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. 2020 Nov 12;11(11):976. doi: 10.1038/s41419-020-03185-3

Fig. 2. p53 directly binds to the DEPTOR promoter and transactivates its transcription.

Fig. 2

a The three putative p53-binding sites in the DEPTOR promoter. According to the p53 consensus DNA-binding sequence (left), three putative p53-binding sites, with mismatches underlined, were identified upstream of the “start” codon of DEPTOR (right). b p53 is required for the activity of DEPTOR promoter. Cells with or without p53 deletion were co-transfected with plasmids expressing Renilla luciferase and pGL3 or pGL3 containing the DEPTOR promoter (DEPTOR-luc) with three putative p53-binding sites. Luciferase activity was assessed using a luciferase assay kit and normalized to Renilla luciferase activity (mean ± S.E.M, n = 3; *p < 0.05, **p < 0.01). c p53-binding site C is required for the activity of the DEPTOR promoter. Cells were co-transfected with plasmids expressing Renilla luciferase and pGL3 or pGL3 containing the DEPTOR promoter with all the three putative p53-binding sites (WT), with the deletion of sites A and B (∆AB), or with the deletion of site C (∆C), followed by luciferase reporter assay (mean ± S.E.M, n = 3; ***p < 0.001). d, e p53-binding site C is required for DEPTOR expression. U2OS and SJSA cells with site C of the DEPTOR promoter on chromosome 8 deleted using CRISPR-Cas9 technology (sgRNA underlined), were harvested for qRT-PCR analysis d or IB with the indicated Abs e. (mean ± S.E.M, n = 3; ***p < 0.001) d. f p53 directly binds to site C in vivo. U2OS cells were harvested for ChIP analysis. Input samples or samples precipitated with IgG or p53 antibody were amplified using primers specific for putative p53-binding sites A, B, or C on the DEPTOR promoter or for the p53-binding region on the MDM2 promoter (positive control) f, left, and relative enrichment of p53 at the putative p53-binding site C was quantified by qRT-PCR analysis (mean ± S.E.M, n = 3; ***p < 0.001) f, right.