Supplementary Figure 6: Fumarate produced by chromatin-associated FH inhibits KDM2B-mediated demethylation at DSB regions.

(a–c,f) The data represent the mean ± SD (n = 3 independent experiments). (d,e,g) Data represent one out of 3 experiments. (a,b) U2OS cells were transfected with or without a vector expressing I-SceI. (a) ChIP analyses with antibodies for H3K36me2, H3K9me2, H3K9me3 and H3K27me2 were performed at the indicated time points after I-SceI transfection. (b) ChIP analyses with the indicated histone H3 methylation antibodies were performed 20 h after I-SceI transfection. The primers described in Fig. 1f were used for the PCR. Control primers were selected against a specific region of chromosome 12. The y-axis stands for the value of I-SceI-induced fold increase of binding of the methylated H3 (the IP value was normalized to the input). (c) U2OS cells were expressed with the indicated H3 proteins. ChIP analyses with an anti-Ku70 antibody were performed at the indicated time points after I-SceI transfection. Immunoblotting analyses were performed with the indicated antibodies. (d) GSC11 cells were expressed with a vector for control shRNA, FH shRNA (left panel), H2A.Z shRNA (right panel) and were reconstituted with the indicated FH or H2A.Z protein expression. Immunoblotting analyses were performed with the indicated antibodies. (e) U2OS cells were expressed with a vector for control shRNA or KDM2B shRNA. Immunoblotting analyses were performed with the indicated antibodies. (f) U2OS cells with depleted FH and reconstituted expression of the indicated FH proteins or with depleted H2A.Z and reconstituted expression of the indicated H2A.Z proteins were transfected with a vector expressing I-SceI. ChIP analyses with an anti-KDM2B antibody were performed 30 h after I-SceI transfection. # stands for no statistical significance between the indicated samples and the WT counterparts. (g) GSC11 cells with depleted endogenous KDM2B were exposed to IR (10 Gy) and harvested 1 h after IR. Chromatin extracts were incubated with malate (2.5 mM) in the presence or absence of αKG (50 μM or 2.5 mM) for 30 min, followed by incubation with KDM2B.