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Lin28ABindsActivePromotersandRecruitsTet1toRegulateGeneExpression报告人：倪瑞栋MolecularCell,61(1),153-160.IF:13.958摘要背景实验总结个人观点背景[1]1.Shyhchang,etal.CellStemCell,2013.12(4),395.核心功能介绍生理影响背景•OnestudyinC.elegansalsosuggestedbindingofLin28Atothelet-7genomiclocus.[2]•Structurally,Lin28Aiscomposedoftwoclassicalnucleicacidbindingdomains.Notably,CSDdomainsinothermoleculeshavebeenshowntobindDNA[3]•Ten-eleventranslocation(Tet)familyproteinscansequentiallyconvert5-methylcytosine(5mC)to5-hydroxymethylcytosine(5hmC),leadingtoDNAdemethylation[4]2.VanWynsberghePM,etal.NatureStructural&MolecularBiology,2011,18(3):302.3.Hudson,W.H.,andOrtlund,E.A.Nat.Rev.Mol.CellBiol,2014,15,749–760.4.Yao,B,etal.Hum.Mol.Genet,2014,23,1095–1107.背景[1]核心功能介绍生理影响实验•Step1：Lin28ABindstoSpecificGenomicLociinMouseESCsandDirectlyBindsDNA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（coldprobe），没有生物素（Biotin）标记的DNA，实验中不显色实验•Step2：Lin28AIsPrimarilyAssociatedwithTranscribedGenesinmESCs图6：Lin28A敲低（lentivirus-mediatedshRNA）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实验中是结合的，证实Lin28A确实调控基因表达（区别于直接作用RNA）。实验•Step2：Lin28AIsPrimarilyAssociatedwithTranscribedGenesinmESCs图4：Lin28A与PolIIChIP结果有重合图5：组蛋白分析得到Lin28A结合的位点实验•Step3：Lin28AFormsaComplexwithTet1toRecruitTet1toTheirGenomicCo-targetsWecomparedgenome-wideLin28Adistributionwithpublisheddatabases.Interestingly,morethanhalf(49.1%–71.5%)ofLin28AbindingsitesweresharedbyTet1inmESCs.图7：Tet1与Lin28A各自DNA结合区有5632个相同基因位点实验•Step3：Lin28AFormsaComplexwithTet1toRecruitTet1toTheirGenomicCo-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Lin28AKD(敲低)decreasedTet1enrichmentoncommontargetsby30%–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•Step3：Lin28AFormsaComplexwithTet1toRecruitTet1toTheirGenomicCo-targets考虑到Lin28A与PolII酶有相似的DNA结合区。暗示着DNA先结合PolII，再结合Lin28A。Co-immunoprecipitation(co-IP)analysis免疫共沉淀图9：PolII酶抑制剂（Triptolide简写tri）使得Lin28A结合DNA能力下降(co-IP)实验实验•Step3：Lin28AFormsaComplexwithTet1toRecruitTet1toTheirGenomicCo-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BubbubbleDNA结合能力最优。图9：BubbubbleDNA更优结合Lin28A，GST（谷胱甘肽巯基转移酶）为对照组实验•Step4：Lin28ACoordinateswithTet1toRegulateCytosineModificationDynamicsandGeneExpressionOver95%ofup-ordownregulatedgenesboundbyLin28AwerealsoboundbyTet1onpromotersorintra-genicregions图10：Lin28A和Tet1密切联系图11：证实Lin28A征募Tet1达到5-methylcytosine(5mC)to5-hydroxymethylcytosine(5hmC)效果结果