DNA methylation
RNA methylation
Histone methylation
The fifth genetic code 5mC
DNA甲基化的可逆性不如组蛋白甲基化，因为形成的是C-C键而不是C-N键
In mammals, CG 70-80%
In Arabidopsis, CG 24%, CHG 6.7%, CHH 1.7% (H=A,T,C)
non-CG methylation 可能与体细胞的全能性有关
CpG island：promotor上一段没有methylation的CG
DNA methylation并不是普遍的，植物中非常多，动物中哺乳动物有，但是其他动物没有methylation和对应的enzyme。
植物对甲基化突变不敏感。因此常用作甲基化的模式生物。动物很多都胚胎致死。
N6-Methyladenine (m6A) DNA modification
m6A含量非常低，m6A/A = 0.1%，占全碱基估计万分之一
Epialleles are heritable in plants
Biological roles of DNA methylation (5mC)
- controlling the transposons activity, silence transposon
- regulating gene expression (X-chromosome inactivation, genomic imprinting)
- control development
Determine the DNA methylation level
- single locus
- southern blot
- Extract total DNA ⇒ Enzyme digestion ⇒ blot
- simple, fast, useful for repeat region
- chop-PCR
- bisulfite sequencing
- 通过亚硫酸盐将C转变为U，PCR后为T
- 通过测量C含量变化来表征甲基化水平。
- PacBio: 通过mA和A的检测时间差异判断甲基化
- Nanopole：通过精细测量电压差。
- Genome wide
- ChIP-chip
- ChIP-seq
- whole genome bisulfite sequencing
investigate DNA methylation in plants
- isolate mutants
- directly isolate hypo or hyper-methylated mutants
- using report system
- protein-protein interaction
- yeast-2-hybrids
- complex purification
- genome wide sequencing
- different tissue, developmental stage, single cell
- environment (stress)
DDM1 encodes a SWI2/SNF2-like protein
(SWI/SNF system还会参与RNA 3D structure改变、miRNA biogenesis)
甲基化一旦丢失，就没法按照原本的方法修饰回来了（找不到如何修饰的方法了）
RNA directed DNA methylation pathway
DNA methyltransferases in Arabidopsis
- De novo DNA methylation
- In mammal: Dnmt3
- In plant: DRM2, DRM1
- Maintainence of DNA methylation
- CG:
- In mammal: Dnmt1
- In plant: MET1
- CHG: in plant CMT3
- CHH: in plant DRM2 & CMT2
Asymmetric methylation sites require additional info
- RNA-directed DNA methylation (RdDM)
- Pol IV (siRNA) & V (lncRNA)
CMT2 is also required for CHH methylation maintenance
protein that bind methyl-CpG
Reversing DNA methylation: mechanisms, genomics, and biological functions
- passive DNA demethylation: 通过复制等直接丢失，cell cycle-dependent, replication-dependent dilution
- ROS1 is the first confirmed DNA demethylase
- DME, affects gene imprinting
- active DNA demethylation in plants and animals
双胞胎表现不一样大部分是因为在早期胚胎发育时期，甲基化抹去重建过程中出现差别，而很多时候并不是因为所谓生活习惯的不同
绝大部分DNA methylation是抑制基因表达，但是也有促进基因表达的，例如ROS1
Function of Active demethylation in plants
- Stomatal development
- Embryo development
- Tracheary element differentiation
- Pollen germination
- Gluten abundance
- Nodule differentiation
- Disease resistance
- Fruit ripening