A novel mutation in the promoter region of RPL8 regulates milk fat traits in dairy cattle by binding transcription factor Pax6.

ABSTRACT

Ribosomal protein L8 (RPL8) was considered as a promising candidate gene for the milk fat percentage trait in dairy cattle in our previous genome-wide association studies, but the mechanism remains to be determined. Here we investigated the molecular mechanism underlying the effect of bovine RPL8 on milk fat percentage. We demonstrated that RPL8 silencing in bovine mammary epithelial cells affected the expression of genes encoding fat-related enzymes (ACACA, FASN, ACSS1, FABP3, SREBP-1, DGAT1, GPAM, PLIN2, PLIN5 and CIDEA). Furthermore, we showed here that a single nucleotide polymorphism, g.-931G > T (chr141508300, UMD3.1) in the putative RPL8 promoter region significantly reduced its promoter activity. Interestingly, this decrease in activity was paralleled by lower RPL8 expression in mammary gland tissues of dairy cattle with the homozygous TT genotype compared to that of cattle with the wild-type homozygous GG genotype. Importantly, we found g.-931G > T added a paired box 6 (Pax6)-binding site and this mutation located in the presumed Pax6-binding site. EMSA and co-immunoprecipitation (Co-IP) assays confirmed the interaction between RPL8 and Pax6 and the T allele exhibited a higher affinity of DNA/protein interactions than G allele, suggesting that Pax6 is an important transcription factor for RPL8 expression. In addition, lactating cows with the GG and GT genotypes presented a significant decrease in milk fat percentage compared to cows with TT genotypes. Altogether, our study indicated that g.-931G > T at RPL8 promoter altered its expression by affecting the interplay between Pax6 and RPL8, which may account for the association with milk fat traits. Findings herein first elucidated the biological function of RPL8 gene in milk fat and the identified SNP g.-931G > T may be considered as genetic makers for breeding in dairy cattle.