Présentation lors du séminaire de lancement du 9 mai 2012

Transcription

MINISTÈRE DE L’ ÉCOLOGIE
DU DÉVELOPPEMENT
DURABLE, DU TRANSPORT
ET DE LA MER
Séminaire de présentation des projets de l’APR 2010
MethylED: Impact of Endocrine Disruptors on genomic
DNA methylation patterns in the germline
Michael WEBER,CNRS UMR7242 Biotechnologie et Signalisation
Cellulaire, Strasbourg
Jesus del Mazo, Centro de Investigaciones Biológicas, Madrid
PNRPE- Séminaire de présentation des projets de l’APR 2010, 9 mai 2012
Introduction: DNA methylation in mammals
DNA+Histones=Chromatin
Long-lasting
cellular
memory
Genome
Epi-genome
ON
ON
OFF
OFF
Zygote
Stem cells
Differentiated cells
Heritable epigenetic marks create a memory of gene expression states
NH2
Cytosine
N
C
O
N
Methylation
(DNMT)
NH2
CH3
N
5mC
O
N
5-Methylcytosine
NH2
Cytosine
N
C
O
N
Methylation
(DNMT)
Passive
demethylation
NH2
CH3
N
5mC
O
N
5-Methylcytosine
NH2
Cytosine
N
C
Passive
demethylation
or DNA repair
O
N
Methylation
(DNMT)
Passive
demethylation
NH2
NH2
OH
CH3
N
N
5hmC
O
N
Oxydation
(TET)
5-Hydroxymethylcytosine
5mC
O
N
5-Methylcytosine
VERTEBRATES
Plants (Arabidopsis thaliana)
Yes
Fungi (Saccharomyces cerevisiae)
(Neurospora crassa)
Porifera (sponge)
Cnidaria (jellyfish)
Nematodes (Caenorhabditis elegans)
Insects (Drosophila melanogaster)
(Apis mellifera)
Molluscs
Echinoderms (Sea urchin)
Urochordates (Sea squirt)
Cephalochordates (Amphioxus)
Reptiles
Birds
Mammals
Amphibians
Fishes
No
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
YES
YES
YES
YES
YES
Methylation occurs on cytosines of CpG
dinucleotides, which are unevenly
distributed in mammalian genomes.
CpG island
2-5% methylated CpG islands in
normal cells.
TF=Transcription
Factor
TF
MBD
MBD=Methyl
Binding Domain
Protein
a) Essential for mammalian
development
WT
Dnmt1-/-
9.5dpc
Lei H et al,
Development (1996)
WT
Dnmt3a-/-3b-/-
b) Perturbed in many pathologies
Growth and neuro-developmental
syndromes
-ICF syndrome
-Beckwith-Wiedemann syndrome
-Prader-Willi/Angelman’s syndrome
Auto-immune and metabolic diseases
All types of
cancer
9.5dpc
Okano M et al,
Cell (1999)
Diagnostic markers
Therapeutic targets
Objective: Test the impact of Endocrine Disruptors on
Primordial germ cells undergo massive DNA methylation reprogramming and are
therefore susceptible to epigenetic alterations
high
Cytosine
methylation
PGCs
low
2-cell
E0.5
4-cell
8-cell Morula Blastocyst
E3.5
E7.5
E13.5 E15.5
Birth
Adult
Primordial germ cells undergo massive DNA methylation reprogramming and are
therefore susceptible to epigenetic alterations
Changes in DNA methylation in rodents after in utero maternal exposure to
chemicals, diet, alcohol
Ho SM et al., Cancer Res 2006
Dolinoy DC et al., PNAS 2007
Zama AM et al., Endocrinology 2009
Bromer JG et al., Endocrinology 2009
Waterland R et al., Mol Cell Biol 2003
Kaminen-Ahola N et al., PLoS Genet 2010
Multigenerational non-mendelian effects suggest possible transmission of altered
DNA methylation through generations
Anway MD et al., Science 2005
Stouder C et al., Reproduction 2010
Cropley JE et al., PNAS 2006
EDs
F0
F1
F2
To evaluate the potential effect of environmental factors on epigenetic states, it is
first necessary to describe the « normal » epigenetic landscape
Our results indicate that a small fraction of the genome escapes methylation
reprogramming during development, which suggests possible transmission of
epimutations through generations Borgel J et al, Nat Genet (2010)
Guibert S et al, Genome Res (2012)
high
Cytosine
methylation
PGCs
low
2-cell
E0.5
4-cell
8-cell Morula Blastocyst
E3.5
E7.5
E13.5 E15.5
Birth
Adult
Methodologies
CD1 mice (outbread)
mating
E13.5
birth
F1-F3
EDs
(2 doses)
MEHP
ZEA
VINC
Embryo
Adult
Controls
Male PGCs
(F1-F3)
Spermatogonia
(F1-F3)
Liver +Testis
+Spermatozoa
(F1-F3)
Gene/miRNA expression
DNA methylome
Methodologies
RRBS (Reduced Representation Bisulfite Sequencing)
Sodium Bisulfite
Illumina
Hi-Seq 2000
Quantitative
methylation information
for 1,200,000 CpGs
Expected results and deliverables
Tasks
Deliverables
months
6
Task 1 Isolation of E13.5 PGCs from mice of the F1-F3
generations.
Task 1
Isolation of spermatogonia, testis and spermatozoa from
mice of the F1-F3 generations.
Sample collection from EDs/control mice
JdM
Task 2
Task 3
DNA methylation
biomarkers in F1
mRNA/miRNA
expression in F1
JdM
MW
Task 4
Transmission of DNA methylation
biomarkers in F2/3
Isolation of somatic cells (liver) from mice of the F1-F3
generations.
Task 2 RRBS/MeDIP-Seq profiles in spermatozoa from control
and exposed F1 mice
Comparative analysis of altered DNA methylation in
relation to ED compound and dosage
Validation of DNA methylation biomarkers in PGCs,
spermatogonia, spermatozoa and somatic cells
Task 3 Transcriptome expression profiles in PGCs,
spermatogonia, and testis from control and exposed F1
mice
miRNA expression profiles in PGCs, spermatogonia,
spermatozoa and testis from control and exposed F1
mice
Selection and validation of gene expression biomarkers
MW
Comparative analysis between DNA methylation and
gene expression abnormalities
Task 4 Analysis of DNA methylation biomarkers in germ cells
and somatic cells of unexposed F2/F3 individuals
Analysis of altered mRNA/miRNA expression at
candidate biomarkers in unexposed F2/F3 individuals
Understand epigenetic mechanisms of action of EDs
Relate epigenetic effects to doses and compound ID
Identify novel DNA methylation biomarkers related to ED exposure
12
18
24
30
36
Acknowledgements
UMR 7242 “Biotechnology and Cell Signalling”
CNRS/University of Strasbourg
Julie Borgel
Sylvain Guibert
Centro de Investigationes Biológicas,
Madrid, Spain
Jesus del MAZO
Ghislain Auclair
Elouan Cleroux
Ambre Bender

Présentation lors du séminaire de lancement du 9 mai 2012

Transcription

Documents pareils

Appel à candidatures « Jeunes Chercheurs » 2016 – Session

Liste des réductions d`ancienneté pour les assistants

DÉVELOPPEMENT ÉCONOMIQUE