Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions

Ana Martín-Pardillos, Anastasia Tsaalbi-Shtylik, Si Chen, Seka Lazare, Ronald P. van Os, Albertina Dethmers-Ausema, Nima Borhan Fakouri, Matthias Bosshard, Rossana Aprigliano, Barbara van Loon, Daniela C. F. Salvatori, Keiji Hashimoto, Celia Dingemanse-van der Spek, Masaaki Moriya, Lene Juel Rasmussen, Gerald de Haan, Marc H. G. P. Raaijmakers and Niels de Wind

Data supplements

Article Figures & Data


  • Figure 1.

    Rev1 hematopoietic stem cell (HSC) display competitive and proliferative defects (see also supplemental Figure 1). The involvement of TLS in tolerance of endogenous DNA damage in the hematopoietic system was investigated by analyzing Rev1 blood and bone marrow, by competitive repopulation experiments, and by culture of hematopoietic stem and progenitor cells (HSPCs) in vitro. *P < .05; **P < .01; ***P < .001; ****P < .0001. Data are mean ± standard error of the mean (SEM). (A) Helix-distorting nucleotide lesions (blue spheres) can be repaired by ggNER, dependent on the Xpc gene. In case a lesion escapes timely repair, it arrests processive replication (black rectangle), resulting in replication stress and DNA damage signaling. The lesion can be bypassed postreplicatively by Rev1-dependent DNA TLS (zig-zag line). Thereby, TLS prevents the induction of replication stress and double-stranded DNA (dsDNA) breaks. TLS frequently misincorporates (in red) opposite the damaged nucleotide, which originates nucleotide substitution mutations. (B) Cytopenia in 26- to 30-month-old Rev1 mice (n = 11), compared with age-matched WT mice (n = 6). (C) Relative contribution of myeloid and lymphoid cells in the WT and Rev1 blood at 3 months of age (3m) and when moribund (MB). N = 10. (D) Frequencies of LSK, LSK34-, and LSK-SLAM cells in bone marrow of 5-month-old Rev1 (n = 5) and WT mice (n = 5). Frequencies are depicted as percent of mononuclear cells. (E) Impaired function of Rev1-deficient HSCs as demonstrated by competitive repopulation assays. Scheme of competitive transplantation experiments (top). Competitive transplantation of WT (n = 9) and Rev1 HSCs (n = 8) (bottom). (See also supplemental Figure 1.) (F) Impaired proliferative capacity of HSPCs as demonstrated by reduced CAFC numbers from 5-month-old WT (n = 4) and Rev1 (n = 4) mice. (G) Sizes of colonies after single-cell sorting of LSK-SLAM cells from 5-month-old Rev1 (n = 3) and WT (n = 3) mice.

  • Figure 3.

    Rev1 protects against replication stress and genomic breaks in the hematopoietic system (see also supplemental Figure 4). We investigated the induction of DNA breaks in the absence of Rev1-mediated TLS at endogenous helix-distorting DNA lesions in blood and bone marrow. *P < .05; **P < .01; ***P < .001; ****P < .0001. Data are mean ± SEM. (A) Chromosome fragments (Howell-Jolly bodies, arrowheads) in erythrocytes of 3-month-old Rev1 and Rev1Xpc mice. Bar represents 10 μm. Right panel: quantification. WT: 3 m (n = 5), MB (n = 6). Xpc: 3 m (n = 5), MB (n = 6). Rev1: 3 m (n = 5), MB (n = 9). Rev1Xpc: 3 m (n = 6), MB (n = 8). (B) Chromosome breaks outside of S phase, measured by single-cell alkaline comet gel electrophoresis of bone marrow of 3-month-old mice. WT (n = 4), Xpc (n = 4), Rev1 (n = 4), Rev1Xpc (n = 4). Comet intensities of BrdU-negative cells are shown. Increased DNA breaks in bone marrow hematopoietic cells of Rev1 and Rev1Xpc mice as demonstrated by γH2AX (C) and 53BP1 (D) immunostaining. The fraction of positive cells shown was normalized relative to 3-month-old WT. WT: 3 m (n = 8), MB (n = 6). Xpc: 3 m (n = 7), MB (n = 6). Rev1: 3 m (n = 6), MB (n = 6). Rev1Xpc: 3 m (n = 5-6), MB (n = 9).

  • Figure 4.

    Rev1 protects against endogenous DNA damage–induced senescence and apoptosis (see also supplemental Figure 5). Proliferation, replication, senescence, and apoptosis were quantified in bone marrow of all 4 genotypes. *P < .05; **P < .01; ***P < .001; ****P < .0001. Data are mean ± SEM. Reduced proliferation (Ki67 immunostaining) (A) and replication (BrdU and EdU incorporation) (B) in the bone marrow of moribund Rev1Xpc mice. WT: 3 m (n = 7-9), MB (n = 5-6). Xpc: 3 m (n = 6-8), MB (n = 6). Rev1: 3 m (n = 7), MB (n = 6). Rev1Xpc: 3 m (n = 6), MB (n = 9). Increased senescence and apoptosis in the bone marrow of Rev1Xpc mice as demonstrated by immunostaining for Dec1 (C), p16 (D), and caspase-3 (E). WT: 3 m (n = 4-8), MB (n = 5-6). Xpc: 3 m (n = 5-8), MB (n = 5-6). Rev1: 3 m (n = 3-7), MB (n = 6). Rev1Xpc: 3 m (n = 3-6), MB (n = 6-8). m, months; MB, moribund (see Figure 2A for survival data). The fraction of positive cells shown was normalized relative to 3-month-old WT bone marrow.

  • Figure 5.

    Rev1-dependent TLS and ggNER converge on helix-distorting oxidative DNA lesions resulting from progressive mitochondrial dysfunction (see also supplemental Figure 6). We investigated the involvement of Rev1-dependent TLS at helix-distorting lipid peroxidation–derived nucleotide adduct by treating Rev1 HSCs with a radical scavenger, by using an in cellulo TLS assay, by investigating the sensitivity of Rev1 cells to a lipid peroxidation–derived aldehyde, to oxidative stress, and by measuring oxidative stress in bone marrow. We then characterized the quantity and functionality of Rev1Xpc mitochondria. *P < .05; **P < .01; ***P < .001; ****P < .0001. Data are mean ± SEM. (A) DNA breaks (γH2AX) in cultured HSCs (LSK-SLAM), WT (n = 3), and Rev1 (n = 3), treated or nontreated with the ROS scavenger NAC. The fraction of positive cells shown was normalized relative to WT. (B) The prototypic DNA-reactive lipid peroxidation–derived aldehyde 4-ONE and its adduction to a cytosine base (H-εdC). (C) Top: TLS assay at a site-specific H-εdC. MEFs were transfected with the substrate, followed by incubation to allow TLS, and by recovery of covalently closed progeny plasmids in Escherichia coli. The fraction of recovered substrate, compared with an undamaged internal control, is a measure of TLS activity of the MEFs. Bottom: Relative efficiency and mutation spectrum of TLS events at a site-specific H-εdC lesion. (D) Clonal survival of WT, Rev1, Xpc, and Rev1Xpc MEFs in response to the addition of the mitochondrial poison paraquat to the growth medium. (E) Clonal survival of Xpc and Rev1Xpc MEFs in response to the addition of 4-HNE to the growth medium. Oxidative stress in the bone marrow of Rev1Xpc mice as evidenced by: lipofuscin accumulation (brown inclusions) in bone marrow of moribund mice (F), 4-HNE-positive cells (G), activation of p38 signaling [phospho (γ)p38 staining] (H), and accumulation of free radical-induced oxidative DNA lesions (OHdG-positive cells) (I) in Rev1Xpc mice: 1m (n = 3-4), 3m (n = 3-5), MB (n = 5-6). The fraction of positive cells shown was normalized relative to 3-month-old WT bone marrow. (J) Relative mtDNA contents, as determined by real-time PCR, in bone marrow from Xpc (n = 5-6) and Rev1Xpc (n = 5-6) mice. All mtDNA levels were normalized to those in 3-month-old Xpc mice. (K) Western blot of mitochondrial complexes I to IV in bone marrow from Xpc and Rev1Xpc mice (4 mice per group). Lamin B1: internal standard. (L) Expression of the mitochondrial stress proteins UCP2 and PGC-1α in WT, Xpc, Rev1, and Rev1Xpc bone marrow. Lamin B: internal standard. wk, weeks; m, months; MB, moribund (see Figure 2C for survival data). (M) Mitochondrial membrane potentials in bone marrow of Xpc and Rev1Xpc mice. All potentials in Rev1Xpc bone marrow were normalized to those in Xpc bone marrow of the same age. 1 m (n = 4), 1.5 m (n = 4), 3 m (n = 4-6), MB (n = 3-4). (N) Basal oxygen consumption rates in viable cells from bone marrow from Xpc and Rev1Xpc mice. All oxygen consumption rates in Rev1Xpc bone marrow were normalized to those in Xpc bone marrow of the same age. 2wk (n = 5-6), 3 m (n = 3).

  • Figure 6.

    Model for the role of mutagenic TLS in maintenance of the hematopoietic system. (A) Genomic nucleotides, damaged by endogenous sources or by chemical decay, form a threat to DNA transactions such as transcription or replication, in case they remain unrepaired. (B) Processive replication is arrested by a nucleotide, damaged by a helix-distorting oxidative nucleotide lesion. (C) The damaged nucleotide is bypassed by TLS. This prevents replication stress, but at the expense of the frequent incorporation of an incorrect nucleotide opposite the lesion (in red). (D) Subsequent repair of the damaged nucleotide, or replication of the bottom DNA strand, fixates the mutation. This contributes to the acquisition of clonal mutations in the aging hematopoietic system. Mutations in hematopoietic cells acquired during aging have been associated with the development of myeloid neoplasms in humans. (E) Stalled replicons that are not released by TLS can collapse to dsDNA breaks. DNA damage signaling at ssDNA gaps opposing the lesions and at dsDNA breaks induces senescence or apoptosis, ultimately resulting in collapse of the hematopoietic system. (F) We hypothesize that failure to release arrested replicons may underlie the observed mitochondrial dysfunction, possibly via depletion of NAD+ that is required simultaneously at DNA breaks and for mitochondrial respiration. This may lead to increased ROS production and to the induction of additional oxidative DNA lesions. A positive feedback loop between replication stress at the nuclear genome and mitochondrial dysfunction is proposed to further accelerate the collapse of the hematopoietic system.