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 low-level 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

°Des facteurs en trans communs: les CTCF CTCF: CCCTC-binding factor 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/MB11-85,52,13/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

°Des différences de composition de la chromatine, allèles spécifiques 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.

Une reprogrammation épigénétique aléatoire 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 thérapeutique 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

Épigénétiques altérations ou cancer en premier? 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)