RESUMEN
Genomic imprinting is an epigenetic modification that results in expression from only one of the two parental copies of a gene. Differences in methylation between the two parental chromosomes are often observed at or near imprinted genes. Beckwith-Wiedemann syndrome (BWS), which predisposes to cancer and excessive growth, results from a disruption of imprinted gene expression in chromosome band 11p15.5. One third of individuals with BWS lose maternal-specific methylation at KvDMR1, a putative imprinting control region within intron 10 of the KCNQ1 gene, and it has been proposed that this epimutation results in aberrant imprinting and, consequently, BWS1, 2. Here we show that paternal inheritance of a deletion of KvDMR1 results in the de-repression in cis of six genes, including Cdkn1c, which encodes cyclin-dependent kinase inhibitor 1C. Furthermore, fetuses and adult mice that inherited the deletion from their fathers were 20-25% smaller than their wildtype littermates. By contrast, maternal inheritance of this deletion had no effect on imprinted gene expression or growth. Thus, the unmethylated paternal KvDMR1 allele regulates imprinted expression by silencing genes on the paternal chromosome. These findings support the hypothesis that loss of methylation in BWS patients activates the repressive function of KvDMR1 on the maternal chromosome, resulting in abnormal silencing of CDKN1C and the development of BWS.
Asunto(s)
Síndrome de Beckwith-Wiedemann/genética , Encéfalo/metabolismo , Impresión Genómica , Análisis de Secuencia por Matrices de Oligonucleótidos , Alelos , Animales , Northern Blotting , Mapeo Cromosómico , Regulación hacia Abajo , Padre , Femenino , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Datos de Secuencia Molecular , Madres , Mapeo Físico de Cromosoma , Regulación hacia ArribaRESUMEN
Imprinting control regions (ICRs) are known to repress genes by utilizing one of two mechanisms, CTCF-mediated insulation or the transcription of non-coding RNAs (ncRNAs). The KvDMR1 ICR contains both the promoter for the Kcnq1ot1 ncRNA and two CTCF-binding sites located within sequences exhibiting repressive activity in enhancer-blocking assays. Deletion of KvDMR1 results in ubiquitous biallelic expression of eight maternal-specific genes in distal chromosome 7. Here we report that while truncation of the Kcnq1ot1 transcript results in the loss of imprinted expression of these genes in the placenta, it does not affect imprinted expression of Cdkn1c in a subset of embryonic tissues despite universal loss of paternal-specific methylation at Cdkn1c. Consistent with tissue-specific loss of imprinted expression, growth deficiency of these mutant mice was less severe than that observed previously in mice with deletion of KvDMR1. This study demonstrates that the KvDMR1 locus can silence Cdkn1c by a mechanism independent of Kcnq1ot1 transcription, perhaps by CTCF-associated repression, making it the first example of an ICR capable of silencing the same gene by two distinct mechanisms.
Asunto(s)
Silenciador del Gen/fisiología , Impresión Genómica/fisiología , Proteínas de la Membrana/genética , Animales , Factor de Unión a CCCTC , Células Cultivadas , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Femenino , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Canales de Potasio con Entrada de Voltaje/biosíntesis , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/fisiología , Proteínas Represoras/genética , Proteínas Represoras/fisiologíaRESUMEN
Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of the Kcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.