Monoubiquitination of H2AX protein regulates DNA damage response signaling

Abstract

Double strand breaks (DSBs) are the most deleterious of the DNA lesions that initiate genomic instability and promote tumorigenesis. Cells have evolved a complex protein network to detect, signal, and repair DSBs. In mammalian cells, a key component in this network is H2AX, which becomes rapidly phosphorylated at Ser(139) (γ-H2AX) at DSBs. Here we show that monoubiquitination of H2AX mediated by the RNF2-BMI1 complex is critical for the efficient formation of γ-H2AX and functions as a proximal regulator in DDR (DNA damage response). RNF2-BMI1 interacts with H2AX in a DNA damage-dependent manner and is required for monoubiquitination of H2AX at Lys(119)/Lys(120). As a functional consequence, we show that the H2AX K120R mutant abolishes H2AX monoubiquitination, impairs the recruitment of p-ATM (Ser(1981)) to DSBs, and thereby reduces the formation of γ-H2AX and the recruitment of MDC1 to DNA damage sites. These data suggest that monoubiquitination of H2AX plays a critical role in initiating DNA damage signaling. Consistent with these observations, impairment of RNF2-BMI1 function by siRNA knockdown or overexpression of the ligase-dead RNF2 mutant all leads to significant defects both in accumulation of γ-H2AX, p-ATM, and MDC1 at DSBs and in activation of NBS1 and CHK2. Additionally, the regulatory effect of RNF2-BMI1 on γ-H2AX formation is dependent on ATM. Lacking their ability to properly activate the DNA damage signaling pathway, RNF2-BMI1 complex-depleted cells exhibit impaired DNA repair and increased sensitivity to ionizing radiation. Together, our findings demonstrate a distinct monoubiquitination-dependent mechanism that is required for H2AX phosphorylation and the initiation of DDR.

Figures

FIGURE 1.

RNF2-BMI1 interacts with H2AX in response to DNA damage. A, silver staining of the H2AX complex separated by SDS-PAGE. 293T cells were transfected with the indicated plasmids, and FLAG-H2AX complexes were purified from whole cell extracts and analyzed by mass spectrometry. B, left, co-immunoprecipitation of RNF2 and BMI1 with H2AX from 293T cells transfected with Myc-RNF2, HA-BMI1, and FLAG-H2AX was analyzed by Western blot analysis at the indicated time points after IR (4 gray). Right, co-immunoprecipitation of BMI1 with RNF2 from 293T cells transfected with HA-RNF2 15 min after IR (4 gray). C, co-immunoprecipitation of ATM and RNF2 with BMI1 from 293T cells transfected with HA-BMI1 15 min after IR (4 gray). D, U2OS cells were transfected with the indicated siRNAs. Seventy-two h later, cells were exposed to IR (4 gray) and analyzed by immunofluorescence assay with FK2 and BMI1 antibodies.

FIGURE 2.

Monoubiquitination of H2AX mediated by RNF2-BMI1 regulates DNA damage signaling. A, wild-type (left) and mutant GST-H2AX (right) proteins were incubated with adenosine triphosphate, E1, E2 along with GST, or GST-RNF2 or GST-BMI1 proteins for in vitro ubiquitination of H2AX. B, FLAG-H2AX was overexpressed in U2OS cells transfected with control siRNA, RNF2 siRNA, or BMI1 siRNA. Fifteen min after IR (4 gray), in vivo ubiquitination of H2AX was detected by the indicated antibodies after immunoprecipitation. C, H2AX-deficient MEFs were stably reconstituted with wild-type H2AX and harvested at the indicated time points after IR (4 gray). D, H2AX-deficient MEFs were transiently reconstituted with the indicated H2AX constructs and monoubiquitination of H2AX was detected 15 min after IR (4 gray). E, H2AX-deficient MEFs were stably reconstituted with wild-type H2AX or the H2AX K119R/K120R mutant. The γ-H2AX expression level was detected at the indicated time points after IR (4 gray). F, H2AX-deficient MEFs were stably reconstituted with wild-type H2AX or the H2AX K119R/K120R mutant. Chromatin fractionation was harvested at the indicated time points (IR 4 gray) to detect chromatin-bound ATM. G, radiation-induced nuclear foci formation was analyzed by using antibodies against p-ATM and γ-H2AX after IR (4 gray, 15 min) in H2AX-deficient MEFs stably reconstituted with wild-type H2AX or H2AX K119R/K120R mutant.

FIGURE 3.

RNF2-BMI1 is involved in regulating DNA damage response. A, U2OS cells were transfected with the indicated siRNAs. Analysis of H2AX phosphorylation was performed by immunoblotting at the indicated time points after exposure to IR (4 gray). B, U2OS cells were transfected with control siRNA and RNF2 siRNA. Seventy-two hours later, cells were exposed to IR (4 gray) and analyzed by immunofluorescence assay with the indicated antibodies 15 min after IR. C, U2OS cells were transfected with wild-type or ligase-dead mutant RNF2 tagged by hemagglutinin (HA). Forty-eight hours later, cells were exposed to IR (4 gray), and 15 min after that, p-ATM (right) or MDC1 (left) foci formation were analyzed with immunofluorescence assay. Expression of ectopic HA-tagged protein in cells was identified by anti-HA antibody. D, A-T human fibroblast cells were transfected with control siRNA or BMI1 siRNA. Seventy-two hours later, cells were analyzed by immunofluorescence assay with γ-H2AX antibody 15 min after IR (4 gray). E, co-immunoprecipitation of H2AX, MDC1, MDC1, and ATM from U2OS cells transfected with control siRNA or RNF2 siRNA with FLAG-H2AX at the indicated the time points after IR (4 gray).

FIGURE 4.

RNF2 depletion causes impaired DSB repair and hypersensitivity to IR. A, neutral comet assay was performed in U2OS cells transfected with control, BMI1 siRNA, or RNF2 siRNA at the indicated time points after exposure to IR; representative images (left) and quantitative analysis (right) are shown. Each value represents the mean ± S.E. of three independent experiments using Student's t test. B, U2OS cells were transfected with the indicated siRNAs and exposed to IR at the indicated doses. Fourteen days later, cells were stained with crystal violet. Colonies containing more than 50 cells were counted. Each value represents the mean ± S.D. of three independent experiments. C, a proposed model of DNA damage signaling involving RNF2-BMI1-mediated H2AX monoubiquitination and phosphorylation. The recruitment of the RNF2-BMI1 complex at the damaged site mediates monoubiquitination of H2AX, which facilitates the recruitment of p-ATM, γ-H2AX formation, and in turn recruiting downstream DNA damage responsive proteins such as MDC1 to the damaged loci to activate the DNA damage signaling pathway.