p53 regulates diverse tissue-specific outcomes to endogenous DNA damage in mice

DNA repair deficiency can lead to segmental phenotypes in humans and mice, in which certain tissues lose homeostasis while others remain seemingly unaffected. This may be due to different tissues facing varying levels of damage or having different reliance on specific DNA repair pathways. However, we find that the cellular response to DNA damage determines different tissue-specific outcomes. Here, we use a mouse model of the human XPF-ERCC1 progeroid syndrome (XFE) caused by loss of DNA repair. We find that p53, a central regulator of the cellular response to DNA damage, regulates tissue dysfunction in Ercc1-/- mice in different ways. We show that ablation of p53 rescues the loss of hematopoietic stem cells, and has no effect on kidney, germ cell or brain dysfunction, but exacerbates liver pathology and polyploidisation. Mechanistically, we find that p53 ablation led to the loss of cell-cycle regulation in the liver, with reduced p21 expression. Eventually, p16/Cdkn2a expression is induced, serving as a fail-safe brake to proliferation in the absence of the p53-p21 axis. Taken together, our data show that distinct and tissue-specific functions of p53, in response to DNA damage, play a crucial role in regulating tissue-specific phenotypes

Primordial germ cell DNA demethylation and development require DNA translesion synthesis

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals

Mouse SLX4 Is a Tumor Suppressor that Stimulates the Activity of the Nuclease XPF-ERCC1 in DNA Crosslink Repair

SLX4 binds to three nucleases (XPF-ERCC1, MUS81-EME1, and SLX1), and its deficiency leads to genomic instability, sensitivity to DNA crosslinking agents, and Fanconi anemia. However, it is not understood how SLX4 and its associated nucleases act in DNA crosslink repair. Here, we uncover consequences of mouse Slx4 deficiency and reveal its function in DNA crosslink repair. Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool. The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents. Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks. Mini-SLX4-XPF-ERCC1 also vigorously stimulates dual incisions around a DNA crosslink embedded in a synthetic replication fork, an essential step in the repair of this lesion. These observations define vertebrate SLX4 as a tumor suppressor, which activates XPF-ERCC1 nuclease specificity in DNA crosslink repairope

Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia

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Endogenous formaldehyde is a hematopoietic stem cell genotoxin and metabolic carcinogen

Endogenous formaldehyde is produced by numerous biochemical pathways fundamental to life, and it can crosslink both DNA and proteins. However, the consequences of its accumulation are unclear. Here we show that endogenous formaldehyde is removed by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), and Adh5−/− mice therefore accumulate formaldehyde adducts in DNA. The repair of this damage is mediated by FANCD2, a DNA crosslink repair protein. Adh5−/−Fancd2−/− mice reveal an essential requirement for these protection mechanisms in hematopoietic stem cells (HSCs), leading to their depletion and precipitating bone marrow failure. More widespread formaldehyde-induced DNA damage also causes karyomegaly and dysfunction of hepatocytes and nephrons. Bone marrow transplantation not only rescued hematopoiesis but, surprisingly, also preserved nephron function. Nevertheless, all of these animals eventually developed fatal malignancies. Formaldehyde is therefore an important source of endogenous DNA damage that is counteracted in mammals by a conserved protection mechanism

Fanconi anemia DNA crosslink repair factors protect against LINE-1 retrotransposition during mouse development.

Acknowledgements: We thank M. Grompe for Fancd2-deficient mice. We thank W. An for the gift of SN1 LINE-1 mice. We thank J. Wysocka for the kind gift of K562 cells carrying the LINE-1 reporter. We thank J. V. Moran for providing the pJJ101/L1.3 and pJJ101/L1.3(D702A) plasmids. We thank K. Burns for the kind gift of the LINE-1 ORF2p antibody. We thank H. Lans and W. Vermeulen for XPF and XPF(D715A) cDNAs. We thank P. Alcón and L. A. Passmore for FANCA and FANCC cDNAs. We thank the Fanconi Anemia Research Foundation for providing the PD20, PD220 and PD331 cell lines. We thank the Human Research Tissue Bank (National Institute for Health Research Cambridge Biomedical Research Centre) for processing the histology. We thank M. Hodskinson for purifying recombinant proteins and assisting in biochemical experiments. We thank L. Mulderrig for his assistance in performing in vitro retrotransposition assays and F. Langevin for his assistance in generating K562 knockout cell lines. We thank R. Hill for his assistance with embryo dissection, primordial germ cell quantification, histology and ploidy assays. We are grateful to J. Clark, D. Karpinski, M. Wiacek, C. Knox and Ares and Biomedical Staff for their help with mouse experimental work. We thank M. Daly, P. Andrée Penttilä, F. Zhang and the Flow Cytometry Core staff for their technical assistance. N.B. is supported by the César Milstein Studentship from the Darwin Trust of Edinburgh. This work was supported by the Medical Research Council as part of UK Research and Innovation (file reference no. MC_UP_1201/18).Long interspersed nuclear element 1 (LINE-1) is the only autonomous retrotransposon in humans and new integrations are a major source of genetic variation between individuals. These events can also lead to de novo germline mutations, giving rise to heritable genetic diseases. Recently, a role for DNA repair in regulating these events has been identified. Here we find that Fanconi anemia (FA) DNA crosslink repair factors act in a common pathway to prevent retrotransposition. We purify recombinant SLX4-XPF-ERCC1, the crosslink repair incision complex, and find that it cleaves putative nucleic acid intermediates of retrotransposition. Mice deficient in upstream crosslink repair signaling (FANCA), a downstream component (FANCD2) or the nuclease XPF-ERCC1 show increased LINE-1 retrotransposition in vivo. Organisms limit retrotransposition through transcriptional silencing but this protection is attenuated during early development leaving the zygote vulnerable. We find that during this window of vulnerability, DNA crosslink repair acts as a failsafe to prevent retrotransposition. Together, our results indicate that the FA DNA crosslink repair pathway acts together to protect against mutation by restricting LINE-1 retrotransposition

p53 regulates diverse tissue-specific outcomes to endogenous DNA damage in mice.

Acknowledgements: We thank the Human Research Tissue Bank for processing histological samples. The Human Research Tissue Bank is supported by the NIHR Cambridge Biomedical Research Centre. We would like to thank C. Knox, C. Watson, L. Tredgett, J. Clark, the Ares and Biomed staff for assistance with animal procedures and experimentation. We thank M. Daly, F. Zhang, P. Penttila and the Flow Cytometry Core staff for their technical assistance. We would like to thank C. Perez for assisting with the generation of RNA sequencing libraries. Funding was provided by the Medical Research Council as part of UK Research and Innovation file reference no. MC_UP_1201/18 (G.P.C., R.J.H, N.B., H.W. and A.C.) and Hubrecht Institute (J.I.G.), Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research) project number OCENW.M20.113 (J.S.).DNA repair deficiency can lead to segmental phenotypes in humans and mice, in which certain tissues lose homeostasis while others remain seemingly unaffected. This may be due to different tissues facing varying levels of damage or having different reliance on specific DNA repair pathways. However, we find that the cellular response to DNA damage determines different tissue-specific outcomes. Here, we use a mouse model of the human XPF-ERCC1 progeroid syndrome (XFE) caused by loss of DNA repair. We find that p53, a central regulator of the cellular response to DNA damage, regulates tissue dysfunction in Ercc1-/- mice in different ways. We show that ablation of p53 rescues the loss of hematopoietic stem cells, and has no effect on kidney, germ cell or brain dysfunction, but exacerbates liver pathology and polyploidisation. Mechanistically, we find that p53 ablation led to the loss of cell-cycle regulation in the liver, with reduced p21 expression. Eventually, p16/Cdkn2a expression is induced, serving as a fail-safe brake to proliferation in the absence of the p53-p21 axis. Taken together, our data show that distinct and tissue-specific functions of p53, in response to DNA damage, play a crucial role in regulating tissue-specific phenotypes