RESUMEN
Recent studies, primarily in mouse embryonic stem cells, have highlighted the unique chromatin state of pluripotent stem cells, including the incorporation of histone variants into specific genomic locations, and its role in facilitating faithful expression of genes during development. However, there is little information available on the expression and subcellular localisation of histone variants in human embryonic stem cells (hESCs). In this study, we confirmed the expression of a panel of histone variant genes in several hESC lines and demonstrated the utility of transfection of in vitro transcribed, epitope-tagged mRNAs to characterise the subcellular localisation of these proteins. The subcellular localisations of variant histone H3 (CENP-A, H3.3), H2A (MACROH2A, H2AX, H2AZ, H2ABBD) and H1 (H1A, HB, H1C, H1D) were examined, revealing distinct nuclear localisation profiles for each protein. These data highlight the differences between murine (m) ESCs and hESCs, including the presence of a MACROH2A-enriched inactive X chromosome in undifferentiated XX hESC lines. We also provide the first evidence for MACROH2A accumulation on the Y-chromosome in XY hESCs.
Asunto(s)
Células Madre Embrionarias/fisiología , Histonas , Isoformas de Proteínas , ARN Mensajero , Transfección , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Línea Celular , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/citología , Histonas/genética , Histonas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Transfección/métodos , Inactivación del Cromosoma XRESUMEN
The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric alpha-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I-dependent manner. The nucleolar targeting of CENPC1 and INCENP requires alpha-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following alpha-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds alpha-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and "RNA replenishment" experiments, we show that alpha-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. Our data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis.
Asunto(s)
Nucléolo Celular/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ARN/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Nucléolo Celular/efectos de los fármacos , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Dactinomicina/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Datos de Secuencia Molecular , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción GenéticaRESUMEN
The ability to create fully functional human chromosome vectors represents a potentially exciting gene-delivery system for the correction of human genetic disorders with several advantages over viral delivery systems. However, for the full potential of chromosome-based gene-delivery vectors to be realized, several key obstacles must be overcome. Methods must be developed to insert therapeutic genes reliably and efficiently and to enable the stable transfer of the resulting chromosomal vectors to different therapeutic cell types. Research to achieve these outcomes continues to encounter major challenges; however recent developments have reiterated the potential of chromosome-based vectors for therapeutic gene delivery. Here we review the different strategies under development and discuss the advantages and problems associated with each.