TFIID and SAGA roles in transcription machinery

1. TFIID and SAGA rolesin transcription machinery Inna WeinerReading group in Computational Molecular Biology16/11/06

2. Core promoter Proximal promoter Enhancer/silencer Global structure of RNA polymerase II promoters TSS

3. The core promoter (Butler and Kadonaga, 2002)

4. How to recruit RNA Polymerase? Assembly of general transcription factors is required to recruit RNA Polymerase to promoters A9.30

5. General transcription factors • Factors that permit efficient selective initiation by Pol II in vitro: • Promoters recognition • DNA melting • Isolated by biochemical fractionation experiments. • TFIIA, TFIIB, TFIID, TFIIE,TFIIF and TFIIH complexes were isolated and are sufficient to permit in vitro specific transcription.

6. TBP TATA box - Binding Protein • TBP is a single polypeptide that sits astride the TATA box as a molecular ''saddle,'' inducing a sharp bend in the DNA • Regarded as a universal transcription factor, essential for initiation by RNA Pol I, II, and III

7. TAFs (TBP Associated Factors) The role of the TAFs in the TFIID complex: - Specific interactions with activators - Binding to the promoter Inr and DPE elements - Modifying chromatin

8. Pre Initiation Complex (PIC) formation Transition from PIC formation into active transcription: 1. Initiation: one of TFIIH subunits has an helicase activity and is able to melt the double helices DNA. 2. Elongation: this step is triggered by the phosphorilation of the CTD repeats of the polymerase by one of the TFIIH subunit that has a kinase activity.

9. Pol II starts mRNA synthesis Very few factors accompany the elongating transcript

10. Summary Localization to promoters Melting the DNA, transcription initiation and elongation

11. Is the general transcription machinery really general? (Holstege et al., 1998)

12. Outline • Introduction to general transcription machinery • TFIID and SAGA – unique or redundant function? • TFIID and SAGA roles in transcription regulation

14. TFIID and SAGA • Multi-unit complexes • Perform two actions essential for Pol II initiation: • Contain a subunit with histone acetyltransferase activity • Possess TBP binding activity • Question: Are the functions of TFIID and SAGA in vivo unique or overlapping?

15. Percentage of genome dependent on subunits of SAGA and TFIID

16. Whole genome analysis of shared TAFIIs mutations

17. Shared vs Specific TAFIIs Influence ~30% of the genome is dependent on TFIID specific sub-units ~12% of the genome is dependent on SAGA specific sub-units ~70% of the genome depends on one or more of the shared TAFs

18. How do TFIID and SAGA interact? TFIID and SAGA have compensatory functions

19. Genetic Interactions X ∆X Y ∆X Y ∆Y ∆X ∆X Y w.t. X Y Y X Y ∆X Y<∆X*∆Y ∆X Y=∆X*∆Y ∆X Y>∆X *∆Y Aggravating interaction No interaction Alleviating interaction Slide by Ariel

20. How do TFIID and SAGA interact? TFIID and SAGA have compensatory functions

21. Conclusions • There are distinct requirements for specific sub-units of TFIID and SAGA in global expression • The functions of TAFII145 and GCN5 are redundant

23. Research Objective • TFIID and SAGA • Share a common set of TAFs • Regulate chromatin • Deliver TBP to promoters • What is their distinct function and relationship in genome-wide regulatory network? • Tested organism: S.cerevisiae

24. Experimental Setup • Create single and double-mutants: • SAGA-specific mutant: GCN5, SPT3 • TFIID-specific mutant: TAF1 (=TAFII145) • Compare gene expression of single and double mutants by performing high-throughput analysis

25. GCN5 mutants rbp1-1: gene expression without pol II activity • gcn5Δ strain displayed a general decrease in expression • Over 60% of the genes decreased expression by >4 standard devations • Gcn5hat did not change expression significantly  GCN5 makes a positive modest contribution to the expression of most of the genes  HAT activity of GCN5 plays a redundant or minor role

26. SPT3 mutants and TAF1 mutants • spt3Δ and spt3E240K strains do not differ substantially from wild type  spt3 plays a small or redundant role • Taf1ts2 strain causes leftward shift of the distribution • 84% of the genome decreased depression by >4 std’s • But population shift is not as severe as for rbp1-1  taf1 makes a positive contribution to genes expression, but its action is not absolute

27. GCN5/TAF1 double mutants • TAF1/GCN5 double mutants shift the population like rbp1-1 • TAF1 and GCN5Δ interaction is expected • GCN5hat sensitivity suggests that HAT activity of GCN5 is important when TAF1 is absent  GCN5 makes a positive modest contribution to the expression of most of the genes  HAT activity of GCN5 plays a redundant or minor role  GCN5 and TAF1 are associated to the same HAT activity

28. SPT3 mutants and TAF1 mutants  spt3 plays a small or redundant role • 97% of genes in the TAF1/SPT3Δ mutant decreased by >4 std’s: nearly complete shutdown of transcription both TAF1 and SPT3 contribute to the expression of all measurable genes  SAGA and TFIID are the only redundant complexes in transcriptional general activity  taf1 makes a positive contribution to genes expression, but its action is not absolute

29. Conclusions • TFIID and SAGA each contribute to the expression of nearly all genes

30. TFIID or SAGA dominated genes • TFIID-dominated genes: 90% that showed greater dependency on TAF1 than SPT3 • SAGA-dominated genes: 10% that are SPT3-dependent • TAF1 appears to be inactive in SAGA-dominated genes

31. Conclusions • TFIID and SAGA each contribute to the expression of nearly all genes • TFIID dominates at ~90% of all genes, and SAGA Dominates at ~10%

32. Stress-Induced Genes Tend to be SAGA Dominated • Enrichment in stress-dependent conditions • Genes that are commonly upregulated biased (p_value < 10-30) to SAGA-dominated • Genes that are commonly downregulated biased (p_value < 10-10) to TFIID-dominated • What advantage might SAGA provide in environmental stress response that TFIID does not?

33. Conclusions • TFIID and SAGA each contribute to the expression of nearly all genes

34. Histone Acetylation • Acetylation of H3 and H4 is associated with transcriptional activation • H4 under-acetylated regions were biased to SAGA-dominated genes whereas H4 overacetylated regions biased to TFIID-dominated genes • Hda1 and Rpd3 appear to assist in keeping low acetylation at SAGA-dominated genes and high acetylation at TFIID-dominated genes

35. Conclusions • TFIID and SAGA each contribute to the expression of nearly all genes

36. SAGA-dominated genes are largely TAF-independent • TAFs (TBP Associated Factors) are subunits of TFIID but a subset are also present in SAGA • Genes that are positively regulated by TAFs were biased toward the TFIID-dominated class • TAF-independent promoters are likely to be SAGA-dominated

37. SAGA-dominated genes are highly regulated • How do other transcription factors function? • SRB10 phosphorilates a number of stress response regulators. Genes that are most inhibited by SRB10 are SAGA-dominated • Genes regulated by stress activators Msn2 and Msn4 are also SAGA-dominated There is a coordinated stress response pathway that is up-regulated by gene-specific activators like Msn2/4 and down-regulated by Srb-10 - regulated phosphorylation and histone de-acetylation

38. SAGA-dominated genes are Coordinately Regulated Stress-induced gened

39. Summary • TFIID and SAGA each contribute to the expression of nearly all genes

40. Two distinct mechanisms