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.,單個(gè)堿基被測序的平均次數(shù),測序獲得的序列占整個(gè)基因組的比例,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