Identificación y Análisis Funcional de Nuevos Reguladores Transcripcionales Implicados en

la Respuesta Celular al Estrés

Rosa M. Marion

Erin K. O’Shea LabHoward Hughes Medical Institute

oxygen availability,temperature,salt concentration,DNA damaging agents,nutrient starvation,pH………..

signaling pathway

stress inducedgene expression

program

STRESS!!!!!!!!STRESS!!!!!!!!

transcriptionfactor

~200

~6000

Transcriptional Regulatory NetworksTranscriptional Regulatory Networks

The transcriptional response to stress

ESRrepressed

ESRinduced

Transcriptional Regulatory NetworksTranscriptional Regulatory Networks

Transcription factor-GFP fusion

Change in localization?

STRESSSTRESS

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

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16 15

10

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Msn2-GFP

0.5 M NaCl

Large-Scale Analysis of Transcription Factor Subcellular

Localization Upon Stress

0.4 mM H202

Sfp1-GFP relocalizes in response to different stresses

Before stress

Afterstress

0.1%MMS2mM DTT0.5 M NaCl

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 inribosome biogenesis

Regulation of ribosome biosynthesis

200000 ribosomes /cell 2000ribosomes/minute70% 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 cell’s 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

nuclear Sfp1

cytosolic Sfp1 ribosomal gene transcription

ribosomal genetranscription

ON

OFF

Does Sfp1 localization correlate with RP gene expression?

HYPOTHESIS: Sfp1 is a regulator of ribosomal gene expression

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

0.5 M NaCl

1. Sfp1 relocalizes to the cytoplasm under environmental stress

Ctrl

ON OFFRibosomal genetranscription

Stationary phaseLog phase

ON OFFRibosomal genetranscription

2. Sfp1 relocalizes to the cytoplasm when cells reach stationary phase

Ctrl Rapamycin

ON OFF

Powers T. and Walter P.Mol. Biol. Cell (1999)

ribosomal genetranscription

3. Sfp1 relocalizes to the cytoplasm when cells are treated with rapamycin

SD complete S ethanol +glucoseS ethanol

ON OFF ON

4. Sfp1 relocalizes to the cytoplasm when cells are starved for glucose

Tunicamycin (3h 30’)Ctrl

ON OFFribosomal genetranscription

5. Sfp1 relocalizes to the cytoplasm when the secretory pathway is interrupted

Sfp1 binds RP promoters

0

1

2

3

4

50' rapamycin30' rapamycin60' rapamycin

Sfp1

Occ

upan

cy

Sfp1-HA Chromatin IP and Quantitative PCR

Quantitavive PCR for target genes(promoter area)

Defect in regulation of RP gene expression in sfp1 mutants

WT vs. WT + rap1

sfp1 vs. sfp1 + rap2

WT vs. sfp13

WT + rap vs. sfp1 + rap4

1 2 3 4

Pathways that control ribosomal protein gene transcription

TOR PKA PKC

ribosomal protein genetranscription

Nutrients Glucose

ON

Adapted from Jacinto E. and Hall M. N. Nature Reviews Molecular Cell Biology 4, 117-126 (2003)

SFP1Ribosomal Biogenesis Genes

TOR and RP gene expression

TOR CONTROLS LOCALIZATION OF SFP1

WTSfp1-GFP

tor1-1Sfp1-GFP

Ctrl Rapamycin

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

Ctrl Rapamycin

PKA and RP gene expression

Low PKA High PKA

RP gene expression OFF

RP gene expression ON

starvation Glucose (cAMP)

Bcy1(regulatory)

Tpk1,2,3(catalitic)

wtSfp1 GFP

bcy1Sfp1GFP

Ctrl Rapamycin

PKA CONTROLS LOCALIZATION OF SFP1

 Schmelzle T. and Hall M. N. Mol. Cell. Biol. (2004)

 Schmelzle T. and Hall M. N. Mol. Cell. Biol. (2004)

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RPL1B RPL27B RPS0B RPL11A RPL2B

WT

bcy1

Sfp1-HA Chromatin IP and Quantitative PCR

Sfp1

Occ

upan

cy

TOR can control Sfp1 localization independently of PKA

Stress stimuli can be transmitted to Sfp1 independently of PKA

TORrapamycin

nutrients

TAP42/SIT4RAS/cAMP

PKA

SFP1RP Promoter ON

glucose

Stress?

-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 nuclearlocalization

-Stresses such as oxidative or osmotic stress can control Sfp1 localization Independently of PKA

Summary

TORrapamycin

nutrients

TAP42/SIT4RAS/cAMP

PKA

SFP1RP Promoter ON

glucose

Stress?

SFP1

RP PromoterFHL1

IFH1

RAP1

-localization of Sfp1 binding site in RP promoters