Bmi1 regulates mitochondrial function and the DNA damage response pathway

Abstract

Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1(-/-) phenotype. Here we demonstrate that cells derived from Bmi1(-/-) mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1(-/-) mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.

Figure 1. The absence of Bmi1 increases ROS levels and alters mitochondrial function

a, Levels of ROS as assessed by DCFDA fluorescence in purified wild-type (WT) or Bmi1^–/– bone marrow cells. b, ROS levels in freshly isolated thymocytes. c, Quantitative rtPCR expression analysis of gene products involved in redox homeostasis in either WT or Bmi1^–/– thymocytes. Results are normalized to Gapdh expression (n = 3 animals per group, *P < 0.05). d, Oxygen consumption in intact thymocytes under basal conditions, following the addition of the mitochondrial inhibitor oligomycin (0.5 μg ml^–1) or in the presence of the uncoupler FCCP (1 μM). Cells were obtained from n = 4 animals per genotype; *P < 0.02. e, Relative ATP levels in freshly isolated thymocytes. Measurements were made in triplicate (mean and s.d.) with 4 animals per group; *P < 0.01. f, Representative oxygen consumption from isolated heart mitochondria. g, Measured state III respiration for either complex-I- or complex-II-dependent respiration (mean and s.d.; n = 4 animals per group; *P < 0.05, **P < 0.01). h, NAD(P)H levels as assessed by endogenous fluorescence in WT and Bmi1^–/– thymocytes. i, ROS levels in Bmi1^–/– thymocytes in the presence or absence of the complex I inhibitor rotenone (Rot), the chemical uncoupler FCCP or the NADPH oxidase inhibitor DPI (mean and s.d.; n = 3; *P < 0.05). j, Analysis of thymocytes using the redox fluorophore MitoSox Red.

Figure 2. Antioxidant treatment rescues Bmi1^–/– thymocytes

a, ROS in thymocytes isolated from four-week-old wild-type (WT) mice or Bmi1^–/– mice randomized to NAC treatment for one week before collection. b, Overall thymus size in 4-week-old mice either treated for one week with NAC (+) of left untreated (–). c, Total number of thymocytes recovered in 4-week-old Bmi1^–/– mice either treated for one week with NAC or left untreated (mean and s.d.; n = 4 animals per group). d, Representative assessment of thymocyte maturation (CD4⁺CD8⁺ cells) in 4-week-old WT or Bmi1^–/– mice that were treated with NAC for one week or left untreated before collection. e, Quantitative assessment of thymocyte maturation in 4-week-old mice randomized for one week of antioxidant treatment (mean and s.d.; n = 4 animals per group). f, Weight of Bmi1^–/– mice with or without antioxidant treatment beginning after weaning (mean and s.d.; n = 5–7 animals per group). *P < 0.05.

Figure 3. Activation of the DDR pathway in Bmi1^–/– thymocytes occurs through a redox-sensitive pathway

a, Quantitative rtPCR analysis of Ink4a/Arf expression in thymocytes obtained from mice randomized to antioxidant therapy (mean and s.d.; n = 3 animals per group). NS, not significant. b, ROS levels in thymocytes obtained from WT, Bmi1^–/– or combined Bmi1/p16^Ink4a-deleted mice. c, Levels of the oxidatively modified nucleotide 8-oxoguanine in isolated thymocytes. DAPI (4,6-diamidino-2-phenylindole) staining (blue) was used to visualize nuclei. d, 53BP1 nuclear foci thymocytes obtained from mice treated for one week before collection with the antioxidant NAC or left untreated. Overall percentage of thymocytes demonstrating activation of the DDR in each condition is shown. e, Western blot analysis for Chk2 activation in thymocytes obtained from WT or Bmi1^–/– mice. f, Assessment of primary thymocyte survival in culture 24 h after isolation.

Figure 4. Inhibition of the DDR pathway by Chk2 deletion rescues multiple defects in Bmi1^–/– mice

a, Chk2 deletion restores in vivo thymocyte maturation of Bmi1^–/– mice. WT, wild type. b, Architecture of the thymus in WT, Bmi1^–/– and combined Bmi1^–/–Chk2^–/– mice. The arrows point to a normal medulla region. c, Representative TUNEL staining in the cortex region of the thymus. d, Quantitative rtPCR analysis of Ink4a/Arf induction in Bmi1^–/– or combined Bmi1^–/–Chk2^–/– thymocytes (mean and s.d.; n = 3 animals per group). e, Frequency of LSK cells intotal bonemarrow (*P < 0.01; mean and s.d., n = 6 animals per group). f, In vivo CFU-S in lethally irradiated mice infused with equal numbers of bone marrow cells obtained from WT, Bmi1^–/– or combined Bmi1^–/–Chk2^–/– mice (*P < 0.01; mean and s.d., n = 5 animals per group). g, Peripheral blood composition four months after competitive repopulation in which equal amounts of WT competitor bone marrow cells (CD45.1) and combined Bmi1^–/–Chk2^–/– donor bone marrow (CD45.2) were transplanted into an irradiated host (CD45.2). We observed little to no contribution of the donor-derived Bmi1^–/–Chk2^–/– cells after such competitive repopulation experiments. h, Representative appearance of WT, Bmi1^–/– or combined Bmi1^–/–Chk2^–/– mice. i, Analysis of body weight at 6 weeks of age (*P < 0.01; mean and s.d.; n = 6 per group). j, Defects in cerebellar architecture observed in Bmi1^–/– mice are rescued by Chk2 deletion. k, Lifespan of Bmi1^–/– mice with varying Chk2 status. P < 0.05 Chk2^+/– versus Chk2^+/+ and P < 0.001 for Chk2^–/– versus Chk2^+/+; n ≥ 15 animals per genotype. l, The Polycomb protein Bmi1 normally simultaneously represses the Ink4a/Arf locus leading to reduced p16^Ink4a and p19^Arf expression as well as modulating mitochondrial function to lower ROS levels and suppress activation of the DDR pathway. Activation of both Ink4a/Arf and the DDR have been separately linked to tumour suppression and stem cell ageing.