RESUMO
In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.
Assuntos
Núcleo Celular/genética , Núcleo Celular/fisiologia , Impressão Genômica/genética , Fator de Crescimento Insulin-Like II/genética , RNA não Traduzido/genética , Receptor IGF Tipo 2/genética , Ovinos/genética , Alelos , Animais , Sequência de Bases , Metilação de DNA , Epigênese Genética/genética , Evolução Molecular , Feminino , Humanos , Íntrons/genética , Masculino , RNA Longo não Codificante , LínguaRESUMO
We identified a maternally methylated CpG island at the mouse Zac1 locus on chromosome (Chr.) 10 in a screen for imprinted genes. The homologous human gene ZAC (also known as LOT1 and PLAGLI) is a candidate gene for transient neonatal diabetes (TNDM), an imprinted disorder associated with paternal duplication for 6q24 and characterized by intrauterine growth retardation and insulin dependence. A mouse model would be indispensable to investigate the basis of the disorder, however, there is apparently no similar phenotype in mice with the corresponding chromosome anomaly. To begin to understand this difference, we have undertaken a comparative analysis of the mouse and human genes. We show that the CpG island is far upstream of the coding body of mouse Zac1, that Zac1 transcripts initiate in a conserved region in the CpG island, and transcripts undergo complex splicing--all properties shared with the human gene. CpG island methylation is present in oocyte DNA and constitutes a germline-specific epigenetic mark. Mice with uniparental disomy (UPD) for Chr. 10 exhibit appropriate parent-of-origin dependent expression of Zac1, indicating that the absence of phenotypes comparable to aspects of human TNDM is not because imprinting of Zac1 is relaxed in these UPD mice.
Assuntos
Proteínas de Ciclo Celular/genética , Genes Supressores de Tumor , Impressão Genômica , Transativadores/genética , Fatores de Transcrição , Região 5'-Flanqueadora/genética , Processamento Alternativo , Animais , Sequência de Bases , Northern Blotting , Sequência Conservada/genética , Ilhas de CpG/genética , Éxons , Expressão Gênica , Genes/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Proteínas Supressoras de Tumor , Dissomia UniparentalRESUMO
A characteristic of imprinted genes is that the maternal and paternal alleles show differences in methylation. To perform a genome-wide screen for novel imprinted loci, we applied methylation-sensitive representational difference analysis (Me-RDA) to parthenogenetic mouse embryos, to identify differentially methylated regions (DMRs) methylated specifically on the maternal allele. We isolated a total of 26 distinct clones from known and novel DMRs and identified three novel imprinted genes. Nap1l5 is located on proximal chromosome 6 and encodes a protein with homology with nucleosome assembly proteins (NAPs); it has tissue-specific imprinting with expression from the paternal allele. We identified two DMRs on chromosome 15, a chromosome that was not thought to contain imprinted loci, and demonstrated that each is associated with a paternally expressed transcript. Peg13 gives rise to a noncoding RNA that is highly expressed in the brain and imprinted in all tissues examined. A DMR was also identified at the chromosome 15 Slc38a4 gene, which encodes a system A amino acid transporter; we show that Slc38a4 is imprinted in a tissue-specific manner. Interestingly, two of the three novel genes identified in this screen are located within the introns of other genes; their identification indicates that such "microimprinted" domains may be more common than previously thought.