MCB 317 Genetics and Genomics MCB 317 Topic
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 what’s going on in vivo? Chromatin Immunoprecipitation (Ch. IP) 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
Because formaldehyde crosslinks protein-DNA and protein -protein, each of the different proteins, A, B, C and D, will “Ch. IP” to the DNA that is bound by A. C D B 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 Ch. IP
Performing Ch. IP on mutant strains can give insight into the arrangement of proteins in the complex relative to DNA D C C D C B A Wild-type Ch. IP signal from: A, B, C, D B A Gene B deleted Ch. IP signal from: A only A Gene D deleted Ch. IP signal from: A, B and C
What is order of action in vivo?
An Imaginary Yeast Gene t = 0 min UAS Pr YFG 1 ORF Act TBP UAS Pr Act t = 5 min t = 10 min YFG 1 ORF
UAS Pr YFG 1 ORF Primer Set 1 Primer Set 2 PCR on Total Purified Genomic DNA (not Ch. IP): Primer Set 1 Primer Set 2 Set 1 + Set 2
Strain 1 = Activator is Epitope Tagged t = 0 min UAS Pr YFG 1 ORF Act TBP UAS Pr Act t = 5 min t = 10 min YFG 1 ORF
Strain 1 = Activator is Epitope Tagged t = 0 min t = 5 min UAS Pr YFG 1 ORF Act UAS Set 1 Pr YFG 1 ORF Set 2 t = 10 min Act TBP UAS Pr YFG 1 ORF Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn 0 5 10 Time (min)
Strain 2 = TBP is Epitope Tagged t = 0 min t = 5 min UAS Pr YFG 1 ORF Act UAS Set 1 Pr YFG 1 ORF Set 2 t = 10 min Act TBP UAS Pr YFG 1 ORF Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn 0 5 10 Time (min)
Strain 3 = Mediator is Epitope Tagged What Would You Conclude? Set 1 Set 2 C 5 10 0 Time (min) 15 Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn C = Control = Pruified Genomic DNA (no Ch. IP)
Combine Data from 3 Strains -> Model t = 0 min t = 5 min t = 10 min UAS Pr YFG 1 ORF Act TBP UAS Pr Act YFG 1 ORF Mediator t = 15 min Act TBP UAS Pr YFG 1 ORF
Order of events/action at the GAL 1 promoter
Components UAS Pr Also Gal 4 activator protein GAL 1 ORF
Binding in vitro Gal 4 TBP UAS Pr Gal 4 activator protein GAL 1 ORF
Strategy: GAL 1 OFF in Glucose -> ON in Galactose Grow in Glucose -> shift to Galactose -> Ch. IP each component at various time points to determine when they bind control region Glucose Galactose UAS Pr GAL 1 ORF Ch. IP at 1 min, 2 min, 3 min, etc…. .
GAL 1 Ch. IP UAS Pr GAL 1 ORF PCR Perform Ch. IP for each component at each time point. NOTE: Each Component = different strain Primer does not distinguish binding at UAS from binding at the Promoter
Ch. IP Resolution Limited by Fragment Size UAS 75 bp Pr GAL 1 ORF PCR Shear DNA 500 -1000 bp Fragments U P U P U P
Conclusions from Ch. IP of GAL 1 Control Region Resolution could not distinguish binding at UAS vs. Promoter 1. 2. 3. 4. 5. Gal 4 bound constitutively Gal 4 recruits SAGA and Mediator independently SAGA does not recruit mediator Recruitment of mediator is not sufficient to recruit the basal factors Mediator bound before RNAPII
Model of Recruitment at Gal 1 Saga RNAPII and Basal Factors Gal 4 Mediator Dashed arrows = not addressed by this experiment
“Surprises” at Gal 1 1. Gal 4 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 URS 2 Pr HO ORF Both URS 1 and URS 2 are required for txn of HO RNAPII Basal Factors Mediator Swi/Snf chromatin remodeling complex SAGA (co-activator) SBF activator Swi 5 activator
Proteins that bind HO control region in vitro: Swi 5 SBF TBP et al URS 1 URS 2 Pr HO ORF RNAPII Basal Factors Mediator Swi/Snf = chromatin remodeling complex SAGA = co-activator, histone acetylase SBF = activator Swi 5 = activator
Primer Set 1 = S 1 = URS 1 Primer Set 2 = S 2 = URS 2 Primer Set 3 = S 3 = Pr URS 1 URS 2 Pr Set 1 Set 2 Set 3 PCR = Genomic DNA (not Ch. IP) Lane 1 = Set 1 only Lane 2 = Set 2 only Lane 3 = Set 3 only Lane 4 = Set 1 + 2 + 3 “Multiplex PCR” HO ORF S 1 S 3 S 2 1 2 Primer sets can resolve URS 1, URS 2 and Pr in Ch. IP analysis 3 4
URS 1 URS 2 Pr Set 1 Set 2 Set 3 HO ORF Swi 5 -tag S 1 S 3 S 2 1 2 3 4 5 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min
URS 1 URS 2 Pr HO ORF Set 1 Set 2 Set 3 Mediator-tag Swi 5 -tag Swi/Snf-tag S 1 S 3 S 2 1 2 3 4 5 1 2 3 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min; Lane 5 = 8 min Conclusions? ? ? 4 5
Model Derived From Data on Previous Slide t = 0 min t = 2 min URS 1 URS 2 Pr HO ORF Swi 5 URS 1 URS 2 Pr HO ORF t = 6 and 8 min Swi/Snf Mediator Swi 5 t = 4 min URS 1
Is Swi/Snf Chromatin remodeling complex required to recruit Mediator or is Swi 5 sufficient? t = 0 min t = 2 min URS 1 URS 2 Pr HO ORF Swi 5 URS 1 URS 2 Pr HO ORF t = 6 and 8 min Swi/Snf Mediator Swi 5 t = 4 min URS 1
Swi 2 = No functional Swi/Snf chromatin remodeling complex URS 1 URS 2 Pr HO ORF Set 1 Set 2 Set 3 Mediator-tag Swi 5 -tag Swi/Snf-tag S 1 S 3 S 2 1 2 3 4 5 1 2 3 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min 4 5
Swi 2 = No Swi/Snf chromatin remodelling complex t = 0 min t = 2 min t = 4 min t = 6 and 8 min URS 1 URS 2 Pr HO ORF Swi 5 URS 1 URS 2 Pr HO ORF Swi 5
One Model Derived From Data on WT and snf 2 strains t = 0 min t = 2 min URS 1 URS 2 Pr HO ORF Swi 5 URS 1 URS 2 Pr HO ORF Mediator t = 4 min Swi 5 Swi/Snf URS 1 Mediator t = 6 and 8 min Swi/Snf URS 1
Order of initial events at HO in vivo
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. 2. 3. 4. 5. Swi 5 binds transiently Mediator at URS 1, URS 2 and Promoter Swi/Snf and mediator stay bound at URS 1 after Swi 5 is no longer bound-- how? Swi/Snf and Saga arrive at URS 2 before SBF -- how? SBF recruited to URS 2 by Saga (activator recruited by a co-activator)
1. 2. 3. 4. 5. Swi 5 binds transiently Mediator at URS 1, URS 2 and Promoter Swi/Snf and mediator stay bound at URS 1 after Swi 5 is no longer bound- how? Swi/Snf and Saga arrive at URS 2 before SBF- how? SBF recruited to URS 2 by Saga (activator recruited by a co-activator)
PIR 1, CLN 2 and HO puzzles
Lodish 11 -32
UAS = enhancer URS = silencer URS = Upstream Repressor Sequence Not Regulatory Seq as in HO Lodish 11 -32
“Writers” and “Readers” of the Histone Code
Ch. IP of Histones Antibodies against different modified forms of the Histone Tails
Use antibodies that are specific for histone modifications
Lodish 11 -33 Puzzle? Why does Ch. IP using Abs to the histone tails work? What do the histone tails do?
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