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

2. What is order of action in vivo?

4. 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?

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

10. 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

11. 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

12. What is order of action in vivo?

13. An Imaginary Yeast Gene UASPrYFG1 ORF Act TBP UASPrYFG1 ORF Act UASPrYFG1 ORF t = 0 min t = 5 min t = 10 min

14. 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

15. Strain 1 = Activator is Epitope Tagged UASPrYFG1 ORF TBP UASPrYFG1 ORF Act UASPrYFG1 ORF t = 0 min t = 5 min t = 10 min Act

16. 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) 0 510 Set1 Set2 Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn

17. 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) 0 510 Set1 Set2 Primer Set 1 = UAS; Primer Set 2 = Pr Both Sets of Primers are in each PCR rxn

18. Strain 3 = Mediator is Epitope Tagged What Would You Conclude? Time (min) 0 510 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

19. 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

20. Order of events/action at the GAL1 promoter

21. Components Also Gal4 activator protein UASPrGAL1 ORF

22. Binding in vitro Gal4 activator protein UASPrGAL1 ORF Gal4 TBP

23. 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…..

24. 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

25. ChIP Resolution Limited by Fragment Size UASPrGAL1 ORF PCR 75 bp Shear DNA 500-1000 bp Fragments PU PU PU PU PU PU

26. 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

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

28. “Surprises” at Gal1 1.Gal4 bound constitutively 2.Mediator binds independently of RNAPII

29. Order of events/action at the HO promoter

30. 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

31. 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

32. Primer Set 1 = S1 = URS1 Primer Set 2 = S2 = URS2 Primer Set 3 = S3 = Pr URS2PrHO ORFURS1 Set 1Set 2Set 3 S1 S3 S2 1 234 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

33. URS2PrHO ORFURS1 Set 1Set 2Set 3 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min S1 S3 S2 1 234 Swi5-tag 5

34. 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 S2 1 234 Swi5-tag Mediator-tagSwi/Snf-tag 5 1 2345 1 2345 Conclusions???

35. 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

36. 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?

37. URS2PrHO ORFURS1 Set 1Set 2Set 3 Lane 1 = 0 min; Lane 2 = 2 min; Lane 3 = 4 min; Lane 4 = 6 min S1 S3 S2 1 234 Swi5-tag Mediator-tagSwi/Snf-tag 5 1 2345 1 2345 Swi2  = No functional Swi/Snf chromatin remodeling complex

38. 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

39. 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

40. Order of initial events at HO in vivo

41. What evidence might lead you to draw this arrow?

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

43. “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)

44. 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)

45. PIR1, CLN2 and HO puzzles

46. Lodish 11-32

47. URS = Upstream Repressor Sequence Not Regulatory Seq as in HO UAS = enhancer URS = silencer

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

50. ChIP of Histones Antibodies against different modified forms of the Histone Tails

51. Use antibodies that are specific for histone modifications

53. Lodish 11-33 Puzzle? Why does ChIP using Abs to the histone tails work? What do the histone tails do?