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. 2017 Aug 10;45(18):10614–10633. doi: 10.1093/nar/gkx715

Figure 7.

Figure 7.

The chromatin remodeler Tip60–TRRAP–P400 is required for H2AX-dependent NHEJ. (A) Effect of hypotonic condition (Hypo), sodium butyrate (NaBut, 2 mM) and chloroquine (Chlo, 5 μg/ml) on I-SceI-induced mNHEJ in H2AX+/+ and H2AX–/– BGN reporter cells. Bars here and below represent the mean ± S.D. of three independent experiments, each in triplicates. Student's paired t-test between H2AX+/+ and H2AX–/– BGN reporter cells: P = 0.024 in Control; P = 0.122 in Hypo; P = 0.124 in NaBut; P = 0.076 in Chlo. (B–D) Percentage of I-SceI-induced GFP+ cells from BGN reporter cells transfected with RNAi against Tip60 (B), TRRAP (C) and P400 (D). Student's paired t-test in H2AX+/+ BGN reporter cells: P = 0.032 between ‘siTip60 #1’ and ‘Scramble’ and P = 0.039 between ‘siTip60 #2’ and ‘Scramble’ in (B); P = 0.008 between ‘siTRRAP #1’ and ‘Scramble’ and P = 0.003 between ‘siTRRAP #2’ and ‘Scramble’ in (C); P = 0.032 between ‘siP400 #1’ and ‘Scramble’ and P = 0.039 between ‘siP400 #2’ and ‘Scramble’ in (D). Student's paired t-test in H2AX–/– BGN reporter cells: NS between ‘Scramble’ and any other siRNA. (E) Model for the function of H2AX in NHEJ. H2AX-dependent NHEJ is responsible for about a half of classical but mutagenic NHEJ, contributing to ∼7–25% of total NHEJ. Upon DSBs, ATM can phosphorylate H2AX and help initiate the formation of the γH2AX/MDC1 chromatin domain. This domain may recruit the remodeler Tip60–TRRAP–P400. Alternatively, if DSBs induce transient heterochromatization of the damaged chromatin, the γH2AX/MDC1 chromatin domain may help relax the heterochromatized region via Tip60–TRRAP–P400 recruited. Tip60–TRRAP–P400 thus repositions nucleosomes locally around the breaks and allow sufficient processing of DNA ends for efficient DSB repair by C-NHEJ at the expense of losing a few extra nucleotides at NHEJ junctions. See details in the Discussion.