Transcript No Slide Title

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
STRESS!!!!!!!!
oxygen availability,
temperature,
salt concentration,
DNA damaging agents,
nutrient starvation,
pH………..
signaling pathway
transcription
factor
stress induced
gene expression
program
Transcriptional Regulatory Networks
~200
~6000
The transcriptional
response to stress
ESRrepressed
ESRinduced
Transcriptional Regulatory Networks
Transcription factor-GFP fusion
STRESS
Change in localization?
Construction of a yeast TF-GFP library
-live cells, no fixation needed
-endogenous promoter
251 known or predicted transcription factors
172 had detectable fluorescent signal (70%)
137 nuclear (80%)
35 cytoplasmic (20%)
Large-Scale Analysis of Transcription Factor Subcellular
Localization Upon Stress
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Msn2-GFP
0.5 M NaCl
Sfp1-GFP relocalizes in response to different stresses
0.4 mM H202
Before
stress
After
stress
0.5 M NaCl
2mM DTT
0.1%MMS
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
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
HYPOTHESIS: Sfp1 is a regulator of ribosomal gene
expression
Does Sfp1 localization correlate with RP gene expression?
nuclear Sfp1
cytosolic Sfp1
ribosomal gene
transcription
ON
ribosomal gene
transcription
OFF
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
1. Sfp1 relocalizes to the cytoplasm under environmental stress
Ribosomal gene
transcription
Ctrl
0.5 M NaCl
ON
OFF
2. Sfp1 relocalizes to the cytoplasm when cells reach stationary phase
Log phase
Ribosomal gene
transcription
ON
Stationary phase
OFF
3. Sfp1 relocalizes to the cytoplasm when cells are
treated with rapamycin
Ctrl
Rapamycin
ON
OFF
Powers T. and Walter P.
Mol. Biol. Cell (1999)
ribosomal gene
transcription
4. Sfp1 relocalizes to the cytoplasm when cells are starved for glucose
SD complete
ON
S ethanol
OFF
S ethanol
+glucose
ON
5. Sfp1 relocalizes to the cytoplasm when the secretory pathway
is interrupted
Ctrl
ribosomal gene
transcription
ON
Tunicamycin (3h 30’)
OFF
Sfp1 binds RP promoters
Quantitavive PCR for target genes
(promoter area)
Sfp1-HA Chromatin IP and Quantitative PCR
0' rapamycin
30' rapamycin
60' rapamycin
4
3
2
1
R
YE
F3
SS
B2
N
SR
1
N
AN
1
IM
D
4
PL
1B
PL
27
R B
PS
0B
R
PL
11
R A
PL
2B
0
R
Sfp1 Occupancy
5
Defect in regulation of RP
gene expression in sfp1D
mutants
1
WT vs. WT + rap
2
sfp1D vs. sfp1D + rap
3
WT vs. sfp1D
4
WT + rap vs. sfp1D + rap
1 2
3 4
Pathways that control ribosomal protein
gene transcription
Nutrients
Glucose
TOR
PKA
ribosomal protein gene
transcription
PKC
ON
TOR and RP gene expression
SFP1
Ribosomal Biogenesis
Genes
Adapted from Jacinto E. and Hall M. N.
Nature Reviews Molecular Cell Biology 4, 117-126 (2003)
TOR CONTROLS LOCALIZATION OF SFP1
WT
Sfp1-GFP
tor1-1
Sfp1-GFP
Ctrl
Rapamycin
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
PKA and RP gene expression
Tpk1,2,3
(catalitic)
Bcy1
(regulatory)
starvation
Low PKA
RP gene expression
OFF
Glucose (cAMP)
High PKA
RP gene expression
ON
PKA CONTROLS LOCALIZATION OF SFP1
Ctrl
Schmelzle T. and Hall M. N. Mol.
Cell. Biol. (2004)
wt
Sfp1 GFP
Dbcy1
Sfp1GFP
Rapamycin
9
WT
8
bcy1
Sfp1 Occupancy
7
Schmelzle T. and Hall M. N. Mol.
Cell. Biol. (2004)
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5
4
3
2
1
0
RPL1B
RPL27B
RPS0B
RPL11A
RPL2B
Sfp1-HA Chromatin IP and Quantitative PCR
TOR can control Sfp1 localization independently of PKA
Stress stimuli can be transmitted to Sfp1
independently of PKA
nutrients
TOR
rapamycin
TAP42/SIT4
glucose
RAS/cAMP
PKA
Stress
?
RP Promoter
SFP1
ON
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
nutrients
TOR
rapamycin
TAP42/SIT4
glucose
RAS/cAMP
PKA
Stress
?
RP Promoter
SFP1
ON
IFH1
SFP1
RP Promoter
FHL1
RAP1
-localization of Sfp1 binding site in RP promoters