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TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis.

Gao L, Li D, Ma K, Zhang W, Xu T, Fu C, Jing C, Jia X, Wu S, Sun X, Dong M, Deng M, Chen Y, Zhu W, Peng J, Wan F, Zhou Y, Zon LI, Pan W - PLoS Genet. (2015)

Bottom Line: Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs.Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment.Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

No MeSH data available.


Related in: MedlinePlus

Subcellular mislocalization and defective ATR/Chk1 activation link to defects in HSPCs in the topbp1cas003 mutants.(A) Confocal imaging analysis of anti-FLAG immunostaining (green) and DAPI (blue) in HeLa cells transfected with FLAG tagged TopBP1WT (WT), TopBP1cas003 (cas003) and TopBP1cas003-NLS (cas003-NLS). TopBP1WT is mainly localized in the nucleus (left column), cytosol mislocalization of TopBP1cas003 (middle column) can be corrected by the additional SV40 NLS on its C-terminus (TopBP1cas003-NLS, right column). Scale bars represent 25 μm. (B-F) WISH analysis of hematopoiesis in siblings and topbp1cas003 mutant embryos with Tol2-transposase mediated topbp1WT and topbp1cas003-NLS transgenesis at 4dpf, which are quantified in F (the numbers of embryos are shown above). Defective c-myb expression in topbp1cas003 mutants can be rescued by topbp1cas003–NLS as well as topbp1WT. (B-C, B’-C’) 28/32 siblings and 12/15 topbp1cas003 mutants show indicated WISH results. (D-E, D’-E’) WISH results of well rescued mutants. (B’-E’) Enlarged views of CHT region in the left column. (G) Western blot with pChk1 antibody in control morphants and hydroxyurea (HU) treated control or topbp1 morphants. The morphants were treated with 250mM HU or mock from 60hpf to 76hpf. topbp1 knockdown could abrogate the Chk1 phosphorylation in the tail region upon HU treatment. (H) Immunoblotting analysis showing reduced phospho-Chk1 level in tail region of topbp1cas003 mutants upon HU treatment, comparing to that in wild-type siblings. The embryos were treated with 250mM HU or mock from 60hpf to 76hpf. (I) Schematic diagram of variant forms of TopBP1, including wild-type (WT), cas003, cas003-NLS, ΔAAD, W1156R, R122E and R669E mutation. The regions associated with ATR activation and Rad9 or MDC1 interactions are indicated. After Tol2-mediated transient transgenesis of variant forms of TopBP1 into topbp1cas003 mutant embryos, quantitative analysis of the c-myb expression in the CHT region at 4dpf was performed for the evaluation of rescue capability (n>20). “+” (rescue); “-” (not rescue effect). Except WT and cas003-NLS, all TopBP1 mutation forms are unable to rescue hematopoietic defects in topbp1cas003 mutants.
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pgen.1005346.g006: Subcellular mislocalization and defective ATR/Chk1 activation link to defects in HSPCs in the topbp1cas003 mutants.(A) Confocal imaging analysis of anti-FLAG immunostaining (green) and DAPI (blue) in HeLa cells transfected with FLAG tagged TopBP1WT (WT), TopBP1cas003 (cas003) and TopBP1cas003-NLS (cas003-NLS). TopBP1WT is mainly localized in the nucleus (left column), cytosol mislocalization of TopBP1cas003 (middle column) can be corrected by the additional SV40 NLS on its C-terminus (TopBP1cas003-NLS, right column). Scale bars represent 25 μm. (B-F) WISH analysis of hematopoiesis in siblings and topbp1cas003 mutant embryos with Tol2-transposase mediated topbp1WT and topbp1cas003-NLS transgenesis at 4dpf, which are quantified in F (the numbers of embryos are shown above). Defective c-myb expression in topbp1cas003 mutants can be rescued by topbp1cas003–NLS as well as topbp1WT. (B-C, B’-C’) 28/32 siblings and 12/15 topbp1cas003 mutants show indicated WISH results. (D-E, D’-E’) WISH results of well rescued mutants. (B’-E’) Enlarged views of CHT region in the left column. (G) Western blot with pChk1 antibody in control morphants and hydroxyurea (HU) treated control or topbp1 morphants. The morphants were treated with 250mM HU or mock from 60hpf to 76hpf. topbp1 knockdown could abrogate the Chk1 phosphorylation in the tail region upon HU treatment. (H) Immunoblotting analysis showing reduced phospho-Chk1 level in tail region of topbp1cas003 mutants upon HU treatment, comparing to that in wild-type siblings. The embryos were treated with 250mM HU or mock from 60hpf to 76hpf. (I) Schematic diagram of variant forms of TopBP1, including wild-type (WT), cas003, cas003-NLS, ΔAAD, W1156R, R122E and R669E mutation. The regions associated with ATR activation and Rad9 or MDC1 interactions are indicated. After Tol2-mediated transient transgenesis of variant forms of TopBP1 into topbp1cas003 mutant embryos, quantitative analysis of the c-myb expression in the CHT region at 4dpf was performed for the evaluation of rescue capability (n>20). “+” (rescue); “-” (not rescue effect). Except WT and cas003-NLS, all TopBP1 mutation forms are unable to rescue hematopoietic defects in topbp1cas003 mutants.

Mentions: To further understand the molecular mechanism of HSPC apoptosis which was induced by this particular defective TopBP1 without its 8th BRCT domain and the putative NLS domain, we analyzed the subcellular localization of TopBP1cas003. Confocal imaging showed that flag-tagged TopBP1WT was predominantly localized in the nucleus of transfected HeLa cells (Fig 6A, left column). However, TopBP1cas003 was mistakenly localized in cytoplasm (Fig 6A, middle column), which was consistent with our previous sequence analysis on the lack of putative NLS in TopBP1cas003 (Fig 3C) and immunoblotting analysis on TopBP1WT/TopBP1cas003 protein in cytoplasmic and nucleus fractions of pooled embryos from heterozygote incrossing (S5L Fig). Moreover, addition of SV40 NLS at C terminus of TopBP1cas003 was sufficient to correct TopBP1cas003 subcellular localization defect (Fig 6A, right column). To test whether the hematopoietic deficiency in topbp1cas003 mutants could also be rescued by the nuclear localized TopBP1cas003, we carried out transient transgenesis of topbp1cas003-NLS or topbp1WT (as the positive control) in the topbp1cas003 mutants. WISH results of c-myb at 4dpf indicated that ectopic expression of topbp1cas003-NLS could rescue c-myb expression in topbp1cas003 mutants, as efficient as transgenesis with topbp1WT (Fig 6B–E’, quantified in F). Collectively, we concluded that the loss of NLS in TopBP1cas003 and the failure of nuclear localization directly caused HSPCs deficiency in topbp1cas003 mutants.


TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis.

Gao L, Li D, Ma K, Zhang W, Xu T, Fu C, Jing C, Jia X, Wu S, Sun X, Dong M, Deng M, Chen Y, Zhu W, Peng J, Wan F, Zhou Y, Zon LI, Pan W - PLoS Genet. (2015)

Subcellular mislocalization and defective ATR/Chk1 activation link to defects in HSPCs in the topbp1cas003 mutants.(A) Confocal imaging analysis of anti-FLAG immunostaining (green) and DAPI (blue) in HeLa cells transfected with FLAG tagged TopBP1WT (WT), TopBP1cas003 (cas003) and TopBP1cas003-NLS (cas003-NLS). TopBP1WT is mainly localized in the nucleus (left column), cytosol mislocalization of TopBP1cas003 (middle column) can be corrected by the additional SV40 NLS on its C-terminus (TopBP1cas003-NLS, right column). Scale bars represent 25 μm. (B-F) WISH analysis of hematopoiesis in siblings and topbp1cas003 mutant embryos with Tol2-transposase mediated topbp1WT and topbp1cas003-NLS transgenesis at 4dpf, which are quantified in F (the numbers of embryos are shown above). Defective c-myb expression in topbp1cas003 mutants can be rescued by topbp1cas003–NLS as well as topbp1WT. (B-C, B’-C’) 28/32 siblings and 12/15 topbp1cas003 mutants show indicated WISH results. (D-E, D’-E’) WISH results of well rescued mutants. (B’-E’) Enlarged views of CHT region in the left column. (G) Western blot with pChk1 antibody in control morphants and hydroxyurea (HU) treated control or topbp1 morphants. The morphants were treated with 250mM HU or mock from 60hpf to 76hpf. topbp1 knockdown could abrogate the Chk1 phosphorylation in the tail region upon HU treatment. (H) Immunoblotting analysis showing reduced phospho-Chk1 level in tail region of topbp1cas003 mutants upon HU treatment, comparing to that in wild-type siblings. The embryos were treated with 250mM HU or mock from 60hpf to 76hpf. (I) Schematic diagram of variant forms of TopBP1, including wild-type (WT), cas003, cas003-NLS, ΔAAD, W1156R, R122E and R669E mutation. The regions associated with ATR activation and Rad9 or MDC1 interactions are indicated. After Tol2-mediated transient transgenesis of variant forms of TopBP1 into topbp1cas003 mutant embryos, quantitative analysis of the c-myb expression in the CHT region at 4dpf was performed for the evaluation of rescue capability (n>20). “+” (rescue); “-” (not rescue effect). Except WT and cas003-NLS, all TopBP1 mutation forms are unable to rescue hematopoietic defects in topbp1cas003 mutants.
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pgen.1005346.g006: Subcellular mislocalization and defective ATR/Chk1 activation link to defects in HSPCs in the topbp1cas003 mutants.(A) Confocal imaging analysis of anti-FLAG immunostaining (green) and DAPI (blue) in HeLa cells transfected with FLAG tagged TopBP1WT (WT), TopBP1cas003 (cas003) and TopBP1cas003-NLS (cas003-NLS). TopBP1WT is mainly localized in the nucleus (left column), cytosol mislocalization of TopBP1cas003 (middle column) can be corrected by the additional SV40 NLS on its C-terminus (TopBP1cas003-NLS, right column). Scale bars represent 25 μm. (B-F) WISH analysis of hematopoiesis in siblings and topbp1cas003 mutant embryos with Tol2-transposase mediated topbp1WT and topbp1cas003-NLS transgenesis at 4dpf, which are quantified in F (the numbers of embryos are shown above). Defective c-myb expression in topbp1cas003 mutants can be rescued by topbp1cas003–NLS as well as topbp1WT. (B-C, B’-C’) 28/32 siblings and 12/15 topbp1cas003 mutants show indicated WISH results. (D-E, D’-E’) WISH results of well rescued mutants. (B’-E’) Enlarged views of CHT region in the left column. (G) Western blot with pChk1 antibody in control morphants and hydroxyurea (HU) treated control or topbp1 morphants. The morphants were treated with 250mM HU or mock from 60hpf to 76hpf. topbp1 knockdown could abrogate the Chk1 phosphorylation in the tail region upon HU treatment. (H) Immunoblotting analysis showing reduced phospho-Chk1 level in tail region of topbp1cas003 mutants upon HU treatment, comparing to that in wild-type siblings. The embryos were treated with 250mM HU or mock from 60hpf to 76hpf. (I) Schematic diagram of variant forms of TopBP1, including wild-type (WT), cas003, cas003-NLS, ΔAAD, W1156R, R122E and R669E mutation. The regions associated with ATR activation and Rad9 or MDC1 interactions are indicated. After Tol2-mediated transient transgenesis of variant forms of TopBP1 into topbp1cas003 mutant embryos, quantitative analysis of the c-myb expression in the CHT region at 4dpf was performed for the evaluation of rescue capability (n>20). “+” (rescue); “-” (not rescue effect). Except WT and cas003-NLS, all TopBP1 mutation forms are unable to rescue hematopoietic defects in topbp1cas003 mutants.
Mentions: To further understand the molecular mechanism of HSPC apoptosis which was induced by this particular defective TopBP1 without its 8th BRCT domain and the putative NLS domain, we analyzed the subcellular localization of TopBP1cas003. Confocal imaging showed that flag-tagged TopBP1WT was predominantly localized in the nucleus of transfected HeLa cells (Fig 6A, left column). However, TopBP1cas003 was mistakenly localized in cytoplasm (Fig 6A, middle column), which was consistent with our previous sequence analysis on the lack of putative NLS in TopBP1cas003 (Fig 3C) and immunoblotting analysis on TopBP1WT/TopBP1cas003 protein in cytoplasmic and nucleus fractions of pooled embryos from heterozygote incrossing (S5L Fig). Moreover, addition of SV40 NLS at C terminus of TopBP1cas003 was sufficient to correct TopBP1cas003 subcellular localization defect (Fig 6A, right column). To test whether the hematopoietic deficiency in topbp1cas003 mutants could also be rescued by the nuclear localized TopBP1cas003, we carried out transient transgenesis of topbp1cas003-NLS or topbp1WT (as the positive control) in the topbp1cas003 mutants. WISH results of c-myb at 4dpf indicated that ectopic expression of topbp1cas003-NLS could rescue c-myb expression in topbp1cas003 mutants, as efficient as transgenesis with topbp1WT (Fig 6B–E’, quantified in F). Collectively, we concluded that the loss of NLS in TopBP1cas003 and the failure of nuclear localization directly caused HSPCs deficiency in topbp1cas003 mutants.

Bottom Line: Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs.Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment.Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

No MeSH data available.


Related in: MedlinePlus