Identificación y Análisis Funcional de Nuevos Reguladores Transcripcionales Implicados en la Respuesta Celular al Estr

1. Identificación y Análisis Funcional de Nuevos Reguladores Transcripcionales Implicados en la Respuesta Celular al Estrés Rosa M. Marion Erin K. O’Shea Lab Howard Hughes Medical Institute

2. oxygen availability, temperature, salt concentration, DNA damaging agents, nutrient starvation, pH……….. STRESS!!!!!!!! signaling pathway transcription factor stress induced gene expression program

3. ~200 ~6000 Transcriptional Regulatory Networks

4. ESRrepressed ESRinduced The transcriptional response to stress

5. Transcription factor-GFP fusion STRESS Change in localization? Transcriptional Regulatory Networks

6. 251 known or predicted transcription factors 172 had detectable fluorescent signal (70%) 137 nuclear (80%) 35 cytoplasmic (20%) Construction of a yeast TF-GFP library -live cells, no fixation needed -endogenous promoter

7. 1 2 3 4 5 6 7 8 9 10 11 12 24 23 22 21 20 19 18 17 16 15 14 13 Msn2-GFP 0.5 M NaCl Large-Scale Analysis of Transcription Factor Subcellular Localization Upon Stress

8. Sfp1-GFP relocalizes in response to different stresses 0.4 mM H202

9. Before stress After stress 2mM DTT 0.1%MMS 0.5 M NaCl

10. Sfp1 controls ribosomal biogenesis Jorgensen P. and Tyers M. Science (2002) sfp1D has a very small cell size (~40% smaller than WT) and overexpression increases cell size propose that Sfp1 is a regulator of genes involved in ribosome biogenesis

11. Regulation of ribosome biosynthesis -Regulated mainly at the level of transcription: transcription of genes involved in ribosome biosynthesis is tightly coordinated -Involves the activity of Pol I, Pol II and Pol III -It consumes an enormous amount of the cell’s resources: RNA:80%rRNA (60% of total transcription) 15%tRNA 5% mRNA (50% pol II activity dedicated to RP) 200000 ribosomes /cell 2000ribosomes/minute 70% splicing activity Ribosome formation is coupled with the protein-synthetic needs of the cell and the availability of nutrients Not known how signaling pathways regulate RP gene expression

12. HYPOTHESIS: Sfp1 is a regulator of ribosomal gene expression Does Sfp1 localization correlate with RP gene expression? ribosomal gene transcription nuclear Sfp1 ON ribosomal gene transcription cytosolic Sfp1 OFF

13. Regulation of ribosomal protein gene expression RP gene expression is down regulated : 1. under environmental stress 2. when cells reach stationary phase 3. when cells are treated with rapamycin 4. when cells are starved for glucose 5. when there is a defect in the secretory pathway

14. 1. Sfp1 relocalizes to the cytoplasm under environmental stress Ctrl 0.5 M NaCl Ribosomal gene transcription ON OFF

15. 2. Sfp1 relocalizes to the cytoplasm when cells reach stationary phase Log phase Stationary phase Ribosomal gene transcription ON OFF

16. Sfp1 relocalizes to the cytoplasm when cells are • treated with rapamycin Ctrl Rapamycin Powers T. and Walter P. Mol. Biol. Cell (1999) ON OFF ribosomal gene transcription

17. 4. Sfp1 relocalizes to the cytoplasm when cells are starved for glucose S ethanol SD complete S ethanol +glucose ON OFF ON

18. 5. Sfp1 relocalizes to the cytoplasm when the secretory pathway is interrupted Ctrl Tunicamycin (3h 30’) ON OFF ribosomal gene transcription

19. Sfp1 binds RP promoters Quantitavive PCR for target genes (promoter area) Sfp1-HA Chromatin IP and Quantitative PCR Sfp1 Occupancy

20. 4 4 3 3 2 2 1 1 WT vs. WT + rap sfp1D vs. sfp1D + rap WTvs.sfp1D WT + rap vs. sfp1D + rap Defect in regulation of RP gene expression in sfp1D mutants

22. Pathways that control ribosomal protein gene transcription Nutrients Glucose TOR PKA PKC ribosomal protein gene transcription ON

23. Ribosomal Biogenesis Genes TOR and RP gene expression SFP1 Adapted fromJacinto E. and Hall M. N. Nature Reviews Molecular Cell Biology 4, 117-126 (2003)

24. TOR CONTROLS LOCALIZATION OF SFP1 WT Sfp1-GFP tor1-1 Sfp1-GFP Ctrl Rapamycin

25. Dmsn2, 4 Sfp1-GFP Dgat1 Sfp1-GFP Dgln3 Sfp1-GFP Dtip41 Sfp1-GFP Dpph21 Sfp1-GFP Drtg2 Sfp1-GFP Drtg3 Sfp1-GFP Drtg1 Sfp1-GFP Dmks1 Sfp1-GFP Dsit4 Sfp1-GFP Ctrl Rapamycin

26. PKA and RP gene expression Tpk1,2,3 (catalitic) Bcy1 (regulatory) starvation Glucose (cAMP) Low PKA High PKA RP gene expression OFF RP gene expression ON

27. PKA CONTROLS LOCALIZATION OF SFP1 Ctrl Rapamycin wt Sfp1 GFP Dbcy1 Sfp1GFP

28. Sfp1 Occupancy Sfp1-HA Chromatin IP and Quantitative PCR

29. TOR can control Sfp1 localization independently of PKA

30. Stress stimuli can be transmitted to Sfp1 independently of PKA

31. Stress ? nutrients TOR rapamycin glucose RAS/cAMP PKA TAP42/SIT4 SFP1 RP Promoter ON

32. Summary -Sfp1 is an activator of RP gene transcription -Sfp1 relocalizes from the nucleus to the cytoplasm in response to diverse environmental stimuli and stresses, coincident with lower expresssion of RP genes -Sfp1 binds to the RP promoters and this binding is regulated by stress -Cells lacking Sfp1 show a defect in regulation of RP gene transcription in response to stress, indicating that Sfp1 is required for the proper downregulation of RP genes in response to stress -The TOR pathway controls localization of Sfp1. TOR activity induces Sfp1 nuclear localization -PKA controls localization of Sfp1. PKA activity induces Sfp1 nuclear localization -Stresses such as oxidative or osmotic stress can control Sfp1 localization Independently of PKA

34. Stress ? nutrients TOR rapamycin glucose RAS/cAMP PKA TAP42/SIT4 SFP1 RP Promoter ON

35. IFH1 FHL1 RAP1 SFP1 RP Promoter

37. -localization of Sfp1 binding site in RP promoters