Extended Data 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, HCT116 ASS1-EV (EV) and ASS1-KO cells were incubated with (Dox) or without Dox (Ctrl) for 2 hrs, and incubated for 48 hrs. RNA was extracted and SMARCC1 RNA levels were detected using quantitative real-time PCR. (n = 3 biologically independent samples) b, HCT116 ASS1-EV (EV) and ASS1-KO cells were incubated with Dox for 2 hrs, and after 48 hrs, fractionated and the nuclear fraction was immunoblotted for succination (SC). Prior to succinaton staining, the membrane was stained with Ponceau for total protein. (n = 3 biologically independent samples) c, Mapping of read counts of promoter accessibility using ATAC-Seq around peaks of SMARCC1 binding to chromatin in HeLa cells from the ENCODE project (ENCODE experiment ENCFF971JGA). d, Mapping of read counts of promoter accessibility using ATAC-Seq around peaks of H3K27ac enhancer-associated histone marks, when considering only intergenic peaks, as annotated by HOMER59, data from the ENCODE project (ENCODE experiment ENCSR661KMA). e, Left panel: Heat-map analysis of transcriptomic data comparing changes in gene expression between EV Ctrl and ASS1-KO Ctrl cells. (n = 5 biologically independent samples) Right panels: RNA expression levels of selected cell cycle and survival genes regulated by p53. (P-value: ZNF385A: <0.0001; GADD45A: <0.0001) (n = 5 biologically independent samples) f, RNA expression levels of p53-related genes are down-regulated in EV treated with Dox vs. untreated, while their expression is increased in ASS1 KO cells treated with Dox compared to treated EV. (n = 5 biologically independent samples) g, SKOV3 cells were transfected with GFP, SMARCC1 C520 or SMARCC1 C520E plasmids for 48 hrs, and subsequently real-time PCR for SMARCC1 and ASS1 was performed to verify transfection. (n = 4 biologically independent samples) Data are represented as the mean ± s.e.m. ns, not significant. P values were determined by unpaired two-tailed Student’s t-test (a, e, and g).
