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

Abstract

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.

A Comprehensive Analysis of the Myocardial Transcriptome in ZBED6-Knockout Bama Xiang Pigs

“…After ZBED6 knockout, the IGV of RNA-seq results showed that IGF2 was significantly upregulated in myocardial tissue. Previous studies have confirmed with RT–qPCR that IGF2 expression is significantly higher in ZBED6 -KO group than in the ZBED6 -WT group [ 26 ], which is consistent with the results in mice and cattle [ 4 , 6 , 7 ]. We again confirmed the reliability of our data.…”

“…Previous histological analysis of myocardial HE-stained sections showed that knockout of ZBED6 resulted in thicker myocardial muscle fibers and a reduction in connective tissue. These findings are complementary to the heart weight results, indicating that ZBED6 knockout could promote the growth and development of pig hearts to a certain extent [ 26 ].…”

“…ZBED6 can inhibit the expression of IGF2 , reduce the deposition of subcutaneous fat, promote the growth of skeletal muscle [ 26 ] and also affect the growth and development of the myocardium [ 40 ]. By knocking in the same IGF2 intron 3–3072 site, ZBED6 improved meat production in Chinese Bama Xiang pigs [ 41 ].…”

“…In our previous research, the significantly DEGs (differentially expressed genes) were enriched in signals of muscle development and the immune response between the ZBED6 -KO ( ZBED6 knockout) Bama Xiang pigs and the ZBED6 -WT (wild-type) group. This indicated that ZBED6 was likely to affect the related functions of heart tissue [ 26 ]. Although our early study provided clues for the ZBED6 gene in the heart development of domestic pigs, how lncRNAs affect heart development and the underlying regulatory mechanism in ZBED6 -KO pigs remain unclear.…”

Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System

“…The G-to-A mutation or ZBED6-binding site deletion of IGF2 disrupts the binding of the transcriptional repressor ZBED6, thereby increasing the expression of IGF2 [ 12 , 13 ]. Both Zbed6 -knockout mice and ZBED6-binding-site-mutant mice showed enlargement of muscles and internal organs, similar to the findings in pigs with corresponding mutations [ 14 , 15 ]. Furthermore, the muscle enlargement in mice was significantly greater than that in pigs (15–20% vs. 3–4%, respectively), probably because of the long-term selection of pigs for muscle growth [ 15 ].…”