In this study, we employed a dual-luciferase reporter assay and electrophoretic mobility shift analysis (EMSA) in vitro to explore whether a 12-base pair (bp) insertion/deletion (InDel) variant (namely g.14798187_14798188insTCCCTGCCCCCT) within intron 2 of the chicken BMP2 gene, which was significantly associated with chicken abdominal fat weight and abdominal fat percentage, is a functional marker and its potential regulatory mechanism. The reporter analysis demonstrated that the luciferase activity of the deletion allele was extremely significantly higher than that of the insertion allele (p < 0.01). A bioinformatics analysis revealed that compared to the deletion allele, the insertion allele created a transcription factor binding site of nuclear factor-kappa B (NF-κB), which exhibited an inhibitory effect on fat deposition. A dual-luciferase reporter assay demonstrated that the inhibitory effect of NF-κB on the deletion allele was stronger than that on the insertion allele. EMSA indicated that the binding affinity of NF-κB for the insertion allele was stronger than that for the deletion allele. In conclusion, the 12-bp InDel chicken BMP2 gene variant is a functional variant affecting fat deposition in chickens, which may partially regulate BMP2 gene expression by affecting the binding of transcription factor NF-κB to the BMP2 gene.

Reducing abdominal fat (AF) in broilers is a key objective in broiler breeding, as excessive AF deposition has an unfavorable effect on reproductive and growth traits. Fatty acid synthase gene (FASN) encodes the fatty acid synthase, which promotes fatty acid synthesis and lipid synthesis. Our previous study identified a 7-bp InDel variant (g.4944396_4944402delGACTTCA, rs1060467633) in intron 42 of chicken FASN, demonstrating a marked association with chicken AF. To investigate the underlying mechanism of this association, we conducted dual-luciferase reporter assay, bioinformatics analysis and electrophoretic mobility shift assay (EMSA). Luciferase reporter showed that the deletion allele had significantly higher activity than the insertion allele (p < 0.05), indicating its potential role in regulating gene expression. Bioinformatics analysis and EMSA indicated that the insertion allele created binding site for cAMP-responsive element-binding protein 1 (CREB1). Luciferase reporter confirmed that the insertion allele suppresses FASN expression through CREB1, with a stronger inhibitory effect than the deletion allele. In conclusion, rs1060467633, as a functional variant, regulates FASN expression and AF deposition by altering the binding to CREB1 in chickens.