Tet1蛋白在ES细胞未分化状态维持和内细胞团特化中的功能

10.Knock down Tet1 or not cause different proteins’ expression in ICM ways:RT-qPCR inject H2B-MFP with siTet staning Oct4 or Cdx2,respectively result:Tet’s knockdown bring Oct4&Cdx2’s expression increased
Tet Tet dioxygenase TDG
5mC
5hmC
5caC
C
How to prove the existence of 5mC and 5hmC

⑴SMRT(single-molecule real-time PCR) ⑵Specific enzyme and some relative antibodies
4.Knockdown of Tet1 cause morphological abnormality(Tet1’s function in maintaining) 5.Tet1’s lack reduce ES cell growth rate.(Some factors’ level down) 6.Tet1’s knockdown make several factors level-up,lead to the cells differentiation 7.Tet1 binding place in Nanog’s promoter 8.Tet1 can cause methylation 9.Tet1 Nanog ES cells
《Nature》
role of Tet proteins in 5mC to 5hmC conversion ES-cell selfrenewal and inner cell mass specification
Conclusion of the paper
①.
Tet
｝co-effect
DNMT
DNMT3a
methylation
Mutation
by《DNMT3a Mutations in Acute Myeloid Leukemia 》 《
Both 5mC and 5hmC can inhibit the activity of transcription
The transfer pathway
5.Tet1’s lack reduce ES cell growth rate.(Some factors’ level down) ways:Measure the level of some key stem cell factors Result:Nanog ↓,Oct4 ↓,Sox2 ↓ Q:LIF’s lack make Tet and its mRNA ↓ ,Why do they study these?
7.Tet1 binding place in Nanog’s promoter ways:ChIP-real-time qPCR result:Tet1 binded to some place near Nanog’s promoter
8.Tet1 can cause methylation ways:two group to compare (DNMT lack or not).Let Tet1 down,observed Nanog’s expression result:In normal ones Tet1↓Nanog ↓ In DNMT defected ones Tet1 ↓Nanog’s level changed a little That means Nanog is Tet1’s direct target
9.Tet1
Nanog
ES cells
ways:Knocked down Tet1 add Nanog, measured the morphological feature and Cdx2&Gata6’s quantities result:without Tet1,only with Nanog can make ES cell renewed partly.
2.Over –expression of Tet1,2 5hmC（only in wild-type） ways:Use immunostaining to find whether 5hm C generated when Tets worked result:Tet1,2 can converted 5mC to 5hmC;Tet3 only add the 5hmC’s level without reduce 5mC’s quantities
Ⅲ.Tet1’s function on ICM
10.Knock down Tet1 or not cause different proteins’ expression in ICM
How to testify the results
1.Tet protein’s activity ways: Make Tet protein over-expressed then observed the 5mc staining level result:Tet1,2 reduced the staining but Tet3 wild-type worked well but the mutants ones
Nanog蛋白研究新进展
来自于爱丁堡大学的Ian Chambers博士 的研究小组。在未分化的干细胞中这种因 子表达的水平是上下波动的。而且研究人 员还发现自我更新与Nanog表达并没有关系， 无Nanog仍然可以进行自我更新。
通过观察胚胎干细胞中的基因活性，日本京都大学山中伸 弥教授发现了一组4个基因经由病毒插入细胞时具有重组 成体细胞的能力，这4个基因是：Oct3/4、SOX2、c-Myc 和KLF4。
Nanog expression
ES cell self-renewed
Tet enhanced Nanog’s expression DNMT inhibited Nanog’s expression
② Tet have a essential role in maintaining ES cell fate
3.Verifying the vitro activity of Tet ways:TLC and end labeled result:The generation of 5hmc followed by the enhanced expression of Tet 4. Knockdown of Tet1 cause morphological abnormality(Tet1’s function in maintaining) ways:Real-time PCR 、 shRNA to knock the Tet down result:Tet1,2 expressed in ES cell,but not Tet3;Knockdown of Tet1,but not Tet2,3 caused morphological abnormality.
The outline
Ⅰ.Tet’s activity
1.Tet protein’s activity 2.Over –expression of Tet1,2 type） 3.Verifying the vitro activity of Tet 5hmC（only in wild-
Ⅱ. Tet’s function
The supplement : 1.AID is involved in the procedure of demethylation4. Synonyms:AICDA 198 AA length It can make Cytidine + H2O to uridine + NH3. 2.Transcriptional elongator is required for paternal genome demethylation in zygotes. 3.Some 5hm C -specific DNA glycosylase can help 5hm C to C
6.Tet1’s knockdown make several factors level-up,lead to the cells differentiation ways:Knock down Tet1 to observe SSEA1’s change.Staning Cdx2 Hand1 Gata6… result:SSEA-1 ↓ Cdx2,Hand1,Gata6↑