Fig. 2. Chemically induced multimerization partially restores the transcriptional activity of AireΔCARD.

a Schematic of mAire∆CARD fused with four tandem repeats of FKBP (FKBP4-ΔCARD). Proteins with FKBP repeats are known to multimerize upon addition of chemical dimerizer AP1903⁴⁰. b Transcriptional activity of ∆CARD fused with 1–4 tandem repeats of FKBP (FKBP1–4) in the presence and absence of AP1903. Fusion constructs were transiently expressed (using 1.25 μg/ml DNA) in 293 T cells and DMSO or AP1903 (5 μM) was added 24 h later. Cells were harvested 48 h after transfection, followed by RT-qPCR of the respective Aire-dependent genes (represented by CD4 and IGFL1). The relative mRNA level of an Aire-independent gene, HPRT1, was also shown as a negative control. Data are representative of at least three independent experiments and presented as mean ± s.d., n = 3. See Supplementary Fig. 2a, b for additional target genes and western blot (WB) showing protein expression levels. P-values (two-tailed t-test) were calculated in comparison to empty vector (EV) + AP1903. *p < 0.05; **p < 0.01; p > 0.05 is not significant (ns). Exact p-values are provided in the Source Data File. c, d Representative fluorescence microscopy images of FLAG-tagged ∆CARD fusion variants in 4D6. Note that 4D6 cells were used as these cells are flatter than 293 T, allowing more robust analysis of Aire nuclear localization. Fusion constructs were transiently expressed and treated with DMSO or AP1903 as in (b). 48 h after transfection, cells were immunostained with anti-FLAG (mAire) and anti-PML. See Supplementary Fig. 2c, d for endogenous PML immunostaining.