RESUMEN
Many lineage-specific developmental regulator genes are transcriptionally primed in embryonic stem (ES) cells; RNA Pol(II) is bound at their promoters but is prevented from productive elongation by the activity of polycomb repressive complexes (PRC) 1 and 2. This epigenetically poised state is thought to enable ES cells to rapidly execute multiple differentiation programs and is recognized by a simultaneous enrichment for trimethylation of lysine 4 and trimethylation of lysine 27 of histone H3 (bivalent chromatin) across promoter regions. Here we show that the chromatin profile of this important cohort of genes is progressively modified as ES cells differentiate toward blood-forming precursors. Surprisingly however, neural specifying genes, such as Nkx2-2, Nkx2-9, and Sox1, remain bivalent and primed even in committed hemangioblasts, as conditional deletion of PRC1 results in overt and inappropriate expression of neural genes in hemangioblasts. These data reinforce the importance of PRC1 for normal hematopoietic differentiation and reveal an unexpected epigenetic plasticity of mesoderm-committed hemangioblasts.
Asunto(s)
Células Madre Embrionarias/metabolismo , Epigénesis Genética , Hemangioblastos/fisiología , Proteínas de Homeodominio/genética , Proteínas Represoras/fisiología , Factores de Transcripción SOXB1/genética , Factores de Transcripción/genética , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Cromatina/genética , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/citología , Proteínas Fetales/genética , Proteínas Fluorescentes Verdes/genética , Histonas/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/metabolismo , Integrasas/metabolismo , Ratones , Ratones Noqueados , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1/antagonistas & inhibidores , Factores de Transcripción SOXB1/metabolismo , Proteínas de Dominio T Box/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Proteínas de Pez CebraRESUMEN
Mono-, di-, and trimethylation of specific histone residues adds an additional level of complexity to the range of histone modifications that may contribute to a histone code. However, it has not been clear whether different methylated states reside stably at different chromatin sites or whether they represent dynamic intermediates at the same chromatin sites. Here, we have used recently developed antibodies that are highly specific for mono-, di-, and trimethylated lysine 9 of histone H3 (MeK9H3) to examine the subnuclear localization and replication timing of chromatin containing these epigenetic marks in mammalian cells. Me1K9H3 was largely restricted to early replicating, small punctate domains in the nuclear interior. Me2K9H3 was the predominant MeK9 epitope at the nuclear and nucleolar periphery and colocalized with sites of DNA synthesis primarily in mid-S phase. Me3K9H3 decorated late-replicating pericentric heterochromatin in mouse cells and sites of DAPI-dense intranuclear heterochromatin in human and hamster cells that replicated throughout S phase. Disruption of the Suv39h1,2 or G9a methyltransferases in murine embryonic stem cells resulted in a redistribution of methyl epitopes, but did not alter the overall spatiotemporal replication program. These results demonstrate that mono-, di-, and trimethylated states of K9H3 largely occupy distinct chromosome domains.
Asunto(s)
Núcleo Celular/metabolismo , ADN/biosíntesis , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Epigénesis Genética , Epítopos , Colorantes Fluorescentes , Heterocromatina/metabolismo , Humanos , Indoles , Metilación , Ratones , Microscopía Confocal , Fase SRESUMEN
Histone modifications are central to epigenetic regulation and must be reestablished with each round of DNA replication. Here we describe methods to localize these modifications within mammalian nuclei and to relate them to specific spatiotemporal patterns of DNA replication.