Détermination de la structure secondaire d'une région de l'ARN Xist nécessaire à l'inactivation du chromosome X, la région des A-repeats, et identification de ses partenaires protéiques ayant un rôle structural ou fonctionnel dans l'inactivation

Abstract : Silencing of one X chromosome (XCI) in cells of mammalian female ensures sex chromosome dosage compensation between male and female. The 17kb Xist ncRNA plays an essential role in XCI. Its spread along the future inactivated X chromosome is associated with major modifications of the epigenetic status of this chromosome, including histone H3K27 methylations mediated by PRC2 complex. One key part of Xist necessary for XCI initiation is the phylogenetically conserved A region. It lies at the 5' end of the Xist molecule and contains 8 of a 24-nucleotides motif. Female mouse embryos carrying a mutated Xist deleted for the A region are selectively lost during embryogenesis, which underlines the importance of this element. We performed the first experimental analysis of the structure of the entire A region in solution. By the use of chemical and enzymatic probes and FRET experiments, using oligonucleotides carrying fluorescent dyes, we established a 2D structure for the A region that contains two long stem-loop structures each including 4 repeats which interact together two by two. By immunoprecipitation assays and mass spectrometry analysis, we identified the protein partners of the A region. We demonstrated that the A region associate with PRC2 components which is responsible for the apposition of epigenetic modifications of X inactive chromosome. Others proteins which would have a role in A region function were also identified (PTB, KSRP, Sam68, Vigiline, RHA, TIAR, DEK, H1, BRML1, Rod1, Lin28).
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Sylvain Maenner. Détermination de la structure secondaire d'une région de l'ARN Xist nécessaire à l'inactivation du chromosome X, la région des A-repeats, et identification de ses partenaires protéiques ayant un rôle structural ou fonctionnel dans l'inactivation. Médecine humaine et pathologie. Université Henri Poincaré - Nancy 1, 2009. Français. ⟨NNT : 2009NAN10092⟩. ⟨tel-01775836⟩

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