RESUMEN
BACKGROUND: Topors is a nuclear protein that co-localizes with promyelocytic leukemia bodies and has both ubiquitin and SUMO E3 ligase activity. Expression studies implicated Topors as a tumor suppressor in various malignancies. To gain insight into the function of Topors, we generated a Topors-deficient mouse strain. RESULTS: Mice homozygous for a mutant Topors allele exhibited a high rate of perinatal mortality and decreased lifespan. In addition, heterozygotes were found to have an increased incidence of malignancy, involving a variety of tissues. Consistent with this finding, primary embryonic fibroblasts lacking Topors exhibited an increased rate of malignant transformation, associated with aneuploidy and defective chromosomal segregation. While loss of Topors did not alter sensitivity to DNA-damaging or microtubule-targeting agents, cells lacking Topors exhibited altered pericentric heterochromatin, manifested by mislocalization of HP1alpha and an increase in transcription from pericentric major satellite DNA. Topors-deficient cells exhibited a transcriptional profile similar to that of cells treated with histone deacetylase inhibitors, and were resistant to the anti-proliferative effects of the histone deacetylase inhibitor trichostatin A. CONCLUSION: These results indicate a unique role for Topors in the maintenance of genomic stability and pericentric heterochromatin, as well as in cellular sensitivity to histone deacetylase inhibitors.
Asunto(s)
Neoplasias/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Homólogo de la Proteína Chromobox 5 , Fibroblastos , Inestabilidad Genómica , Heterocigoto , Inhibidores de Histona Desacetilasas , Humanos , Ácidos Hidroxámicos , Ratones , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Topors was identified recently as a human protein that binds to topoisomerase I and p53. Topors contains a highly conserved RING domain and localizes in promyelocytic leukemia nuclear bodies. Relatively little is known regarding topors expression patterns or function. We now demonstrate that topors mRNA and protein are widely expressed in normal human tissues. By contrast, topors mRNA and protein levels are decreased or undetectable in colon adenocarcinomas relative to normal colon tissue, and expression of the topors protein is not detectable in several colon cancer cell lines. The human TOPORS gene is located on chromosome 9p21, with loss of heterozygosity in this region frequently observed in several different malignancies. While we were unable to detect loss of heterozygosity of the TOPORS gene in 16 sporadic colon cancer cases, increased methylation of a CpG island in the TOPORS promoter was evident in colon adenocarcinoma specimens relative to matched normal tissues. Additional studies indicate that forced expression of topors inhibits cellular proliferation and is associated with an accumulation of cells in the G(0)/G(1) phase of the cell cycle. This effect is independent of the topors RING domain and maps to a C-terminal region of the protein. These results suggest that topors functions as a negative regulator of cell growth, and possibly as a tumor suppressor.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Genes Supresores de Tumor , Proteínas de Neoplasias , Proteínas Nucleares/biosíntesis , Factores de Transcripción/biosíntesis , Adenocarcinoma/metabolismo , Ciclo Celular , División Celular , Colon/metabolismo , Neoplasias del Colon/metabolismo , Islas de CpG , Metilación de ADN , Citometría de Flujo , Fase G1 , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Pérdida de Heterocigocidad , Proteínas Luminiscentes/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Fase de Descanso del Ciclo Celular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina/metabolismo , Factores de Tiempo , Distribución Tisular , Proteína p53 Supresora de Tumor/metabolismo , Células U937 , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
TOPORS is the first example of a protein with both ubiquitin and SUMO-1 E3 ligase activity and has been implicated as a tumor suppressor in several different malignancies. To gain insight into the cellular role of TOPORS, a proteomic screen was performed to identify candidate sumoylation substrates. The results indicate that many of the putative substrates are involved in chromatin modification or transcriptional regulation. Transfection studies confirmed mammalian Sin3A as a sumoylation substrate for TOPORS. These findings suggest that TOPORS may function as a tumor suppressor by regulating mSin3A and other proteins involved in chromatin modification.
Asunto(s)
Cromatina/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína SUMO-1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3 , Transfección , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The human topoisomerase I- and p53-binding protein topors contains a highly conserved, N-terminal C3HC4-type RING domain that is homologous to the RING domains of known E3 ubiquitin ligases. We demonstrate that topors functions in vitro as a RING-dependent E3 ubiquitin ligase with the E2 enzymes UbcH5a, UbcH5c, and UbcH6 but not with UbcH7, CDC34, or UbcH2b. Additional studies indicate that a conserved tryptophan within the topors RING domain is required for ubiquitination activity. Furthermore, both in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. Similar to MDM2, overexpression of topors results in a proteasome-dependent decrease in p53 protein expression in a human osteosarcoma cell line. These results are similar to the recent finding that a Drosophila topors orthologue ubiquitinates the Hairy transcriptional repressor and suggest that topors functions as a ubiquitin ligase for multiple transcription factors.