Fig. 4: Directing Aire to PML bodies inhibits transcriptional activity, while dispersing PML bodies increases Aire activity.

a Representative fluorescence microscopy images of FLAG-tagged WT mAire and mAire N-terminally fused with SUMO interaction motifs (SIMs) from RNF4 (SIM-mAire). Right, quantification of Aire foci colocalized with endogenous PML bodies. n = number of Aire foci examined per sample. ***p (two-tailed Student’s t-test) = 1.68e-71. b Transcriptional activity of mAire and SIM-mAire. Experiments are presented as mean ± s.d., n = 3. P-values (two-tailed t-test) were calculated in comparison to WT mAire where *p = 0.031; **p = 0.0035 and 0.0014 for KRT14 and S100A9 respectively; ***p = 0.0004; p = 0.68 is not significant (ns). c Representative fluorescence microscopy images of WT mAire-FLAG with and without co-expression with SIM-mAire (no tag) in 4D6 cells. Right, quantification of the Aire foci colocalized with endogenous PML bodies as in (a). ***p (two-tailed Student’s t-test) = 4.39e-7. d Transcriptional activity of mAire (black circle) with and without co-expression of SIM-mAire (green circle) in 293 T cells. Each circle represents 0.6 μg/ml DNA transfected. Experiments are presented as mean ± s.d., n = 3. See Supplementary Fig. 4a for additional Aire-dependent target genes. P-values (two-tailed t-test) were calculated in comparison to WT mAire where **p = 0.002; p = 0.88 is not significant (ns). e Representative fluorescence microscopy images of FLAG-tagged mAire co-expressed with CMV IE1 (0.5 μg/ml DNA each construct) in 4D6 cells. Cells were immunostained with anti-FLAG and anti-PML. See Supplementary Fig. 4c for the impact of IE1 on PML body dispersion. f Transcriptional activity of mAire, K53A/E54A and SIM-mAire (0.5 μg/ml DNA) with an increasing concentration of IE1 (0, 0.17, 0.5 μg/ml) in 293 T cells. Values are normalized against WT mAire without IE1. Experiments are presented as mean ± s.d., n = 3. Fold-changes in transcriptional activities upon addition of IE1 are indicated. P-values (two-tailed t-test) were calculated in comparison to no IE1. *p < 0.05; **p < 0.01; p > 0.05 is not significant (ns). Exact p-values are provided in the Source Data File.