Integrated multi-omics reveals epigenetic regulation of beef color by the HMOX2 gene

Abstract Background Among the critical quality attributes of fresh meat, color serves as the primary factor driving consumer purchase decisions. Building on our prior findings that loose housing cattle produce darker semitendinosus muscle (lower CIE L*) than do tie stalls cattle, we aimed to elucidate the molecular mechanisms underlying this housing system-induced variation in meat color lightness. Methods Test cattle fitted with smart collars to quantify exercise duration and intensity. An integrated multi-omics approach, combining transcriptomics, nontargeted metabolomics, and reduced representation bisulfite sequencing, was employed to profile differences between cattle with distinct meat color phenotypes. Key findings were subsequently validated using real-time quantitative PCR, western blotting, and bisulfite amplicon sequencing. Results Our analyses revealed that tie stalls cattle, which experience restricted exercise (3.61 h/day vs. 6.76 h/day) and inferior animal welfare conditions, showed significant transcriptional changes compared with loose housing cattle. Specifically, genes associated with heme oxidation ( HMOX1 and HMOX2 ) and misfolded protein binding ( DNAJB9 ) were significantly upregulated, whereas those related to chemokine activity ( CCL21 and CXCL10 ) were downregulated. Furthermore, insulin resistance pathway genes ( IRS2 , PPARGC1A , and SOCS3 ) were upregulated and key exercise-responsive pathways, including cGMP-PKG, Notch, calcium, and PPAR signaling, were downregulated in tie stalls cattle. Metabolomics revealed that biliverdin was a critical metabolite whose abundance was negatively correlated with the expression of HMOX1 and HMOX2 . Critically, we detected and validated that HMOX2 was significantly hypermethylated in tie stalls cattle. Conclusions We propose a methylation-mediated mechanism in which restricted exercise and inferior animal welfare conditions induce HMOX2 hypermethylation, increase its transcription and HO-2 protein synthesis, and accelerate heme degradation. This process enhances meat color lightness, albeit at the cost of some oxygen-carrying capacity, while mitigating oxidative stress, inflammation, and cellular damage.