Protein ubiquitination regulates protein stability and modulates the composition of signaling complexes. A20 is a negative regulator of inflammatory signaling, but the molecular mechanisms involved are ill understood. Here, we generated Tnfaip3 gene-targeted A20 mutant mice bearing inactivating mutations in the zinc finger 7 (ZnF7) and ZnF4 ubiquitin-binding domains, revealing that binding to polyubiquitin is essential for A20 to suppress inflammatory disease. We demonstrate that a functional ZnF7 domain was required for recruiting A20 to the tumor necrosis factor receptor 1 (TNFR1) signaling complex and to suppress inflammatory signaling and cell death. The combined inactivation of ZnF4 and ZnF7 phenocopied the postnatal lethality and severe multiorgan inflammation of A20-deficient mice. Conditional tissue-specific expression of mutant A20 further revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells. Collectively, these results demonstrate that the anti-inflammatory and cytoprotective functions of A20 are largely dependent on its ubiquitin-binding properties.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Catrysse, L., Vereecke, L., Beyaert, R. & van Loo, G. A20 in inflammation and autoimmunity. Trends Immunol. 35, 22–31 (2014).
Martens, A. & van Loo, G. A20 at the crossroads of cell death, inflammation, and autoimmunity. Cold Spring Harb. Perspect. Biol. 12, a036418 (2020).
Wertz, I. E. et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430, 694–699 (2004).
Lu, T. T. et al. Dimerization and ubiquitin mediated recruitment of A20, a complex deubiquitinating enzyme. Immunity 38, 896–905 (2013).
De, A., Dainichi, T., Rathinam, C. V. & Ghosh, S. The deubiquitinase activity of A20 is dispensable for NF-κB signaling. EMBO Rep. 15, 775–783 (2014).
Wertz, I. E. et al. Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation. Nature 528, 370–375 (2015).
Lee, E. G. et al. Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice. Science 289, 2350–2354 (2000).
Tokunaga, F. et al. Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved in NF-κB regulation. EMBO J. 31, 3856–3870 (2012).
Draber, P. et al. LUBAC-recruited CYLD and A20 regulate gene activation and cell death by exerting opposing effects on linear ubiquitin in signaling complexes. Cell Rep. 13, 2258–2272 (2015).
Verhelst, K. et al. A20 inhibits LUBAC-mediated NF-κB activation by binding linear polyubiquitin chains via its zinc finger 7. EMBO J. 31, 3845–3855 (2012).
Polykratis, A. et al. A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain. Nat. Cell Biol. 21, 731–742 (2019).
Turer, E. E. et al. Homeostatic MyD88-dependent signals cause lethal inflammation in the absence of A20. J. Exp. Med. 205, 451–464 (2008).
Vereecke, L. et al. Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis. J. Exp. Med. 207, 1513–1523 (2010).
Priem, D. et al. A20 protects cells from TNF-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms. Cell Death Dis. 10, 692 (2019).
Matmati, M. et al. A20 (TNFAIP3) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis. Nat. Genet. 43, 908–912 (2011).
Vande Walle, L. et al. Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis. Nature 512, 69–73 (2014).
Bosanac, I. et al. Ubiquitin binding to A20 ZnF4 is required for modulation of NF-κB signaling. Mol. Cell 40, 548–557 (2010).
Graham, R. R. et al. Genetic variants near TNFAIP3 on 6q23 are associated with systemic lupus erythematosus. Nat. Genet. 40, 1059–1061 (2008).
Adrianto, I. et al. Association of a functional variant downstream of TNFAIP3 with systemic lupus erythematosus. Nat. Genet. 43, 253–258 (2011).
Sokhi, U. K. et al. Dissection and function of autoimmunity-associated TNFAIP3 (A20) gene enhancers in humanized mouse models. Nat. Commun. 9, 658 (2018).
Wang, S., Wen, F., Wiley, G. B., Kinter, M. T. & Gaffney, P. M. An enhancer element harboring variants associated with systemic lupus erythematosus engages the TNFAIP3 promoter to influence A20 expression. PLoS Genet. 9, e1003750 (2013).
Compagno, M. et al. Mutations of multiple genes cause deregulation of NF-κB in diffuse large B-cell lymphoma. Nature 459, 717–721 (2009).
Kato, M. et al. Frequent inactivation of A20 in B-cell lymphomas. Nature 459, 712–716 (2009).
Schmitz, R. et al. TNFAIP3 (A20) is a tumor suppressor gene in Hodgkin lymphoma and primary mediastinal B cell lymphoma. J. Exp. Med. 206, 981–989 (2009).
Zhou, Q. et al. Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease. Nat. Genet. 48, 67–73 (2016).
Clausen, B. E., Burkhardt, C., Reith, W., Renkawitz, R. & Forster, I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 8, 265–277 (1999).
Madison, B. B. et al. cis Elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J. Biol. Chem. 277, 33275–33283 (2002).
Adachi, O. et al. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9, 143–150 (1998).
Armaka, M., Ospelt, C., Pasparakis, M. & Kollias, G. The p55TNFR–IKK2–Ripk3 axis orchestrates arthritis by regulating death and inflammatory pathways in synovial fibroblasts. Nat. Commun. 9, 618 (2018).
Baird, D., Murray, D., Payne, R. & Soutar, D. An Introduction to GenStat for Windows v.19 (Genstat, 2017).
We thank D. Huyghebaert, L. Bellen and D. Vanhede for animal care and A. Fossoul and M. Gennadi for excellent technical assistance. We also thank the InfrafrontierGR infrastructure (ERDF and NSRF 2007–2013 and 2014–2020) for providing histology and mCT facilities. A.M. is supported by a grant from the ‘Concerted Research Actions’ (GOA) of the Ghent University. Research in the G.v.L. laboratory is supported by research grants from The Research Foundation—Flanders (FWO), the ‘Geneeskundige Stichting Koningin Elisabeth’ (GSKE), the CBC Banque Prize, the Charcot Foundation, the ‘Belgian Foundation against Cancer’, ‘Kom op tegen Kanker’ and the GOA of Ghent University. The M.A. laboratory is supported by a start-up grant from the Stavros Niarchos Foundation to BSRC ‘Alexander Fleming’.
M.L. is an employee of Janssen Pharmaceutica. All other authors declare no competing interests.
Editor recognition statement: L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Schematic depiction of the A20/Tnfaip3 locus indicating the sequence of the single-stranded oligonucleotide used for mutating the ZnF7 domain that was introduced by pronuclear injection into mouse zygotes, and sequencing result of the wild-type (WT) and of the targeted ZnF7 knock-in allele. Boxes indicate exons 3 to 9 (E3–E9). b, Birth and survival rates of control (A20+/+), A20ZnF7/+ and A20ZnF7/ZnF7 offspring from A20ZnF7/+ x A20ZnF7/+ breeding couples. c, Gross appearance of A20ZnF7/+ and A20ZnF7/ZnF7 mice. d, Representative pictures of spleen and inguinal lymph nodes from 28-week-old control (A20+/+) and A20ZnF7/ZnF7 littermate mice. e, Representative pictures of hindpaws of 15-week-old control (A20+/+) and A20ZnF7/ZnF7 littermates, showing extensive swelling of the toes of the A20ZnF7/ZnF7 mice. f, Representative micro-CT pictures of hindpaws (left) and knees (right) of 28-week-old control (A20+/+) and A20ZnF7/ZnF7 littermates. g, Representative hematoxylin-eosin-stained histological images of ankle joints (left) and toes (right) from 28-week-old control (A20+/+) and A20ZnF7/ZnF7 littermates. Scale bar, 500 µm.
a–c, General gating strategy as applied for immune cell populations described in Fig. 1g. (a) Lymphocytes, singlets, live, CD3−CD19 + (B cells), CD3 + CD19− (T cells) and CD3−CD19−NK1.1+ (NK cells); (b) non-debris, singlets, live, lineage− (CD3−CD19−NK1.1-), F4/80+, CD64 + and autofluorescent; (c) non-debris, singlets, live, lineage−, Ly6G+CD11b + (neutrophils) and Ly6G−SiglecF−Ly6ChiCD11b + (monocytes). FSC: forward scatter, SSC: side scatter, A: Area, H: height, W: width, L/D: live/dead. d–f, Bar graphs representing absolute numbers of total (left) and naive (right) CD4 T cells (d), total (left) and naive (right) CD8 T cells (e) and yd T cells (f) as measured by flow cytometry in the spleens of A20+/+, A20ZnF/+ and A20ZnF7/ZnF7 animals. Data are expressed as mean ± SEM. *, ** represent p < 0.05 and p < 0.01 (Two-sided non-parametric Mann-Whitney test between indicated genotypes).
Extended Data Fig. 3 MyD88-dependent mechanisms contribute to the local inflammatory pathology in A20ZnF7 mice.
a, b, Gross appearance (a) and body weight (b) of of 10 week-old A20ZnF7/+MyD88+/-, A20ZnF7/ZnF7MyD88+/- and A20ZnF7/ZnF7 MyD88-/- mice. Each dot represents a biologically independent mouse (A20ZnF7/+MyD88+/-, n = 9; A20ZnF7/ZnF7MyD88+/-, n = 13 and A20ZnF7/ZnF7 MyD88-/-, n = 6). Data are expressed as mean ± SEM. * and **** represent p < 0.05 and p < 0.0001, respectively (parametric two-way ANOVA between indicated genotypes). c, Representative hematoxylin-eosin-stained sections of liver from 18-week-old A20ZnF7/ZnF7MyD88+/+ and A20ZnF7/ZnF7MyD88-/- littermates. Scale bar, 50 μm. Picture representative for 3 biologically independent mice. d, Representative pictures of hindpaws of 10-week-old A20ZnF7/ZnF7MyD88+/+ and A20ZnF7/ZnF7MyD88-/- littermates. Pictures representative for 3 biologically independent mice e, Levels of IL-6 and TNF in serum of A20+/+ MyD88+/+, A20+/+ MyD88-/-, A20ZnF7/ZnF7MyD88+/+ and A20ZnF7/ZnF7MyD88-/- mice. Each dot represents a biologically independent mouse (A20+/+ MyD88+/+, n = 9; A20+/+ MyD88-/-, n = 3; A20ZnF7/ZnF7MyD88+/+, n = 13 and A20ZnF7/ZnF7MyD88-/-, n = 6). Data are expressed as mean ± SEM. *, ** represent p < 0.05 and p = 0.0033 respectively (parametric one-way ANOVA between indicated genotypes).
Western blot analysis of whole cell lysates from A20+/+, A20ZnF7/ZnF7 and A20myel-KO BMDMs stimulated with TNF as indicated. β-tubulin is shown as a loading control. Figure representative for 3 independent experiments.
a, Schematic depiction of the A20/Tnfaip3 locus indicating the position of ZnF4 and ZnF7 mutations. Boxes indicate exons 3 to 9 (E3–E9). Sequences of the donor vector, containing ~1 kb 5′ and 3′ homologous arms around the Cys-to-Ala mutations used for mutating the ZnF4 and ZnF7 domains, that were introduced by pronuclear injection into mouse zygotes. Sequencing result of the wild-type (WT) allele and of the targeted ZnF4 and ZnF7 knock-in alleles. b, Gross appearance of 2-week old control (A20+/+), A20ZnF4ZnF7/+ and A20ZnF4ZnF7/ZnF4ZnF7 mice. c, Gross appearance of 2-week old A20ZnF4ZnF7/ZnF4ZnF7MyD88-/- mice compared to A20ZnF4ZnF7/ZnF4ZnF7MyD88+/-mice.
a, Targeting scheme showing the LoxP-flanked (Floxed) and knock-in A20 alleles. Boxes indicate exons 3 to 9 (E3-E9). LoxP sites are indicated by arrowheads. b, Gross appearance of 2 week-old control (Tnfaip3ZnF4ZnF7/ZnF4ZnF7CreDel+/+) and Tnfaip3ZnF4ZnF7/ZnF4ZnF7CreDelTg/+ littermate mice. c, Representative histological images of ankle joints from 30-week-old littermate mice with the indicated genotypes. Bone erosion was detected by tartrate-resistant acid phosphatase (TRAP) staining of osteoclast activity, and cartilage destruction was assessed by proteoglycan staining with toluidine blue. H/E, haematoxylin and eosin. Scale bar: 500 μm. Pictures representative for 5 biologically independent mice. d, Clinical score, based on loss in body weight, stool consistency, and presence of fecal blood, of 30 week-old Tnfaip3ZnF4ZnF7/ZnF4ZnF7vilCreTg/+ (n = 4) and control (Tnfaip3ZnF4ZnF7/ZnF4ZnF7vilCre+/+, n = 4) littermate mice treated with 1.5 % DSS. The experiment was stopped at day 5 since Tnfaip3ZnF4ZnF7/ZnF4ZnF7vilCreTg/+ started dying. Data are expressed as mean ± SEM. * represents p = 0.0204 (2-way ANOVA with Sidak’s multiple comparison) e, Intestinal permeability assay using FITC-labelled dextran in 30-week-old Tnfaip3ZnF4ZnF7/ZnF4ZnF7vilCreTg/+ (n = 4) and control (Tnfaip3ZnF4ZnF7/ZnF4ZnF7vilCre+/+, n = 4) mice before and after 5 days of DSS treatment. Data are expressed as mean ± SEM. * represents p = 0.0143 (2-way ANOVA with Sidak’s multiple comparison).
About this article
Cite this article
Martens, A., Priem, D., Hoste, E. et al. Two distinct ubiquitin-binding motifs in A20 mediate its anti-inflammatory and cell-protective activities. Nat Immunol 21, 381–387 (2020). https://doi.org/10.1038/s41590-020-0621-9
Cell Death & Differentiation (2021)
Arthritis Research & Therapy (2020)
Nature Immunology (2020)
Scientific Reports (2020)