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Research Article
The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia
Nadia Danilova, Elena Bibikova, Todd M. Covey, David Nathanson, Elizabeth Dimitrova, Yoan Konto, Anne Lindgren, Bertil Glader, Caius G. Radu, Kathleen M. Sakamoto, Shuo Lin
Disease Models & Mechanisms 2014 7: 895-905; doi: 10.1242/dmm.015495
Nadia Danilova
1Department of Molecular, Cell & Developmental Biology, University of California, Los Angeles, CA 90095, USA.
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  • For correspondence: ndanilova@ucla.edu shuolin@ucla.edu
Elena Bibikova
2Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5208, USA.
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Todd M. Covey
2Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5208, USA.
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David Nathanson
3Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095, USA.
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Elizabeth Dimitrova
3Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095, USA.
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Yoan Konto
2Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5208, USA.
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Anne Lindgren
1Department of Molecular, Cell & Developmental Biology, University of California, Los Angeles, CA 90095, USA.
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Bertil Glader
2Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5208, USA.
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Caius G. Radu
3Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095, USA.
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Kathleen M. Sakamoto
2Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5208, USA.
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Shuo Lin
1Department of Molecular, Cell & Developmental Biology, University of California, Los Angeles, CA 90095, USA.
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  • For correspondence: ndanilova@ucla.edu shuolin@ucla.edu
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  • Fig. 1.
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    Fig. 1.

    Zebrafish embryos switch from a salvage to a de novo pathway of dNTP synthesis during development. (A) dNTPs are synthesized de novo by the RNR enzyme or via the nucleoside salvage pathways. ADA, adenosine deaminase; NDP, nucleoside diphosphate; dNMP, dNDP and dNTP are deoxynucleoside mono-, di- and triphosphate, respectively; XDH, xanthine dehydrogenase; HPRT1, hypoxanthine phosphoribosyltransferase; TK1, thymidine kinase; DCK, deoxycytidine kinase. (B) Embryos have maternal supplies of dNTPs to support rapid cell division. The amount of free dATP in embryos decreased with age. hpf, hours post-fertilization. Means of three replicates were used to generate the graph. (C) De novo synthesis was low in early embryos; rrm1 expression increased through development, as measured using RT-qPCR. The fold change of expression was calculated relative to the expression at 0 hpf. qPCR in panels C and D was performed in triplicate, and the means were used to generate the graphs. (D) The expression of dck, encoding the salvage enzyme, was high in early-stage embryos and decreased with age. The results of RT-qPCR analyses are shown. The fold change of expression was calculated relative to the expression at 0 hpf.

  • Fig. 2.
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    Fig. 2.

    The expression of genes involved in nucleotide metabolism changes in RP-deficient zebrafish. (A) ada and xdh, which are involved in nucleotide catabolism, were upregulated in Rpl11 mutants. Genes encoding enzymes involved in nucleotide biosynthesis, such as ppat (phosphoribosyl pyrophosphate amidotransferase) and cad (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase) were also upregulated. With regards to salvage enzymes, expression of hprt was increased. The levels of tk1 and dck, which are expressed only in proliferating cells, were decreased. Gene expression was measured by using RT-qPCR analyses of embryos at 48 hpf. The fold change of expression in Rpl11 mutants was calculated relative to the expression in wild-type siblings. (B) Embryos that had been injected with a morpholino targeting Rps19 had similar changes in expression to those in Rpl11 mutants. RT-qPCR analyses were performed at 24 hpf. The fold change of gene expression in Rps19 morphants was calculated relative to expression of the gene wild-type embryos. (C) An increase in ada expression in Rps19-deficient embryos was detected, starting at 19 hpf. Red, embryos injected with a morpholino against Rps19 (Rps19 MO); blue, uninjected control (wt). Analyses were performed by using RT-qPCR. The fold change of expression was calculated relative to gene expression at 0 hpf. In panels C,E,F,G, qPCR was performed in triplicate and the means were used to generate the graphs. (D) rrm1 was upregulated in Rpl11 mutants at 48 hpf and in Rps19 morphants at 24 hpf. Analyses were performed by using RT-qPCR. The fold change of gene expression for Rpl11 mutants was calculated relative to expression in wild-type siblings. The fold change in ada expression in Rps19 morphants was calculated relative to expression in wild-type embryos. In A,B,D, the means±s.d. are shown. (E) Timecourse of expression of a transactivating isoform of tp53 (TAp53) in wild-type embryos (wt, blue) and in embryos that had been injected with Rps19 morpholino (MO, red). The results of RT-qPCR analyses are shown. The fold change of gene expression was calculated relative to expression at 0 hpf. (F) rrm1 expression was increased at 3.5 and 6 hpf in embryos that had been injected with an Rps19-specific morpholino (red) but not in embryos that had been injected with a 5-base-mismatch morpholino (mis MO, orange). Expression of the gene in wild-type embryos is shown in blue. The results of RT-qPCR analyses are shown. The fold change of expression was calculated relative to expression of the gene at 0 hpf. (G) Upregulation of rrm1 was also observed in tp53−/− embryos that had been injected with an Rps19-specific morpholino (Rps19 MO, red), expression in control tp53−/− embryos is in blue. The results of RT-qPCR analyses are shown. The fold change of expression was calculated relative to the expression at 0 hpf.

  • Fig. 3.
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    Fig. 3.

    RP-deficient zebrafish show activation of the DNA damage checkpoint pathway. (A) Phosphorylation of residue Ser139 of histone H2A.X was induced in embryos that had been injected with an Rps19-specific morpholino (MO). Western blotting was performed at 24 hpf. Staining of tubulin was used for a loading control. Molecular masses are shown on the right. Wt, wild type. (B) Phosphorylation of residue Ser345 in Chk1 kinase was increased in Rps19-deficient embryos (MO). Western blotting was performed at 24 hpf. (C) Embryos that had been injected with an Rps19 morpholino had increased levels of p53; the treatment of morphants with 3 nM of Chk1 inhibitor PF477736 (PF), 10 nM of the ATR and ATM inhibitor CGK733 (GCK), or 3 nM of the ATM inhibitor KU60019 (KU) reduced p53 levels. Western blotting was performed at 24 hpf. (D) Treatment of Rpl11 mutants with inhibitors of the ATR-ATM-Chk1 pathway resulted in downregulation of the p53 targets p21 and puma. Gene expression was analyzed by using RT-qPCR. The fold change of gene expression was calculated relative to expression in wild-type siblings. Means±s.d. are shown. (E) Embryos that had been injected with a morpholino against Rps19 (Rps19 MO) had few red blood cells at 3.5 days post-fertilization. The treatment of Rps19 morphants with PF477736 partially rescued this hematopoietic defect.

  • Fig. 4.
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    Fig. 4.

    Human RPS19 deficient cells have an activated DNA damage checkpoint. Intracellular phosphoflow cytometry of RPS19-deficient human CD34+ cells from fetal liver 5 days after transduction with lentiviral vectors expressing shRNA against RPS19. A vector targeting luciferase was used as a control. All vectors also expressed GFP. Histograms show GFP-positive gated cells from luciferase control (Luc, black) or RPS19-knockdown cells (RPS19, red). Bar graphs show the median fluorescent intensity obtained from the histograms for each antibody-fluorochrome conjugate. We used primary antibodies against phosphorylated p53 at residue S37 [p-p53 (S37)], total p53, phosphorylated p53 at residue S15 [p-p53 (S15)] that were conjugated to Alexa Fluor 647. We also used the following unconjugated antibodies: mouse against phosphorylated ATM at residue S1981 [p-ATM (S1981)], rabbit against phosphorylated 53BP1 at residue S1778 [p-53BP1 (S1778)], rabbit against phosphorylated Chk1 at S345 [p-Chk1 (S345)], and rabbit against phosphorylated Chk2 at T68 [pChk2 (T68)] with a secondary labeling step using either anti-mouse IgG antibody conjugated to PE or anti-rabbit IgG antibody conjugated to PE.

  • Fig. 5.
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    Fig. 5.

    RP-deficiency results in an imbalanced dNTP pool, ATP depletion and AMPK activation. (A) Levels of dNTPs in Rpl11 mutants (m, black) and wild-type siblings (s, gray) at 48 hpf. dTTP was increased in Rpl11 mutants. Student’s t-test, *P<0.01. (B) At 18 hpf, dTTP was increased in embryos that had been injected with an Rps19 morpholino (MO, black). Student’s t-test, *P<0.05. wt (gray), wild-type embryos. (C) The ATP levels in Rpl11 mutants (mut Rpl11) and Rps19 morphants (MO Rps19) were decreased. The ATP level is shown relative to that of siblings for Rpl11 mutants, and relative to that of wild-type embryos for Rps19 morphants. ATP levels were measured 48 hpf in mutants and 24 hpf in morphants. In A–C, the means±s.d. are shown. (D) Rps19 deficiency led to the activating phosphorylation of AMPK at Thr172 (p-AMPK), as analyzed by western blotting. Treatment with nucleosides reduced AMPK phosphorylation. wt, wild-type embryos; MO, embryos that had been injected with a morpholino against Rps19. Molecular masses are indicated on the right.

  • Fig. 6.
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    Fig. 6.

    Rescue of RP-deficient zebrafish with exogenous nucleosides. (A) At 48 hpf, Rpl11 mutants incorporated more [3H] deoxycytidine into DNA than their wild-type siblings (P<0.002). The count adjusted to 4 μg of genomic DNA is shown. (B) At 48 hpf, embryos that had been injected with a morpholino against Rps19 incorporated more [3H]deoxycytidine into DNA than wild-type embryos (P<0.005). The count adjusted to 2 μg of genomic DNA is shown. (C) Supplementation with exogenous nucleosides (NS) decreased the expression of tp53, p21 and puma in zebrafish embryos that had been injected with a morpholino against Rps19 (MO). L, treatment with 0.5 mg/ml leucine was used for comparison. RT-qPCR analyses were performed at 22 hpf. The fold change in gene expression was calculated relative to expression in wild-type embryos. (D) The addition of exogenous nucleosides reduced the expression of tp53, p21 and puma in Rpl11-mutant zebrafish embryos. RT-qPCR analyses were performed at 48 hpf. The fold change in expression was calculated relative to expression in wild-type siblings. (E) Nucleoside treatment decreased the level of p53 protein in Rps19-deficient zebrafish embryos. 22 hpf. Western blot. (F) The addition of exogenous nucleosides normalized the altered expression of genes that are involved in nucleotide metabolism – rrm1 and ada. RT-qPCR analyses were performed at 22 hpf. In A–D and F, means±s.d. are shown. (G) Treatment with nucleosides decreased the amount of apoptosis. Embryos were injected with a morpholino against Rps19, and half of these were treated with nucleosides. Representative images are shown of fish at 28 hpf. Acridine orange staining shows fewer apoptotic cells in morphants that had been treated with nucleosides. (H) The addition of exogenous nucleosides increased the amount of red blood cells in Rpl11 mutants. Representative images are shown of O-dianizidine staining of fish at 80 hpf. Rpl11 mutants were confirmed by genotyping after staining. Arrows point to erythroid cells.

  • Fig. 7.
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    Fig. 7.

    Schematic of the changes that are induced in cells by a deficiency of RPs. The nascent rRNA cannot be processed correctly in the absence of some RPs, which hypothetically leads to replication stress and DNA damage. Alongside this stress, global changes in nucleotide metabolism arise from (i) the necessity to catabolise defective rRNAs; (ii) the need to produce more rRNAs; (iii) the need to produce more dNTPs for DNA repair; (iv) the decreased availability of ATP and precursors for biosynthesis, caused by suppressed glycolysis; and (v) ADA activity destroying ATP. Altogether, metabolism perturbations lead to dNTP imbalance and ATP depletion. These factors can further exacerbate replication stress and DNA damage. The pattern of p53 phosphorylation that we observed is consistent with inputs from several kinases from the ATR-ATM-Chk1-Chk2 pathway. In addition, the activation of AMPK, caused by low ATP levels, can contribute to p53 activation. During replication stress and DNA damage, ATR kinase activates RNR (de novo pathway) to increase production of dNTPs, which are necessary for DNA repair. At the same time, ATM kinase activates the salvage enzyme DCK to produce more dNTPs through salvage pathways. Salvage pathways are barely used in healthy cells, but for stressed cells they are much more important, as illustrated by the increased incorporation of radioactively labeled deoxycytidine into the DNA of zebrafish that are deficient in RPs. Exogenous nucleosides rescue cells that are deficient in RPs by decreasing replication stress through providing additional dNTPs for DNA repair.

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Keywords

  • Ribosomal protein deficiency
  • Rps19
  • Rpl11
  • p53
  • ATR
  • RNR
  • Chk1
  • ATP
  • AMPK
  • Exogenous nucleosides

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Research Article
The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia
Nadia Danilova, Elena Bibikova, Todd M. Covey, David Nathanson, Elizabeth Dimitrova, Yoan Konto, Anne Lindgren, Bertil Glader, Caius G. Radu, Kathleen M. Sakamoto, Shuo Lin
Disease Models & Mechanisms 2014 7: 895-905; doi: 10.1242/dmm.015495
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Research Article
The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia
Nadia Danilova, Elena Bibikova, Todd M. Covey, David Nathanson, Elizabeth Dimitrova, Yoan Konto, Anne Lindgren, Bertil Glader, Caius G. Radu, Kathleen M. Sakamoto, Shuo Lin
Disease Models & Mechanisms 2014 7: 895-905; doi: 10.1242/dmm.015495

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