Integrative epigenomic and transcriptomic profiling reveals organ-specific and coordinated cold stress responses in the brain and gill of Nile tilapia

Abstract

Cold stress is a major environmental challenge limiting the survival and productivity of tropical aquaculture species such as Nile tilapia (Oreochromis niloticus). The brain and gill represent two key organs that orchestrate systemic and environmental responses: the brain serves as the central thermosensory integrator and neuroendocrine control center, while the gill serves as the primary interface for respiration, ion regulation, and immune defense. However, the molecular mechanisms underlying their tissue-specific and potentially coordinated responses to cold remain unclear. Here, we applied integrative ATAC-seq and RNA-seq analyses to systematically investigate chromatin accessibility and gene expression dynamics in tilapia brain and gill tissues under cold stress. We identified thousands of differentially expressed genes and accessible regions, with significant correlations between transcriptional changes. Transcription factor footprinting revealed that Fra1 and Nrf act as key tissue-specific regulators, governing immune, apoptotic, and metabolic reprogramming in the brain and gill, respectively. Notably, the Fra1 module in the brain activated signaling pathways associated with stress response, neurodevelopment, and metabolic regulation which may influence peripheral responses by coordinating systemic physiological adjustments under cold stress, while Nrf-mediated regulation in the gill supported local homeostasis through redox and transport-related mechanisms. These findings highlight the hierarchical and organ-specific transcriptional control underlying cold adaptation in ectotherms. Our study provides the first chromatin accessibility atlas of cold-responsive regulatory networks across central and peripheral organs in fish, offering mechanistic insight and molecular targets for breeding cold-tolerant aquaculture strains.

Keywords

Cold stress / Chromatin accessibility / Transcriptional regulation / Epigenetic transcriptional adaptation / Nile tilapia

References

[1] Azuma M, Toyama R, Laver E, Dawid IB. Perturbation of rRNA synthesis in the bap28 mutation leads to apoptosis mediated by p53 in the zebrafish central nervous system. J Biol Chem, 2006, 281: 13309-13316

[2] Beisaw A, Kuenne C, Guenther S, Dallmann J, Wu CC, Bentsen M, Looso M, Stainier DYR. AP-1 contributes to chromatin accessibility to promote sarcomere disassembly and cardiomyocyte protrusion during zebrafish heart regeneration. Circ Res, 2020, 126: 1760-1778

[3] Bell AM, Utting C, Dickie AC, Kucharczyk MW, Quillet R, Gutierrez-Mecinas M, Razlan ANB, Cooper AH, Lan Y, Hachisuka J, Weir GA, Bannister K, Watanabe M, Kania A, Hoon MA, Macaulay IC, Denk F, Todd AJ. Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons. Proc Natl Acad Sci U S A, 2024, 121: e2314213121

[4] Bentsen M, Goymann P, Schultheis H, Klee K, Petrova A, Wiegandt R, Fust A, Preussner J, Kuenne C, Braun T, Kim J, Looso M (2020) ATAC-seq footprinting unravels kinetics of transcription factor binding during zygotic genome activation. Nat Commun 11:4267. https://doi.org/10.1038/s41467-020-18035-1

[5] Blasco FR, Taylor EW, Leite CAC, Monteiro DA, Rantin FT, McKenzie DJ. Tolerance of an acute warming challenge declines with body mass in Nile tilapia: evidence of a link to capacity for oxygen uptake. J Exp Biol, 2022, 225: jeb244287

[6] Chang RJA, Celino-Brady FT, Seale AP. Changes in cortisol and corticosteroid receptors during dynamic salinity challenges in Mozambique tilapia. Gen Comp Endocrinol, 2023, 342: 114340

[7] Chen S, Zhou Y, Chen Y, Gu J. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34: i884-i890

[8] Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, Workman C, Christmas R, Avila-Campilo I, Creech M, Gross B, Hanspers K, Isserlin R, Kelley R, Killcoyne S, Lotia S, Maere S, Morris J, Ono K, Pavlovic V, Pico AR, Vailaya A, Wang PL, Adler A, Conklin BR, Hood L, Kuiper M, Sander C, Schmulevich I, Schwikowski B, Warner GJ, Ideker T, Bader GD. Integration of biological networks and gene expression data using Cytoscape. Nat Protoc, 2007, 2: 2366-2382

[9] Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM, Li H. Twelve years of SAMtools and BCFtools. Gigascience, 2021, 10: giab008

[10] Diver MM, King JVL, Julius D, Cheng Y. Sensory TRP channels in three dimensions. Annu Rev Biochem, 2022, 91: 629-649

[11] El-Sayed AM, Khaled AA, Hamdan AM, Makled SO, Hafez EE, Saleh AA. The role of antifreeze genes in the tolerance of cold stress in the Nile tilapia (Oreochromis niloticus). BMC Genomics, 2023, 24: 476

[12] Endo S, Miyagi N, Matsunaga T, Ikari A. Rabbit dehydrogenase/reductase SDR family member 11 (DHRS11): its identity with acetohexamide reductase with broad substrate specificity and inhibitor sensitivity, different from human DHRS11. Chem Biol Interact, 2019, 305: 12-20

[13] Fathi N, Nirouei M, Salimian Rizi Z, Fekrvand S, Abolhassani H, Salami F, Ketabforoush AHME, Azizi G, Saghazadeh A, Esmaeili M, Almasi-Hashiani A, Rezaei N. Clinical, immunological, and genetic features in patients with NFKB1 and NFKB2 mutations: a systematic review. J Clin Immunol, 2024, 44: 160

[14] Gao S, Chang YM, Zhao XF, Sun B, Zhang LM, Liang LQ, Dong ZG. The effect of different bicarbonate alkalinity on the gill structure of Amur ide ( Leuciscus waleckii ). AHS, 2022, 44: 736-743

[15] Geletu TT, Zhao J. Genetic resources of Nile tilapia (Oreochromis niloticus Linnaeus, 1758) in its native range and aquaculture. Hydrobiologia, 2023, 850: 2425-2445

[16] George M, Reddy AP, Reddy PH, Kshirsagar S. Unraveling the NRF2 confusion: distinguishing nuclear respiratory factor 2 from nuclear erythroid factor 2. Ageing Res Rev, 2024, 98: 102353

[17] Grandi FC, Modi H, Kampman L, Corces MR. Chromatin accessibility profiling by ATAC-seq. Nat Protoc, 2022, 17: 1518-1552

[18] Gu Z, Eils R, Schlesner M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics, 2016, 32: 2847-2849

[19] Hannemann N, Cao S, Eriksson D, Schnelzer A, Jordan J, Eberhardt M, Schleicher U, Rech J, Ramming A, Uebe S, Ekici A, Cañete JD, Chen X, Bäuerle T, Vera J, Bogdan C, Schett G, Bozec A. Transcription factor Fra-1 targets arginase-1 to enhance macrophage-mediated inflammation in arthritis. J Clin Invest, 2019, 129: 2669-2684

[20] Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng JX, Murre C, Singh H, Glass CK. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell, 2010, 38: 576-589

[21] Hu P, Liu M, Zhang D, Wang J, Niu H, Liu Y, Wu Z, Han B, Zhai W, Shen Y, Chen L. Global identification of the genetic networks and cis-regulatory elements of the cold response in zebrafish. Nucleic Acids Res, 2015, 43: 9198-9213

[22] Hu P, Liu M, Liu Y, Wang J, Zhang D, Niu H, Jiang S, Wang J, Zhang D, Han B, Xu Q, Chen L. Transcriptome comparison reveals a genetic network regulating the lower temperature limit in fish. Sci Rep, 2016, 6: 28952

[23] Huang J, Zhou Q, Gao M, Nowsheen S, Zhao F, Kim W, Zhu Q, Kojima Y, Yin P, Zhang Y, Guo G, Tu X, Deng M, Luo K, Qin B, Machida Y, Lou Z. Tandem deubiquitination and acetylation of SPRTN promotes DNA-protein crosslink repair and protects against aging. Mol Cell, 2020, 79: 824-835.e5

[24] Huang S, Zhao W, Yan C, Qi J, Li X, Wu Z, Chan J, Chen L, Hu P. Elucidating the chromatin-driven transcription regulatory networks response to Streptococcus agalactiae infection under low temperature in Nile tilapia. Fish Shellfish Immunol, 2025, 164: 110464

[25] Jiao H, Huang S, Zhang M, Huang Q, Yan C, Qi J, Cheng J, Xu Y, Zhai X, Li X, Zhan S, Li W, Wu Z, Chan J, Chen L, Hu P. Uncovering the chromatin-mediated transcriptional regulatory network governing cold stress responses in fish immune cells. J Genet Genomics, 2025

[26] Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol, 2019, 37: 907-915

[27] Kristiansen TS, Fernö A, Pavlidis MA, van de Vis H (Eds) (2020) The Welfare of Fish. In: Animal Welfare. Springer, Cham. https://doi.org/10.1007/978-3-030-41675-1

[28] Liao Y, Smyth GK, Shi W. Featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 2014, 30: 923-930

[29] Liu H, Wang L, Weng X, Chen H, Du Y, Diao C, Chen Z, Liu X. Inhibition of Brd4 alleviates renal ischemia/reperfusion injury-induced apoptosis and endoplasmic reticulum stress by blocking FoxO4-mediated oxidative stress. Redox Biol, 2019, 24: 101195

[30] Liu R, Liu R, Song G, Li Q, Cui Z, Long Y. Mitochondria dysfunction and cell apoptosis limit resistance of Nile tilapia (Oreochromis niloticus) to lethal cold stress. Animals, 2022, 12: 2382

[31] Liu X, Bie XM, Lin X, Li M, Wang H, Zhang X, Yang Y, Zhang C, Zhang XS, Xiao J. Uncovering the transcriptional regulatory network involved in boosting wheat regeneration and transformation. Nat Plants, 2023, 9: 908-925

[32] Long Y, Li L, Li Q, He X, Cui Z. Transcriptomic characterization of temperature stress responses in larval zebrafish. PLoS ONE, 2012, 7: e37209

[33] Los DA, Murata N. Membrane fluidity and its roles in the perception of environmental signals. Biochim Biophys Acta, 2004, 1666: 142-157

[34] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol, 2014, 15: 550

[35] Mendoza DD. Temperature sensing by membranes. Annu Rev Microbiol, 2014, 68: 101-116

[36] Mommsen TP, Vijayan MM, Moon TW. Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation. Rev Fish Biol Fish, 1999, 9: 211-268

[37] Murphy MR, Ramadei A, Doymaz A, Varriano S, Natelson DM, Yu A, Aktas S, Mazzeo M, Mazzeo M, Zakusilo G, Kleiman FE. Long non-coding RNA generated from CDKN1A gene by alternative polyadenylation regulates p21 expression during DNA damage response. Nucleic Acids Res, 2023, 51: 11911-11926

[38] Mykkänen OT, Kirjavainen PV, Pulkkinen L, Mykkänen H, Törrönen R, Kolehmainen M, Adriaens M, Laaksonen DE, Puupponen-Pimiä R, Poutanen K (2014) Reduction of subacute endotoxemia could mediate beneficial effects of Vaccinium myrtillus in metabolic syndrome - a PBMC gene expression study. Acta Horticul 1017: 369–379. https://doi.org/10.17660/ActaHortic.2014.1017.45

[39] Powell S, Forslund K, Szklarczyk D, Trachana K, Roth A, Huerta-Cepas J, Gabaldón T, Rattei T, Creevey C, Kuhn M, Jensen LJ, von Mering C, Bork P. eggNOG v4.0: nested orthology inference across 3686 organisms. Nucleic Acids Res, 2014, 42: D231-D239

[40] Ramírez F, Dündar F, Diehl S, Grüning BA, Manke T. Deeptools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res, 2014, 42: W187-W191

[41] Reid CH, Patrick PH, Rytwinski T, Taylor JJ, Willmore WG, Reesor B, Cooke SJ. An updated review of cold shock and cold stress in fish. J Fish Biol, 2022, 100: 1102-1137

[42] Rossi A, Bacchetta C, Cazenave J. Effect of thermal stress on metabolic and oxidative stress biomarkers of Hoplosternum littorale (Teleostei, Callichthyidae). Ecol Indic, 2017, 79: 361-370

[43] Sokolova I. Bioenergetics in environmental adaptation and stress tolerance of aquatic ectotherms: linking physiology and ecology in a multi-stressor landscape. J Exp Biol, 2021, 224: jeb236802

[44] Stark R, Brown G (2012) DiffBind: Differential binding analysis of ChIP-Seq peak data.

[45] Su K, Ma Y, Zhou M, Liu Y, Li C, Jiang Y, Wu Q, Peng G, Wang Y, Fan S. De novo missense variants of UNC13A are implicated in epileptic encephalopathies and neurodevelopmental disorders. Genes Dis, 2024, 12: 101315

[46] Szeto AC, Clark PA, Ferreira AC, Heycock M, Griffiths EL, Jou E, Mannion J, Luan SL, Storrar S, Knolle MD, Kozik P, Jolin HE, Fallon PG, McKenzie AN. Mef2d potentiates type-2 immune responses and allergic lung inflammation. Science, 2024, 384: eadl0370

[47] Topal A, Özdemir S, Arslan H, Çomaklı S. How does elevated water temperature affect fish brain? (A neurophysiological and experimental study: Assessment of brain derived neurotrophic factor, cFOS, apoptotic genes, heat shock genes, ER-stress genes and oxidative stress genes). Fish Shellfish Immunol, 2021, 115: 198-204

[48] Verleih M, Rebl A, Köllner B, Korytář T, Köbis JM, Kühn C, Wimmers K, Goldammer T. Iron-sulfur cluster scaffold (ISCU) gene is duplicated in salmonid fish and tissue and temperature dependent expressed in rainbow trout. Gene, 2013, 512: 251-258

[49] Wan Q, Su J. Transcriptome analysis provides insights into the regulatory function of alternative splicing in antiviral immunity in grass carp (Ctenopharyngodon idella). Sci Rep, 2015, 5: 12946

[50] Wen B, Jin SR, Chen ZZ, Gao JZ. Physiological responses to cold stress in the gills of discus fish (Symphysodon aequifasciatus) revealed by conventional biochemical assays and GC-TOF-MS metabolomics. Sci Total Environ, 2018, 640: 1372-1381

[51] Wood CM, Eom J. The osmorespiratory compromise in the fish gill. Comp Biochem Physiol A Mol Integr Physiol, 2021, 254: 110895

[52] Wu SM, Liu JH, Shu LH, Chen CH. Anti-oxidative responses of zebrafish (Danio rerio) gill, liver and brain tissues upon acute cold shock. Comp Biochem Physiol A Mol Integr Physiol, 2015, 187: 202-213

[53] Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, Feng T, Zhou L, Tang W, Zhan L, Fu X, Liu S, Bo X, Yu G. ClusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation, 2021, 2: 100141

[54] Yang H, Luan Y, Liu T, Lee HJ, Fang L, Wang Y, Wang X, Zhang B, Jin Q, Ang KC, Xing X, Wang J, Xu J, Song F, Sriranga I, Khunsriraksakul C, Salameh T, Li D, Choudhary MNK, Topczewski J, Wang K, Gerhard GS, Hardison RC, Wang T, Cheng KC, Yue F. A map of cis-regulatory elements and 3D genome structures in zebrafish. Nature, 2020, 588: 337-343

[55] York JM. Temperature activated transient receptor potential ion channels from Antarctic fishes. Open Biol, 2023, 13: 230215

[56] York JM, Zakon HH. Evolution of transient receptor potential (TRP) ion channels in Antarctic fishes (Cryonotothenioidea) and identification of putative thermosensors. Genome Biol Evol, 2022, 14: evac009

[57] Yu H, Shen Y, Sun J, Xu X, Wang R, Xuan Y, Lu L, Li J. Molecular cloning and functional characterization of the NFIL3/E4BP4 transcription factor of grass carp, Ctenopharyngodon idella. Dev Comp Immunol, 2014, 47: 215-222

[58] Yu G, Wang LG, He QY. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics, 2015, 31: 2382-2383

[59] Zeng F, He J, Jin X, Liao Q, Chen Z, Peng H, Zhou Y. FRA-1: a key factor regulating signal transduction of tumor cells and a potential target molecule for tumor therapy. Biomed Pharmacother, 2022, 150: 113037

[60] Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, Liu XS. Model-based analysis of ChIP-Seq (MACS). Genome Biol, 2008, 9: R137

[61] Zhao T, Zhang J, Lei H, Mu C, Chen L, Liu Q, Luo Q, Zhang C, Long Y, Su J, Wang Y, Li Z, Sun J, Chen G, Li Y, Liao X, Shang Y, Hu G, Chen Q, Zhu Y. NRF1-mediated mitochondrial biogenesis antagonizes innate antiviral immunity. EMBO J, 2023, 42: e113258

[62] Zuo J, Wu D, Zhang Y, Luo H, Jing G, Yuan M, Fang Q, Yang C, Wang X, Wu X, Song X. VCPIP1 negatively regulates NF-κB signaling pathways by deubiquitinating and stabilizing Erbin in MDP-stimulated macrophages. Int Immunopharmacol, 2024, 143: 113622

Funding

Innovative Research Group Project of the National Natural Science Foundation of China(No. 32373113)

National Natural Science Foundation of China(No. 32403003)

RIGHTS & PERMISSIONS

The Author(s)