PDF - 2.3 Mo - Master Sciences et Technologie

Régulation épigénétique :
modifications de la chromatine
Frédéric Bantignies
08.09.2014
Génétique versus Epigénétique
Génétique
Mutations
Stables,
souvent irréversibles
Epigénétique
Méthylation de lADN
Modifications des histones
Réarrangements structuraux
des Chromosomes
Etats régulatoires
nucléocytoplasmique
Moindre stabilité, processus réversibles ou modulables
Phénomènes adaptatifs ?
Adapté de larticle de F.Kepes dans M/S, avril 2005
Une définition moderne de
l’Epigenetique
(Cold Spring Harbor Laboratory Press, 1996)
« Létude des changements
héréditaires dans lexpression des gènes,
ayant lieu sans modification de la séquence
dADN »
De tels mécanismes pouvant permettre à
des cellules génétiquement identiques
dêtre phénotypiquement distinctes…
Le but actuel de lEpigénétique
Comprendre comment des cellules
peuvent avoir les mêmes gènes (le
même génome), exprimer les mêmes
protéines, mais adopter des états
fonctionnels différents…
Plan du cours
1.  Rappels sur Les bases moléculaires de
lépigénétique
- La chromatine, les histones et leurs modifications posttraductionnelles (PTMs)
- notion de « writer » et « reader »
- « cross-talk » entre PTMs des histones
- influence sur l’activité des gènes
2. Deux exemples de régulations épigénétiques
- Régulation des gènes homéotiques au cours du développement
par les protéines des groupes Polycomb et Trithorax
- La différenciation cellulaire (cellules pluripotentes versus
cellules différenciées) mettant en jeu des mécanismes
dynamiques de régulation chromatinienne
10,000 nm/ 0.01mm
A human
interphasic
nucleus
11 nm
1bp (0.33nm)
One human chromosome of 100 Mb = 33,000,000 nm/33 mm
The Chromatin fiber
30 nm
Chromatin fiber
More compact structure
11 nm
(beads)
Less compact structure
In vitro
30nm
11nm
The nucleosome:
The fundamental unit of the chromatin
Histone
octamer
145 bp
of DNA
+
Nucleosome
An octamer (8 units)
of histones:
4 core histones
H2A, H2B, H3, H4
(X2)
--> The histone octamer organizes 145 bp of DNA in 1 3/4 helical turn of DNA:
48 nm of DNA packaged in a disc of 6 x 11nm
The cristal structure of the nucleosome (1997)
reveals that the N-termini of the histones are highly exposed
Luger, Mader, Richmond, Sargent & Richmond
Nature 389, 251-260 (1997)
H2A
H2B
H3
H4
Modifications post-traductionnelles (PTMs)
des queues histones
et leur fonction dans la régulation génique
Various PTMs
Review from Gardner, Allis and Strahl et al, J.Mol.Biol. 2011
Lysine residues on Histone tails
H2A
H2B
H3
H4
Histone Methylation on
Lysine or Arginine
Lysine
S-adenosylmethyionine
N
C C γ C ε
N+
O C α β C δ C
HMT (Histone
Methyl-Transferase)
Histone
Demethylase
N
ε-N-monomethyl-Lysine
C
C C
O
C
C ε N+
C
C
S-adenosylmethyionine
Histone
Demethylase
HMT (Histone
Methyl-Transferase)
N
ε-N-dimethyl-Lysine
S-adenosylmethyionine
C
C C
O
C
HMT (Histone
Methyl-Transferase)
C
N
ε-N-trimethyl-Lysine
C C
O
C
C ε N+
C
C
C
C ε N+ C
C C
C
Histone
Demethylase
Conséquences moléculaires des PTMs des histones
concept de « writer » et « reader »
Writer:
HAT: Histone acetyl-transferases
HMT: Histone methyl-transferases
Reader:
Specific factors
or macromolecular complexes
Eraser:
HDAC: Histone deacetylases
KDM: Histone demethylases
Review from Gardner, Allis and Strahl et al, J.Mol.Biol. 2011
Mise en place de complexes multiprotéiques
(spécifiques) au niveau de la chromatine
Review from Gardner, Allis and Strahl et al, J.Mol.Biol. 2011
H3-K9me and H3-K27me recognition by the chromodomain
proteins HP1 and Polycomb
Polycomb
HP1
SUV39H1/2
(HMT)
EZH1/2
(HMT)
H3-K9me2/3
H3-K27me3
PRC1 PC
Constitutive Heterochromatin
Polycomb silencing
(Facultative
Heterochromatin)
Histone Marks and Their Writers
UTX, JMJD3
H3-K4me3
H3-K9me3
Activation
Repression
H3-K36me3
H3-K27me3
From Kouzarides, SnapShot, Cell 2007
Histone Marks and Their Readers
Readers identified by SILAC (Stable Isotope Labeling by Amino acids in mammalian Cell culture)
from Vermeulen et al, Cell 2010
Chromatin Immunoprecipitation (ChIP)
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$" Hybridation des échantillons sur des puces: ChIP-on-chip
ou Séquençage Haut-débit: ChIP-seq
High-resolution profiling of Histone Methylations in the Human Genome
Active versus Inactive genes
Data from Barski et al, Cell 2007
ChIP-Seq profiles of three histone modifications and readers
across the Active Eif3B gene on human chromosome 7
H3K36me3
N-PAC
H3K27me3
H3K4me3
SGF29
TRRAP
BAP18
GATAD1
PHF8
EIF3B
Data from Vermeulen et al, Cell 2010
Targeting of the LSD1 H3K4 demethylase (eraser)
to specific genomic regulatory sequences (enhancers)
TALEN fusion protein
ChIP-seq tracks at the SCL locus
(gene expressed in K562 cells)
Data from Mendenhall et al, Nature Biotech 2013
Targeting of the LSD1 H3K4 demethylase (eraser)
to specific genomic regulatory sequences
affect gene expression
Fig.3: TALE-LSD1 fusions to endogenous enhancers affect proximal gene expression
(analyzed by RNA-seq)
Data from Mendenhall et al, Nature Biotech 2013
Plan du cours
1.  Rappels sur Les bases moléculaires de
lépigénétique
- La chromatine, les histones et leurs modifications posttraductionnelles (PTMs)
- notion de « writer » et « reader »
- « cross-talk » entre PTMs des histones
- influence sur l’activité des gènes
2. Deux exemples de régulations épigénétiques
- Régulation des gènes homéotiques au cours du développement
par les protéines des groupes Polycomb et Trithorax
- La différenciation cellulaire (cellules pluripotentes versus
cellules différenciées) mettant en jeu des mécanismes
dynamiques de régulation chromatinienne
Repression by the Polycomb-group (PcG) proteins
An example of cooperation between histone marks
PRC2
Nurf55
E(z)
Esc
Su(z)12
Gene
silencing
PRE
Ph
Psc
Pc
dRing
PRC1
H3K27me3
H2AK119ub
PRC2: Polycomb repressive complex 2
PRC1: Polycomb repressive complex 1
From Bantignies and Cavalli, Trends in Genetics 2011
Cross-talk between H3K27 and H2AK119
From Whitcomb et al, J Biol Chem 2012
Fig.2: Western blot analysis of H3K27me3 in HMTase reactions with PRC2
From Kalb et al, nsmb 2014
Cooperation between histone marks for the recruitment
of PcG complexes on chromatin
From Kalb et al, nsmb 2014
Histone modifications by the PcG proteins lead to
chromatin compaction and Transcriptional repression
PRC1
PRC2
H3K27me3
H2AK119ub
PRC1
In vitro
Control
PRC1
Illustration from Bantignies and Cavalli, Trends in Genetics 2011
Data from Francis et al, Science 2004
Activation by the Trithorax-group (TrxG) proteins
Another example of cooperation between histone marks
TAC1 complexe
Gene
activation
3
3
3
H3K4me3 also allow the recruitment of ISWI-dependant
chromatin remodeling complexes leading to chromatin opening
Histone modifications by TrxG proteins lead to
chromatin opening and Transcriptional activation
Opposing functions of Polycomb group (PcG) and trithorax group (trxG) complexes on chromatin
Histone Acetylation
and Methylation
(TAC1 and ASH1
complexes)
PRE
Nucleosome remodeling
H3K27Ac (BRM complex)
H4-Ac
H3K4me3
H3K36me3
ON
trxG
Maintenance of
active states
(open chromatin)
H3K4me3
H3K36me3
H3K27Ac
H4-Ac
Target
gene
Histone Methylation
and Ubiquitylation
(PRC2 and PRC1
complexes)
OFF
H3K27me3
PcG
Maintenance of
repressed states
(compact chromatin)
H2AK119ub
Chromatin
compaction
H3K27me3
H2AK119ub
The developmental profiles of H3K27me3 and H3K27ac are
dynamic and complementary
Fig 3: Complementary changes in H3K27ac and H3K27me3 during embryogenesis
From Tie et al, Development 2009
Antagonization between histone marks at the H3K27 residue
S2 Drosophila cells
(0.15, 0.3 and 0.45 million cells)
Fig 4: Knockdown of E(Z) leads to reciprocal changes in H3K27me3 and H3K27ac
From Tie et al, Development 2009
Precise Regulation at the H3K27 residue
From Tie et al, Development 2009
Sg4 cells – Abd-B active
BG3 cells – Abd-B repressed
H3K27me3
Pc
Trx-C
Trx-N
Ash1
H3K27ac
PolII
H3K4me3
Figure 1 from
Abd-B
Schwartz et al,
PlosGenetics 2010
Régulation des gènes homéotiques (Hox)
chez la drosophile
Développement
A
P
Détermination du
Profil dexpression
des gènes Hox au cours
de lembryogénèse
Maintien du
Profil dexpression des
gènes Hox
Protéines du groupe
Polycomb
Protéines du groupe
trithorax
PcG
trxG
Maintien de l’état réprimé
Maintien de l’état activé
Maintien/Mémoire Epigénétique à
travers les divisions cellulaires
PcG
TrxG
Maintenance of
repressed states
(compact chromatin)
Maintenance of
active states
(open chromatin)
Illustration of the epigenetic memory by the PcG proteins
for the regulation of the Hox Ubx gene in Drosophila
Larval Discs
Construction transgénique
Early embryo
Late embryo
head
wing
haltere
P
A
“Polycomb Response Element”
Legend: The correct expression pattern is created in the embryo and epigenetically maintained
throughout development when all three elements are combined: the embryonic enhancer sets the
pattern, the PRE maintains the repressed or non-repressed state and the imaginal disc now remains
active only posterior to parasegment 6, not in the head or wings.
Illustration from a Review of Schwartz and Pirrotta
Use of the UAS-Gal4 system in Drosophila
to demonstrate the epigenetic memory by the PcG/TrxG system
Illustration from a Review of Steffen and Ringrose, nrm 2014
Hérédité Epigénétique des états chromatiniens réprimés et
activés à travers le développement
hsp70
PRE
Fab-7
Gal4
HS
UASG lacZ
GAL4
white
3,6 kb
Maintenance
of the
repressed state
by
lacZ repressed
white repressed
Maintenance
of the
active state
by
+HS
lacZ active
Data from Cavalli and Paro, Cell 1998
PcG
proteins
white active
TrxG
proteins
Régulation des gènes Hox
chez les mammifères
Relative H3K27me3
4
27
4
27
From a Review of Lyons and Lomvardas, BBA 2014
Importance de la régulation épigénétique au
cours de la différenciation des cellules
(ES cells)
Cellules
pluripotentes
Cellules
progénitrices
Cellules différenciées
ES
(Pluripotent cells)
Progenitor cells
Terminally
differentiated cells
Housekeeping
genes
Pluripotency
genes
Early
differentiation
genes
Late
differentiation
genes
Chromatine Condensée
Chromatine Ouverte
ES cells
vs.
NPCs
(Neural progenitor cells)
vs.
MEFs
(Embryonic fibroblasts)
« b v l n d m i »:
Poises the gene in a permissive active or
repressed state able to be resolved
during the next steps of differentiation
H3K4me3: the gene is active
H3K27me3: the gene is silenced
Data (ChIP-seq) from Mikkelsen et al, Nature 2007
ES cells
vs.
NPCs
(Neural progenitor cells)
vs.
MEFs
(Embryonic fibroblasts)
bv l n d m i
bv l n d m i
H3K27me3
Data (ChIP-seq) from Mikkelsen et al, Nature 2007
ES cells
Pluripotency
genes
Early
differentiation
genes
Polycomb Group
target genes
Late
differentiation
genes
Polycomb Group
target genes
ON
K4me3
K4me3
OFF
Neuronal
Progenitors
OFF
ON
(cell lineage
specific)
DNA
met
OFF
DNA
met
m
K4me3
OFF
DNA
met
K27me3
K
bv l n d m i s
OFF
K27me3
(unrelated
cell lineage)
K27me3
D
DNA
met
K4me3
K27me3
OFF
Terminal differentiated
cells (neurons)
OFF
OFF
DNA
met
ON
(cell fate
specific)
K4me3
OFF
(unrelated
cell fate)
K27me3
K27me3
K
bv l n d m i s
Reviewed in Roure and Bantignies, Epigenomics 2009: Illustration of the results of Mohn et al, Mol.Cell 2008
Pour en savoir plus…
http://www.igh.cnrs.fr/equip/cavalli
http://www.epigenesys.eu/
http://epigenmed.fr
Et quelques revues récentes:
- Roure and Bantignies, Epigenomics, vol.1(2): 301-318, 2009
- Margueron and Reinberg, Nature, vol.469: 343-9, 2011
- Beisel and Paro, Nat Rev Genet, vol.12(2): 123-35, 2011
- Fisher and Fisher, Current Opinion in Genetics & Development, vol.21:1-7, 2011
- Bantignies and Cavalli, Trends in Genetics, 2011
- Lyons and Lomvardas, Biochimica et Biophysica Acta, 2014
- Steffen and Ringrose, Nature Reviews MolCellBio, 2014
[email protected]