Fig. 1: Filament assembly of Aire CARD mediates nuclear foci formation and transcriptional activity.

a Schematic of murine Aire domain architecture with previously characterized functions of individual domains. b Representative EM image of wild-type (WT) mAire CARD. While fully formed CARD filaments (i) were major species, thin protofilaments (ii) were also observed. Right, representative images of the two types of filaments. 2D class averages were also shown for fully formed CARD filaments. c, d Representative absorption spectra of Congo Red (CR, 15 μM) (c) or fluorescence emission spectra (excitation at 430 nm) of Thioflavin (ThT, 50 μM) (d) in the presence of WT mAire CARD filaments or known amyloids of RIP1/3-RHIM⁷⁰. 5 μM monomeric concentration was used for both proteins. The experiments were performed two independent times. e 3D homology model of mAire CARD using the program FUGUE 2.01. Putative CARD:CARD interfaces were identified from the sequence alignment with other CARDs (Supplementary Fig. 1a) and are highlighted green. f Representative EM images of recombinant mAire CARD. WT protein and those with mutations in the putative CARD:CARD interfaces in (e) were compared. g Transcriptional activity of WT mAire and mutants, as measured by the relative mRNA levels of Aire-dependent genes (represented by CD4, IGFL1, KRT14 and S100A9). An Aire-independent gene, HPRT1, was also examined as a negative control. Aire variants were transiently expressed (using 1.25 μg/ml DNA) in 293 T cells for 48 h prior to RT-qPCR analysis. Data are representative of at least three independent experiments and presented as mean ± s.d., n = 3. Right, western blot (WB) showing the expression levels of FLAG-tagged mAire compared to endogenous levels of Histone H3 (anti-H3). See Supplementary Fig. 1c for experiments in 4D6. P-values (two-tailed t-test) were calculated in comparison to WT mAire. *p < 0.05; **p < 0.01; p > 0.05 is not significant (ns). Exact p-values are provided in the Source Data File. h Representative fluorescence images of FLAG-tagged mAire in 293 T cells using anti-FLAG. See Supplementary Fig. 1d for experiments in 4D6.