Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome

Abstract

Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN) is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation betweenOPNmRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcineOPNwas frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that theOPNpromoter region −2615 to −2239 nucleotides (nt) may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation ofOPNpromoter was found in dose-dependent response of 5-aza-dc treatment and followed theOPNmRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation ofOPNpromoter activity. These data suggested that methylation in theOPNpromoter plays a crucial role in the regulation ofOPNexpression that we found in cloned pigs genome.