生物文献汇报

1、Lin28A Binds Active Promoters and Recruits Tet1 to Regulate Gene Expression,报告人：倪瑞栋,Molecular Cell,61(1), 153-160. IF: 13.958,摘要,背景,实验,总结,个人观点,背景1,1.Shyhchang , et al. Cell Stem Cell,2013. 12(4), 395.,核心功能介绍,生理影响,背景,One study in C. elegans also suggested binding of Lin28A to the let-7 genomic locus.

2、2 Structurally, Lin28A is composed of two classical nucleic acid binding domains. Notably, CSD domains in other molecules have been shown to bind DNA3 Ten-eleven translocation (Tet) family proteins can sequentially convert 5-methylcytosine (5mC ) to 5-hydroxymethylcytosine (5hmC), leading to DNA dem

3、ethylation 4,2.Van Wynsberghe P M, et al. Nature Structural & Molecular Biology, 2011, 18(3):302. 3.Hudson, W.H., and Ortlund, E.A. Nat. Rev. Mol. Cell Biol, 2014,15, 749760. 4. Yao, B , et al. Hum. Mol. Genet, 2014, 23, 10951107.,背景1,核心功能介绍,生理影响,实验,Step 1：Lin28A Binds to Specific Genomic Loci in Mo

4、use ESCs and Directly Binds DNA,图2：ChIP得到Lin28A结合motif,图3：EMSA证实motif与Lin28A结合,图1：ChIP集中TSS,说明：冷探针（cold probe），没有生物素（Biotin）标记的DNA，实验中不显色,实验,Step 2：Lin28A Is Primarily Associated with Transcribed Genes in mESCs,图6：Lin28A敲低 （ lentivirus-mediated shRNA ）再进行RNA-seq。 用qPCR证明敲低操作是成功的。有许多基因是和DNA在ChIP实验中是结

5、合的，证实Lin28A确实调控基因表达（区别于直接作用RNA）。,实验,Step 2：Lin28A Is Primarily Associated with Transcribed Genes in mESCs,图4：Lin28A与Pol II ChIP结果有重合,图5：组蛋白分析得到Lin28A结合的位点,实验,Step 3：Lin28A Forms a Complex with Tet1 to Recruit Tet1 to Their Genomic Co-targets,We compared genome-wide Lin28A distribution with published

6、 databases. Interestingly, more than half (49.1%71.5%) of Lin28A binding sites were shared by Tet1 in mESCs.,图7：Tet1与Lin28A各自DNA结合 区有5632个相同基因位点,实验,Step 3：Lin28A Forms a Complex with Tet1 to Recruit Tet1 to Their Genomic Co-targets,图8：无论敲低Lin28A还是Tet1都会导致另外一个蛋白质富集DNA能力下降,Lin28A KD(敲低) decreased Tet1

7、 enrichment on common targetsby30%60%. 研究Lin28A和Tet1和DNA的结合关系，发现二者有密切联系。但敲低Tet1不影响Lin28A与DNA结合能力,实验,Step 3：Lin28A Forms a Complex with Tet1 to Recruit Tet1 to Their Genomic Co-targets,考虑到Lin28A与Pol II酶有相似的DNA结合区。暗示着DNA先结合Pol II，再结合Lin28A。 Co-immunoprecipitation (co-IP) analysis免疫共沉淀,图9：Pol II酶抑制剂（Tr

8、iptolide简写tri）使得Lin28A结合DNA能力下降,(co-IP),实验,实验,Step 3：Lin28A Forms a Complex with Tet1 to Recruit Tet1 to Their Genomic Co-targets,考虑到Lin28A拥有结合DNA和RNA的能力、结合空腔，设计不同类型DNA，得到Bub bubble DNA结合能力最优。,图9：Bub bubble DNA更优结合Lin28A，GST（谷胱甘肽巯基转移酶）为对照组,实验,Step 4：Lin28A Coordinates with Tet1 to Regulate Cytosine Modification Dynamics and Gene Expression,Over 95% of up- or downregulated genes bound by Lin28A were also bound by Tet1 on pro