CLOCK is an Epigenetic Integrator of Circadian Rhythm and T Cell Immunity

CLOCK is an Epigenetic Integrator of Circadian Rhythm and T Cell Immunity | 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 Article CLOCK is an Epigenetic Integrator of Circadian Rhythm and T Cell Immunity Khashayarsha Khazaie, Shirin Eyvazi, Abu Osman, Abdulrahman Saadalla, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9107841/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The circadian clock imposes a critical yet incompletely understood layer of regulation on adaptive immunity. T helper 17 (Th17) antimicrobial immune responses including expression of IL-17A by lamina propria CD4⁺ T cells, exhibit diurnal variation and are sensitive to circadian disruption. While the core circadian regulators CLOCK and BMAL1 canonically function as a heterodimer to drive rhythmic gene expression, emerging evidence suggests they may have distinct regulatory roles. Here we integrated ChIP-seq, ATAC-seq, and RNA-seq analyses in naïve CD4⁺ T cells to reveal that the CLOCK-BMAL1 complex controls circadian and metabolic programs through promoter binding, whereas exclusive CLOCK binding at promoters, together with CLOCK-BMAL1 binding at enhancers, regulates immune-associated genes. Using the mutant CLOCKΔ19, which lacks the transactivation domain, we observed disrupted circadian transcription and globally reduced chromatin accessibility, alongside increased accessibility at Th17-associated loci that their altered temporal regulation. Functionally, ClockΔ19 produces β-catenin stabilization in T cells, pronounced expansion of RORγt⁺ CD4⁺ T cells and Treg cells, impaired Treg suppression of Th17 responses, heightened Th17 responses, and reduced IFN-γ production during viral infection. Collectively, these findings define BMAL1 dependent and independent CLOCK functions that program naïve CD4⁺ T cell fate, restrain Th17 differentiation, and preserve immune homeostasis. Biological sciences/Immunology/Mucosal immunology Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte differentiation Epigenetics CLOCK BMAL1 Circadian Rhythm Immune Signaling Naïve CD4 T Cell Lineage Specification Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable2.uppathwaystranscriptionClock19vswild.csv Gene Set Enrichment Analysis (GSEA) results showing pathways upregulated in ClockΔ19 compared with wild-type (WT) samples. SupplementaryTable4.geneslistforchipseq.csv Genes associated with ChIP-seq peaks identified through Venn diagram comparison. SupplementaryTable3.geneslistforATACSeq.csv Genes associated with ATAC-seq peaks from the AZ4 and AZ16 conditions identified through Venn diagram comparison. SupplementaryTable1.downpathwaystranscriptionClock19vswild.csv Gene Set Enrichment Analysis (GSEA) results showing pathways downregulated in ClockΔ19 compared with wild-type (WT) samples. Cite Share Download PDF Status: Under Review Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-9107841","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":612239300,"identity":"b6c96fc9-e465-43ab-86f1-6ea3acd1f24a","order_by":0,"name":"Khashayarsha 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T helper 17 (Th17) antimicrobial immune responses including expression of IL-17A by lamina propria CD4⁺ T cells, exhibit diurnal variation and are sensitive to circadian disruption. While the core circadian regulators CLOCK and BMAL1 canonically function as a heterodimer to drive rhythmic gene expression, emerging evidence suggests they may have distinct regulatory roles. Here we integrated ChIP-seq, ATAC-seq, and RNA-seq analyses in naïve CD4⁺ T cells to reveal that the CLOCK-BMAL1 complex controls circadian and metabolic programs through promoter binding, whereas exclusive CLOCK binding at promoters, together with CLOCK-BMAL1 binding at enhancers, regulates immune-associated genes. Using the mutant CLOCKΔ19, which lacks the transactivation domain, we observed disrupted circadian transcription and globally reduced chromatin accessibility, alongside increased accessibility at Th17-associated loci that their altered temporal regulation. Functionally, ClockΔ19 produces β-catenin stabilization in T cells, pronounced expansion of RORγt⁺ CD4⁺ T cells and Treg cells, impaired Treg suppression of Th17 responses, heightened Th17 responses, and reduced IFN-γ production during viral infection. 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