Tet Methylcytosine Dioxygenase 3 Promotes Cardiovascular Senescence by DNA 5-Hydroxymethylcytosine-Mediated Sp1 Transcription Factor Expression

Abstract

Cellular senescence is a significant contributor to various age-related diseases. Tet methylcytosine dioxygenase 3 (TET3) is a pivotal regulator of epigenetic modifications, and this study aimed to elucidate its role in cellular senescence. The study utilized replication and paraquat (PQ)-induced senescent endothelial cells, as well as TET3 heterozygous, p53 heterozygous, and PQ-induced senescent mice as experimental models. Senescent endothelial cells were analyzed using hydromethylated DNA immunoprecipitation sequencing, β-galactosidase staining, real-time PCR, western blotting, immunofluorescence staining, dot blot, chromatin immunoprecipitation assay, and luciferase reporter assays. These analyses were conducted following TET3 knockdown and gene overexpression. TET3 is instrumental in the elevation of 5-hydroxymethylcytosine (5-hmC) levels in both replication and PQ-induced senescent endothelial cells, as well as in the cardiovascular systems of PQ-induced aging mice. TET3 significantly promoted cellular senescence in PQ-induced endothelial cells and mice. TET3 facilitates the upregulation of the Sp1 transcription factor (SP1) through 5-hmC modification, leading to a synergistic interaction between SP1 and ETS proto-oncogene 1 that further enhances p53 expression. Moreover, p53 not only promotes cellular senescence in vitro and in vivo but also reciprocally enhances TET3 and 5-hmC levels. These findings underscore the critical role of elevated TET3 and 5-hmC levels in cellular senescence.

Keywords

cellular senescence / DNA 5-hydroxymethylcytosine / Sp1 transcription factor / Tet methylcytosine dioxygenase 3 / tumor protein p53

References

[1] C. Lopez-Otin, M. A. Blasco, L. Partridge, M. Serrano, and G. Kroemer, “Hallmarks of Aging: An Expanding Universe,” Cell 186, no. 2 (Jan( 2023): 243-278.

[2] J. H. Yang, M. Hayano, P. T. Griffin, et al., “Loss of Epigenetic Information as a Cause of Mammalian Aging,” Cell 186, no. 2 (Jan( 2023): 305-326 e327.

[3] G. Ficz, M. R. Branco, S. Seisenberger, et al., “Dynamic Regulation of 5-hydroxymethylcytosine in Mouse ES Cells and During Differentiation,” Nature 473, no. 7347 (May( 2011): 398-402.

[4] K. P. Koh, A. Yabuuchi, S. Rao, et al., “Tet1 and Tet2 Regulate 5-hydroxymethylcytosine Production and Cell Lineage Specification in Mouse Embryonic Stem Cells,” Cell Stem Cell 8, no. 2 (Feb 2011): 200-213.

[5] H. Wu, A. C. D'Alessio, S. Ito, et al., “Dual Functions of Tet1 in Transcriptional Regulation in Mouse Embryonic Stem Cells,” Nature 473, no. 7347 (May( 2011): 389-393.

[6] Q. Shen, Q. Zhang, Y. Shi, et al., “Tet2 promotes Pathogen Infection-induced Myelopoiesis Through mRNA Oxidation,” Nature 554, no. 7690 (Feb 2018): 123-127.

[7] Q. Zhang, K. Zhao, Q. Shen, et al., “Tet2 is Required to Resolve Inflammation by Recruiting Hdac2 to Specifically Repress IL-6,” Nature 525, no. 7569 (Sep( 2015): 389-393.

[8] J. J. Fuster, S. MacLauchlan, M. A. Zuriaga, et al., “Clonal Hematopoiesis Associated With TET2 Deficiency Accelerates Atherosclerosis Development in Mice,” Science 355, no. 6327 (Feb 2017): 842-847.

[9] L. Hu, Z. Li, J. Cheng, et al., “Crystal Structure of TET2-DNA Complex: Insight Into TET-mediated 5mC Oxidation,” Cell 155, no. 7 (Dec 2013): 1545-1555.

[10] D. Wu, D. Hu, H. Chen, et al., “Glucose-regulated Phosphorylation of TET2 by AMPK Reveals a Pathway Linking Diabetes to Cancer,” Nature 559, no. 7715 (Jul( 2018): 637-641.

[11] Y. Wang, S. Sano, Y. Yura, et al., “Tet2-mediated Clonal Hematopoiesis in Nonconditioned Mice Accelerates Age-associated Cardiac Dysfunction,” JCI Insight 5, no. 6 (Mar( 2020).

[12] I. Puig, S. P. Tenbaum, I. Chicote, et al., “TET2 controls Chemoresistant Slow-cycling Cancer Cell Survival and Tumor Recurrence,” Journal of Clinical Investigation 128, no. 9 (Aug 2018): 3887-3905.

[13] A. Kawabe, K. Yamagata, S. Kato, et al., “Role of DNA Dioxygenase Ten-Eleven Translocation 3 (TET3) in Rheumatoid Arthritis Progression,” Arthritis Research & Therapy 24, no. 1 (Sep( 2022): 222.

[14] M. Wagner, J. Steinbacher, T. F. Kraus, et al., “Age-dependent Levels of 5-methyl-, 5-hydroxymethyl-, and 5-formylcytosine in human and Mouse Brain Tissues,” Angewandte Chemie (International ed in English) 54, no. 42 (Oct( 2015): 12511-12514.

[15] S. Dzitoyeva, H. Chen, and H. Manev, “Effect of Aging on 5-hydroxymethylcytosine in Brain Mitochondria,” Neurobiology of Aging 33, no. 12 (Dec 2012): 2881-2891.

[16] Y. Qian, J. Tu, N. L. Tang, et al., “Dynamic Changes of DNA Epigenetic Marks in Mouse Oocytes During Natural and Accelerated Aging,” International Journal of Biochemistry & Cell Biology 67 (Oct 2015): 121-127.

[17] B. Bai, Y. Liang, C. Xu, et al., “Cyclin-dependent Kinase 5-mediated Hyperphosphorylation of Sirtuin-1 Contributes to the Development of Endothelial Senescence and Atherosclerosis,” Circulation 126, no. 6 (Aug 2012): 729-740.

[18] S. V. Brodsky, O. Gealekman, J. Chen, et al., “Prevention and Reversal of Premature Endothelial Cell Senescence and Vasculopathy in Obesity-induced Diabetes by ebselen,” Circulation Research 94, no. 3 (Feb 2004): 377-384.

[19] S. Taddei, A. Virdis, P. Mattei, et al., “Aging and Endothelial Function in Normotensive Subjects and Patients With Essential Hypertension,” Circulation 91, no. 7 (1995): 1981-1987.

[20] M. Abbas, L. Jesel, C. Auger, et al., “Endothelial Microparticles from Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity: Role of the Ang II/AT1 Receptor/NADPH Oxidase-Mediated Activation of MAPKs and PI3-Kinase Pathways,” Circulation 135, no. 3 (Jan( 2017): 280-296.

[21] S. I. Bloom, M. T. Islam, L. A. Lesniewski, and A. J. Donato, “Mechanisms and Consequences of Endothelial Cell Senescence,” Nature Reviews Cardiology 20, no. 1 (Jan( 2023): 38-51.

[22] F. Wunnemann, T. Fotsing Tadjo, M. Beaudoin, et al., “Multimodal CRISPR Perturbations of GWAS Loci Associated With Coronary Artery Disease in Vascular Endothelial Cells,” PLos Genet 19, no. 3 (Mar( 2023): e1010680.

[23] D. Zhang, X. Sun, J. Liu, X. Xie, W. Cui, and Y. Zhu, “Homocysteine Accelerates Senescence of Endothelial Cells via DNA Hypomethylation of human Telomerase Reverse Transcriptase,” Arteriosclerosis, Thrombosis, and Vascular Biology 35, no. 1 (Jan( 2015): 71-78.

[24] K. Dryer, M. Gajjar, N. Narang, et al., “Coronary Microvascular Dysfunction in Patients With Heart Failure With Preserved Ejection Fraction,” American Journal of Physiology. Heart and Circulatory Physiology 314, no. 5 (May( 2018): H1033-H1042.

[25] A. B. Gevaert, H. Shakeri, A. J. Leloup, et al., “Endothelial Senescence Contributes to Heart Failure with Preserved Ejection Fraction in an Aging Mouse Model,” Circulation: Heart Failure 10, no. 6 (Jun( 2017).

[26] F. S. Loffredo, A. P. Nikolova, J. R. Pancoast, and R. T. Lee, “Heart Failure With Preserved Ejection Fraction: Molecular Pathways of the Aging Myocardium,” Circulation Research 115, no. 1 (Jun( 2014): 97-107.

[27] Y. Yamazaki, D. J. Baker, M. Tachibana, et al., “Vascular Cell Senescence Contributes to Blood-Brain Barrier Breakdown,” Stroke; A Journal of Cerebral Circulation 47, no. 4 (Apr 2016): 1068-1077.

[28] M. Saker, L. Lipskaia, E. Marcos, et al., “Osteopontin, a Key Mediator Expressed by Senescent Pulmonary Vascular Cells in Pulmonary Hypertension,” Arteriosclerosis, Thrombosis, and Vascular Biology 36, no. 9 (Sep( 2016): 1879-1890.

[29] S. J. Chinta, G. Woods, M. Demaria, et al., “Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson's Disease,” Cell Reports 22, no. 4 (Jan( 2018): 930-940.

[30] Q. Chen, G. F. Vasse, K. O. Nwozor, et al., “FAM13A regulates Cellular Senescence Marker p21 and Mitochondrial Reactive Oxygen Species Production in Airway Epithelial Cells,” American Journal of Physiology. Lung Cellular and Molecular Physiology 325, no. 4 (Oct( 2023): L460-L466.

[31] Z. S. Chang, J. B. Xia, H. Y. Wu, et al., “Forkhead Box O3 Protects the Heart Against Paraquat-induced Aging-associated Phenotypes by Upregulating the Expression of Antioxidant Enzymes,” Aging Cell 18, no. 5 (Oct( 2019): e12990.

[32] R. R. Alcendor, S. Gao, P. Zhai, et al., “Sirt1 regulates Aging and Resistance to Oxidative Stress in the Heart,” Circulation Research 100, no. 10 (May( 2007): 1512-1521.

[33] T. P. Truong, M. Sakata-Yanagimoto, M. Yamada, et al., “Age-Dependent Decrease of DNA Hydroxymethylation in Human T Cells,” Journal of Clinical and Experimental Hematopathology 55, no. 1 (2015): 1-6.

[34] E. Valentini, M. Zampieri, M. Malavolta, et al., “Analysis of the Machinery and Intermediates of the 5hmC-mediated DNA Demethylation Pathway in Aging on Samples From the MARK-AGE Study,” Aging (Albany NY) 8, no. 9 (Aug 2016): 1896-1922.

[35] A. Herrmann, C. Lahtz, J. Song, et al., “Integrin alpha6 Signaling Induces STAT3-TET3-mediated Hydroxymethylation of Genes Critical for Maintenance of Glioma Stem Cells,” Oncogene 39, no. 10 (Mar( 2020): 2156-2169.

[36] Y. Tsukada, T. Akiyama, and K. I. Nakayama, “Maternal TET3 Is Dispensable for Embryonic Development but Is Required for Neonatal Growth,” Scientific Reports 5 (Oct 2015): 15876.

[37] N. Kitsera, J. Allgayer, E. Parsa, et al., “Functional Impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a Single Hemi-modified CpG Dinucleotide in a Gene Promoter,” Nucleic Acids Research 45, no. 19 (Nov( 2017): 11033-11042.

[38] P. Zhang, X. Chen, Y. Zhang, et al., “Tet3 enhances IL-6 Expression Through Up-regulation of 5-hmC in IL-6 Promoter in Chronic Hypoxia Induced Atherosclerosis in Offspring Rats,” Life Sciences 232 (Sep 2019): 116601.

[39] T. Sato and K. Furukawa, “Sequential Action of Ets-1 and Sp1 in the Activation of the human Beta-1,4-galactosyltransferase V Gene Involved in Abnormal Glycosylation Characteristic of Cancer Cells,” Journal of Biological Chemistry 282, no. 38 (Sep( 2007): 27702-27712.

[40] H. Nakatsuka, T. Sokabe, K. Yamamoto, et al., “Shear Stress Induces Hepatocyte PAI-1 Gene Expression Through Cooperative Sp1/Ets-1 Activation of Transcription,” American Journal of Physiology Gastrointestinal and Liver Physiology 291, no. 1 (Jul( 2006): G26-34.

[41] N. Lu, R. Heuchel, M. Barczyk, W. M. Zhang, and D. Gullberg, “Tandem Sp1/Sp3 Sites Together With an Ets-1 Site Cooperate to Mediate alpha11 Integrin Chain Expression in Mesenchymal Cells,” Matrix Biology 25, no. 2 (Mar( 2006): 118-129.

[42] M. Y. Liu, M. Eyries, C. Zhang, F. S. Santiago, and L. M. Khachigian, “Inducible Platelet-derived Growth Factor D-chain Expression by Angiotensin II and Hydrogen Peroxide Involves Transcriptional Regulation by Ets-1 and Sp1,” Blood 107, no. 6 (Mar( 2006): 2322-2329.

[43] F. S. Santiago and L. M. Khachigian, “Ets-1 Stimulates Platelet-derived Growth Factor A-chain Gene Transcription and Vascular Smooth Muscle Cell Growth via Cooperative Interactions With Sp1,” Circulation Research 95, no. 5 (Sep( 2004): 479-487.

[44] F. H. Xiao, Q. Yu, Z. L. Deng, et al., “ETS-1 Acts as a Regulator of human Healthy Aging via Decreasing Ribosomal Activity,” Science Advances 8, no. 17 (Apr 2022): eabf2017.

[45] S. P. O'Hara, P. L. Splinter, C. E. Trussoni, et al., “ETS Proto-oncogene 1 Transcriptionally Up-regulates the Cholangiocyte Senescence-associated Protein Cyclin-dependent Kinase Inhibitor 2A,” Journal of Biological Chemistry 292, no. 12 (Mar( 2017): 4833-4846.

[46] T. J. Huot, J. Rowe, M. Harland, et al., “Biallelic Mutations in p16(INK4a) Confer Resistance to Ras- and Ets-induced Senescence in human Diploid Fibroblasts,” Molecular and Cellular Biology 22, no. 23 (Dec 2002): 8135-8143.

[47] D. Xu, T. J. Wilson, D. Chan, et al., “ETS-1 Is Required for p53 Transcriptional Activity in UV-induced Apoptosis in Embryonic Stem Cells,” Embo Journal 21, no. 15 (Aug 2002): 4081-4093.

[48] A. Rufini, P. Tucci, I. Celardo, and G. Melino, “Senescence and Aging: The Critical Roles of p53,” Oncogene 32, no. 43 (Oct( 2013): 5129-5143.

[49] M. P. Baar, R. M. C. Brandt, D. A. Putavet, et al., “Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging,” Cell 169, no. 1 (Mar( 2017): 132-147 e116.

[50] M. Bachman, S. Uribe-Lewis, X. Yang, M. Williams, A. Murrell, and S. Balasubramanian, “5-Hydroxymethylcytosine Is a Predominantly Stable DNA Modification,” Nature Chemistry 6, no. 12 (Dec 2014): 1049-1055.

[51] M. Iurlaro, G. Ficz, D. Oxley, et al., “A Screen for Hydroxymethylcytosine and Formylcytosine Binding Proteins Suggests Functions in Transcription and Chromatin Regulation,” Genome Biology 14, no. 10 (2013): R119.

[52] C. G. Spruijt, F. Gnerlich, A. H. Smits, et al., “Dynamic Readers for 5-(hydroxy)methylcytosine and Its Oxidized Derivatives,” Cell 152, no. 5 (Feb 2013): 1146-1159.

[53] I. Choi, R. Kim, H. W. Lim, K. H. Kaestner, and K. J. Won, “5-hydroxymethylcytosine Represses the Activity of Enhancers in Embryonic Stem Cells: A New Epigenetic Signature for Gene Regulation,” BMC Genomics [Electronic Resource] 15, no. 1 (Aug 2014): 670.

[54] A. A. Serandour, S. Avner, F. Oger, et al., “Dynamic Hydroxymethylation of Deoxyribonucleic Acid Marks Differentiation-associated Enhancers,” Nucleic Acids Res. 40, no. 17 (Sep( 2012): 8255-8265.

[55] L. Liu, J. Wang, S. Wang, M. Wang, Y. Chen, and L. Zheng, “Epigenetic Regulation of TET1-SP1 during Spermatogonia Self-Renewal and Proliferation,” Frontiers in Physiology 13 (2022): 843825.

[56] B. Tampe, D. Tampe, C. A. Muller, et al., “Tet3-mediated Hydroxymethylation of Epigenetically Silenced Genes Contributes to Bone Morphogenic Protein 7-induced Reversal of Kidney Fibrosis,” Journal of the American Society of Nephrology 25, no. 5 (May( 2014): 905-912.

[57] J. L. Kirkland and T. Tchkonia, “Cellular Senescence: A Translational Perspective,” EBioMedicine 21 (Jul 2017): 21-28.

[58] J. Birch and J. Gil, “Senescence and the SASP: Many Therapeutic Avenues,” Genes & development 34, no. 23-24 (Dec 2020): 1565-1576.

[59] P. R. Coryell, B. O. Diekman, and R. F. Loeser, “Mechanisms and Therapeutic Implications of Cellular Senescence in Osteoarthritis,” Nature Reviews Rheumatology 17, no. 1 (Jan( 2021): 47-57.

[60] H. Sugimoto, K. Okamura, S. Sugimoto, et al., “Sp1 is a co-activator With Ets-1, and Net is an Important Repressor of the Transcription of CTP:Phosphocholine Cytidylyltransferase Alpha,” Journal of Biological Chemistry 280, no. 49 (Dec 2005): 40857-40866.

[61] C. L. Jorcyk, D. K. Watson, G. J. Mavrothalassitis, and T. S. Papas, “The human ETS-1 Gene: Genomic Structure, Promoter Characterization and Alternative Splicing,” Oncogene 6, no. 4 (Apr 1991): 523-532.

[62] T. Oka, A. Rairkar, and J. H. Chen, “Structural and Functional Analysis of the Regulatory Sequences of the Ets-1 Gene,” Oncogene 6, no. 11 (Nov( 1991): 2077-2083.

[63] L. Jackson-Grusby, C. Beard, R. Possemato, et al., “Loss of Genomic Methylation Causes p53-dependent Apoptosis and Epigenetic Deregulation,” Nature Genetics 27, no. 1 (Jan( 2001): 31-39.

[64] K. I. Leonova, L. Brodsky, B. Lipchick, et al., “p53 cooperates With DNA Methylation and a Suicidal Interferon Response to Maintain Epigenetic Silencing of Repeats and Noncoding RNAs,” PNAS 110, no. 1 (Jan( 2013): E89-98.

[65] A. Tovy, A. Spiro, R. McCarthy, et al., “p53 is Essential for DNA Methylation Homeostasis in Naive Embryonic Stem Cells, and Its Loss Promotes Clonal Heterogeneity,” Genes & Development 31, no. 10 (May( 2017): 959-972.

[66] D. Munoz-Espin, M. Canamero, A. Maraver, et al., “Programmed Cell Senescence During Mammalian Embryonic Development,” Cell 155, no. 5 (Nov( 2013): 1104-1118.

[67] M. Storer, A. Mas, A. Robert-Moreno, et al., “Senescence Is a Developmental Mechanism That Contributes to Embryonic Growth and Patterning,” Cell 155, no. 5 (Nov( 2013): 1119-1130.

[68] Y. Dang, D. Hu, J. Xu, et al., “Comprehensive Analysis of 5-hydroxymethylcytosine in zw10 Kinetochore Protein as a Promising Biomarker for Screening and Diagnosis of Early Colorectal Cancer,” Clinical and Translational Medicine 10, no. 3 (Jul( 2020): e125.

[69] J. Pan, F. Liu, X. Xiao, et al., “METTL3 promotes Colorectal Carcinoma Progression by Regulating the m6A-CRB3-Hippo Axis,” Journal of Experimental & Clinical Cancer Research 41, no. 1 (Jan( 2022): 19.

[70] Y. Dang, R. Xu, J. Pan, et al., “Dynamic Changes in DNA Methylation and Hydroxymethylation Revealed the Transformation of Advanced Adenoma Into Colorectal Carcinoma,” Clinical and Translational Medicine 13, no. 3 (Mar( 2023): e1202.

[71] W. Gan, Y. Dang, X. Han, et al., “ERK5/HDAC5-mediated, Resveratrol-, and Pterostilbene-induced Expression of MnSOD in human Endothelial Cells,” Molecular Nutrition & Food Research 60, no. 2 (Feb 2016): 266-277.

RIGHTS & PERMISSIONS

2025 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.