Figure 2.

GFI1 modulates the induction of apoptosis by p53 through CTD and core domain PTMs. (A) Thymocytes from GFI1 WT and KO mice were exposed to 5Gy IR or left untreated. Nuclear extracts from these cells were immunoprecipitated with p53-me2K370 or p53-acK117 specific antibodies and blotted for total p53. (B) Nuclear extracts from GFI1 WT, KO and P2A thymocytes were immunoprecipitated with p53-me2K370 or p53-meK372 specific antibodies and blotted for total p53. (C) Nuclear extracts as in B were immunoprecipitated with a p53-acK117 specific antibody and blotted for total p53. (D) 293T cells were transfected with the indicated combinations of p53-Flag, SMYD2, GFI1 and LSD1 expression vectors. Nuclear extracts were immunoprecipitated with an anti-Flag antibody and blotted for p53-me2K370 and total p53. (E) 293T cells were transfected with the indicated combinations of p53-HA, SMYD2, GFI1, GFI1-P2A and GFI1-ΔSNAG expression vectors. Nuclear extracts were immunoprecipitated with anti-HA or anti-GFI1 antibodies and blotted as indicated. (F) Thymocytes were extracted from GFI1 WT and KO mice, exposed to 5Gy IR or left untreated and fixed with formaldehyde after 2 hours. Chromatin immunoprecipitation was performed to isolate p53-bound DNA fragments. Fold enrichment of the indicated target promoters relative to a non-specific IgG control is shown. Error bars represent s.d. P values: *=<0.05, **=<0.01, ***=<0.001, calculated from a Welch corrected t-test. (G) Thymocytes were extracted from mice carrying combinations of Gfi1 KO and p53 KO. Cells were exposed to 5Gy IR or left untreated and were stained for Annexin V 4 hours later. The proportion of Annexin V positive cells as measured by FACS is shown. Statistical significance was calculated using Fisher’s exact test. (H) mRNA was extracted from thymocytes as in (G). The levels of the indicated genes were measured at the indicated time points after IR by qPCR relative to Gapdh.