表观遗传修饰新发现.ppt
Critical roles of DNA dmethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomoto fruit,PNAS Published online May 15,2017,1,郝雪峰,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.,2,2.1 Research Background,Cytosine methylation levels&gene activation5-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）,3,4,2.2 Research Background,ROS1 preferentially targets TEsTEs（Transposable Elements）When ROS1 targeted TEs are located in the promoter regions of genes,ROS1 can protect gene expression by demethylating the adjacent TEs.Many studies have demonstrated that,by demethylating nearby TEs,active DNA demethylation is critical for gene activation or for the prevention of silencing.,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.,5,2.4 Research Background,Tomato contains four putative DNA demethylases:SlDML 14SlDML 1/2：most closely related to AtROS1SlDMLs play critical roles in fruit ripening.RNAi against the conserved HhH-GPD domainWhich SlDML(s)is required for fruit ripening?What impact dose active DNA demethylation have on the genome-wide during fruit ripening?,6,3.Research Contents,Generated two mutant alleles of SlDML2.in the tomato cultivar Micro-Tomusing CRISPR/Cas 9 systemDetermined the genome-wide effect of SlDML2 on DNA pared the DNA methylomes of WT and sidml2 mutant fruits;Analyzed the transcriptome of sidml2 mutant fruits.,4.Result,4.1 Generation of stable loss-of-function mutant alleles of SlDML2 using the CRISPR/Cas9 gene-editing system.,8,4.Result,4.2 The sldml2 mutations inhibit fruit ripening.,9,4.Result,4.3 The sldml2 mutations cause genome-wide DNA hypermethylation.Whole-genome bisulfite sequencing was performed to investigate the genetic function of SlDML2 in DNA demethylation during fruit ripening.,单个碱基被测序的平均次数,测序获得的序列占整个基因组的比例,The results is consistent with the presumed function of SlDML2 in DNA demethylaton.,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.,DNA methylation levels of several sldml2 hyper-DMRs(boxed regions)in WT and sldml2.,13,(A)The compositions of hyer-DMRs.(B)Density plots of 15.,14,Chromosomal distribution of SlDML2-targeted TEs and total TEs.,(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 2000-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 2000-bp upstream sequences in Arabidopsis,tomato,maize,and rice.,18%,24%,31%,39%,58%,77%,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.,(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,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:,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).,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.,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.,21,22,
