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1、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 2017.10.18,1. Abstract,DNA methylation is a conserved epigenetic mark important for genome integrity, development, and en

2、vironmental 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 ripe

3、ning-repressed genes.,2,2.1 Research Background,Cytosine methylation levels 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 r

4、ipening.,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 fu

5、nction 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

6、.,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 d

7、istances 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 I

8、G 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%,2

9、4%,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 s

10、equence 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

11、 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 im