RESUMO
Argonaute proteins (AGOs), which play an essential role in cytosolic post-transcriptional gene silencing, have been also reported to function in nuclear processes like transcriptional activation or repression, alternative splicing and, chromatin organization. As most of these studies have been conducted in human cancer cell lines, the relevance of AGOs nuclear functions in the context of mouse early embryonic development remains uninvestigated. Here, we examined a possible role of the AGO1 protein on the distribution of constitutive heterochromatin in mouse embryonic stem cells (mESCs). We observed a specific redistribution of the repressive histone mark H3K9me3 and the heterochromatin protein HP1α, away from pericentromeric regions upon Ago1 depletion. Furthermore, we demonstrated that major satellite transcripts are strongly up-regulated in Ago1_KO mESCs and that their levels are partially restored upon AGO1 rescue. We also observed a similar redistribution of H3K9me3 and HP1α in Drosha_KO mESCs, suggesting a role for microRNAs (miRNAs) in the regulation of heterochromatin distribution in mESCs. Finally, we showed that specific miRNAs with complementarity to major satellites can partially regulate the expression of these transcripts.
Assuntos
MicroRNAs , Células-Tronco Embrionárias Murinas , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Homólogo 5 da Proteína Cromobox , Fatores de Iniciação em Eucariotos , Heterocromatina/genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Interferência de RNA , Fatores de Transcrição/genéticaRESUMO
In mouse, although four Argonaute (AGO) proteins with partly overlapping functions in small-RNA pathways exist, only Ago2 deficiency causes embryonic lethality. To investigate the role of AGO2 during mouse early development, we generated Ago2-deficient mouse embryonic stem cells (mESCs) and performed a detailed characterization of their differentiation potential. Ago2 disruption caused a global reduction of microRNAs, which resulted in the misregulation of only a limited number of transcripts. We demonstrated, both in vivo and in vitro, that AGO2 is dispensable for the embryonic germ-layer formation. However, Ago2-deficient mESCs showed a specific defect during conversion into extra-embryonic endoderm cells. We proved that this defect is cell autonomous and can be rescued by both a catalytically active and an inactive Ago2, but not by Ago2 deprived of its RNA binding capacity or by Ago1 overexpression. Overall, our results suggest a role for AGO2 in stem cell differentiation.
Assuntos
Proteínas Argonautas/genética , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias Murinas/citologia , Animais , Linhagem Celular , Endoderma/citologia , Endoderma/crescimento & desenvolvimento , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/citologia , Camadas Germinativas/crescimento & desenvolvimento , Camundongos , MicroRNAs/genéticaRESUMO
Complex gene regulation systems ensure the maintenance of cellular identity during early development in mammals. Eukaryotic small RNAs have emerged as critical players in RNA interference (RNAi) by mediating gene silencing during embryonic stem cell self-renewal. Most of the proteins involved in the biogenesis of small RNAs are essential for proliferation and differentiation into the three germ layers of mouse embryonic stem cells. In the last decade, new functions for some RNAi proteins, independent of their roles in RNAi pathways, have been demonstrated in different biological systems. In parallel, new concepts in stem cell biology have emerged. Here, we review and integrate the current understanding of how RNAi proteins regulate stem cell identity with the new advances in the stem cell field and the recent non-canonical functions of the RNAi proteins. Finally, we propose a reevaluation of all RNAi mutant phenotypes, as non-canonical (small non-coding RNA independent) functions may contribute to the molecular mechanisms governing mouse embryonic stem cells commitment.