Fig. 7: PML colocalization explains the dominant negative effect of C302Y and C311Y.

a Transcriptional activity of hAire (black circle) and its changes upon co-expression with C302Y (orange circle) and C311Y (blue circle) in 293 T cells. Each circle represents 0.6 μg/ml transfected DNA. Data are presented as mean ± s.d., n = 3. b Representative fluorescence microscopy images of hAire WT-FLAG co-expressed with hAire C302Y-HA (0.6 μg/ml DNA each) in 4D6 cells. Cells were immunostained with anti-FLAG (WT hAire) and anti-PML. Right, quantitation of Aire foci colocalized with PML bodies. n = number of Aire foci examined per sample. Statistical significance comparison was calculated using a two-tailed Student’s t-test for two population proportions where each population consists of all individual Aire foci examined. ***p = 1.24e-9 (WT + C302Y) and 5.45e-05 (WT + C311Y). c Representative fluorescence microscopy images of hAire WT-FLAG co-expressed with hAire C302Y-HA (0.3 μg/ml DNA each) in the presence or absence of IE1-HA (0.5 μg/ml) in 4D6 cells. Cells were immunostained with anti-FLAG (WT hAire) and anti-PML. d Transcriptional activity of hAire WT (0.3 μg/ml) co-expressed with empty vector (0.3 μg/ml left panel) or hAire C302Y (0.3 μg/ml, right panel) and the impact of IE1 co-expression (0, 0.17, 0.5 μg/ml). Data are presented as mean ± s.d., n = 3. Fold-changes in transcriptional activities with IE1 are indicated. P-values (two-tailed t-test) were calculated in comparison to WT mAire. **p < 0.01; ***p < 0.001. Exact p-values are in the Source Data File. Right, western blot showing the expression levels of FLAG-tagged hAire variants co-expressed with or without IE1. e Model for PML body-mediated regulation of Aire function. Proper Aire function requires large homo-multimerization, but this property inevitably subjects Aire to PML body-associated protein quality control and transcriptional regulation. Note that PML body localization does not require protein mis-folding of Aire, and that the locations of Aire foci are not pre-defined. Instead, PML body localization can be induced by multiple factors, including altered multimerization property (through mutations in CARD), loss of chromatin interaction (through mutations in PHD1) or by interaction with PML colocalizing alleles.