Two distinct ubiquitin-binding motifs in A20 mediate its anti-inflammatory and cell-protective activities

Abstract

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.

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Fig. 1: A20ZnF7/ZnF7 knock-in mice develop spontaneous inflammatory pathology and are sensitized to TNF-induced toxicity.
Fig. 2: ZnF7 is critical for A20-mediated suppression of inflammatory signaling and cell death.
Fig. 3: A20ZnF4ZnF7/ZnF4ZnF7 knock-in mice phenocopy A20 knockout mice.
Fig. 4: Tissue-specific A20ZnF4ZnF7 expression phenocopies tissue-specific A20 deficiency.

Data availability

Source data for Fig. 2 and Extended Data Fig. 4 are included with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

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’.

Author information

A.M., D.P., E.H., J.V., S.R., L.C., S.V., L.D., M.S., H.V., K.S., T.H. and K.I. performed the experiments. A.M., D.P., E.H., J.V., L.C., S.V., A.W., S.J., M.L., R.B., M.A., M.J.M.B. and G.v.L. analyzed the data. G.v.L. provided ideas and coordinated the project. A.M. and G.v.L. wrote the manuscript.

Correspondence to Geert van Loo.

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Competing interests

M.L. is an employee of Janssen Pharmaceutica. All other authors declare no competing interests.

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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.

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Extended data

Extended Data Fig. 1 A20ZnF7 knock-in mice.

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.

Extended Data Fig. 2 FACS immunophenotyping of spleen of control and A20ZnF7 mice.

ac, General gating strategy as applied for immune cell populations described in Fig. 1g. (a) Lymphocytes, singlets, live, CD3CD19 + (B cells), CD3 + CD19 (T cells) and CD3CD19NK1.1+ (NK cells); (b) non-debris, singlets, live, lineage (CD3CD19NK1.1-), F4/80+, CD64 + and autofluorescent; (c) non-debris, singlets, live, lineage, Ly6G+CD11b + (neutrophils) and Ly6GSiglecFLy6ChiCD11b + (monocytes). FSC: forward scatter, SSC: side scatter, A: Area, H: height, W: width, L/D: live/dead. df, 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).

Extended Data Fig. 4 ZnF7 is critical for A20-mediated suppression of inflammatory signaling.

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. Source data

Extended Data Fig. 5 A20ZnF4ZnF7 knock-in mice.

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.

Extended Data Fig. 6 Conditional ‘floxed’ A20ZnF4/ZnF7 knock-in 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).

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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

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