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Cullin-RING ligases (CRLs), the largest family of ubiquitin ligases, require neddylation on the scaffold protein Cullins for their ligase activity. The role of neddylation\u2013CRL system in thymus development and fate determination of \u03b1\u03b2\/\u03b3\u03b4 T cells remains elusive. Here, we generated conditional knockout mouse models with thymus individual deletion of Ube2m or Ube2f, 2 neddylation E2-conjugating enzymes, and Rbx1 or Sag, 2 dual neddylation and ubiquitylation E3 ligases. We found that only Rbx1, but not Ube2m\/Ube2f, nor Sag, plays an essential role in thymus development and fate determination of \u03b1\u03b2\/\u03b3\u03b4 T cells. Specifically, Rbx1 loss causes shrinkage of the thymus, delayed T cell development, increased \u03b3\u03b4 T cells in the thymus, increased the ratio of immature\n            <jats:italic>Gzma<\/jats:italic>\n            <jats:sup>+<\/jats:sup>\n            \u03b3\u03b4 T cells, and decreased the ratio of the proliferative subpopulation. Some of these phenotypes were moderately rescued by simultaneous Bim deletion. Mechanistically, Rbx1 loss alters the Akt, NF-\u03baB, and metabolic pathways in progenitor \u03b3\u03b4 T cells\/DN3a cells. Finally,\n            <jats:italic>Rbx1<\/jats:italic>\n            loss altered the \u03b3\u03b4 T1\/T17 cell population in the thymus, suggesting that Rbx1 controls the fate of \u03b3\u03b4 T cells.\n          <\/jats:p>","DOI":"10.34133\/research.0774","type":"journal-article","created":{"date-parts":[[2025,6,17]],"date-time":"2025-06-17T01:27:17Z","timestamp":1750123637000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark_01","source":"Crossref","is-referenced-by-count":1,"title":["E3 Ligase Rbx1 Orchestrates Thymus Development and Fate Determination of \u03b1\u03b2-\u03b3\u03b4 T Cells"],"prefix":"10.34133","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2188-0073","authenticated-orcid":false,"given":"Di","family":"Wu","sequence":"first","affiliation":[{"name":"Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education) of the Second Affiliated Hospital, and Institute of Translational Medicine, \rZhejiang University School of Medicine, Hangzhou 310029, China."},{"name":"Cancer Center of Zhejiang University, Hangzhou 310029, China."},{"name":"Research Center for Life Science and Human Health, \rBinjiang Institute of Zhejiang University, Hangzhou 310053, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiuxu","family":"Chen","sequence":"additional","affiliation":[{"name":"Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education) of the Second Affiliated Hospital, and Institute of Translational Medicine, \rZhejiang University School of Medicine, Hangzhou 310029, China."},{"name":"Cancer Center of Zhejiang University, Hangzhou 310029, China."},{"name":"Research Center for Life Science and Human Health, \rBinjiang Institute of Zhejiang University, Hangzhou 310053, China."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingjia","family":"Tan","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, \rUniversity of Michigan, Ann Arbor, MI 48109, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5841-4287","authenticated-orcid":true,"given":"Yi","family":"Sun","sequence":"additional","affiliation":[{"name":"Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education) of the Second Affiliated Hospital, and Institute of Translational Medicine, \rZhejiang University School of Medicine, Hangzhou 310029, China."},{"name":"Cancer Center of Zhejiang University, Hangzhou 310029, China."},{"name":"Research Center for Life Science and Human Health, \rBinjiang Institute of Zhejiang University, Hangzhou 310053, China."},{"name":"State Key Laboratory of Transvascular Implantation Devices, the Second Affiliated Hospital, \rZhejiang University School of Medicine, Hangzhou 310029, China."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","published-online":{"date-parts":[[2025,7,10]]},"reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/nri1257"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molimm.2024.10.002"},{"key":"e_1_3_4_4_2","doi-asserted-by":"crossref","DOI":"10.3389\/fimmu.2022.900556","article-title":"COVID-19 vaccination influences subtypes of \u03b3\u03b4-T cells during pregnancy","volume":"13","author":"Wang L","year":"2022","unstructured":"Wang L, Li J, Jiang S, Li Y, Guo R, Chen Y, Chen Y, Yu H, Qiao Q, Zhan M, et al. COVID-19 vaccination influences subtypes of \u03b3\u03b4-T cells during pregnancy. Front Immunol. 2022;13: Article 900556.","journal-title":"Front Immunol"},{"key":"e_1_3_4_5_2","doi-asserted-by":"crossref","first-page":"tkad054","DOI":"10.1093\/burnst\/tkad054","article-title":"Luminol-conjugated cyclodextrin biological nanoparticles for the treatment of severe burn-induced intestinal barrier disruption","volume":"12","author":"Song Y","year":"2024","unstructured":"Song Y, Li Y, Hu W, Li F, Sheng H, Huang C, Gou X, Hou J, Zheng J, Xiao Y. Luminol-conjugated cyclodextrin biological nanoparticles for the treatment of severe burn-induced intestinal barrier disruption. 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Adv Sci. 2022;9(32): Article e2204522.","journal-title":"Adv Sci"},{"issue":"2","key":"e_1_3_4_33_2","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1093\/emboj\/17.2.384","article-title":"Bim: A novel member of the Bcl-2 family that promotes apoptosis","volume":"17","author":"O\u2019Connor L","year":"1998","unstructured":"O\u2019Connor L, Strasser A, O\u2019Reilly LA, Hausmann G, Adams JM, Cory S, Huang DC. Bim: A novel member of the Bcl-2 family that promotes apoptosis. 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ITM2A is induced during thymocyte selection and T cell activation and causes downregulation of CD8 when overexpressed in CD4+CD8+ double positive thymocytes. J Exp Med. 1999;190(2):217\u2013228.","journal-title":"J Exp Med"},{"issue":"1","key":"e_1_3_4_40_2","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1002\/eji.1830180129","article-title":"Murine CD2 gene expression during fetal thymus ontogeny","volume":"18","author":"Owen MJ","year":"1988","unstructured":"Owen MJ, Jenklnson EJ, Brown MH, Sewell WA, Krissansen GW, Crumpton MJ, Owen JJT. Murine CD2 gene expression during fetal thymus ontogeny. Eur J Immunol. 1988;18(1):187\u2013189.","journal-title":"Eur J Immunol"},{"issue":"2","key":"e_1_3_4_41_2","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1385\/IR:21:2-3:315","article-title":"The thymus and negative selection","volume":"21","author":"Kishimoto H","year":"2000","unstructured":"Kishimoto H, Sprent J. The thymus and negative selection. 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Sci Immunol. 2018;3(25): Article eaas9818.","journal-title":"Sci Immunol"},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.3109\/10428194.2011.564696"},{"key":"e_1_3_4_50_2","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1007\/978-3-030-36422-9_10","article-title":"Molecular mechanisms of Notch signaling in lymphoid cell lineages development: NF-\u03baB and beyond","volume":"1227","author":"Tsaouli G","year":"2020","unstructured":"Tsaouli G, Barbarulo A, Vacca A, Screpanti I, Felli MP. Molecular mechanisms of Notch signaling in lymphoid cell lineages development: NF-\u03baB and beyond. 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