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MCB 317 Genetics and Genomics

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 ( ChIP ) Does a specific protein of interest bind to a specific site on a chromosome in vivo ?.

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MCB 317 Genetics and Genomics

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  1. MCB 317Genetics and Genomics MCB 317 Topic 10, part 6 A Story of Transcription

  2. What is order of action in vivo?

  3. How do we get at what’s going on in vivo?Chromatin Immunoprecipitation(ChIP)Does a specific protein of interest bind to a specific site on a chromosome in vivo?

  4. UNLESS STATED OTHERWISE, WE ONLY LOOK AT PCR PRODUCTS FROM THE PPT

  5. 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. 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 ChIP

  6. Performing ChIP on mutant strains can give insight into the arrangement of proteins in the complex relative to DNA D C C D C B B A A A 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

  7. What is order of action in vivo?

  8. An Imaginary Yeast Gene t = 0 min UAS Pr YFG1 ORF Act t = 5 min UAS Pr YFG1 ORF Act TBP t = 10 min UAS Pr YFG1 ORF

  9. UAS Pr YFG1 ORF Primer Set 2 Primer Set 1 PCR on Total Purified Genomic DNA (not ChIP): Primer Set 1 Primer Set 2 Set 1 + Set 2

  10. Act Act Strain 1 = Activator is Epitope Tagged t = 0 min UAS Pr YFG1 ORF t = 5 min UAS Pr YFG1 ORF TBP t = 10 min UAS Pr YFG1 ORF

  11. Act Act Strain 1 = Activator is Epitope Tagged t = 0 min UAS Pr YFG1 ORF Set1 t = 5 min UAS Pr YFG1 ORF Set2 TBP t = 10 min 0 5 10 UAS Pr YFG1 ORF Time (min) Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn

  12. Strain 2 = TBP is Epitope Tagged t = 0 min UAS Pr YFG1 ORF Act Set1 t = 5 min UAS Pr YFG1 ORF Set2 Act TBP t = 10 min 0 5 10 UAS Pr YFG1 ORF Time (min) Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn

  13. Strain 3 = Mediator is Epitope TaggedWhat Would You Conclude? Set1 Set2 5 10 15 C 0 Time (min) Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn C = Control = Pruified Genomic DNA (no ChIP)

  14. Combine Data from 3 Strains -> Model t = 0 min UAS Pr YFG1 ORF Act t = 5 min UAS Pr YFG1 ORF Act TBP t = 10 min UAS Pr YFG1 ORF Mediator Act TBP t = 15 min UAS Pr YFG1 ORF

  15. Order of events/action at the GAL1 promoter

  16. Components UAS Pr GAL1 ORF Also Gal4 activator protein

  17. Binding in vitro Gal4 TBP UAS Pr GAL1 ORF Gal4 activator protein

  18. UAS UAS Pr Pr GAL1 ORF GAL1 ORF Strategy:GAL1 OFF in Glucose -> ON in GalactoseGrow in Glucose -> shift to Galactose -> ChIP each component at various time points to determine when they bind control region Glucose Galactose ChIP at 1 min, 2 min, 3 min, etc…..

  19. GAL1 ChIP UAS Pr GAL1 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

  20. ChIP Resolution Limited by Fragment Size 75 bp UAS Pr GAL1 ORF PCR Shear DNA 500-1000 bp Fragments U P U P U P U P U P U P

  21. 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

  22. Model of Recruitment at Gal1 Saga RNAPII and Basal Factors Gal4 Mediator Dashed arrows = not addressed by this experiment

  23. “Surprises” at Gal1 • Gal4 bound constitutively • Mediator binds independently of RNAPII

  24. Order of events/action at the HO promoter

  25. URS1 URS2 Pr HO ORF HO txn is cell cycle regulated: OFF in M -> ON in G1 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

  26. Proteins that bind HO control region in vitro: TBP et al Swi5 SBF URS1 URS2 Pr HO ORF RNAPII Basal Factors Mediator Swi/Snf = chromatin remodeling complex SAGA = co-activator, histone acetylase SBF = activator Swi5 = activator

  27. URS1 URS2 Pr HO ORF Set 1 Set 2 Set 3 S1 S3 S2 1 2 3 4 Primer Set 1 = S1 = URS1 Primer Set 2 = S2 = URS2 Primer Set 3 = S3 = Pr PCR = Genomic DNA (not ChIP) Lane 1 = Set 1 only Lane 2 = Set 2 only Lane 3 = Set 3 only Lane 4 = Set 1 + 2 + 3 “Multiplex PCR” Primer sets can resolve URS1, URS2 and Pr in ChIP analysis

  28. URS1 URS2 Pr HO ORF Set 1 Set 2 Set 3 Swi5-tag S1 S3 S2 1 2 3 4 5 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min

  29. URS1 URS2 Pr HO ORF Set 1 Set 2 Set 3 Mediator-tag Swi/Snf-tag Swi5-tag S1 S3 S2 1 1 1 2 3 4 5 2 3 4 5 2 3 4 5 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min; Lane 5 = 8 min Conclusions???

  30. Model Derived From Data on Previous Slide t = 0 min URS1 URS2 Pr HO ORF Swi5 t = 2 min URS1 URS2 Pr HO ORF Swi/Snf Mediator Swi5 t = 4 min URS1 URS2 Pr HO ORF Swi/Snf Mediator t = 6 and 8 min URS1 URS2 Pr HO ORF

  31. Is Swi/Snf Chromatin remodeling complex required to recruit Mediator or is Swi5 sufficient? t = 0 min URS1 URS2 Pr HO ORF Swi5 t = 2 min URS1 URS2 Pr HO ORF Swi/Snf Mediator Swi5 t = 4 min URS1 URS2 Pr HO ORF Swi/Snf Mediator t = 6 and 8 min URS1 URS2 Pr HO ORF

  32. URS1 URS2 Pr HO ORF Set 1 Set 2 Set 3 Swi2 = No functional Swi/Snf chromatin remodeling complex Mediator-tag Swi/Snf-tag Swi5-tag S1 S3 S2 1 1 1 2 3 4 5 2 3 4 5 2 3 4 5 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min

  33. Swi2 = No Swi/Snf chromatin remodelling complex t = 0 min URS1 URS2 Pr HO ORF Swi5 t = 2 min URS1 URS2 Pr HO ORF Swi5 t = 4 min URS1 URS2 Pr HO ORF t = 6 and 8 min URS1 URS2 Pr HO ORF

  34. One Model Derived From Data on WT and snf2 strains t = 0 min URS1 URS2 Pr HO ORF Swi5 t = 2 min URS1 URS2 Pr HO ORF Mediator Swi/Snf Swi5 t = 4 min URS1 URS2 Pr HO ORF Mediator Swi/Snf t = 6 and 8 min URS1 URS2 Pr HO ORF

  35. Order of initial events at HO in vivo

  36. Order of initial events at HO in vivo What evidence might lead you to draw this arrow?

  37. In vivo order of events leading to txn of the HO gene

  38. “Surprises” at HO • Swi5 binds transiently • Mediator at URS1, URS2 and Promoter • Swi/Snf and mediator stay bound at URS1 after Swi5 is no longer bound-- how? • Swi/Snf and Saga arrive at URS2 before SBF -- how? • SBF recruited to URS2 by Saga (activator recruited by a co-activator)

  39. Swi5 binds transiently • Mediator at URS1, URS2 and Promoter • Swi/Snf and mediator stay bound at URS1 after Swi5 is no longer bound- how? • Swi/Snf and Saga arrive at URS2 before SBF- how? • SBF recruited to URS2 by Saga (activator recruited by a co-activator)

  40. PIR1, CLN2 and HO puzzles

  41. Lodish 11-32

  42. URS = Upstream Repressor Sequence Not Regulatory Seq as in HO UAS = enhancer URS = silencer Lodish 11-32

  43. “Writers” and “Readers”of the Histone Code

  44. ChIP of HistonesAntibodies against different modified forms of the Histone Tails

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