We have shown that BMI1 is recruited to sites of DNA damage. Several types of DNA lesions are able to recruit BMI, including DNA DSBs induced by laser scissors, ZFNs, and IR, and collapsed replication forks induced by HU and aphidicolin. BMI1 is rapidly localized to sites of laser scissors induced breaks, and remains present for up to 24 h (see Fig. S1 in the supplemental material). The sustained localization of BMI1, assayed at 30 min after DNA damage, is dependent on H2AX phosphorylation and intact ATM/ATR (). Sustained localization of BMI1 is also reduced in Rnf8−/− MEFs but is intact in 53bp1−/− cells and in Brca1 mutant cells. This suggests that sustained BMI1 localization to DNA damage sites requires signaling through ATM/ATR pathway but is independent of 53BP1 and proximal to BRCA1.
Of note, BMI1 localization is still present at very early time points (5 to 10 min) after DNA damage in H2AX−/−
MEFs (see Fig. S3 in the supplemental material). This observation suggests that initial transient recruitment of BMI1 may occur independently of H2AX phosphorylation. Recent reports suggest that some polycomb factors, including MEL18, may be recruited transiently to sites of DNA damage in a PARP-dependent fashion (10
). BMI1 localization to laser scissors-induced DNA damage is still present in PARP1 deficient cells, both at early and late time points (see Fig. S6 in the supplemental material). Thus, initial, transient recruitment of BMI1 to DNA breaks may be independent of both PARP1 and H2AX; however, sustained localization of BMI1, and subsequent ubiquitination of H2A-K119, requires intact ATM/ATR, H2AX phosphorylation, and RNF8 recruitment. The differing dynamics of MEL18 and BMI1 to sites of DNA damage suggest that MEL18 and BMI1 may be differentially regulated in the DNA repair process. Thus, different polycomb factors may be present in several independent complexes and function in different signaling pathways during the DNA damage response.
DNA damage-induced ubiquitination of H2A-K119 is dependent on BMI1. However, DNA damage-induced polyubiquitination of other substrates is not impaired and RAP80 recruitment is intact in Bmi1−/−
cells (). This suggests that, unlike RNF8, BMI1 is specifically required for H2A-K119 ubiquitination but not required for polyubiquitination of other substrates. Western blot data suggest that the monoubiquitination of H2A-K119 is the predominant modification induced by DNA damage at this residue; however, we cannot exclude the possibility that polyubiquitination of H2A-K119 may also be present. These data are consistent with the presence of multiple, topologically distinct ubiquitin species at DSBs (34
). Since BMI1 is required for efficient HR-mediated repair (), these data further suggest that BMI1-mediated H2AK119 ubiquitination at sites of DNA breaks plays a role in facilitating HR-mediated repair.
These data support a model in which DNA damage-induced activation of ATM and ATR leads to local H2AX phosphorylation and recruitment of MDC1 and RNF8. These events are required for sustained localization of BMI1 to sites of DNA breaks, where, together with RING1B, BMI1 ubiquitinates H2A-K119 () and contributes to efficient HR-mediated DNA repair. The exact function of ubiquitinated H2A-K119 in facilitating HR-mediated DNA repair remains to be determined. It is also possible that other substrates of BMI1 ubiquitin-ligase activity, such as TOP2, may also play a role in the DNA repair pathway (1
). Since H2A-K119 ubiquitination has a role in transcriptional silencing, BMI1-associated H2A-K119 ubiquitination at DNA breaks may also play a role in mediating local transcriptional silencing during the DNA repair response (45
Model showing the role for Bmi1 in the DNA damage response pathway. See the text for details.
A role for BMI1 in the DNA damage response has been previously reported (10
). BMI1 was shown to modulate mitochondrial production of reactive oxygen species (ROS). Loss of BMI1 leads to greater generation of ROS-mediated DNA damage, resulting in checkpoint activation (29
). BMI1 has also been recently reported to localize at sites of DNA damage, interact with ATM, and contribute to radioresistance in glioblastoma cells (13
). Our work demonstrates a direct and specific role of BMI1 in local H2A-K119 ubiquitination at sites of DNA damage and functional role of BMI1 in HR-mediated DNA repair. We show that cells lacking BMI1 are sensitive to ionizing radiation () (13
). How much of this phenotype is due to direct impairment of HR-mediated repair and how much is due to the potential role of BMI1 in regulating ROS metabolism is not clear at present. It is also possible the increased ROS-mediated DNA damage seen in cells lacking BMI1 may be partly due to impaired DNA repair pathways.
Unlike the findings presented here, Facchino et al. found that localization of BMI1 to sites of IR-induced damage was not dependent upon ATM activation. The differences between our findings and those of Facchino et al. may be due to different cell types analyzed and differing methods of generating and visualizing DNA breaks. Our data are in agreement with Ismail et al., who recently reported that BMI1 was recruited to sites of DNA damage and involved in histone ubiquitination (25
). These researchers found that an initial recruitment of BMI1 is still present in H2AX−/−
cells 5 min after induction of damage. Our data also show that initial recruitment is partly independent of H2AX. However, we demonstrate that both sustained localization of BMI1 at time points greater than 20 min, and H2A-K119 ubiquitination is abolished in H2AX−/−
cells. These data suggest that there may be biphasic recruitment of BMI1, with an immediate transient phase that is independent of H2AX and a later sustained phase that is dependent upon ATM/ATR and H2AX phosphorylation.
We have demonstrated that BMI1, CBX2, and RING1B can all localize to sites of DNA breaks. It is not clear what other polycomb-related proteins are involved in the DNA repair response. Both PHF1 and heterochromatin protein 1 (HP1) family members have been shown to be recruited to sites of UV damage and DNA DSBs in human cells (21
). The histone methyltransferase EZH2 has also been shown to be recruited to sites of DNA breaks induced by ISCE1-induced DNA breaks in mammalian cells (38
). A recent report has demonstrated that a variety of polycomb proteins, including MEL18, CBX6, CBX7, and CBX8, are recruited transiently to sites of DNA damage in a PARP-dependent manner (10
). It is possible that distinct complexes of polycomb group proteins may be involved in transient early PARP-dependent DNA damage response and the sustained ATM/ATR-dependent DNA damage response.
BMI1 and related polycomb group proteins are thought to play an essential role in mediating chromatin compaction and transcriptional silencing during development. The recruitment of BMI1 to sites of DNA breaks suggests that similar effects may be occurring near sites of DNA damage. DNA DSBs have been shown to induce local ATM-dependent transcriptional silencing in nearby chromatin regions that is associated with H2A-K119 ubiquitination and chromatin compaction (45
). Transcriptional silencing associated with DNA methylation of CpG islands and associated changes in histone methylation also been shown to occur near sites of DNA breaks (38
). The data present here would suggest a role for BMI1 and related polycomb proteins in mediating these processes. Thus, BMI1 and associated polycomb proteins may link chromatin changes associated with DNA repair response to localized transcriptional repression near sites of DNA damage. Whether this can result in long-term epigenetic changes at sites of DNA damage, i.e., a chromatin memory of prior to DNA damage, under certain conditions remains to be determined.
BMI1 has been implicated in playing a crucial role in stem cell maintenance. Loss of BMI1 is associated with cell cycle arrest and premature senescence in many cell types (53
). Dysfunction of DNA repair factors also induces similar checkpoint activation and cellular senescence, due to the accumulation of unrepaired DNA lesions. It is possible that at least part of the phenotype associated with the loss of BMI1 is due to the role of BMI1 in the DNA damage response.
In summary, these data demonstrate that BMI1 is recruited to sites of DNA damage, where it is required for ubiquitination of histone H2A. This function of BMI1 is downstream of ATR/ATM and RNF8 and is required for efficient HR-mediated DNA repair. These findings may give new insight into the role of BMI1 in mediating chromatin changes at sites of DNA damage and the impact of its dysfunction in both stem cells and somatic cells.