The 67-bp Variable Duplication in the Bovine ADIPOQ Promoter Is Associated with Fat-Related Carcass Traits and Modulates Promoter Activity through a HES5-Responsive Motif

Abstract Background: The ADIPOQ gene is involved in lipid metabolism, energy homeostasis, and fat deposition in cattle. Although previous studies have suggested that regulatory variants in the ADIPOQ promoter may contribute to phenotypic variation, the functional and phenotypic significance of the 67-bp variable duplication in the bovine ADIPOQ promoter remains incompletely understood. This study aimed to characterize the regulatory effect of this duplication and evaluate its association with carcass-related traits in Gaoqing Black cattle. Results: Analysis of public cattle transcriptomic data showed that ADIPOQ had an adipose-enriched expression pattern. Genotyping of 461 Gaoqing Black cattle identified three genotypes at the 67-bp duplication locus, with 1D/1D being predominant, 1D/2D present at a low frequency, and 2D/2D rare. Dual-luciferase reporter assays in 293T and CHO cells showed that increasing copy number of the 67-bp duplication reduced ADIPOQ promoter activity, indicating a copy number-dependent repressive effect. Transcription factor prediction and functional validation suggested that HES5 may contribute to this repression through a binding site within the duplicated sequence, and mutation of the predicted binding motif abolished the HES5-associated inhibitory effect. Association analyses further showed that, compared with 1D/1D cattle, 1D/2D males had lower brisket fat weight and shank meat weight, while 1D/2D females had lower carcass fat weight. Conclusions: The 67-bp variable duplication in the bovine ADIPOQ promoter acts as a functional regulatory variant that suppresses promoter activity and is associated with selected carcass and fat-related traits in Gaoqing Black cattle. These findings provide insight into the regulatory architecture of ADIPOQ and suggest that this promoter variant may be useful for understanding genetic variation in fat deposition-related traits in cattle.