An Anticlastogenic Function for the Polycomb Group Gene Bmi1
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An anticlastogenic function for the Polycomb Group gene Bmi1 Jalila Chagraouia, Josée Héberta,b,c, Simon Girarda, and Guy Sauvageaua,b,c,1 aLaboratory of Molecular Genetics of Hematopoietic Stem Cells, Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC, Canada H3C 3J7; and bDivision of Hematology, Department of Medicine and cLeukemia Cell Bank of Quebec, Maisonneuve-Rosemont Hospital, Montréal, QC, Canada H1T 2M4 Edited by Suzanne Cory, The Walter and Eliza Hall Institute, Melbourne, VI, Australia, and approved February 16, 2011 (received for review September 24, 2010) BMI1 is a key component of multiprotein Polycomb repression to express low levels of the RNF2 PcG protein. Recently, the complex 1 (PRC1), and its disruption in mice induces severe aplastic SUMO E3 ligase PcG gene Pc2, known to interact with BMI1 anemia by early adulthood. The contributing mechanisms respon- and RNF2 within Polycomb repression complex 1 (PRC1), has sible for this phenotype remain elusive. Here we show that trans- been shown to be essential for the sumoylation and nuclear lo- formed human cell lines as well as primitive hematopoietic cells calization of centrin 2 required in the nucleotide-excision repair exhibit a high frequency of spontaneous chromosome breaks process (17). upon BMI1 depletion and are hypersensitive to genotoxic agents. Two recent studies have linked Bmi1 to oxidative metabolism. Consistent with these observations, we found that BMI1 is re- Chatoo et al. (18) reported that Bmi1 prevents intracellular accu- cruited rapidly to DNA damage foci where it blocks transcriptional mulation of reactive oxygen species (ROS) in neurons through re- elongation. We also show that BMI1 contributes to homologous pression of p53 pro-oxidant activity. Liu et al. (19) showed that recombination DNA repair and is required for checkpoint recovery. Bmi1 deficiency leads to increased expression of several genes in- Taken together, our results suggest that BMI1 is critical for the volved in ROS homeostasis and mitochondrial function. They also maintenance of chromosome integrity in both normal and trans- demonstrated that the activation of ROS-mediated DNA damage fi formed cells. response in Bmi1-de cient mice occurs in an ink4a/arf-independent manner. (19) Here, we build on these observations and show that BMI1 ensures the maintenance of chromosome integrity in both Bmi1 he Polycomb group gene (PcG) is known as a key de- normal and transformed cells by promoting transcriptional re- Tterminant of normal and leukemic hematopoietic stem cell pression at the lesions. We also document that the loss of Bmi1 (HSC) function. In its absence HSCs fail to self-renew, leading recapitulates several phenotypes associated with genomic instability to bone marrow failure and profound anemia in young mice. syndromes (i.e., cell-cycle defects, DNA damage sensitivity, and Although many functions have been ascribed to BMI1, the mo- ROS accumulation) that often result in bone marrow failure, im- lecular mechanisms underlying its role in HSCs remain un- munodeficiency, congenital abnormalities, and growth retardation. certain. In mouse and human fibroblasts, Bmi1 genetically ink4a arf interacts with p16 and/or p19 to prevent senescence (1–4). Results BMI1 binds the loci together directly with other PcG proteins Bmi1−/− fi Correlation Between Survival of Mice and Activity of Long- leading to changes in histone modi cations compatible with gene Term Repopulating HSC. Deletion of Bmi1 leads to axial skeleton repression (5, 6). Evidence suggests that the inactivation of patterning and hematopoietic defects, severe ataxia, and seizures. ink4a/arf is not the sole mechanism by which BMI1 regulates − − Although Bmi1-deficient mice survive after birth, about half of HSC activity. In support of this evidence, Bmi1 / leukemia cell ink4a arf them die before the weaning period (3 wk old) (20). Mice that do lines lacking expression of p16 and p19 still require the survive the first days die within 4–10 wk. By simplifying young ectopic expression of Bmi1 to generate leukemia in vivo (7). ataxic animals’ access to water and nutrients, we were able to Moreover, the demonstration that Bmi1 genetically interacts extend the median and maximum lifespan of Bmi1-deficient mice with E4 transcription factor 1 (E4f1), an atypical p53 E3 ubiq- significantly, to 150 d (Fig. S1A), at which time a phenotype of uitin ligase (8), to regulate the repopulating activity of HSCs in very severe bone marrow failure was observed. Considering the an INK4a/ARF-independent manner further substantiates this 4- to 5-mo transition period typically associated with HSCs that hypothesis (9). show short- or intermediate-term versus long-term repopulation Biochemical studies recently showed that the PcG protein ring (LTR) potential (21), these data are consistent with the hypoth- fi nger protein 2 (RNF2) is an E3 ubiquitin ligase exhibiting esis that Bmi1 deficiency is not compatible with the maintenance monoubiquitination activity toward histone H2A. BMI1 has been of LTR-HSC activity (Fig. S1B). proposed to protect RNF2 from degradation and to enhance its −/− monoubiquitinating activity (10). Cell-Cycle Progression Defect in Bmi1 HSCs. By conducting a se- Several studies suggested that PcG proteins, including BMI1, ries of genetic complementation studies (Fig. S1C), we showed have a role in the DNA damage response/repair process (11–13). that disruption of p53/pRb pathways (E6–E7), prevention of Hong et al. (14) recently reported the immediate recruitment of premature senescence (T-box 2, TBX2) (22) or reduction of the Polycomb repressive complex 2 (PRC2)-associated PHD fin- ROS levels (N-acetyl cysteine, NAC) failed to restore the LTR − − ger protein 1 (PHF1) to double-strand breaks (DSB) following activity of Bmi1 / HSCs (Fig. S1 D and E). Although freshly − − irradiation. PHF1 interacts directly with KU70/KU80 and RAD50 isolated Bmi1 / fetal liver cells can be fully rescued by Bmi1 or proteins, and its knockdown sensitizes cells to irradiation. Simi- larly, Rouleau et al. (15) showed that poly(ADP ribose)poly- merase 3 (PARP-3), which interacts with proteins typically found Author contributions: J.C. and G.S. designed research; J.C. performed research; J.H. and at DSBs in the nonhomologous end-joining (NHEJ) pathways, is S.G. contributed new reagents/analytic tools; J.C., J.H., and S.G. analyzed data; and J.C. part of the PRC2 complex that includes enhancer of zeste ho- and G.S. wrote the paper. molog 2 (EZH2) and suppressor of zeste 12 homolog (SUZ12). The authors declare no conflict of interest. Together these observations strongly suggest a role for the PRC2 This article is a PNAS Direct Submission. in the NHEJ repair pathway. 1To whom correspondence should be addressed. E-mail: [email protected]. Using RNAi, Bergink et al. (16) demonstrated that UV- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. induced H2A monoubiquitination is reduced in cells engineered 1073/pnas.1014263108/-/DCSupplemental. 5284–5289 | PNAS | March 29, 2011 | vol. 108 | no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1014263108 Downloaded by guest on September 28, 2021 − − its ΔPEST mutant, it was impossible to rescue Bmi1 / cells that In both cell lines, Bmi1 knockdown resulted in the formation of were kept in culture for 2 d or more. radial chromosome forms reminiscent of some chromosomal in- To gain further insights into this observation, we examined the stability syndromes. We also observed an increase in the rate of − − cell-cycle status of primitive Bmi1 / hematopoietic cells that were spontaneous chromosome breaks in both shBmi1-transfected cell kept in culture under growth conditions that normally support fetal lines (arrows in Fig. S3A). HCT116 cells transfected with a control liver HSC activity (23). As shown in Fig. S1F, cultures initiated vector showed a maximum of two breaks per cell, with 84% of cells with these cells showed a significant increase of both cycling (fewer having no chromosome breaks (black bars in Fig. S3B). In con- cells in G0) and apoptotic (subG1) cells compared with control trast, shBmi1-transfected HCT116 cells were more susceptible to − − HSCs. Strikingly, Bmi1 / HSCs accumulated in G2 (Fig. S1F). spontaneous chromosome breakage, with a maximum of 16 breaks This finding indicates that Bmi1-deficient fetal liver HSCs are per cell and with 56% of cells having more than two breaks (white exquisitely sensitive to ex vivo manipulation and are impaired ir- bars). Similarly, although 62% of control 293T cells showed no reversibly after a few days in culture. Overall, these data suggest break, 84% of shBmi1-transfected 293T cells displayed more than − − that the severe phenotype observed in Bmi1 / cells is likely to be two breaks per cell (Fig. S3B Right). shBmi1-transfected 293T cells the result of cumulative effects rather than being attributable only exhibited a much higher frequency of chromosome breakage than to deregulation of p53 or pRb pathways. shBmi1-transfected HCT116 cells, with the number of breaks per cell exceeding 20 in 56% of the scored metaphases (Fig. S3B). γ-H2AX Foci Formation in the Absence of BMI1. The multiple cell- Together these results link spontaneous chromosome breakage −/− cycle anomalies observed in cultured Bmi1 cells, together with with reduction in BMI1 protein levels. the growing body of evidence linking PcG genes to DNA damage response, prompted us to investigate further the potential role Loss of Bmi1 Renders Cells Hypersentitive to Clastogenic Agents. To for BMI1 in this process. investigate if Bmi1 impairment affects chromosome integrity upon We first performed a series of time-course experiments to DNA damage, we performed cytogenetic analysis of HCT116 and characterize the appearance of DNA damage-induced γ-H2AX 293T cells engineered to express low levels of Bmi1 and exposed to foci in murine embryonic fibroblasts (MEF) freshly isolated from different clastogens. As shown in Fig. 2A, Bmi1 knockdown −/− wild-type or Bmi1 mice.