gene expression regulation during cellular differentiation · 2019-10-16 · gene expression...
TRANSCRIPT
Gene expression regulation during cellular differentiation
Dom Helmlinger
CRBM
15/10/2019
Outline
1. Gene expression regulation: why and how?
2. Yeast as model systems.
3. Control of sexual differentiation in S. pombe.
4. Regulation of epigenetic factors by nutrient-sensing signaling pathways.
Defining gene expression regulation
and why should you care?
Problem
• Genes alone can account for the extraordinary complexity of a living organism.
• Genes interact with each other and with their environment (medium, cells):
– House-keeping genes: always ON
– Regulated genes: ON or OFF
Gene expression regulation
Difference prokaryotes / eukaryotes
• Prokaryotes: ON is the default state
regulators = repressors
• Eukaryotes: OFF is the default state
regulators = activators
Difference prokaryotes / eukaryotes
• Prokaryotes: ON is the default state
regulators = repressors
although: archeal histones…
• Eukaryotes: OFF is the default state
regulators = activators
although: pervasive transcription…
Importance of regulating gene expression
• Self-renewal or differentiation
• Adaptation to the environment
• Perturbations during oncogenesis
Structure of an eukaryotic gene
exon+1
intron exon
Promoter
Genomic DNA
TerminatorRNA
mRNA
Protein
ncRNA:rRNAtRNAsnRNA, snoRNAmiRNA, siRNA…
Regulating the expression of a gene
exon+1
intron exon
Promoter
Genomic DNA
TerminatorRNA
1. TRANSCRIPTION
Protein
2. PROCESSING3. DEGRADATION
4. TRANSLATION, PROCESSING, PTMs, DEGRADATION
5. ncRNA
Regulating transcription
exon+1
intron exon
1. Chromatin structure
Regulating transcription
exon+1
intron exon
Nucleosomes
1. Chromatin structure
2. Sequences (cis-regulatory elements)
Regulating transcription
exon+1
intron exonTATAUAS
Nucleosomes
1. Chromatin structure
2. Sequences (cis-regulatory elements)
3. DNA-binding factors (trans-acting factors)
Regulating transcription
exon+1
intron exonTATAUAS
Nucleosomes
Acti-vator
1. Structure de la chromatine
2. Séquences (cis)
3. Facteurs DNA-binding (trans)
4. Plein d’autres facteurs!
Regulating transcription
exon+1
intron exonTATAUAS
Nucleosomes
Acti-vator
1. Chromatin structure
2. Sequences (cis-regulatory elements)
3. DNA-binding factors (trans-acting factors)
4. …and many other regulatory proteins!
RNA Pol. II
GeneralTranscription
FactorsCo-activator
GeneralTranscription
FactorsCo-activator
Co-activator
GeneralTranscription
Factors
ElongationFactors
TerminationFactors
ElongationFactors
TerminationFactors
• cis-regulatory elements
• Specific transcription factors:
– DNA binding domain: binds to specific motifs, 6-10 bp, within promoters / enhancers
– Trans-activation domain: recruits co-activators and general transcription factors
• General transcription factors:
– DNA binding: TATA box-binding protein (TBP)
– Initiations (vs. elongators + terminators)
Regulation of transcription initiation 1/2
Regulation of transcription initiation 2/2
• Nucleosomes positioning: ATP-dependent chromatin remodeling complexes
• Histone modifications (‘histone code’): histone-modifying complexes
= transcription co-activators
• Additional layer: non coding RNAs, DNA methylation
= Epigenetic regulation of transcription
Overall objective
To understand the mechanisms of transcription initiation regulation
exon+1
intron exonTATAUAS
Nucleosomes
Acti-vator
RNA Pol. II
GeneralTranscription
FactorsCo-activator
GeneralTranscription
FactorsCo-activato
Co-activator
GeneralTranscription
Factors
TranscriptionElongators
TranscriptionTerminators
TranscriptionElongators
TranscriptionTerminators
EXTERNAL SIGNAL
exon+1
intron exonTATAUASActi-
vator
Nucleosomes
RNA Pol. II
GeneralTranscription
FactorsCo-activator
GeneralTranscription
FactorsCo-activator
Co-activator
GeneralTranscription
Factors
TranscriptionElongators
TranscriptionTerminators
TranscriptionElongators
TranscriptionTerminators
Classical view
Recent discoveries
exon+1
intron exonTATAUAS
Nucleosomes
Acti-vator
RNA Pol. II
GeneralTranscription
FactorsCo-activator
GeneralTranscription
FactorsCo-activator
Co-activator
GeneralTranscription
Factors
TranscriptionElongators
TranscriptionTerminators
TranscriptionElongators
TranscriptionTerminators
EXTERNAL SIGNAL
Open questions
1. How are these transcriptional regulators controlled and coordinated?
2. Mechanisms by which they regulate transcription initiation?
3. How are these large complexes assembled?
Which model systems are available to
address these questions?
Yeast as model systems
• Unicellular Eukaryotes
• Version 1.0 of “higher” Eukaryotes
• Conservation of many factors / mechanisms
Budding yeastSaccharomyces cerevisiae
Fission yeastSchizosaccharomyces pombe
cerevisiae = beer in Latin pombe = beer in Swahili
The tree of all Eukaryotes
Yeast
Animals
Their importance
• Bread, beer & wine (agriculture)• Unicellular - very easy to manipulate• Biomedical research:
– Enzyme (1897)– Cell cycle (2001 Nobel)– Telomeres (2009 Nobel)– Trafficking (2013 Nobel)– Autophagy (2016 Nobel)– Epigenetics: histone-code, RNAi– Role of aneuploidy / genome instability in cancer– Diagnosis tool in breast cancer (BRCA1)– Gene therapy concept
Some numbers
• Cell size: 4-12 mm
• Genome size: 12-14 Mb
• Gene numbers: 5000-6000
• Evolutionary distance:
• Timing:
– Doubling time: few hours
– Getting 10L culture: 24 hrs
– Creating a novel mutant: 2 weeks
Experimental advantages
• Easy to manipulate, store, maintain
• Unlimited biochemistry, genetic approaches
• Tools:
– Genomes very well annotated
– Classical and system wide genetics feasible
– Knock-out, fluorescent tag, purification tag collections
– Best biological characterization of an eukaryotic organism (epigenome, transcriptome, proteome, interactome, metabolome, phenome)
– Novel techniques first developed in yeast
Gene expression regulation in yeast?
0 0 0 0 0 60 285 0 0 0 12 060 0 0 135 0 0 0 3 0
972 elu pat1_processed
0.01
0.1
1
10
100
Normalized Intensity
(log scale)
MergeDC-GR-JM coloured by Venn Diagram
Meiosis
Mitosis
Stressresponse
0 0 0 0 0 60 285 0 0 0 12 060 0 0 135 0 0 0 3 0
972 elu pat1_processed
0.01
0.1
1
10
100
Normalized Intensity
(log scale)
MergeDC-GR-JM coloured by Venn Diagram
Sex
Rock ‘n’ roll
Drugs
Cell fate determination in yeast
• Mating-type switching
• Proliferation vs quiescence vs sexual differentiation
= conjugation ➔meiosis ➔ sporulation
• Dimorphic switch = yeast-to-hyphae, controls virulence in many pathogens, eg. C. albicans
These events are controlled by external cues, from the yeast’s environment, typically nutrient quality.
One goal of my lab
High nutrients
Low nutrients
Proliferation
Differentiation,Quiescence
Geneexpression
Regulatorypathways
External cues Cell fate decisions
To understand how cells sense nutrient availability to
coordinately regulate gene expression and control cell fate.
Regulation of transcription during sexual differentiation in S. pombe:
A model to study how signaling pathways directly control an epigenetic regulator
S. pombe life cycle
M
G1
SG2
High nutrientsPROLIFERATION
S. pombe life cycle
“G0”
Low nutrientsDIFFERENTIATION
M
G1
SG2
High nutrientsPROLIFERATION
S. pombe life cycle
“G0”
Low nutrientsDIFFERENTIATION
M
G1
SG2
High nutrientsPROLIFERATION
meiosissporulation
(haploid)
zygote
conjugation(diploid zygote)
germination
The power of yeast genetics
• Conjugation of haploid yeast cells➔ diploid zygote
• Diploids enter meiosis ➔ 4 haploid spores, physically kept within one bag (ascus)
• Each ascus has the 4 products of ONE meiosis (Mendel laws live!)
• Mutants are easy to create, propagate, and analyze
This the power of yeast genetics!
Regulators of the switch proliferation vs. differentiation
LOW
NUTRIENTS
HIGH
NUTRIENTS
DIFFERENTIATION,
QUIESCENCE
CELL GROWTH,
PROLIFERATION
AMPK
Protein
Synthesis
TORC2
(Rictor)
p38
MAPK
TORC1
(Raptor)
Ste11
cAMP
PKA
Bähler, Hiraoka, Hoffmann, Jones, Kawamukai , Moreno, Murakami, Nielsen, Okayama, Petersen, Russell, Toda, Uritani, Weisman, Yamamoto, Yanagida labs
Regulators of the switch proliferation vs. differentiation
LOW
NUTRIENTS
HIGH
NUTRIENTS
DIFFERENTIATION,
QUIESCENCE
CELL GROWTH,
PROLIFERATION
AMPK
Protein
Synthesis
TORC2
(Rictor)
p38
MAPK
TORC1
(Raptor)
Ste11
cAMP
PKA
?Bähler, Hiraoka, Hoffmann, Jones, Kawamukai , Moreno, Murakami, Nielsen, Okayama, Petersen, Russell, Toda, Uritani, Weisman, Yamamoto, Yanagida labs
Regulators of the switch proliferation vs. differentiation
LOW
NUTRIENTS
HIGH
NUTRIENTS
DIFFERENTIATION,
QUIESCENCE
CELL GROWTH,
PROLIFERATION
AMPK
Protein
Synthesis
TORC2
(Rictor)
p38
MAPK
TORC1
(Raptor)
cAMP
PKA
?SAGA
Tra1/
TRRAP
Spt7
Spt8
Gcn5
Ste11
SAGA: a double agent
Opposing roles of the SAGA complex in the
transcriptional regulation of the switch
proliferation vs. differentiation
The SAGA complex 1/2
• Transcription co-activator
• Multi-functional (19 subunits, 2 MDa)
• Conserved: yeast, plants, insects, mammals
• Functions: development, oncogenesis
gene
RNA Pol. IIGTFs
TBP
Coactivator
TATA +1UAS
Act.
Nucleosome
The SAGA complex 2/2
Multiple, distinct regulatory activities:
• Scaffold: TAFs, Spt7, Ada1
• Histone modifications: Gcn5 (HAT), Ubp8 (DUB)
• TBP recruitment : Spt8, Spt3
• Promoter binding: Tra1
Structural organization
= functional organization
• Never characterized, ‘2nd’ yeast model
• S. pombe is highly divergent from S. cerevisiae: Some processes uniquely conserved
• Similarities: general rule within Eukaryotes
• Differences: novel insights
• 3 initial approaches:– Purifications
– Mutants (KO)
– Transcriptome and phenotypic analyses
Studying SAGA in S. pombe
The S. pombe SAGA complex 1/3
Its subunit composition is identical
between S. pombe, S. cerevisiae and
mammals.
SAGA purification from S. pombe:
Lane 1: no tag
Lane 2: ada1-TAP
1 2
The S. pombe SAGA complex 2/3
Growth phenotypes of viable SAGA deletion mutants
tra1D
WT
spt3D
spt8D
gcn5D
ada2D
ada3D
sgf29D
WT
spt7D
ada1D
spt20D
sgf73D
ubp8D
sgf11D
sus1D
The S. pombe SAGA complex 3/3
Transcriptome
of SAGA
KO mutants
ada1D
spt20D
sgf73D
ubp8D
sgf11D
sus1D
spt8D
gcn5D
ada2D
ada3D
sgf29D
spt7D
tra1D
Transcriptome profile of gcn5D
The transcriptome of gcn5D mutants = that of cells undergoing differentiation (nutrient starved).
p = 3.6 x 10-37
Regulators of the switch proliferation vs. differentiation
LOW
NUTRIENTS
HIGH
NUTRIENTS
DIFFERENTIATION,
QUIESCENCE
CELL GROWTH,
PROLIFERATION
AMPK
Protein
Synthesis
TORC2
(Rictor)
p38
MAPK
TORC1
(Raptor)
Ste11
cAMP
PKA
Bähler, Hiraoka, Hoffmann, Jones, Kawamukai , Moreno, Murakami, Nielsen, Okayama, Petersen, Russell, Toda, Uritani, Weisman, Yamamoto, Yanagida labs
De-repression of Ste11 target genes
Ste11 targets
gcn5D
gcn5Dste11D
Expression of differentiation genes in gcn5D mutants
Differentiation phenotype
h90 gcn5Dh90 gcn5+
DAPI+
DIC
% mating 0% 24%
Gcn5 HAT activity is required of the repression of
differentiation in high nutrient conditions
Time upon starvation (hrs)
genotype 0 4 8 24
wild-type 0 4.6 18 37
gcn5D 16 23 28 59
spt8D 0 0 0 0
ste11D 0 0 0 0
Differentiation phenotype of other SAGA mutants
Opposing regulatory roles of SAGA subunits
in differentiation
Expression of differentiation genes in spt8D mutants
Spt8 is required for the induction of expression of
differentiation genes upon nutrient starvation
richstarved
Subunit composition depending on nutrient availability
SAGA subunit composition is
identical between rich and
starved conditions.
Lane 1: no tag
Lane 2: ada1-TAP
Lane 3: ada1-TAP
Lane 4: no tag
Rich
Starved
1 2 3 4
Are these roles direct?
Chromatin ImmunoPrecipitation (ChIP): is SAGA bound to promoters of differentiation genes?
Are these roles direct?
SAGA binds to promoters of differentiation genes, irrespective of nutrient levels
35
30
25
20
15
10
5
richstarved
ste11+ mei2+
rela
tive
Gcn
5 le
vels
SAGA recruitment
SAGA is recruited by a TF to promoters of differentiation genes, irrespective of nutrients
35
30
25
20
15
10
5
richstarved
ste11+ mei2+
rela
tive
Gcn
5 le
vels
Thus, so far:
With nutrients
Withoutnutrients
SAGA
ste11+
Gcn5
Spt8
SAGA
ste11+
Gcn5
Spt8
WHAT’S GOING ON?
Rst2 Rst2
Which factors act directly?Epistasis analysis (double mutants)
+ nutriments: spt8D suppress gcn5D- nutriments: gcn5D suppress spt8D
Working model
Vegetative growth
SAGA
Gcn5
Spt8
ste11+
Act.
Sexual differentiation
ste11+
SAGA
Gcn5Spt8
Act.
Nutritional
starvation
Conclusions so far
• SAGA directly regulates the expression of
differentiation genes and is a crucial factor for cells to
decide to proliferate or differentiate.
• SAGA switches from a repressor to an activator,
depending on nutrients.
• Open questions:1. Gcn5 repression? Not through histone acetylation!
2. How does SAGA sense nutrient availability to switch from a repressor to an activator of transcription?
Conclusions so far
• SAGA directly regulates the expression of
differentiation genes and is a crucial factor for cells to
decide to proliferate or differentiate.
• SAGA switches from a repressor to an activator,
depending on nutrients.
• Open questions:1. Gcn5 repression? Not through histone acetylation!
2. How does SAGA sense nutrient availability to switch from a repressor to an activator of transcription?
Regulators of the switch proliferation vs. differentiation
LOW
NUTRIENTS
HIGH
NUTRIENTS
DIFFERENTIATION,
QUIESCENCE
CELL GROWTH,
PROLIFERATION
AMPK
Protein
Synthesis
TORC2
(Rictor)
p38
MAPK
TORC1
(Raptor)
cAMP
PKA
?SAGA
Tra1/
TRRAP
Spt7
Spt8
Gcn5
Ste11
?
Direct regulation of the SAGA co-activator
by nutrient availability?
Regulation of SAGA by nutrients
Combine genetic and biochemical approaches:
1. Which nutrient-sensing signaling pathway controls
SAGA?
2. Which SAGA subunit is targeted and how does it
controls its activities?
3. What is the mechanism of this functional switch?
Tsc1
Tsc2TORC1
High
nutrientsDifferentiationRhb1AKT
Gcn5 functions downstream of TORC1
to repress differentiation
Dif
fere
nti
ati
on
ge
ne
ex
pre
ss
ion
High
nutrients
Starved
1.00.5
15.6
36.3
91.7
2.8
147.5
338.8
0.1
1
10
100
1000
WT tsc2Dtsc2Dgcn5Dgcn5D
Gcn5 functions downstream of TORC1
to repress differentiation
Tsc1
Tsc2TORC1
High
nutrientsDifferentiationRhb1AKT
Starvation
High
nutrients
WT tsc2Dtsc2Dgcn5D
gcn5D
Functional interactions between
TORC1, TORC2, and Gcn5
STARVED DIFFERENTIATION,
QUIESCENCE
CELL GROWTH,
PROLIFERATION
Protein
Synthesis
TORC2
(Rictor)
TORC1
(Raptor)
differentiation genesAKT
HIGH
NUTRIENTS
?
?ste11+, mei2+, …
SAGA
Tra1/
TRRAP
Spt7
Spt8
Gcn5
Mechanism?
STARVED DIFFERENTIATION,
QUIESCENCE
CELL GROWTH,
PROLIFERATION
Protein
Synthesis
TORC2
(Rictor)
TORC1
(Raptor)
differentiation genesAKT
HIGH
NUTRIENTS
?
?ste11+, mei2+, …
SAGA
Tra1/
TRRAP
Spt7
Spt8
Gcn5
Functional interactions between
TORC1, TORC2, and Gcn5
Increased phosphorylation of SAGA
in response to nutrient starvation
High Starved High Starved
Differential phosphorylation of the SAGA component Taf12,
in response to nutrient levels
Starved / High:
Differential phosphorylation of the SAGA component Taf12,
in response to nutrient levels
Starved
Hig
h
Differential phosphorylation of the SAGA component Taf12,
in response to nutrient levels
Starved
Hig
h
Discrepancy between genetics and biochemistry:
Taf12 is not phosphorylated when TORC1 is active (high nutrients)
SAGANutrients Differentiation??????TORC1
Discrepancy between genetics and biochemistry:
Taf12 is not phosphorylated when TORC1 is active (high nutrients)
Similar to Gcn5, the PP2A-B55 phosphatase represses
differentiation in rich conditions, downstream of TORC1.
Genetic analyses
SAGANutrients DifferentiationPP2A-Pab1TORC1
SAGANutrients Differentiation??????TORC1
PP2A-B55 de-phosphorylates Taf12
in rich, proliferation conditions
R S R S
WT pab1D
Phosphatase
assay
Rich
TORC2-AKT catalyzes Taf12 phosphorylation upon
nutrient starvation / differentiation
Starved
Kinase assay
Conclusions
PP2A
Pab1Gwl
Igo1
Psk1High
nutrients
Starved
Proliferation
Differentiation
Spt8
Gad8TORC2
TORC1
Gcn5
SAGA
diff.
Taf12P
1. SAGA functions downstream of both the TORC1 and TORC2
signaling pathways to regulate differentiation.
2. Taf12 phosphorylation is tightly and rapidly controlled by the
opposing activities of TORC1-PP2A and TORC2-Gad8AKT.
Taf12 phosphorylation inhibits differentiation
upon nutrient starvation
Increased TORC2 activity negatively regulates
differentiation, through Taf12 phosphorylation
Starved
Both loss of Tor1 AND higher Tor1 activity reduces differentiation.
(Halova D et al., J. Cell Biol., 2013)
Genotype Differentiating cells
WT 39 ± 1%
tor1-T1972A 25 ± 2%
taf12-5A 50 ± 4%
tor1-T1972A taf12-5A 55 ± 2%
Conclusions
1. SAGA functions downstream of both the TORC1 and TORC2
signaling pathways to regulate differentiation.
2. Taf12 phosphorylation is tightly and rapidly controlled by the
opposing activities of TORC1-PP2A and TORC2-AKT.
3. Suggest thats TORC2 both activates and inhibits differentiation,
reminiscent of an INCOHERENT feed-forward loop.
TORC2 P-SAGADifferentiation
Starvation
AND
Incoherent feed-forward loop are used widely
Further readings
• ‘Gene expression & differentiation’:– http://www.nature.com/scitable/topicpage/gene-expression-
regulates-cell-differentiation-931– http://www.nature.com/scitable/topicpage/the-complexity-of-gene-
expression-protein-interaction-34575– http://www.nature.com/scitable/topicpage/environmental-influences-
on-gene-expression-536
• Regulation of meiotic commitment by a lncRNA in S. cerevisiae:http://www.sciencedirect.com/science/article/pii/S0092867406013006
• Signaling to chromatin and cell fate decisions in response to nutrients in S. pombe:https://sites.google.com/site/thehelmlingerlab/