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LES MÉCANISMES EPIGENETIQUES DANS LACTUALITE DE LA SCIENCE I-LES ÉCHECS DU CLONAGE -Reprogrammation épigénétique par méthylation de lADN au cours du développement.

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Présentation au sujet: "LES MÉCANISMES EPIGENETIQUES DANS LACTUALITE DE LA SCIENCE I-LES ÉCHECS DU CLONAGE -Reprogrammation épigénétique par méthylation de lADN au cours du développement."— Transcription de la présentation:

1 LES MÉCANISMES EPIGENETIQUES DANS LACTUALITE DE LA SCIENCE I-LES ÉCHECS DU CLONAGE -Reprogrammation épigénétique par méthylation de lADN 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 lX et imprinting *Définitions, propriétés °Expressions géniques monoalléliques Inactivation dun des deux chromosomes X chez la femelle: -au hasard dans les cellules somatiques -dictée par lorigine parentale (X paternel inactivé) dans des cellules des annexes embryonnaires Imprinting ou empreinte parentale: dictée par lorigine parentale, paternelle ou maternelle, dans les cellules somatiques °Repose sur la formation (initiation+propagation) dhétérochromatine facultative °Inactivation stable et héritable

2 *Un point sur linactivation de lX (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 dexpression 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 dorigine paternelle est inactivé de façon préférentielle A partir des années1990s, il existe un centre dinactivation 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.

3 Xist recrute des partenaires le maintien de linactivation implique des Dnmt ActuellementComment? *Un point sur limprinting Découvert dans les années 1980s chez la souris. Actuellement on connaît plus de 50 gènes soumis à lempreinte parentale. Ce sont les mêmes chez la souris et lhomme (sauf Igf2r) H19/Igf2>100kb BWS>200kb PWS/AS : Prader-Willi&Angelman Syndrome complexBWS : Beckwith Wiedemann Syndrome PWS/AS *Propriétés moléculaires communes à lXCI et limprinting °Organisation des gènes inactivés en cluster °Abondance dARNs anti-sens ou non codant °Des cis-acting switches: des centres de contrôle agissant en cis ICR: imprinting control region

4 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

5 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 dhistones (macro H2A) variations du code histone allèles spécifiques recrutement différentiel de protéines non-histones comme Eed et Ezh2 *transmission dune 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.

6 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. Une reprogrammation épigénétique aléatoire Large-offspring syndrome (LOS) ressemble au beckwith-Wiedeman syndrome 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

7 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. Lavenir du clonage reproductif sans prendre en considération les questions déthique Le choix des cellules qui fournissent le noyau Lanalyse des embryons avant réimplantation (profiling de lexpression génique) Lavenir du clonage thérapeutique Estimer le potentiel de ces cellules à devenir tumorale après réimplantation, à cause des défauts de programmation épigénétique II-MECANISMES EPIGENETIQUES ET CANCER instabilité des chromosomes activations doncogenes silencing de gènes suppresseurs de tumeur inactivation des systèmes de réparation de lADN Propriétés des cellules cancéreuses Causes: génétiques ou épigénétiques

8 Au cours de la cancérogenèse: -une importante hypométhylation générale épigénétique: activation doncogènes génétique: augmente linstabilité des centromères et donc des chromosomes -des hyperméthylations locales dîlots 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? Lœuf avant la poule? Lhyperméthylation serait plutôt associée à la progression tumorale: variabilité cellulaire et capacité à métastaser Des tentatives dinterventions thérapeutiques par utilisation dinhibiteurs des Dnmt (5-aza-2-deoxycytidine=DAC)

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