哺乳动物中Active DNA Demethylation机制

jiabiaohu

应该说哺乳动物Active DNA Demethylation研究还不是很清楚（remain elusive），所以这方面的研究最近几年很热。
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于是有很多种基于一些植物或低等动物的实验结果，有很多种假设被提出来，最近一篇有Zhang Yi（表观遗传研究的牛人） 写的比较好的综述如：Active DNA demethylation: many roads lead to Rome. 2010, Nat Rev Mol Cell Biol 11(9): 607-620.3 A: E# }/ P, j! k, _6 Y

" B) H5 V! Z5 v, m- B: K1. AID介导的Active DNA Demethylation" i. g, \0 ], t$ G6 T) \5 o8 k
美国科学院院士Blau, Helen M.于2010年（Bhutani, N., J. J. Brady, et al. (2010). "Reprogramming towards pluripotency requires AID-dependent DNA demethylation." Nature 463(7284): 1042-1047.）通过将人的成纤维细胞(hMEF)和小鼠的胚胎干细胞(mES)融合得到的异核体（heterokaryons）证明AID参与细胞融合过程中hMEF中Nanog和Oct4的去甲基化：RNA干扰AID后，Nanog和Oct4表达量明显下降，这与Nanog和Oct4的去甲基化程度下降相符；当过表达AID后，再RNA干扰AID时， Nanog和Oct4表达量明显增加而且去甲基化程度很明显；所以AID参与了Active DNA Demethylation 过程；提出的模型如图

2011-5-18 22:40 上传

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/ O" R. ]/ v) f' ^2. Tet1 介导的Active DNA Demethylation
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( w, P- _  w% v5 ~0 [" ^* K+ \( @' k还是Zhang Yi （Ito, S., A. C. D/'Alessio, et al. (2010). "Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification." Nature 466(7310): 1129-1133.）研究组发现, Tet1可以将5mC转化成5hmC（去甲基化的第一步）, 当干扰掉Tet1，会使ES细胞中Nanog启动子的甲基化程度升高，而ES细胞失去自我更新能力；而过表达Nanog却可以rescue Tet1 knock-down 的phenotypes，这些结果表明Tet1直接targets Nanog，并通过调节Nanog 的甲基化水平来调节其表达。. |: X: ?  e( I6 s8 r
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3. AID 和 Tet1介导途径的整合
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# p# R0 E/ m9 p8 G9 G/ j1 H2011年，Johns Hopkins University 的Hongjun Song等发现AID 和Tet1两个可以整合到一起，细胞内首先通过Tet1将5mC转化成5hmC，然后5hmC在AID等的作用下转化成5hmU，最后通过碱基切除修复将5hmU变成C，最终达到去甲基化的目的；具体步骤见图所示

2011-5-18 23:04 上传

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5 A9 E: _. _9 M# I3 v4 O6 S( F以上是鄙人对Active DNA Demethylation研究进展的一点总结，算抛砖引玉吧，希望大家多提供些线索，一些探讨探讨咯·~~~) r% d! I$ C1 w# j

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jiabiaohu

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/ T8 n' {3 F; r) G! R' O& ?7 H& T4 O第三篇文章为cell上的。Guo, Junjie U., Y. Su, et al. (2011). "Hydroxylation of 5-Methylcytosine by TET1 Promotes Active DNA Demethylation in the Adult Brain." Cell 145(3): 423-434.
+ j, `! v2 @! U" t4 F2 S2 o感兴趣的自己下载下来看下吧，我这边上传不了，不知道为什么。

tpwang

回复 jiabiaohu 的帖子) a  `  y# A8 @
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Hydroxylation of 5-Methylcytosine by TET1 Promotes Active DNA Demethylation in t.pdf (789.44 KB)

2011-5-18 23:25 上传

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mcherry.f

Mammalian DNA demethylation is a long term interest for the scientists in this area for many years. And many results are controversial, so someone dubbed it as "a colorful history". For the AID, it is really a star-molecule since its identification in 2000 around by Japanese. It is the molecule, searched by nearly half a century, responsible for the immunoglobulin somatic hypermutation (SHM). It is really intriguing to find its role in DNA demethylation. Actually, considering the studies presented recently, the mechanism of AID in DNA demethylation is a deamination process  from 5meC to T, and then the T is converted to C again by DNA repair (which repair machinery? MMR or BER or others?). The similar mechanism for somatic hypermutation has been revealed, but for the last step, there is no correct DNA repair, which can introduce mutations in the end. So a very interesting question is what is difference between SHM and DNA demethylation when AID plays its role. As I concerned, the level of AID may be the key, because SHM only occurs in B cells, which has high expression level of AID. But for ESCs, the transcriptome data indicates that its level is far less than in B cells.
2 o7 k. T- |* fFor reprogramming, AID is just to diminish the protection of DNA (methylation state makes many genes inactive), while other pluripotent factors function to reprogram the somatic cell into pluripotent one.  

jiabiaohu

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3 u: ^0 n6 `9 E& A( o+ |, Q# |From the aforementioned article, it seems clear that the demethylation is initiated by Tet1 converting 5mC to 5hmC, following AID deaminate the 5hmC to 5hmU, which is substitued by the C through BER(it is confirmed by inhibiting the enzymes in the BER pathway).9 p5 x& g" O0 }0 B% S& @$ I

4 J/ d3 q, g/ i8 w" Z( [! ]The most interesting results of this article I think is integrating the AID and Tet1 to one pathway to demethylate of 5mC. 0 F# ]& g" p! }( G7 _
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As reviewed by Zhang Yi, there may be other means for organisms to remove the methylation modification. Anyway, 5mC---5mhC---5mhU---C will be a promising mechanism for demethylation.

mcherry.f

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Yeah, hydroxylation is essential for 5meC further modification. After there is a base mismatch, why the cell chooses BER instead of MMR is also interesting, because we have already know that MMR will introduce mutations for Ig genes. So the regulation mechanism must be further addressed.

jiabiaohu

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1 J/ p5 h- s$ V' g% a. u4 WYes, there are still many questions remain elusive in most areas, if not all. We just know a corner of the iceberg of life mestry.

zhenhua

在哺乳动物中，发现5hmC的是Anjana Rao lab，他们通过寻找5mC转变成5hmC的酶，发现了哺乳动物中的5hmC。还有另外一个组也发现了。两个工作发在同一期的science上。Anjana Rao 的文章是Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1
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jiabiaohu

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至于最早是谁发现Tet1 参与这种转变的，我觉得应该不重要，现在关键是我们怎么样从这些已知的研究结果中理解哺乳动物细胞中主动去甲基化的机制。不知道Anjana Rao lab 在这方面有没有什么最新的重要进展，有的话，发出来大家共享下咯·~~~

天使

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells