FIGURE 8.

Ubiquitylation of AIRE increases the binding and recruitment of P-TEFb to target genes. A, mutations of Thr-68 and Ser-156 influence the AIRE interaction with P-TEFb. 293T cells expressed WT mHis:AIRE or mutant mHis:AIRE proteins for 48 h. Whole cell lysates were immunoprecipitated (IP) with anti-His antibodies followed by Western blotting with indicated antibodies. B, knockdown of FBXO3 decreases interactions between AIRE and P-TEFb. f:AIRE was expressed in 293T cells with control (siScr) or siFBXO3 (siFBXO3) RNAs for 72 h before harvesting. Total cell lysates were incubated with anti-FLAG beads. AIRE, the endogenous CycT1 and FBXO3 proteins, were examined with indicated antibodies. The top panels contain immunoprecipitated CycT1 and f:AIRE proteins. The lower panels contain 3% input CycT1, f:AIRE, and FBXO3 proteins. C and D, interactions between AIRE and P-TEFb on DNA are decreased by deleterious mutations in AIRE and the depletion of FBXO3. C, presented are ChIPs of AIRE and CycT1 at promoters of TSA genes (KRT14 and S100A8) in cells expressing WT and mutant mHis:AIRE proteins. D, presented are ChIPs of AIRE and CycT1 at the promoter of TSA genes (KRT14 and S100A8) in control or FBXO3-depleted cells. Bars represent the S.E. of three independent experiments (S.E., n = 3). Student's t test was preformed to measure the significance of the data (*, p < 0.05; **, p < 0.01).