RESUMEN
Pluripotency of embryonic stem (ES) cells is controlled by defined transcription factors. During differentiation, mouse ES cells undergo global epigenetic reprogramming, as exemplified by X-chromosome inactivation (XCI) in which one female X chromosome is silenced to achieve gene dosage parity between the sexes. Somatic XCI is regulated by homologous X-chromosome pairing and counting, and by the random choice of future active and inactive X chromosomes. XCI and cell differentiation are tightly coupled, as blocking one process compromises the other and dedifferentiation of somatic cells to induced pluripotent stem cells is accompanied by X chromosome reactivation. Recent evidence suggests coupling of Xist expression to pluripotency factors occurs, but how the two are interconnected remains unknown. Here we show that Oct4 (also known as Pou5f1) lies at the top of the XCI hierarchy, and regulates XCI by triggering X-chromosome pairing and counting. Oct4 directly binds Tsix and Xite, two regulatory noncoding RNA genes of the X-inactivation centre, and also complexes with XCI trans-factors, Ctcf and Yy1 (ref. 17), through protein-protein interactions. Depletion of Oct4 blocks homologous X-chromosome pairing and results in the inactivation of both X chromosomes in female cells. Thus, we have identified the first trans-factor that regulates counting, and ascribed new functions to Oct4 during X-chromosome reprogramming.
Asunto(s)
Emparejamiento Cromosómico , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Represoras/metabolismo , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Cromosoma X/metabolismo , Animales , Factor de Unión a CCCTC , Línea Celular , Femenino , Humanos , Masculino , Ratones , Factor 3 de Transcripción de Unión a Octámeros/deficiencia , Factor 3 de Transcripción de Unión a Octámeros/genética , Unión Proteica , ARN Largo no Codificante , ARN no Traducido/genética , Factores de Transcripción SOXB1 , Activación Transcripcional , Factor de Transcripción YY1/metabolismoRESUMEN
Telomeric regions are known to be transcribed in several organisms. Although originally reported to be transcribed from all chromosomes with enrichment near the inactive X of female cells, we show that telomeric RNAs in fact are enriched on both sex chromosomes of the mouse in a developmentally specific manner. In female stem cells, both active Xs are marked by the RNAs. In male stem cells, both the X and the Y accumulate telomeric RNA. Distribution of telomeric RNAs changes during cell differentiation, after which they associate only with the heterochromatic sex chromosomes of each sex. FISH mapping suggests that accumulated telomeric RNAs localize at the distal telomeric end. Interestingly, telomeric expression changes in cancer and during cellular stress. Furthermore, RNA accumulation increases in Dicer-deficient stem cells, suggesting direct or indirect links to RNAi. We propose that telomeric RNAs are tied to cell differentiation and may be used to mark pluripotency and disease.
Asunto(s)
ARN/genética , Cromosomas Sexuales/genética , Células Madre/metabolismo , Telómero/genética , Animales , Northern Blotting , Línea Celular , Línea Celular Tumoral , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Femenino , Células HeLa , Humanos , Hibridación Fluorescente in Situ/métodos , Masculino , Ratones , Ratones Noqueados , ARN/metabolismo , Ribonucleasa III , Células Madre/citología , Cromosoma X/genética , Inactivación del Cromosoma X , Cromosoma Y/genéticaRESUMEN
With the potential to give rise to all somatic cell types, human embryonic stem cells (hESC) have generated enormous interest as agents of cell replacement therapy. One potential limitation is their safety in vivo. Although several studies have focused on concerns over genomic stability ex vivo, few have analyzed epigenetic stability. Here, we use tools of the epigenetic phenomenon, X-chromosome inactivation (XCI), to investigate their epigenetic properties. Among 11 distinct hESC lines, we find a high degree of variability. We show that, like mouse ESC, hESC in principle have the capacity to recapitulate XCI when induced to differentiate in culture (class I lines). However, this capacity is seen in few hESC isolates. Many hESC lines have already undergone XCI (class II and III). Unexpectedly, there is a tendency to lose XIST RNA expression during culture (class III). Despite losing H3-K27 trimethylation, the inactive X of class III lines remains transcriptionally suppressed, as indicated by Cot-1 RNA exclusion. We conclude that hESC lines are subject to dynamic epigenetic reprogramming ex vivo. Given that XCI and cell differentiation are tightly linked, we consider implications for hESC pluripotency and differentiation potential.
Asunto(s)
Células Madre Embrionarias/metabolismo , Epigénesis Genética , Inactivación del Cromosoma X/genética , Diferenciación Celular , Línea Celular , Núcleo Celular/genética , Células Madre Embrionarias/citología , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Lisina/metabolismo , Metilación , ARN Largo no Codificante , ARN no Traducido/genética , ARN no Traducido/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
X-chromosome inactivation (XCI) ensures the equality of X-chromosome dosages in male and female mammals by silencing one X in the female. To achieve the mutually exclusive designation of active X (Xa) and inactive X (Xi), the process necessitates that two Xs communicate in trans through homologous pairing. Pairing depends on a 15-kb region within the genes Tsix and Xite. Here, we dissect molecular requirements and find that pairing can be recapitulated by 1- to 2-kb subfragments of Tsix or Xite with little sequence similarity. However, a common denominator among them is the presence of the protein Ctcf, a chromatin insulator that we find to be essential for pairing. By contrast, the Ctcf-interacting partner, Yy1 (ref. 8), is not required. Pairing also depends on transcription. Transcriptional inhibition prevents new pair formation but does not perturb existing pairs. The kinetics suggest a pairing half-life of <1 h. We propose that pairing requires Ctcf binding and co-transcriptional activity of Tsix and Xite.