Integrated Multi-Omics Reveals Epigenetic Regulation of Beef Color by the HMOX2 Gene

Integrated Multi-Omics Reveals Epigenetic Regulation of Beef Color by the HMOX2 Gene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Integrated Multi-Omics Reveals Epigenetic Regulation of Beef Color by the HMOX2 Gene Xiao Wang, Xiangmin Yan, Zhen Ma, Lixing Yuan, Na Su, Yuxia Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9256259/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version 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. HMOX2 Methylation Meat color lightness Multi-omics Exercise Housing systems Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial1.xlsx SupplementaryMaterial2.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 27 Apr, 2026 Reviewers invited by journal 08 Apr, 2026 Editor assigned by journal 03 Apr, 2026 Editor invited by journal 31 Mar, 2026 Submission checks completed at journal 31 Mar, 2026 First submitted to journal 31 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9256259","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":621578414,"identity":"e1e9d726-eadd-4bdd-b9ef-595e5e03701b","order_by":0,"name":"Xiao Wang","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Wang","suffix":""},{"id":621578416,"identity":"2476bec8-19dd-4e6a-941e-3fac56ba1cd3","order_by":1,"name":"Xiangmin Yan","email":"","orcid":"","institution":"Xinjiang Academy of Animal 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Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HMOX2, Methylation, Meat color lightness, Multi-omics, Exercise, Housing systems","lastPublishedDoi":"10.21203/rs.3.rs-9256259/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9256259/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Test cattle fitted with smart collars to quantify exercise duration and intensity.\u003c/p\u003e\n\u003cp\u003eAn 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e 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 (\u003cem\u003eHMOX1\u003c/em\u003e and\u003cem\u003e HMOX2\u003c/em\u003e) and misfolded protein binding (\u003cem\u003eDNAJB9\u003c/em\u003e) were significantly upregulated, whereas those related to chemokine activity (\u003cem\u003eCCL21 \u003c/em\u003eand \u003cem\u003eCXCL10\u003c/em\u003e) were downregulated. Furthermore, insulin resistance pathway genes (\u003cem\u003eIRS2\u003c/em\u003e, \u003cem\u003ePPARGC1A\u003c/em\u003e, and \u003cem\u003eSOCS3\u003c/em\u003e) 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 \u003cem\u003eHMOX1\u003c/em\u003eand \u003cem\u003eHMOX2\u003c/em\u003e. Critically, we detected and validated that \u003cem\u003eHMOX2\u003c/em\u003e was significantly hypermethylated in tie stalls cattle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e We propose a methylation-mediated mechanism in which restricted exercise and inferior animal welfare conditions induce \u003cem\u003eHMOX2\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Integrated Multi-Omics Reveals Epigenetic Regulation of Beef Color by the HMOX2 Gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-16 10:29:08","doi":"10.21203/rs.3.rs-9256259/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"107548720190077142104066620168437259304","date":"2026-04-27T23:24:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-08T20:12:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-03T14:26:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-31T18:37:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-31T15:15:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2026-03-31T15:09:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"392f3188-c0d7-4e66-96c7-e8f58735f5e1","owner":[],"postedDate":"April 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-16T10:29:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-16 10:29:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9256259","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9256259","identity":"rs-9256259","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}