Presentation is loading. Please wait.

Presentation is loading. Please wait.

Identificación y Análisis Funcional de Nuevos Reguladores Transcripcionales Implicados en la Respuesta Celular al Estrés Rosa M. Marion Erin K. OShea Lab.

Similar presentations


Presentation on theme: "Identificación y Análisis Funcional de Nuevos Reguladores Transcripcionales Implicados en la Respuesta Celular al Estrés Rosa M. Marion Erin K. OShea Lab."— Presentation transcript:

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

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

3 ~200 ~6000 Transcriptional Regulatory Networks

4 The transcriptional response to stress ESR repressed ESR induced

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

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

7 Msn2-GFP 0.5 M NaCl Large-Scale Analysis of Transcription Factor Subcellular Localization Upon Stress

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

9 Before stress After stress 0.1%MMS2mM DTT 0.5 M NaCl

10 Sfp1 controls ribosomal biogenesis Jorgensen P. and Tyers M. Science (2002) sfp1 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 ribosomes /cell 2000ribosomes/minute 70% splicing activity -Involves the activity of Pol I, Pol II and Pol III -Regulated mainly at the level of transcription: transcription of genes involved in ribosome biosynthesis is tightly coordinated - It consumes an enormous amount of the cells resources: RNA:80%rRNA (60% of total transcription) 15%tRNA 5% mRNA (50% pol II activity dedicated to RP) 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 nuclear Sfp1 cytosolic Sfp1 ribosomal gene transcription ribosomal gene transcription ON OFF Does Sfp1 localization correlate with RP gene expression? HYPOTHESIS: Sfp1 is a regulator of ribosomal gene expression

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

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

15 Stationary phaseLog phase ONOFF Ribosomal gene transcription 2. Sfp1 relocalizes to the cytoplasm when cells reach stationary phase

16 CtrlRapamycin ONOFF Powers T. and Walter P. Mol. Biol. Cell (1999) ribosomal gene transcription 3.Sfp1 relocalizes to the cytoplasm when cells are treated with rapamycin

17 SD completeS ethanol +glucose S ethanol ONOFFON 4. Sfp1 relocalizes to the cytoplasm when cells are starved for glucose

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

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

20 Defect in regulation of RP gene expression in sfp1 mutants WT vs. WT + rap 1 sfp1 vs. sfp1 + rap 2 WT vs. sfp1 3 WT + rap vs. sfp1 + rap

21

22 Pathways that control ribosomal protein gene transcription TOR PKAPKC ribosomal protein gene transcription NutrientsGlucose ON

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

24 TOR CONTROLS LOCALIZATION OF SFP1 WT Sfp1-GFP tor1-1 Sfp1-GFP CtrlRapamycin

25 msn2, 4 Sfp1-GFP gat1 Sfp1-GFP gln3 Sfp1-GFP tip41 Sfp1-GFP pph21 Sfp1-GFP rtg2 Sfp1-GFP rtg3 Sfp1-GFP rtg1 Sfp1-GFP mks1 Sfp1-GFP sit4 Sfp1-GFP CtrlRapamycin

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

27 wt Sfp1 GFP bcy1 Sfp1GFP CtrlRapamycin PKA CONTROLS LOCALIZATION OF SFP1 Schmelzle T. and Hall M. N. Mol. Cell. Biol. (2004)

28 Schmelzle T. and Hall M. N. Mol. Cell. Biol. (2004) Sfp1-HA Chromatin IP and Quantitative PCR Sfp1 Occupancy

29 TOR can control Sfp1 localization independently of PKA

30 Stress stimuli can be transmitted to Sfp1 independently of PKA

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

32 -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 Summary

33

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

35 SFP1 RP Promoter FHL1 IFH1 RAP1

36

37 -localization of Sfp1 binding site in RP promoters


Download ppt "Identificación y Análisis Funcional de Nuevos Reguladores Transcripcionales Implicados en la Respuesta Celular al Estrés Rosa M. Marion Erin K. OShea Lab."

Similar presentations


Ads by Google