表观遗传修饰新发现
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(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
4. Result
(A). DNA methylation levels of sldml2-1 hyper-DMRs in fruits at 46 dpa.
(B). Boxplots showing DNA methylation levels at combined hyper-DMRs.
11
DNA methylation levels of several sldml2 hyper-DMRs (boxed regions) in WT and sldml2.
2
2.1 Research Background
Cytosine methylation levels & gene activation
5-mC levels are dynamically regulated by methylation and demethylation reactions. DNA methylation transcription . is generally associated with inactive
单 个 碱 基 被 测 序 的 平 均 次 数
95.7
96.2 96.4
测序获得的序列占整个基因组的比例
12.5
11.9 13.6
The results is consistent with the presumed function of SlDML2 in DNA demethylaton.
Differentially methylated regions
compared 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.
4
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.
(A) The compositions of hyer-DMRs. (B) Density plots of 1～5.
13
Chromosomal distribution of SlDML2-targeted TEs and total TEs.
14
77% 58% 39% 31%
18%
24%
(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.
(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.
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:
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
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 TEs. Many studies have demonstrated that, by demethylating nearby TEs, active DNA demethylation is critical for gene activation or for the prevention of silencing .
郝雪峰
1
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.
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:
6
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.
Table S1. Statistics of sequencing data
Sample name C in genome # of Cs with coverage >=1 Table S2/3. ListTotal of hyper-DMRs in sldml2-1-46dpa vs. WT-46dpa sldml2-25dpa 251181662 239907749 sldml2-46dpa_rep1 251181662 240673869 sldml2-46dpa-rep1 sldml2-46dpa-rep2 WT-46dpa sldml2-46dpa_rep2 251181662 240478351 hyper-DMRs 21,515 23,026 1645 WT-25dpa 251181662 241742160 hoper-DMRs WT-46dpa 6,643 251181662 6,689 24224334213,141 % of C coverage 95.5 95.8 Depth of C coverage 10.7 14.5
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?
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 gຫໍສະໝຸດ Baidunetic function of SlDML2 in DNA demethylation during fruit ripening.
｛
3,306 hyper-DMRs
13,106 hypo-DMRs ( 8,176 overlap with hyper-DMRs )
•The DNA methylation changes occurred in all three sequence contexts.
C
(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.
4.4 The sldml2 mutations cause genome-wide DNA hypermethylation.
•DNA methylation changes during fruit ripening( ripening-induced DMRs) . 16,412 ri-DMRS
4. Result
(A). DNA methylation levels of sldml2-1 hyper-DMRs in fruits at 46 dpa.
(B). Boxplots showing DNA methylation levels at combined hyper-DMRs.
11
DNA methylation levels of several sldml2 hyper-DMRs (boxed regions) in WT and sldml2.
2
2.1 Research Background
Cytosine methylation levels & gene activation
5-mC levels are dynamically regulated by methylation and demethylation reactions. DNA methylation transcription . is generally associated with inactive
单 个 碱 基 被 测 序 的 平 均 次 数
95.7
96.2 96.4
测序获得的序列占整个基因组的比例
12.5
11.9 13.6
The results is consistent with the presumed function of SlDML2 in DNA demethylaton.
Differentially methylated regions
compared 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.
4
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.
(A) The compositions of hyer-DMRs. (B) Density plots of 1～5.
13
Chromosomal distribution of SlDML2-targeted TEs and total TEs.
14
77% 58% 39% 31%
18%
24%
(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.
(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.
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:
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
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 TEs. Many studies have demonstrated that, by demethylating nearby TEs, active DNA demethylation is critical for gene activation or for the prevention of silencing .
郝雪峰
1
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.
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:
6
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.
Table S1. Statistics of sequencing data
Sample name C in genome # of Cs with coverage >=1 Table S2/3. ListTotal of hyper-DMRs in sldml2-1-46dpa vs. WT-46dpa sldml2-25dpa 251181662 239907749 sldml2-46dpa_rep1 251181662 240673869 sldml2-46dpa-rep1 sldml2-46dpa-rep2 WT-46dpa sldml2-46dpa_rep2 251181662 240478351 hyper-DMRs 21,515 23,026 1645 WT-25dpa 251181662 241742160 hoper-DMRs WT-46dpa 6,643 251181662 6,689 24224334213,141 % of C coverage 95.5 95.8 Depth of C coverage 10.7 14.5
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?
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 gຫໍສະໝຸດ Baidunetic function of SlDML2 in DNA demethylation during fruit ripening.
｛
3,306 hyper-DMRs
13,106 hypo-DMRs ( 8,176 overlap with hyper-DMRs )
•The DNA methylation changes occurred in all three sequence contexts.
C
(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.
4.4 The sldml2 mutations cause genome-wide DNA hypermethylation.
•DNA methylation changes during fruit ripening( ripening-induced DMRs) . 16,412 ri-DMRS