Fig. 4.

UFL1 monoufmylates histone H4 and promotes ATM activation. a Selected proteins identified by mass spectrometry from irradiated 293T cell expressing Flag-His vector or Flag-His-UFM1. N = 1 sample in each group was analyzed. The full list of identified proteins is provided in Supplementary Data 1. Among histone proteins, only H4 is enriched in the Flag-His-UFM1 purification (9 unique/20 total peptides) compared to the Flag-His purification (6 unique/7 total peptides), suggesting that H4 might be ufmylated. b Flag-His-ufmylated proteins were purified from 293T cells before and after IR (2 Gy) after purification with nickel beads and anti-Flag agarose. The immunoprecipitates were detected with indicated antibodies. c Flag-His-ufmylated H4 was purified from control and UFL1 knockdown cells with or without 2 Gy IR and blotted with indicated antibodies. d In vitro ufmylation assay. Purified UBA5, UFC1, UFL1, UFM1, and H4 proteins were incubated together in the presence of ATP and MgCl₂ at 30^°C for 90 min. The reaction products were probed with indicated antibodies. e Wildtype (WT) histone H4 and 11 different single lysine (K) to arginine (R) mutants were transfected into U2OS cells. Flag and His tandem purification was performed and H4 ufmylation was analyzed. f Constructs expressing WT or K31R H4 were transfected into U2OS tet-on UFL1 shRNA expressing cells, and the cells were treated with doxycycline as indicated. Thirty minutes after 2 Gy IR, the cells were harvested and blotted with indicated antibodies. g Colony formation of U2OS cells expressing WT H4 or H4K31R following IR. The data presented are mean ± s.e.m. for n = 3 independent experiments. Statistical significance was calculated using two-way ANOVA. *p < 0.05, **p < 0.01. h U2OS cells stably expressing UFL1 Tet-on shRNA were treated with doxycycline (Dox) and irradiated with 2 Gy IR. Thirty minute later, cells were harvested. Half of the cells were lysed. Chromatin fractions were isolated from other half of cells. The samples were blotted with indicated antibodies. Source data are provided as a Source Data file