MCB 317 Genetics and Genomics MCB 317 Topic 10, part 6 A Story of Transcription What is order of action in vivo? How do we get at whats going on in vivo? Chromatin Immunoprecipitation (ChIP) Does a specific protein of interest bind to a specific site on a chromosome in vivo? UNLESS STATED OTHERWISE, WE ONLY LOOK AT PCR PRODUCTS FROM THE PPT A B CD Because formaldehyde crosslinks protein-DNA and protein- protein, each of the different proteins, A, B, C and D, will ChIP to the DNA that is bound by A. An antibody to A, B, C, OR D will ppt this segment of DNA Also, can use epitope tagged versions of a gene rather than raise antibodies to every protein you want to ChIP A C D Performing ChIP on mutant strains can give insight into the arrangement of proteins in the complex relative to DNA A B CD A B C Wild-type ChIP signal from: A, B, C, D Gene B deleted ChIP signal from: A only Gene D deleted ChIP signal from: A, B and C What is order of action in vivo? An Imaginary Yeast Gene UASPrYFG1 ORF Act TBP UASPrYFG1 ORF Act UASPrYFG1 ORF t = 0 min t = 5 min t = 10 min UASPrYFG1 ORF Primer Set 1 Primer Set 2 PCR on Total Purified Genomic DNA (not ChIP): Primer Set 1 Primer Set 2 Set 1 + Set 2 Strain 1 = Activator is Epitope Tagged UASPrYFG1 ORF TBP UASPrYFG1 ORF Act UASPrYFG1 ORF t = 0 min t = 5 min t = 10 min Act Strain 1 = Activator is Epitope Tagged UASPrYFG1 ORF TBP UASPrYFG1 ORF Act UASPrYFG1 ORF t = 0 min t = 5 min t = 10 min Act Time (min) Set1 Set2 Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn Strain 2 = TBP is Epitope Tagged UASPrYFG1 ORF TBP UASPrYFG1 ORF Act UASPrYFG1 ORF t = 0 min t = 5 min t = 10 min Act Time (min) Set1 Set2 Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn Strain 3 = Mediator is Epitope Tagged What Would You Conclude? Time (min) Set1 Set2 Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn C = Control = Pruified Genomic DNA (no ChIP) 15 C Combine Data from 3 Strains -> Model UASPrYFG1 ORF TBP UASPrYFG1 ORF Act UASPrYFG1 ORF t = 0 min t = 5 min t = 10 min Act UASPrYFG1 ORF TBP t = 15 min Act Mediator Order of events/action at the GAL1 promoter Components Also Gal4 activator protein UASPrGAL1 ORF Binding in vitro Gal4 activator protein UASPrGAL1 ORF Gal4 TBP Strategy: GAL1 OFF in Glucose -> ON in Galactose Grow in Glucose -> shift to Galactose -> ChIP each component at various time points to determine when they bind control region UASPrGAL1 ORFUASPrGAL1 ORF Glucose Galactose ChIP at 1 min, 2 min, 3 min, etc.. GAL1 ChIP UASPrGAL1 ORF PCR Perform ChIP for each component at each time point. NOTE: Each Component = different strain Primer does not distinguish binding at UAS from binding at the Promoter ChIP Resolution Limited by Fragment Size UASPrGAL1 ORF PCR 75 bp Shear DNA bp Fragments PU PU PU PU PU PU Conclusions from ChIP of GAL1 Control Region Resolution could not distinguish binding at UAS vs. Promoter 1. Gal4 bound constitutively 2. Gal4 recruits SAGA and Mediator independently 3.SAGA does not recruit mediator 4.Recruitment of mediator is not sufficient to recruit the basal factors 5.Mediator bound before RNAPII Model of Recruitment at Gal1 Gal4 Saga Mediator RNAPII and Basal Factors Dashed arrows = not addressed by this experiment Surprises at Gal1 1.Gal4 bound constitutively 2.Mediator binds independently of RNAPII Order of events/action at the HO promoter HO txn is cell cycle regulated: OFF in M -> ON in G 1 URS2PrHO ORFURS1 Both URS1 and URS2 are required for txn of HO RNAPII Basal Factors Mediator Swi/Snf chromatin remodeling complex SAGA (co-activator) SBF activator Swi5 activator Proteins that bind HO control region in vitro: URS2PrHO ORFURS1 Swi5SBF TBP et al RNAPII Basal Factors Mediator Swi/Snf = chromatin remodeling complex SAGA = co-activator, histone acetylase SBF = activator Swi5 = activator Primer Set 1 = S1 = URS1 Primer Set 2 = S2 = URS2 Primer Set 3 = S3 = Pr URS2PrHO ORFURS1 Set 1Set 2Set 3 S1 S3 S PCR = Genomic DNA (not ChIP) Lane 1 = Set 1 only Lane 2 = Set 2 only Lane 3 = Set 3 only Lane 4 = Set Multiplex PCR Primer sets can resolve URS1, URS2 and Pr in ChIP analysis URS2PrHO ORFURS1 Set 1Set 2Set 3 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min S1 S3 S Swi5-tag 5 URS2PrHO ORFURS1 Set 1Set 2Set 3 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min; Lane 5 = 8 min S1 S3 S Swi5-tag Mediator-tagSwi/Snf-tag Conclusions??? Model Derived From Data on Previous Slide HO ORFPrURS2URS1 t = 0 min HO ORFPrURS2URS1 t = 2 min HO ORFPrURS2URS1 t = 4 min HO ORFPrURS2URS1 t = 6 and 8 min Swi5 Mediator Swi/Snf Swi5 Mediator Swi/Snf HO ORFPrURS2URS1 t = 0 min HO ORFPrURS2URS1 t = 2 min HO ORFPrURS2URS1 t = 4 min HO ORFPrURS2URS1 t = 6 and 8 min Swi5 Mediator Swi/Snf Swi5 Mediator Swi/Snf Is Swi/Snf Chromatin remodeling complex required to recruit Mediator or is Swi5 sufficient? URS2PrHO ORFURS1 Set 1Set 2Set 3 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min S1 S3 S Swi5-tag Mediator-tagSwi/Snf-tag Swi2 = No functional Swi/Snf chromatin remodeling complex Swi2 = No Swi/Snf chromatin remodelling complex HO ORFPrURS2URS1 t = 0 min HO ORFPrURS2URS1 t = 2 min HO ORFPrURS2URS1 t = 4 min HO ORFPrURS2URS1 t = 6 and 8 min Swi5 One Model Derived From Data on WT and snf2 strains HO ORFPrURS2URS1 t = 0 min HO ORFPrURS2URS1 t = 2 min HO ORFPrURS2URS1 t = 4 min HO ORFPrURS2URS1 t = 6 and 8 min Swi5 Mediator Swi/Snf Swi5 Mediator Swi/Snf Order of initial events at HO in vivo What evidence might lead you to draw this arrow? In vivo order of events leading to txn of the HO gene Surprises at HO 1.Swi5 binds transiently 2.Mediator at URS1, URS2 and Promoter 3.Swi/Snf and mediator stay bound at URS1 after Swi5 is no longer bound-- how? 4.Swi/Snf and Saga arrive at URS2 before SBF -- how? 5.SBF recruited to URS2 by Saga (activator recruited by a co-activator) 1.Swi5 binds transiently 2.Mediator at URS1, URS2 and Promoter 3.Swi/Snf and mediator stay bound at URS1 after Swi5 is no longer bound- how? 4.Swi/Snf and Saga arrive at URS2 before SBF- how? 5.SBF recruited to URS2 by Saga (activator recruited by a co-activator) PIR1, CLN2 and HO puzzles Lodish 11-32 URS = Upstream Repressor Sequence Not Regulatory Seq as in HO UAS = enhancer URS = silencer Writers and Readers of the Histone Code ChIP of Histones Antibodies against different modified forms of the Histone Tails Use antibodies that are specific for histone modifications Lodish Puzzle? Why does ChIP using Abs to the histone tails work? What do the histone tails do?