Porcine ZBED6 regulates growth of skeletal muscle and internal organs via multiple targets

ZBED6 (zinc finger BED domain containing protein 6) is a transcription factor unique to placental mammals and its interaction with the IGF2 (insulin-like growth factor 2) locus plays a prominent role in the regulation of postnatal skeletal muscle growth. Here, we generated lean Bama miniature pigs by generating ZBED6-knockout (ZBED6(−/−)) and investigated the mechanism underlying ZBED6 in growth of muscle and internal organs of placental mammals. ZBED6(−/−) pigs show markedly higher lean mass, lean mass rate, larger muscle fiber area and heavier internal organs (heart and liver) than wild-type (WT) pigs. The striking phenotypic changes of ZBED6(-/-) pigs coincided with remarkable upregulation of IGF2 mRNA and protein expression across three tissues (gastrocnemius muscle, longissimus dorsi, heart). Despite a significant increase in liver weight, ZBED6(-/-) pigs show comparable levels of IGF2 expression to those of WT controls. A mechanistic study revealed that elevated methylation in the liver abrogates ZBED6 binding at the IGF2 locus, explaining the unaltered hepatic IGF2 expression in ZBED6(-/-) pigs. These results indicate that a ZBED6-IGF2-independent regulatory pathway exists in the liver. Transcriptome analysis and ChIP-PCR revealed new ZBED6 target genes other than IGF2, including cyclin dependent kinase inhibitor 1A (CDKN1A) and tsukushi, small leucine rich proteoglycan (TSKU), that regulates growth of muscle and liver, respectively.