Functional Study Of Cip1 Regulating Cell Cycle G1/S Transition In Saccharomyces Cerevisiae

Cell cycle regulation is the key for maintaining normal cell proliferation and genome stability.Cell cycle Gl/S checkpoint is a critical decision-making point,monitoring whether cell enter the division cycle and deciding the start of DNA replication initiation,which is known as " START" in yeast and the " Restriction Point" in mammals.Cdkl is a master kinase for the Gl/S transition in eukaryotes,which is the core in the research field of cell proliferation control.As the simplest eukaryotic model organism,Saccharomyces cerevisiae has made outstanding contributions to the field of cell cycle control for understanding of essential cancer genes.The famous tumor suppressor genes such as Rb,p53,p21 are involved in the regulation of the Gl/S checkpoint,of which mutation usually results in severe cancer.The identification of yeast Whi5 as the homologous of Rb,greatly promotes the elucidation of the tumor suppressor mechanism for Rb.Since the 1980s,scientists have been trying to search for functional analogues of some star tumor suppressors,such as p53 and p21,in the model organisms to simplify the study of tumor suppressor mechanisms.Our group has identified a novel Cdkl inhibitory protein in budding yeast and named it as Cip1(systematic name YPL014W).Cipl acts as a negative protein in cell cycle Gl/S transition.We therefore proposed Cipl as the functional analog of p21.Cipl specifically binds to the Cln3-Cdk1 complex in G1 phase and inhibits its activity.In this study,deletion of Cln3,a known substrate of Cipl,could not rescue the cell cycle arrest caused by Cipl overexpression,suggesting that Cipl may have other substrates redundant to Cln3-Cdkl.cln3? cip1? mutant cells enter S phase earlier than cln3?single mutant,by comparing the expression of CLN2,the marker gene of S phase through western bloting and qPCR.This result demonstrates that Cipl has another downstream regulatory pathway,independent on Cln3-Cdkl during the Gl/S transition.In search of the new downstream pathway of Cipl,a series of interacting proteins of Cipl were identified and characterized by immunoprecipitation and proteomic detection.Two subunits of the Ccr4-Not complex,Ccr4 and Caf120,were confirmed as the novel targets by Cipl,through yeast two-hybrid and immunoprecipitation.A similar interaction between p21 and Ccr4 in human was also confirmed.These results indicate that Ccr4 in both yeast and human cells is a bona fide target of Cipl/p21.Studies have shown that Ccr4 regulates Gl/S transition by modulating the stability of WHI5 mRNA,while the molecular details are still largely unclear.Auxin inducible degron(AID)mediated depletion of Ccr4 generates a clear synthetic lethal to CLN3 deletion,indicating a redundant pathway of Ccr4 in parallel to Cln3.In addition,the current work showed that overexpression of Cln3 or Caf120 partially restored the cell growth defect caused by Cipl overexpression,indicating that Cipl negatively regulated Cln3 and Ccr4.Whi5(Rb homologous protein)is a known Gl/S transcriptional repressor,and its activity is regulated by Cln3 and Ccr4 respectively.Whi5 deletion in this work rescued the cell cycle arrest caused by Cipl overexpression.In summary,Ccr4 and Cln3 are two parallel pathways that jointly inhibit Whi5 and are negatively regulated by Cipl,In addition,through mutant genetic analysis and yeast two-hybrid,Thr135 is indicated as an important phosphorylation site of Cipl,and the phosphorylation may mediate its regulation on Cln3 and Ccr4.This work demonstrate that Cipl has dual repressive control on cell cycel START regulation through negatively regulation on Cln3-Cdkl and Ccr4-Cafl20 complexes.Cln3 and Ccr4 keep Whi5 active and block SBF-mediated Gl/S gene transcr-iption.The new discovery of the working model for Cipl on the Gl/S transition in cell cycle helps our understanding of the eukaryotic cell cycle control,and provides a theoretical basis and new thoughts on the molecular mechanism of human tumor suppressor.