Asunto(s)
Oftalmopatías , Oftalmología , Humanos , Urgencias Médicas , Ojo , Cara , Hospitales , India/epidemiología , Centros de Atención Terciaria , Oftalmopatías/diagnóstico , Oftalmopatías/terapiaRESUMEN
Nucleomethylin (NML), a novel nucleolar protein, is important for mediating the assembly of the energy-dependent nucleolar silencing complex (eNoSC), which also contains SirT1 and SUV39H1. eNoSC represses rRNA transcription during nutrient deprivation, thus reducing energy expenditure and improving cell survival. We found that NML is an RNA binding protein that copurifies with 5S, 5.8S, and 28S rRNA. The SirT1 and RNA binding regions on NML showed partial overlap, and the NML-SirT1 interaction was competitively inhibited by rRNA. Nutrient deprivation triggered downregulation of rRNA transcription, reduced the level of NML-associated rRNA, and stimulated NML-SirT1 binding. Assembly of eNoSC facilitated repression of pre-rRNA transcription. These results suggest that nascent rRNA generates a positive-feedback signal by suppressing the assembly of eNoSC and protecting active ribosomal DNA units from heterochromatin formation. This RNA-mediated mechanism enables the eNoSC to amplify the effects of upstream nutrient-responsive regulators.
Asunto(s)
Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Sirtuina 1/metabolismo , Secuencia de Aminoácidos , Western Blotting , Línea Celular Tumoral , Expresión Génica , Glucosa/farmacología , Células HeLa , Humanos , Metiltransferasas/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Unión Proteica , Interferencia de ARN , ARN Ribosómico/genética , ARN Ribosómico 28S/genética , ARN Ribosómico 28S/metabolismo , ARN Ribosómico 5.8S/genética , ARN Ribosómico 5.8S/metabolismo , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribosomas/efectos de los fármacos , Ribosomas/genética , Homología de Secuencia de Aminoácido , Sirtuina 1/genéticaRESUMEN
The chromosomal region encoding the nuclear NAD(+) synthesis enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT1) is frequently deleted in human cancer. We describe evidence that NMNAT1 interacts with the nucleolar repressor protein nucleomethylin and is involved in regulating rRNA transcription. NMNAT1 binds to nucleomethylin and is recruited into a ternary complex containing the NAD(+)-dependent deacetylase SirT1. NMNAT1 expression stimulates the deacetylase function of SirT1. Knockdown of NMNAT1 enhances rRNA transcription and promotes cell death after nutrient deprivation. Furthermore, NMNAT1 expression is induced by DNA damage and plays a role in preventing cell death after damage. Heterozygous deletion of NMNAT1 in lung tumor cell lines correlates with low expression level and increased sensitivity to DNA damage. These results suggest that NMNAT1 deletion in tumors may contribute to transformation by increasing rRNA synthesis, but may also increase sensitivity to nutrient stress and DNA damage.
Asunto(s)
NAD/biosíntesis , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , ARN Ribosómico/genética , Transcripción Genética , Acetilación/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/metabolismo , Daño del ADN , Regulación hacia Abajo/efectos de los fármacos , Doxorrubicina/farmacología , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Glucosa/farmacología , Humanos , Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , ARN Ribosómico/biosíntesis , Proteínas de Unión al ARN , Sirtuina 1/metabolismoRESUMEN
The NAD-dependent deacetylase SirT1 regulates factors involved in stress response and cell survival and is a potential drug target of activators and inhibitors. Determination of SirT1 function in tumor cells is important for its targeting in cancer therapy. We found that SirT1 knockdown by short hairpin RNA accelerates tumor xenograft formation by HCT116 cells, whereas SirT1 overexpression inhibits tumor formation. Furthermore, pharmacological inhibition of SirT1 stimulates cell proliferation under conditions of growth factor deprivation. Paradoxically, SirT1 inhibition also sensitizes cells to apoptosis by chemotherapy drugs. Immunohistochemical staining revealed high level SirT1 in normal colon mucosa and benign adenomas. SirT1 overexpression was observed in approximately 25% of stage I/II/III colorectal adenocarcinomas but rarely found in advanced stage IV tumors. Furthermore, approximately 30% of carcinomas showed lower than normal SirT1 expression. This pattern is consistent with SirT1 having pleiotropic effects during cancer development (anti-proliferation and anti-apoptotic). These results suggest a rationale for the use of SirT1 activators and inhibitors in the prevention and treatment of colon cancer.
Asunto(s)
Adenocarcinoma/patología , Adenocarcinoma/fisiopatología , Neoplasias del Colon/patología , Neoplasias del Colon/fisiopatología , Sirtuinas/genética , Sirtuinas/metabolismo , Adenocarcinoma/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , División Celular/fisiología , Colon/patología , Colon/fisiología , Neoplasias del Colon/tratamiento farmacológico , Resistencia a Antineoplásicos/fisiología , Femenino , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Inmunohistoquímica , Ratones , Trasplante de Neoplasias , ARN Interferente Pequeño , Sirtuina 1RESUMEN
SUV39H1 is a histone H3K9-specific methyltransferase important for heterochromatin formation, regulation of gene expression, and induction of senescence in premalignant cells. SUV39H1 forms a complex with SirT1, and its activity is stimulated by SirT1 binding. Here we present evidence that the product of the DBC1 (deleted in breast cancer 1) gene disrupts the SUV39H1-SirT1 complex. Furthermore, DBC1 binds to the SUV39H1 catalytic domain and inhibits its ability to methylate histone H3 in vitro and in vivo. Knockdown of endogenous DBC1 increased the level of cellular H3K9 methylation. As expected, DBC1 also binds to SirT1 and inhibits the deacetylase activity of SirT1. These results identify DBC1 as a novel cellular inhibitor of SUV39H1 activity. DBC1 may be an important regulator of heterochromatin formation and genomic stability by disrupting the SUV39H1-SirT1 complex and inactivating both enzymes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Metiltransferasas/genética , Ratones , Complejos Multiproteicos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Sirtuina 1 , Sirtuinas/genética , Sirtuinas/metabolismoRESUMEN
The nicotinamide adenine dinucleotide (NAD)-dependent deacetylase Sir2 (silent information regulator 2) regulates gene silencing in yeast and promotes lifespan extension during caloric restriction. The mammalian homologue of Sir2 (SirT1) regulates p53, NF-kappaB and Forkhead transcription factors, and is implicated in stress response. This report shows that the cell-cycle and apoptosis regulator E2F1 induces SirT1 expression at the transcriptional level. Furthermore, SirT1 binds to E2F1 and inhibits E2F1 activities, forming a negative feedback loop. Knockdown of SirT1 by small interference RNA (siRNA) increases E2F1 transcriptional and apoptotic functions. DNA damage by etoposide causes E2F1-dependent induction of SirT1 expression and knockdown of SirT1 increases sensitivity to etoposide. These results reveal a mutual regulation between E2F1 and SirT1 that affects cellular sensitivity to DNA damage.