RESUMEN
ATP-dependent chromatin remodeling is involved in all DNA transactions and is linked to numerous human diseases. We explored functions of chromatin remodelers during cellular aging. Deletion of ISW2, or mutations inactivating the Isw2 enzyme complex, extends yeast replicative lifespan. This extension by ISW2 deletion is epistatic to the longevity effect of calorie restriction (CR), and this mechanism is distinct from suppression of TOR signaling by CR. Transcriptome analysis indicates that isw2Δ partially mimics an upregulated stress response in CR cells. In particular, isw2Δ cells show an increased response to genotoxic stresses, and the DNA repair enzyme Rad51 is important for isw2Δ-mediated longevity. We show that lifespan is also extended in C. elegans by reducing levels of athp-2, a putative ortholog of Itc1/ACF1, a critical subunit of the enzyme complex. Our findings demonstrate that the ISWI class of ATP-dependent chromatin remodeling complexes plays a conserved role during aging and in CR.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Caenorhabditis elegans/metabolismo , Senescencia Celular/fisiología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Animales , Proteínas de Caenorhabditis elegans/genética , Restricción Calórica , Ensamble y Desensamble de Cromatina/fisiología , Daño del ADN , Reparación del ADN/fisiología , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Técnicas de Inactivación de Genes , Longevidad , Peroxidasas/biosíntesis , Recombinasa Rad51/biosíntesis , Recombinasa Rad51/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico , Factores de Transcripción/genéticaRESUMEN
Cells undergoing developmental processes are characterized by persistent non-genetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone post-translational modifications. Sirtuins, a group of conserved NAD(+)-dependent deacetylases or ADP-ribosyltransferases, promote longevity in diverse organisms; however, their molecular mechanisms in ageing regulation remain poorly understood. Yeast Sir2, the first member of the family to be found, establishes and maintains chromatin silencing by removing histone H4 lysine 16 acetylation and bringing in other silencing proteins. Here we report an age-associated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing ageing models for yeast, may represent an evolutionarily conserved function of sirtuins in regulation of replicative ageing by maintenance of intact telomeric chromatin.
Asunto(s)
Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Acetilación , Acetiltransferasas/metabolismo , División Celular , Cromatina/genética , Cromatina/metabolismo , Epistasis Genética , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Histona Acetiltransferasas , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/deficiencia , Histona Desacetilasas/metabolismo , Histonas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/deficiencia , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2 , Sirtuinas/antagonistas & inhibidores , Sirtuinas/deficiencia , Sirtuinas/metabolismo , Telómero/genética , Telómero/metabolismo , Transcripción GenéticaRESUMEN
p53, the tumour suppressor and transcriptional activator, is regulated by numerous post-translational modifications, including lysine methylation. Histone lysine methylation has recently been shown to be reversible; however, it is not known whether non-histone proteins are substrates for demethylation. Here we show that, in human cells, the histone lysine-specific demethylase LSD1 (refs 3, 4) interacts with p53 to repress p53-mediated transcriptional activation and to inhibit the role of p53 in promoting apoptosis. We find that, in vitro, LSD1 removes both monomethylation (K370me1) and dimethylation (K370me2) at K370, a previously identified Smyd2-dependent monomethylation site. However, in vivo, LSD1 shows a strong preference to reverse K370me2, which is performed by a distinct, but unknown, methyltransferase. Our results indicate that K370me2 has a different role in regulating p53 from that of K370me1: K370me1 represses p53 function, whereas K370me2 promotes association with the coactivator 53BP1 (p53-binding protein 1) through tandem Tudor domains in 53BP1. Further, LSD1 represses p53 function through the inhibition of interaction of p53 with 53BP1. These observations show that p53 is dynamically regulated by lysine methylation and demethylation and that the methylation status at a single lysine residue confers distinct regulatory output. Lysine methylation therefore provides similar regulatory complexity for non-histone proteins and for histones.
Asunto(s)
Oxidorreductasas N-Desmetilantes/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis , Línea Celular , Histona Demetilasas , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metilación , Unión Proteica , Activación Transcripcional , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/química , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Specific sites of lysine methylation on histones correlate with either activation or repression of transcription. The tumour suppressor p53 (refs 4-7) is one of only a few non-histone proteins known to be regulated by lysine methylation. Here we report a lysine methyltransferase, Smyd2, that methylates a previously unidentified site, Lys 370, in p53. This methylation site, in contrast to the known site Lys 372, is repressing to p53-mediated transcriptional regulation. Smyd2 helps to maintain low concentrations of promoter-associated p53. We show that reducing Smyd2 concentrations by short interfering RNA enhances p53-mediated apoptosis. We find that Set9-mediated methylation of Lys 372 inhibits Smyd2-mediated methylation of Lys 370, providing regulatory cross-talk between post-translational modifications. In addition, we show that the inhibitory effect of Lys 372 methylation on Lys 370 methylation is caused, in part, by blocking the interaction between p53 and Smyd2. Thus, similar to histones, p53 is subject to both activating and repressing lysine methylation. Our results also predict that Smyd2 may function as a putative oncogene by methylating p53 and repressing its tumour suppressive function.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Lisina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Humanos , Metilación , RatonesRESUMEN
Covalent histone post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitylation play pivotal roles in regulating many cellular processes, including transcription, response to DNA damage, and epigenetic control. Although positive-acting post-translational modifications have been studied in Saccharomyces cerevisiae, histone modifications that are associated with transcriptional repression have not been shown to occur in this yeast. Here, we provide evidence that histone sumoylation negatively regulates transcription in S. cerevisiae. We show that all four core histones are sumoylated and identify specific sites of sumoylation in histones H2A, H2B, and H4. We demonstrate that histone sumoylation sites are involved directly in transcriptional repression. Further, while histone sumoylation occurs at all loci tested throughout the genome, slightly higher levels occur proximal to telomeres. We observe a dynamic interplay between histone sumoylation and either acetylation or ubiquitylation, where sumoylation serves as a potential block to these activating modifications. These results indicate that sumoylation is the first negative histone modification to be identified in S. cerevisiae and further suggest that sumoylation may serve as a general dynamic mark to oppose transcription.
Asunto(s)
Histonas/metabolismo , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Secuencia de Aminoácidos , Western Blotting , Inmunoprecipitación , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética , Telómero , Ubiquitina/metabolismoRESUMEN
Distinct patterns of posttranslational histone modifications can regulate DNA-templated events such as mitosis, transcription, replication, apoptosis, and DNA damage, suggesting the presence of a "histone code" in these nuclear processes. Phosphorylation of histone H2A S129 at sites of DNA double-strand breaks (DSBs) has been implicated in damage repair in yeast. Here, we describe another phosphorylation event on serine 1 (S1) of histone H4; this event is also associated with MMS- or phleomycin-induced DSBs but not with UV-induced DNA damage. Chromatin-immunoprecipitation (ChIP) studies of an HO-endonuclease-inducible strain show that S1 phosphorylation is specifically enhanced 20- to 25-fold in nucleosomes proximal to the DSB. In addition, we show that casein kinase II (CK2) can phosphorylate H4 S1 in vitro and that null or temperature-sensitive CK2 yeast mutants are defective for induction of H4 S1 phosphorylation upon DNA damage in vivo. Furthermore, H4 S1 phosphorylation and CK2 play a role in DSB re-joining as indicated by a nonhomologous end-joining (NHEJ) plasmid assay. CK2 has been implicated in regulating a DNA-damage response; our data suggest that histone H4 S1 is one of its physiological substrates. These data suggest that this modification is a part of the DNA-repair histone code.