Mechanism of activationModels for mechansim of activationDirect contact in activationSuppression is strong evidence for direct contactHow do distal enhancers work?Does activation require communication between an enhancer and a promoter?Some enhancers increase the rate of transcription initiationLooping vs. trackingDirect contact for activationTracking for activationInteractions may be facilitated by DNA-bending proteinsInterferon beta Enhancer-PromoterCan activation occur without communication between an enhancer and a promoter?Some enhancers increase the probability that a gene is in a transcriptionally competent regionExperiments to look for targets of activatorsWhat proteins bind to the activation domain?GTIFs for RNA polymerase IIGTIFs can be targets of activation domains in vitroAre TAFs required for transcriptional activation?Co-regulatorsActivators and Co-rgulatorsMediator is a co-activatorSome co-regulators work on chromatinClasses of co-regulatorsRegulating the regulatorsRegulation of activator proteinsExample of steroid-hormone receptorsMechanism of activationModels for mechansim of activation•Direct contact between an activator and RNA polymerase or GTF•Indirect interactions–Adaptor–Mediator–Histone modifier complexes–Nucleosome remodelers•No contact between enhancer bound proteins and the target promoter–Open a chromatin domain but not target a promoter–Linking via enhancer-facilitatorsDirect contact in activation•Demonstrated in bacteria and yeast (some genes)•Upstream activation sequences are adjacent to minimal promoters•Examples– lambda repressor activates RNA polymerase at PRM.–cAMP-CAP activates RNA polymerase at lac. Direct contact between cAMP-CAP and the C-treminal domain of the alpha subunit of RNA polymeraseSuppression is strong evidence for direct contact•Hypothesis: an AD makes direct contact with a component of the transcriptional apparatus•Prediction: LOF mutations in the activation domain should be suppressed by appropriate mutations in that component.•E.g. mutations in CAP can be suppressed by mutation in the  subunit of RNA Pol.CTDHow do distal enhancers work?Does activation require communication between an enhancer and a promoter?•If so, expect –An effect on rate of transcription–Specific binding between activator or co-activator and the transcription complex–Mutations in target of binding should abolish activation–Find targets in suppressor screens•If so, is it by looping vs. tracking?–Direct interaction?–Interact via another component?–Tracking?Some enhancers increase the rate of transcription initiationEnhgenepromoterpolymerase & transcriptWith EnhancerWithout EnhancerPolymerase density and amount of transcription increases in all cells in a populationLooping vs. tracking•Communication between enhancer and promoter can be via direct contact–Contact between proteins bound to adjacent sites–Contact between proteins at distal sites, with DNA between them looped out.•Communication can be via tracking–Some component(s) of the transcriptional apparatus enter the chromosome at an enhancer and move along (track) until they act at a distal promoter.Direct contact for activationPol IIaIIDPolIIEnhancerPromoterGTIF? Coactivator?Tracking for activationIIDPolIIEnhancerPromoterInteractions may be facilitated by DNA-bending proteins•Many proteins that bind in the minor groove of DNA also bend the DNA.–TBP, YY1, HMG I(Y)•interferon- gene enhancer–binding sites for 3 conventional txn factors–binding sites for HMG I(Y)–requires binding and bending of DNA by HMGI(Y) for activation by the other proteins bound to the enhancer.Interferon beta Enhancer-PromoterCan activation occur without communication between an enhancer and a promoter?•If so, expect no specific binding between activator and the transcription complex•Possible models:–Open a chromatin domain so that it is more likely to be expressed–Affect probability that gene is in a transcriptionally competent regionSome enhancers increase the probability that a gene is in a transcriptionally competent regionEnhEnhEnhEnhWith enhancerWithout enhancerIncrease in fraction of cells expressing the reporter gene.Amount of expression per expressing cell is same with and without enhancer.Experiments to look for targets of activatorsWhat proteins bind to the activation domain?•Use affinity chromatography, with AD as the ligand•Determine the nuclear proteins that bind specifically to that activation domain.•Find that some GTFs, especially TAFs, bind to either acidic or Q-rich ADs–E.g. the acidic AD of VP16 will bind to TBP, TAFII40 and TFIIB–Q-rich AD of Sp1 binds TAFII130GTIFs for RNA polymerase IITFIIDTBP}TAFsIIBIIAIIEIIFIIHhelicaseprotein kinaseTBPInrIIBIIAPol IIaIIFIIEIIHCTD of large subunit of Pol IIRecognize core promoterTargets Pol II to promoterModulates helicaseHelicaseCTD protein kinaseMany GTIFs are possible targets for activators of transcription.GTIFs can be targets of activation domains in vitro130Are TAFs required for transcriptional activation?•Construct conditional (ts) loss-of-function (LOF) alleles in genes for TAFs in yeast.•Examine the level of expression of various target genes before and after temperature shift (active vs. inactive TAF).•See that many genes are still activated in the absence of TAF function!•TAFS are not required for all activation.•TAFs are important - LOF alleles are lethal. Other functions include cell cycle progression.Co-regulators•Some sequence-specific activators (or repressors) do not regulate transcription by themselves.–Sp1 + TBP + RNA Pol II + other GTFs + promoter DNA: only basal transcription•Co-activators and co-repressors are also needed–Sp1 + TBP + TAFS + RNA Pol II + other GTFs + promoter DNA: get activated transcriptionActivators and Co-rgulatorsLemon & Tjian(2000) Genes &Devel.14:2551Mediator is a co-activator•Yeast RNA Pol II does not respond to activators, but the RNA Pol II holoenzyme does respond to activators–Mediator (SRBs, Rgr1, Gal11, Med 1, 2, 6, 7, etc) is a type of co-activatorpreinitiation complexIIBTATAInrPol IIaIIFIIHIIEIIASRB activator?Some co-regulators work on chromatin•Transcriptional activation in vitro from some promoters requires a chromatin template•Some co-activators and co-repressors covalently modify histones and transctipion factors:–Acetyl transferases (co-activators)–Deacetylases (co-repressors)–Kinases,