Gene expression regulation during cellular differentiation · 2019-10-16 · Gene expression regulation. Difference prokaryotes / eukaryotes •Prokaryotes: ON is the default state

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


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


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-

vator

Nucleosomes

RNA Pol. II


FactorsCo-activator


FactorsCo-activator

Co-activator


Factors





Classical view

Recent discoveries

exon+1

intron exonTATAUAS

Nucleosomes

Acti-vator

RNA Pol. II


FactorsCo-activator


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

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


S. pombe life cycle

“G0”

Low nutrientsDIFFERENTIATION

M

G1

SG2


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


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


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


Growth phenotypes of viable SAGA deletion mutants

tra1D

WT

spt3D

spt8D

gcn5D

ada2D

ada3D

sgf29D

WT

spt7D

ada1D

spt20D

sgf73D

ubp8D

sgf11D

sus1D


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


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?


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:



Starved

Hig

h



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/

Contact info

Dom Helmlinger

[email protected]

Documents

Gene expression regulation during cellular differentiation · 2019-10-16 · Gene expression regulation. Difference prokaryotes / eukaryotes •Prokaryotes: ON is the default state