表观遗传修饰新发现.pptx

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roles of,DNA dmethylation,in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomoto fruit,PNAS,Published online May 15,1,郝雪峰,.10.18,表观遗传修饰新发现专家讲座,第1页,1.Abstract,DNA methylation,is a conserved epigenetic mark important for genome integrity,development,and environmental responses in plants and mammals.,Active DNA demethylaton,in plants is initiated by a family of 5-mC DNA glycosylases/lyases(i.e.,DNA demethylases).,This study suggests,that active DNA demethylation is required for both the,activation,of ripening-induced genes and the,inhibition,of ripening-repressed genes.,2,表观遗传修饰新发现专家讲座,第2页,2.1 Research Background,Cytosine methylation levels&gene activation,5-mC levels,are dynamically regulated by,methylation,and,demethylation,reactions.,DNA methylation,is generally associated with,inactive transcription,.,Active DNA demethylation,is critical for,gene activation,or for,the prevention of silencing,.,DNA methylation site:,In mammals:CG,（,DNMTs,）,In plants:CG,（,MET1,）,/CHH,（,CMT2,DRM2,）,/CNG,（,CMT3,）,3,表观遗传修饰新发现专家讲座,第3页,4,2.2 Research Background,ROS1 preferentially targets TEs,TEs,（,Transposable Elements,）,When ROS1 targeted TEs are located in the promoter regions of genes,ROS1 can,protect gene expression,by demethylating the adjacent TE,s,.,Many studies have demonstrated that,by demethylating nearby TEs,active DNA demethylation is critical for,gene activation,or for the,prevention of silencing,.,表观遗传修饰新发现专家讲座,第4页,2.3 Research Background,In contrast to,Arabidopsis,Tomato,is an economically,important crop,and a,model,for studying fleshy fruit ripening,.,Fruit ripening is associated with many distinct changes,which are driven by,phytohormones,and,developmental factors,.,In addition to,ethylene,and the,TFs,DNA methylation,is another key regulator of fruit ripening.,5,表观遗传修饰新发现专家讲座,第5页,2.4 Research Background,Tomato contains four putative DNA demethylases:,SlDML 1,4,SlDML 1/2,：,most closely related to,AtROS1,SlDMLs play critical roles in fruit ripening.,RNAi against the conserved HhH-GPD domain,Which,SlDML(s)is required for fruit ripening?,What impact,dose active DNA demethylation have on the genome-wide during fruit ripening?,6,表观遗传修饰新发现专家讲座,第6页,3.Research Contents,Generated,two,mutant,alleles of SlDML2.,in the tomato cultivar Micro-Tom,using CRISPR/Cas 9 system,Determined,the,genome-wide effect,of SlDML2 on DNA demethylation.,compared the DNA methylomes of WT and,sidml2,mutant fruits;,Analyzed,the,transcriptome,of,sidml2,mutant fruits.,表观遗传修饰新发现专家讲座,第7页,4.Result,4.1 Generation of stable,loss-of-function mutant alleles of,SlDML2,using the CRISPR/Cas9 gene-editing system.,8,表观遗传修饰新发现专家讲座,第8页,4.Result,4.2 The,sldml2,mutations inhibit fruit ripening.,9,表观遗传修饰新发现专家讲座,第9页,4.Result,4.3,The,sldml2,mutations cause genome-wide DNA hypermethylation.,W,hole-genome bisulfite sequencing,was performed,to i,nvestigate the genetic function of SlDML2,in DNA demethylation during fruit ripening.,Table S1.Statistics of sequencing data,Sample name,Total C in genome,#of Cs with coverage=1,%of C coverage,Depth of C coverage,sldml2,-25dpa,251181662,239907749,95.5,10.7,sldml2,-46dpa_rep1,251181662,240673869,95.8,14.5,sldml2,-46dpa_rep2,251181662,240478351,95.7,12.5,WT-25dpa,251181662,241742160,96.2,11.9,WT-46dpa,251181662,242243342,96.4,13.6,单个碱基,被测序,平均次数,测序取得序列,占,整个基因组,百分比,Table S2/3.List of hyper-DMRs in,sldml2-1,-46dpa,vs.WT-46dpa,sldml2,-46dpa-rep1,sldml2,-46dpa-rep2,WT-46dpa,hyper-DMRs,21,515,23,026,1645,hoper-DMRs,6,643,6,689,13,141,The results is consistent with the presumed function of SlDML2 in DNA demethylaton.,表观遗传修饰新发现专家讲座,第10页,Differentially methylated regions,11,(A).DNA methylation,levels of,sldml2-1,hyper-DMRs,in fruits at 46 dpa.,(B).Boxplots showing,DNA methylation levels,at combined hyper-DMRs.,表观遗传修饰新发现专家讲座,第11页,DNA methylation levels of several,sldml2,hyper-DMRs(boxed regions)in WT and,sldml2,.,表观遗传修饰新发现专家讲座,第12页,13,(A)The,compositions of hyer-DMRs,.(B),Density plots,of 1,5,.,表观遗传修饰新发现专家讲座,第13页,14,Chromosomal distribution of,SlDML2-targeted TEs,and,total TEs,.,表观遗传修饰新发现专家讲座,第14页,(C).,Boxplots showing distances between SIDML2-targeted or nontargeted TE(Left)and IG regions(Right)and their nearest protein-coding genes.,(D).,Boxplot showing the distances between genes and their nearest TEs in Arabidopsis and tomato.,(E).,Percentages of genes that contain TEs in their 500-bp,1000-bp,and-bp upstream sequences in Arabidopsis,tomato,maize,and rice.,15,(C)Boxplots showing distances between SIDML2-targeted or nontargeted TE(Left)and IG regions(Right),and their nearest protein-coding genes.,(D)Boxplot showing the distances between genes and their nearest TEs in Arabidopsis and tomato.,(E)Percentages of genes that contain TEs in their 500-bp,1000-bp,and-bp upstream sequences in,Arabidopsis,tomato,maize,and rice.,18%,24%,31%,39%,58%,77%,表观遗传修饰新发现专家讲座,第15页,4.Result,4.4,The,sldml2,mutations cause genome-wide DNA hypermethylation.,DNA methylation changes,during fruit ripening(ripening-induced DMRs).,3,306,hyper,-DMRs,16,412 ri-DMRS,13,106,hypo,-DMRs(8,176 overlap with hyper-DMRs),The DNA methylation changes,occurred in all three sequence contexts,.,表观遗传修饰新发现专家讲座,第16页,(A/B)Boxplots showing mCG,mCHG,and mCHH levels at ripening-induced hypo-DMRs.,(C)The distribution of log2fold-change of mCG,mCHG,and mCHH levels.,C,表观遗传修饰新发现专家讲座,第17页,18,4.Result,4.5 SlDML2-mediated DNA demethylation,also has silencing functions,.,Using k-means clustering,we assigned the,6,651,hyper-DMR-associated,genes,to three clusters:,表观遗传修饰新发现专家讲座,第18页,19,(A)Boxplots showing changes in DNA methylation levels at different groups of DMRs in sldml2-1-46dpa compared with WT-46dpa(NS,not significant).,表观遗传修饰新发现专家讲座,第19页,20,(B)DNA methylation changes in cluster1 and cluster2 genes in,sldml 2-1,-46dpa,vs.,WT-46dpa,.,(C)DNA methylation changes in cluster1 and cluster2 genes in,ripe fruits,compared with,immature fruits,.,表观遗传修饰新发现专家讲座,第20页,Conclusion,SlDML2,is critical for tomato fruit ripening,because it mediates all ripening-induced DNA demethylation.This genome-wide demthylation is important,not only for activating the expression of ripening factors,but also for silencing hundreds of genes that are no longer needed and must be repressed when the fruit is ripe,.,21,表观遗传修饰新发现专家讲座,第21页,