Fig. 1: p53 trans-activates IGSF9 in breast cancer.

A The canonical binding site of TP53 predicted by JASPAR is manifested by the sequence logograph. B Veen diagram analysis showing 1795 potential target genes of p53. C GeneMANIA was used to identify IGSF9 correlating with TP53. 20 related genes were in the outer circle while two hub genes were in the inner circle. The color of the line shows different kinds of their correlations. D The positive correlation between IGSF9 and TP53 mRNA in breast cancer in TCGA cohort by Pearson correlation coefficient analysis. E The lower level of IGSF9 and p53 protein was detected in breast cancer cells. IGSF9 and p53 protein in breast cancer cells MCF-7 and normal mammary cells MCF-10A were determined by western blot. Relative intensity of IGSF9 and p53 was quantified by Image J and normalized to GAPDH. Data were represented as the mean ± SD. The Student’s t test was used; ***P < 0.001. p53 binds to the promoter of IGSF9. ChIP assay was applied to determine the binding of IGSF9 promoter to p53. p53 antibody enriched the DNA sequence of IGSF9 promoter. PCR (F) and qRT-PCR quantification (G) of the immunoprecipitated DNA were measured. Rabbit IgG was used as a negative control and RNA polymerase II (RNAP II) as a positive control. Values represented enrichment relative to input DNA. Data were represented as the mean ± SD. The Student’s t test was used; **P < 0.01. H −183/−116 nt region of IGSF9 exhibited higher luciferase activity. Luciferase reporter assays were conducted to determine transcription activity of a serial of IGSF9-luc reporter genes ranging from −3000 to +50 bp of putative promoter. ***P < 0.001. I Nucleotide sequence of −183/−116 nt full length, deletion and point mutations were constructed. Transcriptional factor p53-binding sites were showed. J Deletion and mutations within p53 binding site (−137/−131 nt) reduced IGSF9 promoter activity. Data were represented as the mean ± SD. The Student’s t test was used; ***P < 0.001. K p53 showed high binding capability with promoter (−183/−116 nt) of IGSF9. Data were represented as the mean ± SD. The Student’s t test was used; ***P < 0.001. L TP53 knockdown decreased IGSF9 protein expression. IGSF9 was over-expressed and TP53 was knocked down in MCF7 cells as indicated. IGSF9 and p53 protein was determined by western blot. Relative intensity of IGSF9 and p53 was normalized to GAPDH. Data were represented as the mean ± SD. The Student’s t test was used; ***P < 0.001. R157H, but not other mutants (G244D and R248Q) of p53 lost the transcriptional activity on IGSF9. Schematic diagram illustrating the protein domains of p53, and three mutation hotspots as indicated (M). Luciferase assay (N) and western blot (O) were conducted with MCF-7 cell. Quantification (P) was performed by ImageJ. Error bars denote as mean ± SD. The Student’s t test was used; **P < 0.01, ***P < 0.001.