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2.
Nucleic Acids Res ; 41(8): 4447-58, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23444137

RESUMEN

We have previously shown that α-thalassemia mental retardation X-linked (ATRX) and histone H3.3 are key regulators of telomeric chromatin in mouse embryonic stem cells. The function of ATRX and H3.3 in the maintenance of telomere chromatin integrity is further demonstrated by recent studies that show the strong association of ATRX/H3.3 mutations with alternative lengthening of telomeres in telomerase-negative human cancer cells. Here, we demonstrate that ATRX and H3.3 co-localize with the telomeric DNA and associated proteins within the promyelocytic leukemia (PML) bodies in mouse ES cells. The assembly of these telomere-associated PML bodies is most prominent at S phase. RNA interference (RNAi)-mediated knockdown of PML expression induces the disassembly of these nuclear bodies and a telomere dysfunction phenotype in mouse ES cells. Loss of function of PML bodies in mouse ES cells also disrupts binding of ATRX/H3.3 and proper establishment of histone methylation pattern at the telomere. Our study demonstrates that PML bodies act as epigenetic regulators by serving as platforms for the assembly of the telomeric chromatin to ensure a faithful inheritance of epigenetic information at the telomere.


Asunto(s)
Estructuras del Núcleo Celular/metabolismo , Cromatina/metabolismo , Células Madre Embrionarias/metabolismo , Telómero/metabolismo , Animales , Línea Celular Tumoral , Estructuras del Núcleo Celular/química , ADN Helicasas/análisis , Reparación del ADN , Epigénesis Genética , Histonas/análisis , Humanos , Ratones , Células 3T3 NIH , Proteínas Nucleares/análisis , Proteínas Nucleares/fisiología , Fenotipo , Fase S , Proteína Nuclear Ligada al Cromosoma X
3.
Genome Res ; 20(3): 351-60, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20110566

RESUMEN

ATRX (alpha thalassemia/mental retardation syndrome X-linked) belongs to the SWI2/SNF2 family of chromatin remodeling proteins. Besides the ATPase/helicase domain at its C terminus, it contains a PHD-like zinc finger at the N terminus. Mutations in the ATRX gene are associated with X-linked mental retardation (XLMR) often accompanied by alpha thalassemia (ATRX syndrome). Although ATRX has been postulated to be a transcriptional regulator, its precise roles remain undefined. We demonstrate ATRX localization at the telomeres in interphase mouse embryonic stem (ES) cells in synchrony with the incorporation of H3.3 during telomere replication at S phase. Moreover, we found that chromobox homolog 5 (CBX5) (also known as heterochromatin protein 1 alpha, or HP1 alpha) is also present at the telomeres in ES cells. We show by coimmunoprecipitation that this localization is dependent on the association of ATRX with histone H3.3, and that mutating the K4 residue of H3.3 significantly diminishes ATRX and H3.3 interaction. RNAi-knockdown of ATRX induces a telomere-dysfunction phenotype and significantly reduces CBX5 enrichment at the telomeres. These findings suggest a novel function of ATRX, working in conjunction with H3.3 and CBX5, as a key regulator of ES-cell telomere chromatin.


Asunto(s)
ADN Helicasas/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/metabolismo , Telómero/metabolismo , Adenosina Trifosfatasas/química , Animales , Cromatina/metabolismo , Cromatina/fisiología , Ensamble y Desensamble de Cromatina , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , ADN Helicasas/genética , Replicación del ADN/genética , Replicación del ADN/fisiología , Células Madre Embrionarias/metabolismo , Genes , Histonas/genética , Humanos , Discapacidad Intelectual/genética , Interfase/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Ratones , Mutación , Proteínas Nucleares/genética , Proteína Nuclear Ligada al Cromosoma X , Talasemia alfa/genética
4.
Genome Res ; 19(3): 404-14, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19196724

RESUMEN

Little is known about the telomere chromatin dynamics of embryonic stem (ES) cell. Here, we demonstrate localization of histone H3.3 at interphase telomeres and enrichment of Ser31-phosphorylated H3.3 at metaphase telomeres in pluripotent mouse ES cells. Upon differentiation, telomeric H3.3S31P signal decreases, accompanied by increased association of heterochromatin repressive marks and decreased micrococcal nuclease sensitivity at the telomeres. H3.3 is recruited to the telomeres at late S/G2 phase, coinciding with telomere replication and processing. RNAi-depletion of H3.3 induces telomere-dysfunction phenotype, providing evidence for a role of H3.3 in the regulation of telomere chromatin integrity in ES cells. The distinctive changes in H3.3 distribution suggests the existence of a unique and functionally essential telomere chromatin in ES cells that undergoes dynamic differentiation-dependent remodeling during the process of differentiation.


Asunto(s)
Cromatina/fisiología , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Telómero/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Preescolar , Cromatina/metabolismo , Replicación del ADN/genética , Replicación del ADN/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Histonas/fisiología , Humanos , Interfase/genética , Interfase/fisiología , Ratones , Mitosis/genética , Mitosis/fisiología , Células 3T3 NIH , Fosforilación , Protamina Quinasa/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo
5.
Genome Res ; 17(8): 1146-60, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17623812

RESUMEN

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 Genética
6.
J Biol Chem ; 280(5): 3954-62, 2005 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-15557333

RESUMEN

Human neocentromeres are functional centromeres that are devoid of the typical human centromeric alpha-satellite DNA. We have transferred a 60-Mb chromosome 10-derived neocentric marker chromosome, mardel(10), and its truncated 3.5-Mb derivative, NC-MiC1, into mouse embryonic stem cell and have demonstrated a relatively high structural and mitotic stability of the transchromosomes in a heterologous genetic background. We have also produced chimeric mice carrying mardel(10) or NC-MiC1. Both transchromosomes were detected as intact episomal entities in a variety of adult chimeric mouse tissues including hemopoietic stem cells. Genes residing on these transchromosomes were expressed in the different tissues tested. Meiotic transmission of both transchromosomes in the chimeric mice was evident from the detection of DNA from these chromosomes in sperm samples. In particular, germ line transmission of NC-MiC1 was demonstrated in the F1 embryos of the chimeric mice. Variable (low in mardel(10)- or NC-MiC1-containing embryonic stem cells and chimeric mouse tissues and relatively high in NC-MiC1-containing F1 embryos) levels of missegregation of these transchromosomes were detected, suggesting that they are not optimally predisposed to full mitotic regulation in the mouse background, particularly during early embryogenesis. These results provide promising data in support of the potential use of neocentromere-based human marker chromosomes and minichromosomes as a tool for the study of centromere, neocentromere, and chromosome biology and for gene therapy studies in a mouse model system. They also highlight the need to further understand and overcome the factors that are responsible for the definable rates of instability of these transchromosomes in a mouse model.


Asunto(s)
Centrómero , Cromosomas , Ingeniería Genética/métodos , Mitosis/genética , Animales , Línea Celular , Quimera , ADN Satélite , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Híbridas , Ratones , Neomicina , Plásmidos/genética , Inhibidores de la Síntesis de la Proteína , Células Madre/citología
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