Fig. 4: ASS1 regulates p53-related gene transcription following DNA damage via SMARCC1 succination.
From: ASS1 metabolically contributes to the nuclear and cytosolic p53-mediated DNA damage response
a, Control and Dox-treated HCT116 ASS1-EV (EV) and ASS1-KO cells were assayed for the total, soluble and chromatin-bound fraction and immunoblotted for ASS1, SMARCC1, SNF5 and p53. Markers: H3, nuclear; GADPH, cytoplasmic. n = 3 independent experiments. Right panel: quantification of total and soluble SMARCC1 levels relative to GAPDH, and nuclear SMARCC1 levels relative to H3. b, Immunoprecipitation with anti-SMARCC1 of the chromatin-bound fraction of control and Dox-treated HCT116 ASS1-EV (EV) and ASS1-KO cells and immunoblotted for SMARCC1 and SC. n = 3 independent experiments. c, The nuclear fraction of control and Dox-treated HCT116 ASS1-EV (EV) and ASS1-KO cells immunoblotted for general succination (SC). n = 3 biologically independent samples. d, Immunoprecipitation with anti-SMARCC1 or anti-SNF5 of the chromatin-bound fraction of control and Dox-treated HCT116 ASS1-EV (EV) and ASS1-KO immunoblotted for SNF5 or SMARCC1, respectively. n = 3 independent experiments. e, Mapping of read counts of promoter accessibility using ATAC–seq of control and Dox-treated HCT116 ASS1-EV (EV) and ASS1-KO cells. n = 3 biologically independent samples. f, Promoter accessibility of p53 DDR target genes using ATAC–seq of control and Dox-treated HCT116 ASS1-EV (left) and ASS1-KO cells (right). n = 3 biologically independent samples. g, Pathway enrichment analysis of p53 target genes significantly changing between Dox-treated HCT116 ASS1-KO and EV cells. Each bar shows the fold enrichment of a specific pathway. The bars are color-coded according to the different pathway types: yellow, transcription general; purple, cell cycle; orange, death; green, metabolism. n = 5 biologically independent samples. FDR, false discovery rate. h, Real-time PCR for p53 cell cycle genes performed on SKOV3 cells transfected with GFP, SMARCC1 C520 or SMARCC1 C520E plasmids for 24 h or 48 h. n = 3 biologically independent samples for (C520, CCNE2, 24 h), (C520E, CCNA, 48 h), (C520, CCNE2, 48 h); all others, n = 4 biologically independent samples. Data are represented as the mean ± s.e.m. ns, not significant. P values were determined by two-way ANOVA with Tukey’s multiple-comparison test (in a), Wilcoxon rank-sum test (in f) or unpaired two-tailed Student’s t-test (in c and h).
