表观遗传修饰新发现.ppt

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PNAS Published online May 15, 2017
郝雪峰
Critical roles of DNA dmethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomoto fruit
DNA methylation is a conserved epigenetic mark important for genome integrity, development, and environmental responses in plants and mammals.
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Active DNA demethylaton in plants is initiated by a family of 5-mC DNA glycosylases/lyases( ., DNA demethylases).
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This study suggests that active DNA demethylation is required for both the activation of ripening-induced genes and the inhibition of ripening-repressed genes.
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1. Abstract
Research Background
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.
Cytosine methylation levels & gene activation
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In mammals : CG （DNMTs）
In plants : CG（MET1）/CHH（CMT2, DRM2）/CNG（CMT3）
DNA methylation site:
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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 .
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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.
Research Background
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?
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.

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Generation of stable loss-of-function mutant alleles of SlDML2 using the CRISPR/Cas9 gene-editing system.
4. Result
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The sldml2 mutations inhibit fruit ripening.

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.