RESUMEN
Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.
Asunto(s)
Reprogramación Celular/fisiología , Citidina Desaminasa/metabolismo , Metilación de ADN , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , División Celular , Fusión Celular , Línea Celular , Células Cultivadas , Reprogramación Celular/genética , Inmunoprecipitación de Cromatina , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , ADN/química , ADN/genética , ADN/metabolismo , Replicación del ADN , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Pulmón/citología , Pulmón/embriología , Ratones , Modelos Biológicos , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Regiones Promotoras Genéticas/genética , Factores de TiempoRESUMEN
Members of the sirtuin (SIRT) family of NAD-dependent deacetylases promote longevity in multiple organisms. Deficiency of mammalian SIRT6 leads to shortened life span and an aging-like phenotype in mice, but the underlying molecular mechanisms are unclear. Here we show that SIRT6 functions at chromatin to attenuate NF-kappaB signaling. SIRT6 interacts with the NF-kappaB RELA subunit and deacetylates histone H3 lysine 9 (H3K9) at NF-kappaB target gene promoters. In SIRT6-deficient cells, hyperacetylation of H3K9 at these target promoters is associated with increased RELA promoter occupancy and enhanced NF-kappaB-dependent modulation of gene expression, apoptosis, and cellular senescence. Computational genomics analyses revealed increased activity of NF-kappaB-driven gene expression programs in multiple Sirt6-deficient tissues in vivo. Moreover, haploinsufficiency of RelA rescues the early lethality and degenerative syndrome of Sirt6-deficient mice. We propose that SIRT6 attenuates NF-kappaB signaling via H3K9 deacetylation at chromatin, and hyperactive NF-kappaB signaling may contribute to premature and normal aging.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , FN-kappa B/metabolismo , Sirtuinas/metabolismo , Factor de Transcripción ReIA/metabolismo , Acetilación , Animales , Línea Celular , Cromatina/metabolismo , Cruzamientos Genéticos , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Humanos , Longevidad/genética , Ratones , FN-kappa B/genética , Regiones Promotoras Genéticas , Sirtuinas/genética , Factor de Transcripción ReIA/genéticaRESUMEN
The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.
