Fig. 5: p73 binding regulates target gene expression.

a, b p73 binding at the indicated loci in Th1 cells were assayed by ChIP using anti-p73 antibody versus IgG as a control. p73 protein was immunoprecipitated from Th1 cells and visualized by western blotting with anti-p73 (a). A full scan blot image is provided in Supplementary Fig. 9b. The binding was quantified by qPCR (primers to amplify the sites are in Supplementary Table 7; the nonspecific site is from the Il9 gene). Shown is the percentage of total input (b). In b, the experiment was performed n = 3 times n = 2 replicates in each experiment, and representative results are shown. c Schematic of the domain structure of WT and p73-binding motif deletion reporter mutations in the Ifng, Il12rb2, Il2ra, and Tbx21 loci. p73-binding regions from the indicated chromosomal locations were inserted upstream of minimal promoter (minP) and nano-luciferase gene (Nluc). WT reporter constructs retained the indicated p73-binding motifs, whereas the constructs with deletion mutants lacked the indicated nucleotide sequences. For Ifng, two deletion mutants were generated—one lacking the a motif and the other lacking the b motif. For Il12rb2, we tested a deletion mutant corresponding to the peak B motif. d The indicated reporter constructs containing WT or p73-binding motif deletion mutants were transfected into Th1 cells. Reporter activity was normalized against co-transfected control luciferase reporter construct activity and shown as relative activity. e The level of IL-12Rβ2, IL-2Rα (CD25), and T-bet in Th1 cells expressing control vector, TAp73, or DNp73 was measured by flow cytometric staining and shown as MFI. In d, e, experiments were repeated n = 3 times. In the experiment shown in d, the number of replicates was n = 4 for Ifng, Il12rb2-B, and Il2ra and n = 3 for Tbx21. In the experiment shown in e, the number of replicates was n = 3 for IL-12Rβ2 and T-bet and n = 6 for IL-2Rα. Representative results are shown. All data are shown as mean value ± SD and analyzed by a two-tailed unpaired t test. The P values are indicated (**P < 0.01; ***P < 0.001; ****P < 0.0001). Source data for a, b, d, and e are provided in Source Data Files.