RESUMEN
DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(II) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.
Asunto(s)
Ácido Ascórbico/farmacología , Metilación de ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , 5-Metilcitosina/análogos & derivados , Animales , Antioxidantes/farmacología , Blastocisto/metabolismo , Línea Celular , Medios de Cultivo/química , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Ratones , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Pluripotent stem cells are derived from culture of early embryos or the germline and can be induced by reprogramming of somatic cells. Barriers to reprogramming that stabilize the differentiated state and have tumor suppression functions are expected to exist. However, we have a limited understanding of what such barriers might be. To find novel barriers to reprogramming to pluripotency, we compared the transcriptional profiles of the mouse germline with pluripotent and somatic cells, in vivo and in vitro. There is a remarkable global expression of the transcriptional program for pluripotency in primordial germ cells (PGCs). We identify parallels between PGC reprogramming to pluripotency and human germ cell tumorigenesis, including the loss of LATS2, a tumor suppressor kinase of the Hippo pathway. We show that knockdown of LATS2 increases the efficiency of induction of pluripotency in human cells. LATS2 RNAi, unlike p53 RNAi, specifically enhances the generation of fully reprogrammed iPS cells without accelerating cell proliferation. We further show that LATS2 represses reprogramming in human cells by post-transcriptionally antagonizing TAZ but not YAP, two downstream effectors of the Hippo pathway. These results reveal transcriptional parallels between germ cell transformation and the generation of iPS cells and indicate that the Hippo pathway constitutes a barrier to cellular reprogramming.
Asunto(s)
Reprogramación Celular/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes p53 , Células Germinativas/citología , Células Germinativas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Neoplasias de Células Germinales y Embrionarias/etiología , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Interferencia de ARN , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Señalizadoras YAPRESUMEN
Developmental regulatory genes have both activating (H3K4me3) and repressive (H3K27me3) histone modifications in embryonic stem cells (ESCs). This bivalent configuration is thought to maintain lineage commitment programs in a poised state. However, establishing physiological relevance has been complicated by the high number of cells required for chromatin immunoprecipitation (ChIP). We developed a low-cell-number chromatin immunoprecipitation (low-cell ChIP) protocol to investigate the chromatin of mouse primordial germ cells (PGCs). Genome-wide analysis of embryonic day 11.5 (E11.5) PGCs revealed H3K4me3/H3K27me3 bivalent domains highly enriched at developmental regulatory genes in a manner remarkably similar to ESCs. Developmental regulators remain bivalent and transcriptionally silent through the initiation of sexual differentiation at E13.5. We also identified >2,500 "orphan" bivalent domains that are distal to known genes and expressed in a tissue-specific manner but silent in PGCs. Our results demonstrate the existence of bivalent domains in the germline and raise the possibility that the somatic program is continuously maintained as bivalent, potentially imparting transgenerational epigenetic inheritance.