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
1
24
2
23
3
22
4
21
5
20
6
19
7
18
8
17
9
16 15
10
14
11
13
12
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)
0
1
2
3
4
5
6
7
8
9
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