In this report, we have uncovered a novel role for BMI1 in the DDR. Our data reveal that BMI1 and RING2 are rapidly recruited to sites of DSBs and that a BMI1\RING2 complex regulates H2AX ubiquitylation at DNA lesions. The demonstration that BMI1 is necessary for DNA repair, an efficient DDR, and cell viability upon exposure to irradiation suggests that BMI1 plays an early critical role in the DDR. This role for BMI1 in the DDR suggests broader functions than its documented function as a transcriptional repressor.
Recently, cells from mice lacking BMI1 have been shown to have significant mitochondrial dysfunction accompanied by a sustained increase in reactive oxygen species (ROS) that are sufficient to engage the DDR pathway (Liu et al., 2009
). In that study, treatment with an antioxidant or interruption of the DDR by Chk2 deletion substantially improved some, but not all, aspects of the severely defective BMI1 KO mouse phenotype. They observed an induction of the DDR, and this was mediated by increased oxidative stress. Our results indicate the BMI1-null and BMI1-depeleted cells also have defects in efficiently repairing DNA when exogenous DNA-damaging agents such as IR or CLM induce damage. The defect that we have observed may synergize with increased oxidative stress to lead to the accumulation of DNA damage with subsequent effects on viability.
Animals with defective repair pathways are increasingly found to display radiosensitivity and immunodeficiency. BMI1 KO mice develop some phenotypes that are associated with poor DNA repair. For example, BMI1 KO mice share overlapping features with Nijmegen breakage syndrome (NBS) and Fanconi anemia. These are autosomal recessive chromosomal instability disorders characterized by developmental defects, progressive bone marrow failure, and cancer susceptibility (Taniguchi and D’Andrea, 2006
). BMI1 KO mice, born with a hypocellular bone marrow, have a normal number of myeloid cells in the peripheral blood but lower numbers of lymphocytes (van der Lugt et al., 1994
). Also, BMI1-depleted mice are infertile, and develop ataxia and die within 2 mo after birth (van der Lugt et al., 1994
). Consistent with a new role of BMI1 in DSB repair, we found that in the absence of DNA damage, BMI1 KO cells have higher levels of γ-H2AX foci per cell, and we observed an increase in H2AX phosphorylation upon transfecting cells with BMI1-specific shRNA ( and not depicted). Consequently, it may be that some of the phenotypes observed in BMI1 KO mice are related to the new function of BMI1 in DSB repair described here.
Several recent studies implicated other PcG proteins in DNA repair. Overexpression of EZH2 in breast epithelial cells reduced the expression of Rad51 paralogues, which are required for proper repair of DSBs by homologous recombination (Zeidler et al., 2005
). However, the survival of BRCA1-deficient tumor cells requires EZH2 expression, and pharmacological disruption of EZH2 is synthetic lethal in combination with BRCA1 deficiency (Puppe et al., 2009
). The PcG protein PHF1 was found to recruit to DNA damage sites and contribute to the DDR (Hong et al., 2008
). Our data extend these results and suggest a direct role for the BMI1–RING2 E3 ubiquitin ligase complex in DSB repair.
Interplay between PcG complexes and modified histones has been proposed to mediate stable transcriptional repression (Gieni and Hendzel, 2009
). In the prevailing model, PRC2 is recruited to specific genomic locations where it catalyzes H3K27me3. The modified histones, in turn, recruit PRC1, which ubiquitylates H2A histones at lysine 119, which then inhibits RNA polymerase II elongation. We found that PRC2 is not required for the recruitment of BMI1–RING2 complexes to DSB sites. This indicates that the recruitment of the PRC1 complex to the sites of DNA damage does not involve the same mechanism that is involved in gene regulation. Rather, screening several mutant cell lines revealed a requirement for NBS1. The normal recruitment of NBS1 in BMI1 WT and BMI1 KO cells is consistent with NBS1 being upstream of BMI1 in the DDR.
Chromatin regulators with well-established functions in gene regulation such as the ATP-dependent chromatin remodeling complexes INO80, SWR1, RSC, and SWI/SNF, and the histone acetyltransferase complex TRAP-TIP60 also function in DNA repair (Tsukuda et al., 2005
; Murr et al., 2006
). Our results demonstrate that BMI1 is an H2A/H2AX E3 ubiquitin ligase that also has functions in both transcriptional regulation and DNA repair.
It remains formally possible that BMI1 and RING2 function indirectly in the DSB repair process and alter the ubiquitylation levels of γ-H2AX by decreasing the pool of u-H2AX throughout the nucleus. In this case, when cells are treated with IR, DSBs occurring in regions of the genome that contain u-H2AX will generate uγ-H2AX as the H2AX in the vicinity of the DSB is phosphorylated. A reduction in uH2AX throughout the genome will then lead to less uγ-H2AX. This explanation is less likely than the reduced uγ-H2AX observed in BMI1-null or depleted cells to reflect the direct contribution of BMI1–RING2 to ubiquitylation after the induction of DSBs. DSBs are known to be sites of ongoing ubiquitylation of γ-H2AX and H2A. This ubiquitylation turns over rapidly. For example, treatment of cells for 60 min with a proteosome inhibitor, which results in the accumulation of ubiquitin in the cytoplasm, is sufficient to virtually eliminate nuclear ubiquitin and nuclear ubiquitylated chromatin (Dantuma et al., 2006
). Similarly, ubiquitin accumulating at sites of laser microirradiation–induced DNA damage is completely depleted within 60 min of treatment with proteosome inhibitor (Mailand et al., 2007
). Thus, to affect the ubiquitylation levels of γ-H2AX 60 min after IR would appear to necessitate that the E3 ubiquitin ligase activity being measured is predominantly ubiquitylation that has taken place after the induction of DNA damage.
The recruitment of BMI1 and RING2 to IRIF and laser-induced DNA damage support the conclusion that the substantial reduction of uγ-H2AX in the absence of BMI1 reflects the absence of an activity that contributes to ubiquitylation directly at the sites of DSBs. The reduced recruitment of proteins that are known to involve ubiquitin signaling at DSBs further supports a direct contribution of BMI1–RING2 to ubiquitin signaling at DSBs and in response to DNA damage. Whether the reduced rate of DSB repair and the increased sensitivity to IR are determined solely through ubiquitin-dependent mechanisms or involve additional functions of BMI1–RING2 in DSB repair remains to be determined. Regardless of the underlying mechanism, it is clear that BMI1–RING2 function is important for DNA DSB repair.
There are additional unidentified H2AX E3 ubiquitin ligases that are present in cells that are responsible for γ-H2AX ubiquitylation at sites of DNA damage. There is persistent monoubiquitylation of γ-H2AX in RNF8-deficient cells (Huen et al., 2007
; Mailand et al., 2007
; Shao et al., 2009
). Experiments that deplete a deubiquitylase involved in the DDR, BRCC36, in RNF8-null cells enhance the accumulation of γ-H2AX ubiquitylation (Shao et al., 2009
RNF8 is also responsible for the generation of uγ-H2AX. We found that BMI1 and RNF8 independently contribute to radiation resistance but have overlapping roles in the ubiquitylation of H2A/H2AX. The coordinated crosstalk and overlapping activities of ATM and DNA-PK serve as an outstanding precedent for two enzymes with overlapping specificity both being functionally important in the DDR. There is substrate redundancy in the kinase activities of DNA-PK and ATM, including the phosphorylations of histone H2AX, RPA, and even DNA-PKCs itself (Wang et al., 2001
; Chan et al., 2002
; Stiff et al., 2004
; Huen et al., 2007
). Our results show that cells deficient in both BMI1 and RNF8 are more sensitive to radiation than cells lacking either protein individually. This suggests that these proteins represent distinct pathways even though they may share common substrates such as γ-H2AX. The sharing of substrates (H2AX) that is observed between BMI1 and RNF8 without the capacity for functional replacement, therefore, is analogous to the roles that ATM and DNA-PK play in the DDR.