Transcriptional Co-factor Yki Regulates Target Genes Expression Via Recruiting Mediator Complex

Hippo pathway participates in varieties of biological processes,such as embryonic development,organ size regulation,maintaining of adult stem cell,natural immunoregulation and tumorigenesis.Hippo pathway was found by EMS-mediated mutant screening in 2003,and subsequent studies have found that this pathway is highly conserved in revolution.Therefore,the studies of Hippo pathway is significative for understanding development and tumorigenesis.In Drosophila,Hippo pathway regulates the phosphorylation and subcellular localization of Yki by transmitting signals to the co-transcription factor Yki through a series of kinase-coupled reactions.When the Hippo pathway is activated,the kinase Hpo and Sav form an active complex that phosphorylates the downstream kinase Wts.Then,Wts and Mats form a complex that phosphorylates the downstream transcriptional co-factor Yki.Next,phosphorylated Yki binds to microtubule-related proteins 14-3-3 in the cytoplasm and remains in the cytoplasm,which causes Yki to be unable to enter the nucleus for transcriptional activity.In the contrary,when Hippo pathway is inactivated,the phosphorylation of Yki is removed.Then the non-phosphorylated Yki protein enters the nucleus and binds to the transcription factor Sd,and jointly activates the downstream target gene expression.Our latest research also shows that the Hippo pathway not only regulates the phosphorylation and subcellular localization of Yki,but also regulates the stability of Yki.Hippo pathway inhibits the binding between Yki and the deubiquitination enzyme Usp7.It removes the protective effect of Usp7 on Yki and promotes the ubiquitination and degradation of Yki.Therefore,Hippo pathway inhibits Yki activity through a dual mechanism.Thus,Yki is at a key position in the signaling pathway.As a transcription cofactor,Yki has no ability to bind DNA.The mechanism by which Yki regulates the expression of downstream target genes is still unclear.In addition,studies have shown that Yki is enriched in the promoter and enhancer regions of genes,suggesting that Yki not only acts as a transcription cofactor,but also acts as a transcription enhancer.In summary,exploring the function of Yki in Hippo pathway is of great significance to further understand the signal transduction mechanism of Hippo pathway.Kto is an important subunit of transcriptional coactivator Mediator,a multisubunit complex that recruits RNA polymerase II to enhance genes expression.The Mediatorcomplex directly binds to RNA polymerase II and recruit it to the promoter region of the target genes.Many of these subunits act as receptors to recognize numbers of transcription factors and bind these specific transcription factors,then the Mediator activates gene transcription.Mediator complex contains four important components: head,middle,tail and kinase activity center.In this study,our main research object is the kinase active center,which contains four subunits,namely Kto,Skd,CDK8 and Cyc C.We found that the knockout of Mediator complex components Kto and Skd resulted in significantly shrink wing size and eye size in Drosophila.At the same time,knockdown of Kto in posterior compartment of wing disc results in the dramatically downregulation of characteristic target genes of Hippo pathway,diap1's transcription level.Meanwhile,the area of P compartment shrinks simultaneously.Knockdown of these Skd,CDK8,and Cyc C also cause diap1 and ban transcription level decreased,even maldevelopment.Next,we found that kto functions in parallel to Yki or upstream of Yki through genetic epitope analysis.To acquire Kto antibody,the target protein was injected in mice and then the serum was purified to be used.We applied the serum to immunofluorescent stain S2 cell and found that Kto is located in nucleus.Furthermore,Co-IP assay indicates that Kto has protein-protein interaction with Yki,and this process is evolutionarily conserved in mammals.Last,we confirmed that Kto doesn't bind to Sd,and this process is also evolutionarily conserved in mammals.In conclusion,we found that Mediator plays an indispensable role in the expression of Hippo signaling target genes.Yki may promote downstream genes expression by recruiting Mediator complex and recruiting RNA polymerase II to gene promoter or enhancer regions.Our study suggests that Yki plays a bridging role in Hippo signaling,mediating the binding of transcription factor Sd to RNA polymerase II and activating the expression of downstream target genes.We also revealed the mechanism of Yki in Hippo signal transduction.However,how Yki coordinates with Sd binding to RNA polymerase II,and how Yki binding to Kto remains to be further explored.The dysfunction of Hippo pathway is a primary reason of variant human developmental disorders and diseases which makes it an important therapeutic target in modern medicine.We uncovered that Kto cooperates other subunits in Mediator to regulate the stability of Hippo pathway.The discovery will provide what Yki functions in Hippo pathway.This research provides theoretical ideas to the cancer prevention and shows great significance for treatment of diseases.