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Many studies have proved that DNA methylation can regulate gene expression and further affect skeletal muscle growth and development of pig, whereas the mechanisms of how DNA methylation or gene expression alteration ultimately lead to phenotypical differences between the cloned and natural mating pigs remain elusive. This study aimed to investigate genome-wide gene expression and DNA methylation differences between abnormally cloned and normally natural mating piglets and identify molecular markers related to skeletal muscle growth and development in pig. The DNA methylation and genome-wide gene expression in the two groups of piglets were analysed through methylated DNA immunoprecipitation binding high-throughput sequencing and RNA sequencing respectively. We detected 1493 differentially expressed genes between the two groups, of which 382 genes were also differentially methylated. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative and monotonic correlation with gene expression levels around the transcription start site of genes. By contrast, no notable monotonic correlation was observed in other regions. Furthermore, we identified some interesting genes and signalling pathways ( e.g. myosin, heavy chain 7 and mammalian target of rapamycin) which possibly play essential roles in skeletal muscle growth and development. The results of this study provide insights into the relationship of DNA methylation with gene expression in newborn piglets and into the mechanisms in abnormally cloned animals through somatic cell nuclear transfer. [ABSTRACT FROM AUTHOR]

Abnormal phenotypes in cloned pigs can be partly due to changes in epigenetic modifications such as methylation levels of promoter CpG islands. Neuronatin is an imprinted gene, conserved in human, pig, cattle and mouse, which is expressed exclusively from the paternal allele. Three CpG islands located in the promoter region of the porcine neuronatin gene have the potential to regulate the gene expression by cytosine methylation. To illustrate whether neuronatin was differentially expressed among nuclear transfer macroglossia-positive and nuclear transfer macroglossia-negative pigs and in vitro-fertilized pigs, we detected its expression level by qRT-PCR and further quantified methylation levels by pyrosequencing DNA from the liver. The results showed that neuronatin was expressed at a significantly higher level in livers of nuclear transfer macroglossia-positive pigs compared with normal cloned and in vitro-fertilized pigs. Livers of nuclear transfer macroglossia-positive pigs also had a significantly lower methylation level at CpG island 2 and CpG island 3 in the promoter region. [ABSTRACT FROM AUTHOR]