Supplementary Figure 5: Fumarate produced by chromatin-associated FH promotes NHEJ.
From: Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation
(a) Chromatin extracts of thymidine double block-synchronized U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were collected 1 h after IR. Chromatin extracts were subjected to immunoblotting (left panel) or immunoprecipitation analyses (right panel) with the indicated antibodies. Data represent one out of 3 experiments. (b) U2OS cells were expressed with the indicated FH-Flag proteins. FH-Flag proteins were immunoprecipitated and the relative FH activity was measured. The data represent the mean ± SD (n = 3 independent experiments). (c) Immobilized, purified GST-H2A.Z protein was mixed with the indicated purified His-FH proteins, with or without DNA-PK. A GST pull-down assay was performed. Immunoblotting analyses were performed with the indicated antibodies. Data represent one out of 3 experiments. (d) U2OS cells were incubated with the indicated concentrations of monoethyl-fumarate. The relative abundance of intracellular fumarate was measured. The data represent the mean ± SD (n = 3 independent experiments). (e) DR-GFP-expressing U2OS cells with depleted FH and reconstituted expression of the indicated FH proteins were incubated with malate (5 mM) for 20 h after I-SceI transfection. ChIP analyses were performed with an anti-Ku70 antibody. The data represent the mean ± SD (n = 3 independent experiments). (f) DR-GFP-expressing U2OS cells with depleted FH and reconstituted expression of the indicated FH proteins were incubated with malate (5 mM) for 20 h after I-SceI transfection. An NHEJ analysis was performed. A representative image of the PCR products digested by I-SceI and BcgI is presented. The data represent the mean ± SD (n = 3 independent experiments). (g) U2OS cells, with or without FH depletion, were reconstituted with expression of the indicated FH proteins before being harvested. The relative abundance of intracellular fumarate was measured. The data represent the mean ± SD (n = 3 independent experiments).
