gene expression regulation during cellular differentiation · 2019-10-16 · gene expression...

Gene expression regulation during cellular differentiation

Dom Helmlinger

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

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

exon+1

intron exon

Nucleosomes

2. Sequences (cis-regulatory elements)

exon+1

intron exonTATAUAS

Nucleosomes

3. DNA-binding factors (trans-acting factors)

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!

exon+1

intron exonTATAUAS

Nucleosomes

Acti-vator

3. DNA-binding factors (trans-acting factors)

4. …and many other regulatory proteins!

RNA Pol. II

GeneralTranscription

FactorsCo-activator

Co-activator

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

FactorsCo-activator

FactorsCo-activato

Co-activator

Factors

TranscriptionElongators

TranscriptionTerminators

EXTERNAL SIGNAL

exon+1

intron exonTATAUASActi-

Nucleosomes

RNA Pol. II

FactorsCo-activator

Co-activator

Factors

Classical view

Recent discoveries

exon+1

intron exonTATAUAS

Nucleosomes

Acti-vator

RNA Pol. II

FactorsCo-activator

Co-activator

Factors

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

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

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

Normalized Intensity

(log scale)

MergeDC-GR-JM coloured by Venn Diagram

Rock ‘n’ roll

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

High nutrientsPROLIFERATION

S. pombe life cycle

“G0”

Low nutrientsDIFFERENTIATION

S. pombe life cycle

“G0”

Low nutrientsDIFFERENTIATION

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

NUTRIENTS

DIFFERENTIATION,

QUIESCENCE

CELL GROWTH,

PROLIFERATION

Protein

Synthesis

(Rictor)

(Raptor)

Bähler, Hiraoka, Hoffmann, Jones, Kawamukai , Moreno, Murakami, Nielsen, Okayama, Petersen, Russell, Toda, Uritani, Weisman, Yamamoto, Yanagida labs

NUTRIENTS

DIFFERENTIATION,

QUIESCENCE

CELL GROWTH,

PROLIFERATION

Protein

Synthesis

(Rictor)

(Raptor)

?Bähler, Hiraoka, Hoffmann, Jones, Kawamukai , Moreno, Murakami, Nielsen, Okayama, Petersen, Russell, Toda, Uritani, Weisman, Yamamoto, Yanagida labs

NUTRIENTS

DIFFERENTIATION,

QUIESCENCE

CELL GROWTH,

PROLIFERATION

Protein

Synthesis

(Rictor)

(Raptor)

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

RNA Pol. IIGTFs

Coactivator

TATA +1UAS

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

Growth phenotypes of viable SAGA deletion mutants

sgf29D

spt20D

sgf73D

sgf11D

Transcriptome

of SAGA

KO mutants

spt20D

sgf73D

sgf11D

sgf29D

Transcriptome profile of gcn5D

The transcriptome of gcn5D mutants = that of cells undergoing differentiation (nutrient starved).

p = 3.6 x 10-37

NUTRIENTS

DIFFERENTIATION,

QUIESCENCE

CELL GROWTH,

PROLIFERATION

Protein

Synthesis

(Rictor)

(Raptor)

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

gcn5Dste11D

Expression of differentiation genes in gcn5D mutants

Differentiation phenotype

h90 gcn5Dh90 gcn5+

% 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

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

richstarved

ste11+ mei2+

SAGA recruitment

SAGA is recruited by a TF to promoters of differentiation genes, irrespective of nutrients

richstarved

ste11+ mei2+

Thus, so far:

With nutrients

Withoutnutrients

ste11+

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

ste11+

Sexual differentiation

ste11+

Gcn5Spt8

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?

NUTRIENTS

DIFFERENTIATION,

QUIESCENCE

CELL GROWTH,

PROLIFERATION

Protein

Synthesis

(Rictor)

(Raptor)

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

2. Which SAGA subunit is targeted and how does it

controls its activities?

3. What is the mechanism of this functional switch?

Tsc2TORC1

nutrientsDifferentiationRhb1AKT

Gcn5 functions downstream of TORC1

to repress differentiation

nutrients

Starved

1.00.5

WT tsc2Dtsc2Dgcn5Dgcn5D

Gcn5 functions downstream of TORC1

to repress differentiation

Tsc2TORC1

nutrientsDifferentiationRhb1AKT

Starvation

nutrients

WT tsc2Dtsc2Dgcn5D

Functional interactions between

TORC1, TORC2, and Gcn5

STARVED DIFFERENTIATION,

QUIESCENCE

CELL GROWTH,

PROLIFERATION

Protein

Synthesis

(Rictor)

(Raptor)

differentiation genesAKT

NUTRIENTS

?ste11+, mei2+, …

Mechanism?

STARVED DIFFERENTIATION,

QUIESCENCE

CELL GROWTH,

PROLIFERATION

Protein

Synthesis

(Rictor)

(Raptor)

differentiation genesAKT

NUTRIENTS

?ste11+, mei2+, …

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:

Starved

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

TORC2-AKT catalyzes Taf12 phosphorylation upon

nutrient starvation / differentiation

Starved

Kinase assay

Conclusions

Pab1Gwl

Psk1High

nutrients

Starved

Proliferation

Differentiation

Gad8TORC2

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

Incoherent feed-forward loop are used widely

gene expression regulation during cellular differentiation · 2019-10-16 · gene expression...

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