RESUMEN
The cdk inhibitor p57kip2, encoded by the Cdkn1c gene, plays a critical role in mammalian development and in the differentiation of several tissues. Cdkn1c protein levels are carefully regulated via imprinting and other epigenetic mechanisms affecting both the promoter and distant regulatory elements, which restrict its expression to particular developmental phases or specific cell types. Inappropriate activation of these regulatory mechanisms leads to Cdkn1c silencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of Cdkn1c expression requires the binding of the bHLH myogenic factor MyoD to a long-distance regulatory element within the imprinting control region KvDMR1. Interestingly, MyoD binding to KvDMR1 is prevented in myogenic cell types refractory to the induction of Cdkn1c. In the present work, we took advantage of this model system to investigate the epigenetic determinants of the differential interaction of MyoD with KvDMR1. We show that treatment with the DNA demethylating agent 5-azacytidine restores the binding of MyoD to KvDMR1 in cells unresponsive to Cdkn1c induction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and Cdkn1c promoter and, thus, the upregulation of the gene. Analysis of the chromatin status of Cdkn1c promoter and KvDMR1 in unresponsive compared to responsive cell types showed that their differential responsiveness to the MyoD-dependent induction of the gene does not involve just their methylation status but, rather, the differential H3 lysine 9 dimethylation at KvDMR1. Finally, we report that the same histone modification also marks the KvDMR1 region of human cancer cells in which Cdkn1c is silenced. On the basis of these results, we suggest that the epigenetic status of KvDMR1 represents a critical determinant of the cell type-restricted expression of Cdkn1c and, possibly, of its aberrant silencing in some pathological conditions.
Asunto(s)
Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Metilación de ADN/genética , Proteína MioD/genética , Neoplasias/genética , Azacitidina/administración & dosificación , Diferenciación Celular/genética , Línea Celular Tumoral , Cromatina/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Epigénesis Genética/genética , Regulación del Desarrollo de la Expresión Génica , Impresión Genómica/genética , Histona Demetilasas/genética , Humanos , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Proteína MioD/metabolismo , Neoplasias/patología , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Regiones Promotoras GenéticasRESUMEN
Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated in vivo to a c-MYC promoter region recognized as biologically relevant for the transcriptional regulation of the gene. Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation.
Asunto(s)
Cromatina/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , Línea Celular , Cromatina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Poli(ADP-Ribosa) Polimerasa-1 , Procesamiento Proteico-Postraduccional , Activación TranscripcionalRESUMEN
The bHLH transcription factor MyoD, the prototypical master regulator of differentiation, directs a complex program of gene expression during skeletal myogenesis. The up-regulation of the cdk inhibitor p57kip2 plays a critical role in coordinating differentiation and growth arrest during muscle development, as well as in other tissues. p57kip2 displays a highly specific expression pattern and is subject to a complex epigenetic control driving the imprinting of the paternal allele. However, the regulatory mechanisms governing its expression during development are still poorly understood. We have identified an unexpected mechanism by which MyoD regulates p57kip2 transcription in differentiating muscle cells. We show that the induction of p57kip2 requires MyoD binding to a long-distance element located within the imprinting control region KvDMR1 and the consequent release of a chromatin loop involving p57kip2 promoter. We also show that differentiation-dependent regulation of p57kip2, while involving a region implicated in the imprinting process, is distinct and hierarchically subordinated to the imprinting control. These findings highlight a novel mechanism, involving the modification of higher order chromatin structures, by which MyoD regulates gene expression. Our results also suggest that chromatin folding mediated by KvDMR1 could account for the highly restricted expression of p57kip2 during development and, possibly, for its aberrant silencing in some pathologies.
Asunto(s)
Cromatina/química , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Impresión Genómica , Desarrollo de Músculos/genética , Proteína MioD/metabolismo , Elementos Reguladores de la Transcripción , Animales , Células Cultivadas , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/biosíntesis , Canal de Potasio KCNQ1/biosíntesis , Canal de Potasio KCNQ1/genética , Ratones , Regiones Promotoras GenéticasRESUMEN
Poly(ADP-ribose)polymerase-1 (PARP-1) is a predominantly nuclear enzyme that exerts numerous functions in cellular physiology and pathology, from maintenance of DNA stability to transcriptional regulation. Through a proteomic analysis of PARP-1 co-immunoprecipitation complexes, we identified Mitofilin, a mitochondrial protein, as a new PARP-1 interactor. This result prompted us to further investigate the presence and the role of the enzyme in mitochondria. Using laser confocal microscopy and Western blot analysis of purified mitochondria, we demonstrated the mitochondrial localization of a fraction of PARP-1. Further, the effects of overexpressing or down-regulating Mitofilin showed that this protein promotes and is required for PARP-1 mitochondrial localization. We also report several lines of evidence suggesting that intramitochondrial PARP-1 plays a role in mitochondrial DNA (mtDNA) damage signaling and/or repair. First, we show that PARP-1 binds to different regions throughout the mtDNA. Moreover, we demonstrated that the depletion of either PARP-1 or Mitofilin, which abrogates the mitochondrial localization of the enzyme, leads to the accumulation of mtDNA damage. Finally, we show that DNA ligase III, known to be required for mtDNA repair, participates in a PARP-1-containing complex bound to mtDNA. This work highlights a new environment for PARP-1, opening the possibility that at least some of the nuclear functions of the enzyme can be also extended to mtDNA metabolism.
Asunto(s)
ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Western Blotting , Inmunoprecipitación de Cromatina , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Espectrometría de Masas , Microscopía Confocal , Mitocondrias/genética , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/genética , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Proteínas de Unión a Poli-ADP-Ribosa , ARN Interferente Pequeño/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas de XenopusRESUMEN
Poly(ADP-ribose)polymerases are involved in fundamental cellular events as well as they seem to be associated to some viral infection process. In this work, the poly(ADP-ribose)polymerase-1 (PARP-1) role in the polyomavirus life cycle has been investigated. Early viral transcription was reduced by competitive inhibitors of PARPs in Swiss 3T3 cells and almost abolished in PARP-1 knockout fibroblasts and in wild-type fibroblasts when PARP-1 was silenced by RNA interference. In vivo chromatin immunoprecipitation assays showed that poly(ADP-ribosyl)ation (poly(ADP-ribose)) facilitates the release of the capsid protein viral protein 1 (VP1) from the chromatin of infecting virions. In vitro experiments demonstrated that VP1 stimulates the enzymatic activity of PARP-1 and binds non-covalently both protein-free and PARP-1-bound poly(ADP-ribose). Our studies suggest that PARP-1 promotes the complete VP1 displacement from viral DNA favouring the viral early transcription.
Asunto(s)
Proteínas de la Cápside/metabolismo , Regulación Viral de la Expresión Génica , Genes Inmediatos-Precoces/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Poliomavirus/genética , Células 3T3 , Animales , Cápside/metabolismo , Ratones , Modelos Genéticos , Poli(ADP-Ribosa) Polimerasa-1 , Poli Adenosina Difosfato Ribosa/metabolismo , Poliomavirus/patogenicidad , Unión Proteica , Transcripción Genética , Internalización del VirusRESUMEN
In this work, we sought to determine whether the components of the murine polyomavirus capsid establish specific interactions with the minichromosome encapsidated into the mature viral particles by using the cis-diamminedichloroplatinum(II) cross-linking reagent. Our data indicated that VP1, but not minor capsid proteins, interacts with the viral genome in vivo. In addition, semiquantitative PCR assays performed on cross-linked DNA complexes revealed that VP1 binds to all regions of the viral genome but significantly more to the regulatory region. The implications of such an interaction for viral infectivity are discussed.