Effect of polymorphisms in the 5’-flanking sequence of MC1R on feather color in Taihang chickens

Abstract

MC1R plays an important role in the regulation of the formation, transfer, and deposition of melanin in animals and is important for determining coat color. Many studies have reported on single nucleotide polymorphisms (SNPs) in the coding sequence of MC1R. However, few studies have investigated the polymorphisms in the 5’-flanking sequence of MC1R. In this study, we sequenced 2000 bp of the 5’-flanking sequence of MC1R in 300 Taihang chickens with brown feathers (MTH) and 300 Taihang chickens with black feathers (HTH). The sequencing results showed that 4 SNPs (MC1R g.18838722 G > C, g.18838624 T > C, g.18838694 G > A, and g.18838624 C > T) were located in the 5’-flanking sequence of MC1R between the MTH and HTH groups. Association analysis showed that there was a significant correlation between the 4 SNPs and feather color in Taihang chickens. The correlation between MC1R g.18838624 T >C and feather color of Taihang chicken was 100%, of which the CC (E1) genotype is MTH and the TT (E2) genotype is HTH. Furthermore, there was a significant correlation between MC1R g.18838624 T > C and egg production at 302 d. E1 (184.14 ± 0.674) was significantly higher than that in E2 (181.75 ± 0.577) (P < 0.05). Luciferase reporter assays were used to detect the transcriptional activity of MC1R with different SNP genotypes. The results showed that the luciferase activity of E2 was significantly higher than that of E1 (P < 0.05). In addition, transcription factor-binding site predictions showed that E2 creates a new binding site for ZEB1. RT‒qPCR results revealed that the expression of MC1R in E2 was significantly lower than that in E1 (P < 0.05), and the expression of ZEB1 in E2 was significantly higher than that in E1 (P < 0.05). Overexpression and shRNA experiments demonstrated that ZEB1 regulates the expression of MC1R in DF1 cells. ZEB1 has a negative regulatory effect on the transcriptional activity of MC1R; it inhibits the expression of MC1R and affects the feather color of Taihang chickens. This study provides new insight into the molecular mechanism of feather color formation and the transcriptional regulation of MC1R in Taihang chickens.