RESUMEN
ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Impresión Genómica , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Animales , Sitios de Unión/genética , Diferenciación Celular/genética , Islas de CpG/genética , Epigénesis Genética , Sitios Genéticos , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones , Modelos GenéticosRESUMEN
Imprinting Control Regions (ICRs) need to maintain their parental allele-specific DNA methylation during early embryogenesis despite genome-wide demethylation and subsequent de novo methylation. ZFP57 and KAP1 are both required for maintaining the repressive DNA methylation and H3-lysine-9-trimethylation (H3K9me3) at ICRs. In vitro, ZFP57 binds a specific hexanucleotide motif that is enriched at its genomic binding sites. We now demonstrate in mouse embryonic stem cells (ESCs) that SNPs disrupting closely-spaced hexanucleotide motifs are associated with lack of ZFP57 binding and H3K9me3 enrichment. Through a transgenic approach in mouse ESCs, we further demonstrate that an ICR fragment containing three ZFP57 motif sequences recapitulates the original methylated or unmethylated status when integrated into the genome at an ectopic position. Mutation of Zfp57 or the hexanucleotide motifs led to loss of ZFP57 binding and DNA methylation of the transgene. Finally, we identified a sequence variant of the hexanucleotide motif that interacts with ZFP57 both in vivo and in vitro. The presence of multiple and closely located copies of ZFP57 motif variants emerges as a distinct characteristic that is required for the faithful maintenance of repressive epigenetic marks at ICRs and other ZFP57 binding sites.
Asunto(s)
Metilación de ADN , Impresión Genómica , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Represoras/genética , Alelos , Animales , Secuencia de Bases , Línea Celular , Inmunoprecipitación de Cromatina , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Motivos de Nucleótidos/genética , Polimorfismo de Nucleótido Simple , Unión Proteica/genética , Proteínas Represoras/metabolismo , Proteína 28 que Contiene Motivos TripartitoRESUMEN
DNA methylation is a common epigenetic modification of the mammalian genome. Conflicting data regarding the possible presence of methylated cytosines within mitochondrial DNA (mtDNA) have been reported. To clarify this point, we analysed the methylation status of mtDNA control region (D-loop) on human and murine DNA samples from blood and cultured cells by bisulphite sequencing and methylated/hydroxymethylated DNA immunoprecipitation assays. We found methylated and hydroxymethylated cytosines in the L-strand of all samples analysed. MtDNA methylation particularly occurs within non-C-phosphate-G (non-CpG) nucleotides, mainly in the promoter region of the heavy strand and in conserved sequence blocks, suggesting its involvement in regulating mtDNA replication and/or transcription. We observed DNA methyltransferases within the mitochondria, but the inactivation of Dnmt1, Dnmt3a, and Dnmt3b in mouse embryonic stem (ES) cells results in a reduction of the CpG methylation, while the non-CpG methylation shows to be not affected. This suggests that D-loop epigenetic modification is only partially established by these enzymes. Our data show that DNA methylation occurs in the mtDNA control region of mammals, not only at symmetrical CpG dinucleotides, typical of nuclear genome, but in a peculiar non-CpG pattern previously reported for plants and fungi. The molecular mechanisms responsible for this pattern remain an open question.
Asunto(s)
Islas de CpG , Metilación de ADN , ADN Mitocondrial/genética , Región de Control de Posición , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Mitocondrial/sangre , Dioxigenasas/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mitocondrias/genética , Mitocondrias/metabolismo , ADN Metiltransferasa 3BRESUMEN
In the mouse, ZFP57 contains three classical Cys2His2 zinc finger domains (ZF) and recognizes the methylated TGC(met)CGC target sequence using the first and the second ZFs. In this study, we demonstrate that the human ZFP57 (hZFP57) containing six Cys2His2 ZFs, binds the same methylated sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA interaction. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation.