Transcript Diapositive 1 - univ

LES MÉCANISMES EPIGENETIQUES DANS
L’ACTUALITE DE LA SCIENCE
I-LES ÉCHECS DU CLONAGE
-Reprogrammation épigénétique par méthylation de
l’ADN au cours du développement normal
Reprogramming in normal development. The genome of primordial
germ cells is hypomethylated ("reset," white boxes). Reprogramming
and establishment of parent-specific epigenetic marks occur over the
course of gametogenesis so that the genome of sperm and egg is
competent to express the genes that need to be activated in early
embryonic (red hatched box) and later (green hatched box)
development. During cleavage and early postimplantation
development, "embryonic" genes, such as Oct 3/4, become activated
(solid red box) and are repressed at later stages (black boxes) when
tissue-specific genes (green boxes) are activated in adult tissues
(labeled A, B, and C).
-Inactivation de l’X et imprinting
*Définitions, propriétés
°Expressions géniques monoalléliques
Inactivation d’un des deux chromosomes X chez la femelle:
-au hasard dans les cellules somatiques
-dictée par l’origine parentale (X paternel inactivé) dans des
cellules des annexes embryonnaires
Imprinting ou empreinte parentale:
dictée par l’origine parentale, paternelle ou maternelle, dans les
cellules somatiques
°Repose sur la formation (initiation+propagation)
d’hétérochromatine facultative
°Inactivation stable et héritable
*Un point sur l’inactivation de l’X
(X chromosome inactivation XCI)
Mary Lyon, 1961:  un des deux chromosomes X doit être
inactivé au hasard dans chaque cellule somatique
 ce mécanisme doit contribuer à rendre
égaux les niveaux d’expression des gènes du X chez le male et la
femelle
Dans les années 1970s, chez les marsupiaux et dans certains
tissus des annexes embryonnaires murines le X d’origine
paternelle est inactivé de façon préférentielle
A partir des années1990s, il existe un centre d’inactivation du X
 le silencing implique Xist
 Xist est régulé par un « gène
antisens » Tsix
Xist and Tsix expression during early female mouse development. At the single-cell stage of female mouse
embryogenesis (a), Xist expression is undetectable by RT-PCR or FISH. Xist expression commences at the 2-cell
stage at the onset of zygotic transcription. By FISH, cleavage stage embryos (b) exhibit differential biallelic Xist
expression starting at the 2-cell stage, with Xist RNA appearing to coat the Xp at least partially, and a weak Xist
pinpoint signal at the Xm. The late blastocyst (c) consists of the differentiated extraembryonic lineages, the
trophectoderm (d) and primary endoderm (e), and the pluripotent embryonic lineage precursor, the epiblast (g). The
trophectoderm and primary endoderm have undergone imprinted X-inactivation by the mid and late blastocyst stages,
respectively. The Xp (which is now the Xi) has become fully coated by Xist RNA. After the early embryo implants into
the uterus, the extraembryonic tissues derived from the trophectoderm and primary endoderm (f) shut off low-level Xist
expression on the Xm and continue to exhibit Xist RNA coating of the Xp throughout subsequent cell divisions. At the
late blastocyst stage, the cells of the epiblast have reversed the partial Xist RNA coating of the Xp and now exhibit lowlevel Xist RNA pinpoint signals from both the Xm and the Xp. Between implantation and completion of gastrulation,
epiblast cells differentiate into the embryonic germ layers and undergo random X-inactivation. During this time period,
Xist transcription once again displays differential biallelic expression in the embryonic derivatives (h). After completion
of gastrulation, embryonic cells cease to express the Xist pinpoint signal from the Xa. Tsix antisense RNA is
coexpressed whenever low-level pinpoint Xist expression is found, and persists from the Xa for a limited period of time
after Xist RNA shutoff.
 Xist recrute des partenaires
 le maintien de l’inactivation
implique des Dnmt
Actuellement
Comment?
*Un point sur l’imprinting
Découvert dans les années 1980s chez la souris. Actuellement on
connaît plus de 50 gènes soumis à l’empreinte parentale. Ce
sont les mêmes chez la souris et l’homme (sauf Igf2r)
PWS/AS
BWS>200kb
H19/Igf2>100kb
PWS/AS : Prader-Willi&Angelman Syndrome complex
BWS : Beckwith Wiedemann Syndrome
*Propriétés moléculaires communes à l’XCI
et l’imprinting
°Organisation des gènes inactivés en cluster
°Abondance d’ARNs anti-sens ou non codant
°Des cis-acting switches: des centres de contrôle agissant
en cis
ICR: imprinting control region
Similarities among imprinted gene clusters in
mice.Within each cluster, some genes are
omitted for simplicity. ICR, imprinting
control center. Elements shown are not
drawn to scale. The Snurf1/Snrpn/MB1185,52,13/Lpw/Ube3a-as transcript begins at
the PWS ICR, is multicistronic, contains the
antisense of Ube3a, and may continue
beyond Ube3a. Atp10c has so far only been
described in human
°Des îlots CpG méthylés de façon différentielle sur chacun
des homologues
°Des facteurs en trans communs: les CTCF
CTCF: CCCTC-binding factor
Mouse XCI and autosomal imprinting share
CTCF binding sites at the imprinting
center.At the H19/Igf2 locus(left), CTCF
binding to the imprinting control center
(ICR) serves two purposes: chromatin
insulation of shared enhancers against Igf2
and transcriptional activation of H19. At the
Xic (right), the role of CTCF has not yet
been defined. Its position at the 5′ end of
Tsix and the absence (so far) of shared
enhancers for Xist and Tsix make the
transcriptional activation model more
attractive than the chromatin insulation
model.
°Des différences de composition de la chromatine, allèles
spécifiques
utilisation différentielle des variants d’histones (macro H2A)
variations du code histone allèles spécifiques
recrutement différentiel de protéines non-histones comme Eed
et Ezh2
*transmission d’une empreinte parentale
Genomic imprinting during development. The
gamete imprint is erased on both parental alleles
during germ cell development. It is important to
mention that the erasure is normally inherited
from the previous generation and that this is one
of the less clearly understood processes in
genomic imprinting. Then comes the
establishment of new imprinting, usually by the
repression of imprinted genes by allele-specific
DNA methylation. Later, there is the maintenance
of imprinting with mono-allelic expression that
can be propagated to the following generations,
accompanied by cyclic events of erasure,
maintenance, and establishment. Thus, genomic
imprinting can be lost during development,
resulting in abnormal bi-allelic expression. (m)
maternal allele; (p) paternal allele.
Overview of the somatic cell
nuclear-transfer procedure. a
| Chromosomal material is
removed from oocytes in which
metaphase has been arrested. b |
The nucleus from a donor cell that
has been arrested in the G0
PHASE of the cell cycle is
transferred to the ENUCLEATED
oocyte. c | The reconstructed egg
is artificially activated and
development begins. d | The egg
develops to the BLASTOCYST
stage in vitro or in a temporary
recipient. e | The blastocyst is
implanted into the final recipient.
f | The clone, which is genetically
identical to the donor animal, is
born to the recipient
Une reprogrammation épigénétique aléatoire
Reprogramming of a somatic nucleus after nuclear
transfer (NT) may result in (i) no activation of
"embryonic" genes and early lethality, (ii) faulty
activation of embryonic genes and an abnormal
phenotype, or (iii) faithful activation of "embryonic" and
"adult" genes and normal development of the clone.
The latter outcome is the exception, and the
percentage in each category is estimated from data on
cumulus cell NT animals.
Large-offspring syndrome (LOS)
ressemble au beckwith-Wiedeman
syndrome
L’avenir du clonage reproductif sans prendre en considération les
questions d’éthique
Le choix des cellules qui fournissent le noyau
L’analyse des embryons avant réimplantation (profiling de l’expression génique)
L’avenir du clonage thérapeutique
Estimer le potentiel de ces cellules à devenir tumorale après réimplantation, à cause
des défauts de programmation épigénétique
Reproductive versus non-reproductive
cloning
In non-reproductive (therapeutic) cloning,
following somatic cell nuclear transfer
(SCNT; see also Fig. 1) the cloned embryo
develops only to the blastocyst stage. At
this stage, a cluster of cells — the INNER
CELL MASS (ICM) — is evident at one side
of the developing embryo. The ICM is the
source of pluripotent (totipotent)
embryonic stem (ES) cells. These cells can
then be induced to differentiate into
specific cell types, which can be used to
treat diseases that result from the loss
and/or malfunction of particular cells. For
example, myocardial cells that are grown
from ES cells could be used in the
treatment of heart muscle disease.
Crucially, as the nuclear material is derived
from the patient, the transplanted cells will
be immunologically compatible when
transferred back to the patient.
By contrast, in reproductive cloning, after
going through the MORULA stage and
reaching the blastocyst stage, the cloned
embryo is implanted into the uterus and
allowed to develop further. The eventual
aim is for the clone to develop to term and
be born in the same way as non-cloned
offspring.
II-MECANISMES EPIGENETIQUES ET CANCER
Propriétés des cellules cancéreuses
instabilité des chromosomes
activations d’oncogenes
silencing de gènes suppresseurs de tumeur
inactivation des systèmes de réparation de l’ADN
Causes: génétiques ou épigénétiques
Au cours de la cancérogenèse:
-une importante hypométhylation générale
épigénétique: activation d’oncogènes
génétique: augmente l’instabilité des centromères et
donc des chromosomes
-des hyperméthylations locales d’îlots CpG
épigénétique: silencing de gènes suppresseur de
tumeur, expression biallélique de facteur de croissance
normalement imprintés
génétique: augmentation du taux de mutations
ponctuelles
Épigénétiques altérations ou cancer en premier?
L’œuf avant la poule?
L’hyperméthylation serait plutôt associée à la progression
tumorale: variabilité cellulaire et capacité à métastaser
Des tentatives d’interventions thérapeutiques par utilisation
d’inhibiteurs des Dnmt (5-aza-2’-deoxycytidine=DAC)