DNA adenine and cytosine methylations for the purpose to understand their crosstalk and their roles in gene expression regulation with other epigenetic modifications.My thesis work is focused on three different aspects: 1.DNA N6-methyladenine(6m A)prevalence and function in rice epigenome and its complementary to cytosine methyaltion on gene expression regulation;2.function of rice DNA methyltransferase Os DRM2 in genome-wide DNA methyaltion,histone methylation,TE repression,small RNA accumulation,and gene expression;3.DNA cytosine methylation function in establishing programs of laminar joint development.Main results are summaried as below:a.Recent studies revealed the genome-wide distribution of 6m A in different eukaryotes.In addition,6m A plays important roles in eukaryotes development and epigenetic regulation.But its prevalence and genomic function in higher plant are unclear.Using mass spectrometry and immunoprecipitation and validation with analysis of single-molecule real-time sequencing,we observed that: 1)about 0.2% of all adenines are6 m A-methylated in the rice genome;2)6m A occurs most frequently at GAGG motifs and is mapped to about 20% of genes and 14% of TEs(transposable elements);3)In promoters,6m A marks silent genes,but in bodies correlates with gene activity;4)6m A overlaps with 5-methylated cytosine(5m C)at CG sites in gene bodies and is complementary to 5m C at CHH sites in TEs;5)Os ALKBH1 is potentially involved in6 m A demethylation in rice.The results suggest that 6m A is complementary to 5m C as an epigenomic mark in rice and reinforces a distinct role for 6m A as a gene-expression associated epigenomic mark in eukaryotes.b.To study Os DRM2 function,we perform BS-seq(Bisulfite sequencing),RNA-seq(RNA sequencing),s RNA-seq(small RNA sequencing)in wild type(WT)and osdrm2.These results are: 1)Os DRM2 is required for most of the CHH methylation in rice;2)Os DRM2-dependent CHH methylation mainly targets gene-associated small TEs,thereby affecting the methylation of protein-coding genes which are involved in response to stress and metabolic processes;3)a majority of hypo-CHH DMRs(Different Methylation Regions)showed reduced 24-nucleotide si RNA levels in the ordrm2 mutant,indicating a positive correlation between the 24-nucleotide si RNA abundance and Os DRM2-mediated CHH methylation;4)Os DRM2 may cooperate with Os DDM1 for high levels of CHH methylation in the rice genome.Os DRM2 defines distinct DNA methylation pathways in the bulk of DNA cytosine methylation of the rice genome and suggests that DNA methylation mechanisms may vary among different plant species.c.We used yeast two-hybrid system and pull-down experiments to identify Os DRM2 interacting proteins.We show that Os DRM2 could interact with two rice proteins: SDG711,a H3K27me3 methyltransferase;SDG728,a H3K9me2 methyltransferase.Coimmunoprecipitation assays indicate that interaction of Os DRM2 and SDG711 occurred in vivo.In addition,the osdrm2 mutation reduced total H3K27me3 and H3K9me2 levels.H3K27me3 Ch IP-seq(Chromatin Immunoprecipitation sequencing)and BS-seq analysis showed that the osdrm2 mutation also reduced H3K27me3 from many genes that were methylated at CHH sites.We analyzed H3K9me2 and DNA methylation in osdrm2 by Ch IP-q PCR and found that H3K9me2 and DNA methylation were reduced in the Tos17 transposon.These results suggest that Os DRM2 might be involved in SDG711-mediated H3K27me3 and SDG728-mediated H3K9me2 at some loci of the rice genome.d.In rice,MITEs(miniature inverted repeat TEs)are high copy number DNA transposon elements that are found mostly near genes and can influence their expression.The regulation of Os DRM2 on MITEs-associated gene expression is unclear.In this thesis,we showed that deletion of Os DRM2 could increase the angle of laminar joints compared to WT.Paraffin section and morphology experiments show that cell number is increased in osdrm2 adaxial side compared to WT.Furthermore,many protein-coding genes are up-regulated in osdrm2 laminar joints and these genes are involved in lignin and hormone metabolisms.MITEs are enriched in flanks of these genes and their DNA cytosine methylation is decreased in osdrm2.These results suggest that Os DRM2-mediated CHH methylation of MITEs-associated genes involved in lignin and hormone metabolisms is important for rice laminar cell division and development.