Assuntos
Oftalmopatias , Oftalmologia , Humanos , Emergências , Olho , Face , Hospitais , Índia/epidemiologia , Centros de Atenção Terciária , Oftalmopatias/diagnóstico , Oftalmopatias/terapiaRESUMO
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.
Assuntos
Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Sirtuína 1/metabolismo , Sequência de Aminoácidos , Western Blotting , Linhagem Celular Tumoral , Expressão Gênica , Glucose/farmacologia , Células HeLa , Humanos , Metiltransferases/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Ligação Proteica , Interferência de RNA , RNA Ribossômico/genética , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Homologia de Sequência de Aminoácidos , Sirtuína 1/genéticaRESUMO
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.
Assuntos
NAD/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , RNA Ribossômico/genética , Transcrição Gênica , Acetilação/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Dano ao DNA , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina/farmacologia , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Glucose/farmacologia , Humanos , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , RNA Ribossômico/biossíntese , Proteínas de Ligação a RNA , Sirtuína 1/metabolismoRESUMO
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.
Assuntos
Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Neoplasias do Colo/patologia , Neoplasias do Colo/fisiopatologia , Sirtuínas/genética , Sirtuínas/metabolismo , Adenocarcinoma/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Divisão Celular/fisiologia , Colo/patologia , Colo/fisiologia , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Imuno-Histoquímica , Camundongos , Transplante de Neoplasias , RNA Interferente Pequeno , Sirtuína 1RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Humanos , Metiltransferases/genética , Camundongos , Complexos Multiproteicos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Sirtuína 1 , Sirtuínas/genética , Sirtuínas/metabolismoRESUMO
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.