RESUMEN
Recent investigations have implicated long antisense noncoding RNAs in the epigenetic regulation of chromosomal domains. Here we show that Kcnq1ot1 is an RNA polymerase II-encoded, 91 kb-long, moderately stable nuclear transcript and that its stability is important for bidirectional silencing of genes in the Kcnq1 domain. Kcnq1ot1 interacts with chromatin and with the H3K9- and H3K27-specific histone methyltransferases G9a and the PRC2 complex in a lineage-specific manner. This interaction correlates with the presence of extended regions of chromatin enriched with H3K9me3 and H3K27me3 in the Kcnq1 domain in placenta, whereas fetal liver lacks both chromatin interactions and heterochromatin structures. In addition, the Kcnq1 domain is more often found in contact with the nucleolar compartment in placenta than in liver. Taken together, our data describe a mechanism whereby Kcnq1ot1 establishes lineage-specific transcriptional silencing patterns through recruitment of chromatin remodeling complexes and maintenance of these patterns through subsequent cell divisions occurs via targeting the associated regions to the perinucleolar compartment.
Asunto(s)
Cromatina/metabolismo , Silenciador del Gen/fisiología , ARN sin Sentido/fisiología , ARN Mensajero/metabolismo , ARN no Traducido/fisiología , Animales , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Inmunoprecipitación de Cromatina , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Ratones , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio con Entrada de Voltaje/fisiología , ARN Polimerasa II/metabolismo , Estabilidad del ARN , ARN sin Sentido/metabolismo , ARN no Traducido/metabolismoRESUMEN
Establishment of silencing by noncoding RNAs (ncRNAs) via targeting of chromatin remodelers is relatively well investigated; however, their role in the maintenance of silencing is poorly understood. Here, we explored the functional role of the long ncRNA Kcnq1ot1 in the maintenance of transcriptional gene silencing in the one mega-base Kcnq1 imprinted domain in a transgenic mouse model. By conditionally deleting the Kcnq1ot1 ncRNA at different stages of mouse development, we suggest that Kcnq1ot1 ncRNA is required for the maintenance of the silencing of ubiquitously imprinted genes (UIGs) at all developmental stages. In addition, Kcnq1ot1 ncRNA is also involved in guiding and maintaining the CpG methylation at somatic differentially methylated regions flanking the UIGs, which is a hitherto unknown role for a long ncRNA. On the other hand, silencing of some of the placental-specific imprinted genes (PIGs) is maintained independently of Kcnq1ot1 ncRNA. Interestingly, the non-imprinted genes (NIGs) that escape RNA-mediated silencing are enriched with enhancer-specific modifications. Taken together, this study illustrates the gene-specific maintenance mechanisms operational at the Kcnq1 locus for tissue-specific transcriptional gene silencing and activation.
Asunto(s)
Metilación de ADN , Canal de Potasio KCNQ1/genética , ARN no Traducido/genética , Animales , Islas de CpG/genética , Cruzamientos Genéticos , Epigénesis Genética , Femenino , Silenciador del Gen , Impresión Genómica , Heterocromatina/metabolismo , Homocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN/metabolismo , ARN Largo no Codificante , Factores de TiempoRESUMEN
The imprinted Dlk1-Gtl2 region of the mammalian genome - which in sheep encompasses the Callipyge locus, known for its unusual mode of inheritance - encodes a number of maternally expressed miRNAs. Five of these miRNAs, hosted by the antisense transcript antiPeg11, have now been shown to target degradation of the paternally expressed Peg11 mRNA by an RNAi-mediated mechanism.
Asunto(s)
Impresión Genómica/genética , Glicoproteínas/genética , Mamíferos/genética , MicroARNs/genética , Proteínas/genética , Interferencia de ARN , ARN Mensajero/metabolismo , Animales , Componentes del Gen , Humanos , Patrón de Herencia/genética , Mamíferos/crecimiento & desarrollo , MicroARNs/metabolismo , Mutación/genética , ARN Largo no CodificanteRESUMEN
Long noncoding RNAs are implicated in a number of regulatory functions in eukaryotic genomes. The paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1 regulates epigenetic gene silencing in an imprinted gene cluster in cis over a distance of 400 kb in the mouse embryo, whereas the silenced region extends over 780 kb in the placenta. Gene silencing by the Kcnq1ot1 RNA involves repressive histone modifications, including H3K9me2 and H3K27me3, which are partly brought about by the G9a and Ezh2 histone methyltransferases. Here, we show that Kcnq1ot1 is transcribed by RNA polymerase II, is unspliced, is relatively stable and is localised in the nucleus. Analysis of conditional Dicer mutants reveals that the RNAi pathway is not involved in gene silencing in the Kcnq1ot1 cluster. Instead, using RNA/DNA FISH we show that the Kcnq1ot1 RNA establishes a nuclear domain within which the genes that are epigenetically inactivated in cis are frequently found, whereas nearby genes that are not regulated by Kcnq1ot1 are localised outside of the domain. The Kcnq1ot1 RNA domain is larger in the placenta than in the embryo, consistent with more genes in the cluster being silenced in the placenta. Our results show for the first time that autosomal long ncRNAs can establish nuclear domains, which might create a repressive environment for epigenetic silencing of adjacent genes. Long ncRNAs in imprinting clusters and the Xist RNA on the inactive X chromosome thus appear to regulate epigenetic gene silencing by similar mechanisms.
Asunto(s)
Linaje de la Célula , Núcleo Celular/genética , Silenciador del Gen , ARN no Traducido/metabolismo , Animales , Secuencia de Bases , Impresión Genómica , Ratones , Datos de Secuencia Molecular , Interferencia de ARN , Estabilidad del ARN , ARN Largo no Codificante , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genéticaRESUMEN
The mouse Kcnq1 imprinted domain is located on distal chromosome 7 and contains several imprinted genes that are paternally repressed. Repression of these genes is regulated by a non-coding antisense transcript, Kcnq1ot1, which is paternally expressed. Maternal repression of Kcnq1ot1 is controlled by DNA methylation originating in the oocyte. Some genes in the region are imprinted only in the placenta, whereas others are imprinted in both extra-embryonic and embryonic lineages. Here, we show that Kcnq1ot1 is paternally expressed in preimplantation embryos from the two-cell stage, and that ubiquitously imprinted genes proximal to Kcnq1ot1 are already repressed in blastocysts, ES cells and TS cells. Repressive histone marks such as H3K27me3 are present on the paternal allele of these genes in both ES and TS cells. Placentally imprinted genes that are distal to Kcnq1ot1, by contrast, are not imprinted in blastocysts, ES or TS cells. In these genes, paternal silencing and differential histone marks arise during differentiation of the trophoblast lineage between E4.5 and E7.5. Our findings show that the dynamics during preimplantation development of gene inactivation and acquisition of repressive histone marks in ubiquitously imprinted genes of the Kcnq1 domain are very similar to those of imprinted X inactivation. By contrast, genes that are only imprinted in the placenta, while regulated by the same non-coding RNA transcript Kcnq1ot1, undergo epigenetic inactivation during differentiation of the trophoblast lineage. Our findings establish a model for how epigenetic gene silencing by non-coding RNA may depend on distance from the non-coding RNA and on lineage and differentiation specific factors.