Contrôle épigénétique par l'ARN : de la souris aux cellules ES

par Hossein Ghanbarian

Thèse de doctorat en Biologie cellulaire et moléculaire

Sous la direction de Minoo Rassoulzadegan.

Soutenue en 2010

à Nice .


  • Résumé

    Les régulations épigénétiques jouent un rôle déterminant dans le développement normal et pathologique des mammifères. Des travaux antérieurs dans notre laboratoire ont montré que des fragments d’ARN du gène Kit injecté dans des œufs fécondés de souris peuvent produire des anomalies épigénétiques héréditaires, appelés paramutation. Ceci se traduit, dans le cas présent, par un phénotype spécifique à la mutation du gène kit, phénotype qui se maintient chez les adultes pendant plusieurs générations. Ici, nous illustrons l’importance de ce mécanisme de paramutation dans une pathophysiologie du cœur. En effet, la micro-injection dans des œufs fécondés de fragments d’ARN ciblant le gène Cdk9 ou du microRNA miR-1, deux régulateurs clés dans la croissance cardiaque, conduit à des niveaux élevés d'expression du gène Cdk9. Cela entraîne une hypertrophie cardiaque héritée sur plusieurs générations. L’efficacité de la transmission héréditaire est associée avec la présence du microRNA, miR-1, dans le noyau du spermatozoïde. Ces résultats suggèrent que l'ARN est responsable de la production du phénotype paramuté. L’induction d'états épigénétiques par les microARNs implique des mécanismes moléculaires inconnus. Pour les étudier, nous avons analysé l’effet de l’injection de molécules d’ARN, fragment de Cdk9 et miR-1 dans les cellules souches embryonnaires (ES cellules), un système de culture fiable avec la possibilité d’un va-et-vient entre analyses in vivo et in vitro. Nous avons, alors, observée une augmentation du niveau de transcription du gène Cdk9, modification maintenue pendant plusieurs passages. En outre, les cellules modifiées ont montré un potentiel de différenciation préférentiel en cellules cardiaques, tant in vitro en culture cellulaire que in vivo dans des embryons chimériques. Ces résultats mettent en évidence un nouveau mode de régulation et d’hérédité induit par les molécules ARN. Ceci pourrait servir de modèle pour certaines maladies, dont l’hérédité est certaine mais pour lesquelles il n’a pas été possible d’identifier clairement un déterminant chromosomique à transmission mendélienne. Epigenetic regulation shapes normal and pathological mammalian development and physiology. Previous work in our lab showed that Kit RNAs injected into fertilized mouse eggs can produce heritable epigenetic defects, or paramutations, with relevant loss of function pigmentation phenotypes, which affect adult phenotypes in multiple succeeding generations. Here, we illustrate the relevance of paramutation to pathophysiology by injecting fertilized mouse eggs and electroporating embryonic stem cells with RNAs targeting Cdk9, a key regulator of cardiac growth. Microinjecting fragments of either the coding region or the related microRNA miR-1 led to high levels of expression of homologous RNA, resulting in an epigenetic defect, cardiac hypertrophy. Cardiac hypertrophy was inherited in crosses of either male or female miR–1* parents with normal partners for at least 3 generations. At each generation, about 90 per cent of the progeny had enlarged hearts, with variable ratios to body weights and no obvious correlation with the parental heart size. Such a degree of quantitative individual variation rheostat effect is a known feature of plant paramutation. Efficient hereditary transmission correlated with the presence of miR-1 in the sperm nucleus. These findings suggest that RNA is responsible for the production of the paramutated phenotype. Induction of epigenetic states by microRNAs involves still unknown molecular mechanisms, distinct from their known activity in the control of mRNA stability and translation. To investigate in molecular mechanisms of RNA mediated epigenetic modification, we tested paramutation in embryonic stem cells (ES cells), a reliable culture system with back-and forth possibility for in vivo and cell culture analysis. To attempt this goal, we have electroporated miR-1 into ES cell. Interestingly, an increase in the transcription level of Cdk9 was observed and stably maintained for several passages. In addition, run-on assays performed on permeabilized ES cells confirmed an increase in transcription activity in the miR-1 electroporated ES Cells (designated miR–1*). Moreover, the modified cells showed a preferential differentiation potential into cardiac cells, both in cell culture and in chimaeric embryos. A similar epigenetic deregulation was independently induced at the Sox9 locus by a transcript fragment and by the homologous microRNA miR 124, the known inducers of the ‘giant Sox9*’ mouse paramutation. These results highlight the diversity of RNA-mediated epigenetic effects and may provide a paradigm for clinical cases of familial diseases whose inheritance is not fully explained in Mendelian terms.

  • Titre traduit

    RNA-directed epigenetic controls from mice to ES cells : induction cardiac hypertrophy


  • Résumé

    Epigenetic regulation shapes normal and pathological mammalian development and physiology. Previous work in our lab showed that Kit RNAs injected into fertilized mouse eggs can produce heritable epigenetic defects, or paramutations, with relevant loss of function pigmentation phenotypes, which affect adult phenotypes in multiple succeeding generations. Here, we illustrate the relevance of paramutation to pathophysiology by injecting fertilized mouse eggs and electroporating embryonic stem cells with RNAs targeting Cdk9, a key regulator of cardiac growth. Microinjecting fragments of either the coding region or the related microRNA miR-1 led to high levels of expression of homologous RNA, resulting in an epigenetic defect, cardiac hypertrophy. Cardiac hypertrophy was inherited in crosses of either male or female miR–1* parents with normal partners for at least 3 generations. At each generation, about 90 per cent of the progeny had enlarged hearts, with variable ratios to body weights and no obvious correlation with the parental heart size. Such a degree of quantitative individual variation rheostat effect is a known feature of plant paramutation. Efficient hereditary transmission correlated with the presence of miR-1 in the sperm nucleus. These findings suggest that RNA is responsible for the production of the paramutated phenotype. Induction of epigenetic states by microRNAs involves still unknown molecular mechanisms, distinct from their known activity in the control of mRNA stability and translation. To investigate in molecular mechanisms of RNA mediated epigenetic modification, we tested paramutation in embryonic stem cells (ES cells), a reliable culture system with back-and forth possibility for in vivo and cell culture analysis. To attempt this goal, we have electroporated miR-1 into ES cell. Interestingly, an increase in the transcription level of Cdk9 was observed and stably maintained for several passages. In addition, run-on assays performed on permeabilized ES cells confirmed an increase in transcription activity in the miR-1 electroporated ES Cells (designated miR–1*). Moreover, the modified cells showed a preferential differentiation potential into cardiac cells, both in cell culture and in chimaeric embryos. A similar epigenetic deregulation was independently induced at the Sox9 locus by a transcript fragment and by the homologous microRNA miR 124, the known inducers of the ‘giant Sox9*’ mouse paramutation. These results highlight the diversity of RNA-mediated epigenetic effects and may provide a paradigm for clinical cases of familial diseases whose inheritance is not fully explained in Mendelian terms.

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Informations

  • Détails : 1 vol. (102 f.)
  • Annexes : Bibliogr. f. 86-102. Résumé en anglais

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  • Bibliothèque : Université Nice Sophia Antipolis. Service commun de la documentation. Bibliothèque Sciences.
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  • Cote : 10NICE4013
  • Bibliothèque : Bibliothèque interuniversitaire de santé (Paris). Pôle pharmacie, biologie et cosmétologie.
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  • Cote : MFTH 7910
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