FIG. 1.

AIRE cooperates with initiation factors to activate transcription. (A) (Top) Schematic representations of plasmid targets used in CAT assays. pG5CAT contains the CAT gene and five repeats of the GAL4 DNA-binding site (5×UAS) in front of the E1b TATA box (T). pG6SpCAT contains a complete promoter of three Sp1 sites (3×Sp1) and the E1b TATA box in addition to six UASs and the CAT gene. pG6TARCAT contains a TAR RNA structure in addition to the elements present in pG6SpCAT. pA represents the polyadenylation signal. (Bottom) AIRE requires the presence of initiation factors to activate transcription in 1C6 and HeLa-MAGI cells. Cells expressed the indicated plasmid targets alone or with AIRE (lanes 3, 4, 6, 8, 10, 12, and 14) and GalDBD (lanes 2, 4, 11, and 12). Expression levels of AIRE and GalDBD, as determined by Western blotting, are presented below the CAT data. Levels of endogenous GAPDH were determined by Western blotting to validate input for each sample. Error bars denote standard errors of the means of three independent experiments. (B) AIRE activates transcription in a dose-dependent manner. 1C6 cells expressed pG6SpCAT and increasing amounts of AIRE (0.1, 0.25, and 0.8 μg in lanes 2, 3, and 4, respectively). Expression levels of AIRE and GAPDH, as determined by Western blotting, are presented below the CAT data. Error bars denote standard errors of the means of three independent experiments. (C) AIRE induces the elongation of transcription. (Top) Primer combinations for the amplification of primary transcripts. Primers 1 and 2 amplify TAR and thus all transcripts (ST). Primers 1 and 3 amplify only the LT. nt, nucleotides. (Bottom) Total RNA was extracted from HeLa-MAGI cells coexpressing pG6TARCAT and AIRE or GalDBD and was analyzed by RT-qPCR. The Gal.CycT1 chimera was used as the positive control. Data are representative of three independent experiments.