p300 is required for orderly G1/S transition in human cancer cells.

Theroleofthetranscriptionalcoactivatorp300incellcyclecontrolhasnotbeenanalysedindetailduetothelackofappropriateexperimentalsystems.Wehavenowexaminedcellcycleprogressionofp300-deficientcancercell lines, where p300 was disrupted either by genetargeting(p300 cells)orknockeddownusingRNAi.Despite significant proliferation defects under normalgrowthconditions,p300-deficientcellsprogressedrapidlythroughG1withprematureS-phaseentry.AcceleratedG1/Stransitionwasassociatedwithearlyretinoblastoma(RB)hyperphosphorylationandactivationofE2Ftargets.Thep300-acetylaseactivitywasdispensablesinceexpres-sion of a HAT-deficient p300 mutant reversed thesechanges.Co-immunoprecipitationshowedp300/RBinter-actionoccursin vivo duringG1,andthisinteractionhastwopeaks:inearlyG1withunphosphorylatedRBandin late G1 with phosphorylated RB. In vitro kinaseassaysshowedthatp300directlyinhibitscdk6-mediatedRBphosphorylation,suggestingp300actsinearlyG1topreventRBhyperphosphorylationanddelayprematureS-phaseentry.Paradoxically,continuedcyclingofp300cellsdespiteprolongedserumdepletionwasobserved,andthisoccurredinassociationwithpersistentRBhyperphos-phorylation.Altogether,theseresultssuggestthatp300hasanimportantroleinG1/Scontrol,possiblybymodu-latingRBphosphorylation.Oncogene(2007)26,21–29.doi:10.1038/sj.onc.1209771;publishedonline31July2006Keywords:RB; retinoblastoma; hyperphosphorylation;Cdk;HCT116IntroductionThe transcriptional coactivators p300 and CBP arehighlyconservedparalogousproteins,firstidentifiedbytheir interactions with adenoviral E1A and CREB(cAMP response element binding protein) respectively(Chrivia etal., 1993; Eckner etal., 1994). p300/CBPregulate gene expression through interactions withnuclear proteins and participate in a broad spectrumof biological activities, including cell cycle regulation,differentiation, apoptosis and the DNA damage res-ponse (Giordano and Avantaggiati, 1999; GoodmanandSmolik,2000).Bothgenesaretargetedinarangeofcancers: truncating and point mutations in epithelialcancers, and translocations in leukemias. This has ledto the suggestion that p300 and CBP may function asclassical tumor suppressor genes (Gayther etal., 2000;Ozdagetal.,2002;Iyeretal.,2004b;Wardetal.,2005).p300andCBPfunctionasprototypehistoneandfactoracetyltransferases (HAT and FAT) by catalyzing theadditionofanacetylgrouptospecificlysineresiduesonhistones and other proteins, including p53, retinoblas-toma(RB)andE2F(GuandRoeder,1997;Liuetal.,1999; Martinez-Balbas etal., 2000; Chan etal., 2001).Early experiments have suggested that the two homo-logsplayinterchangeable,apparentlyredundantrolesincell physiology. However, it is becoming increasinglyclearthatp300andCBPhavedistinct,nonoverlappingfunctionsinseveralpathways,suchasthep53response(Grossman,2001;Iyeretal.,2004a).Theroleofp300andCBPincellcyclecontrolremainscontroversial,partlyduetothelackofarobustexperi-mental system where cell cycle progression can beanalysedindetail.Severalexperimentshaveimplicatedp300,andtoalesserextentCBP,inG1/Stransition,butwith apparently contradictory results. The inability toculturep300