Haematopoietic stem cells renew blood. Accumulation of DNA damage in these cells promotes their decline, while misrepair of this damage initiates malignancies. Here we describe the features and mutational landscape of DNA damage caused by acetaldehyde, an endogenous and alcohol-derived metabolite. This damage results in DNA double-stranded breaks that, despite stimulating recombination repair, also cause chromosome rearrangements. We combined transplantation of single haematopoietic stem cells with whole-genome sequencing to show that this damage occurs in stem cells, leading to deletions and rearrangements that are indicative of microhomology-mediated end-joining repair. Moreover, deletion of p53 completely rescues the survival of aldehyde-stressed and mutated haematopoietic stem cells, but does not change the pattern or the intensity of genome instability within individual stem cells. These findings characterize the mutation of the stem-cell genome by an alcohol-derived and endogenous source of DNA damage. Furthermore, we identify how the choice of DNA-repair pathway and a stringent p53 response limit the transmission of aldehyde-induced mutations in stem cells.
European Nucleotide Archive
We thank D. Kent for technical advice with single-HSC transplants; R. Berks, A. Middleton, J. Wiles, C. Knox, X. Gong, J. Roe, J. Willems, the ARES staff and Biomed for their help with mouse work; M. Daly, F. Zhang, V. Romashova and M. Balmont for help with flow cytometry; and J. Sale, C. Rada, M. Taylor, Y. L. Wu and members of the Patel laboratory for critical reading of the manuscript. The Human Research Tissue Bank (supported by the NIHR Cambridge Biomedical Research Centre) processed histology. K.J.P. is supported by the MRC and the Jeffrey Cheah Foundation. G.P.C. and L.M. were supported by CRUK. J.I.G. is supported by the Wellcome Trust and King’s College, Cambridge.
Extended data figures
This file contains source gels. a, Fanca genotyping, related to Extended data Fig. 3b. b, Western blots showing lack of Fanca protein, related to Extended data Fig. 3c. c, Agarose gels for the validation of rearrangements by PCR, related to Extended data Fig. 10.
This file contains flow cytometry gating strategies. a, Gating strategy to quantify the frequency of Mn-NCE and Mn-Ret relating to Fig. 2b and g, Fig. 3f and Extended data Fig. 8a. b, Gating strategy for the quantification of LKS and HSCs relating to Fig. 3b-d and Fig. 6b. c, Gating strategy for the sorting and transplantation of single HSCs relating to Fig. 4b and 6d. d, Gating strategy to assess the chimaerism in total peripheral blood, B, T and myeloid cells relating to Fig. 4c.
This file contains generation of Fanca-/-Ku70-/- and FancaF/-Ku70-/- Vav1-iCre mice. a, Fanca+/- crosses in a C57BL/6 background showing that Fanca-/- mice are genotyped at sub-Mendelian ratios 2-3 weeks after birth (13.3% instead of the expected 25%, Fisher’s exact test: P<0.0001). b, Ku70+/- crosses in a C57BL/6 background showing that Ku70-/- mice are genotyped at sub-Mendelian ratios 2-3 weeks after birth (8.5% instead of the expected 25%, Fisher’s exact test: P<0.0001). c, Fanca+/- Ku70+/- crosses were set up to generate Fanca-/-Ku70-/- mice. Although Mendelian segregation predicts 6.25% of the pups to be Fanca-/-Ku70-/-, the actual expected ratio is 1.12% if the sub-Mendelian ratios of both Fanca-/- and Ku70-/- are taken into account. However, no Fanca-/-Ku70-/- pups were found from 463 pups genotyped (Fisher’s exact test: P<0.0307). d, FancaF/F Ku70+/- x Fanca+/- Ku70+/- Vav1-iCre crosses were set up to generate FancaF/-Ku70-/- Vav1-iCre mice in a C57BL/6 background. These mice were born at the expected ratio, which takes into account the sub-Mendelian frequency of Ku70-/- mice (1.6% observed and 2.1% expected, Fisher’s exact test: P<0.6).
This file contains p53 deficiency does not suppress the embryonic lethality of Aldh2-/-Fancd2-/- mice in Aldh2-/- mothers. a, b, In agreement with a previous report 6, Aldh2-/-Fancd2-/- mice on a C57BL/6 x 129S4S6/Sv F1 background could be obtained from Aldh2+/- 129S4S6/Sv mothers but could not be generated from Aldh2-/- 129S4S6/Sv mothers (c). d, Similar crosses were set up to obtain Aldh2-/-Fancd2-/-p53-/- mice on a C57BL/6 x 129S4S6/Sv F1 background, triple mutant mice were born at expected ratios from Aldh2+/- 129S4S6/Sv mothers. e, No Aldh2-/-Fancd2-/-p53-/- mice could be obtained from Aldh2-/- 129S4S6/Sv mothers, showing that lack of p53 is not sufficient to prevent loss of these embryos (P: Fisher’s exact test comparing the number of observed and expected double or triple mutants).