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. 2016 May 4;17(6):874–886. doi: 10.15252/embr.201541685

Figure 7. HUWE1 is a novel ubiquitin ligase for H2AX .

Figure 7

  • A, B
    The impact of HUWE1 on H2AX phosphorylation and ubiquitination, under normal conditions (A) or upon induction of replication stress (2 mM HU for 18 h, or 2 h after exposure to 40 J/m2 UV). Control or HUWE1‐knockout HeLa cells were analyzed.
  • C
    In vitro ubiquitination assay showing that recombinant HUWE1C‐ter can mono‐ubiquitinate H2AX.
  • D
    Wild‐type but not the PIP mutant can correct the defective H2AX ubiquitination in HUWE1‐knockout 293T cells.
  • E
    Chromatin immunoprecipitation showing that γH2AX binding to the common fragile site FRA3B is reduced in HUWE1‐knockout 8988T cells. Cells were treated with 600 nM aphidicolin for 24 h. Binding was quantified relative to input material. Shown is the average of four experiments ± SD. P‐value is 4.4 × 10−6.
  • F
    Western blots showing that several H2AX ubiquitin ligases participate in γH2AX ubiquitination following replication stress. HUWE1, RNF168, and BMI1 were knocked‐down in HeLa cells. The efficiency of the knockdown is shown in Fig EV4. Cells were treated with 600 nM aphidicolin for 24 h, 2 mM HU for 24 h, or analyzed 2 h after exposure to 40 J/m2 UV.
  • G
    Chromatin immunoprecipitation from HeLa cells, showing that knockdown of H2AX ubiquitin ligases can reduce γH2AX binding to FRA3B. Cells were treated with 600 nM aphidicolin for 24 h. Binding was quantified relative to input material. Shown is the average of three experiments ± SD. The P‐values shown indicate the statistical significance relative to siControl (0.0016, 0.0005, and 0.0009, respectively).
  • H
    Chromatin fractionation experiments showing that HUWE1‐knockout HeLa cells, treated with 2 mM HU for 24 h, fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA. In contrast, 53BP1 chromatin association is not induced by HU treatment, and not affected by HUWE1 knockout, and thus can serve as loading control. Shown are blots of whole‐cell extract (WCE) samples, representing input material, and of chromatin pellet samples.