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Altered hematopoiesis in mice lacking DNA polymerase mu is due to inefficient double-strand break repair.

Lucas D, Escudero B, Ligos JM, Segovia JC, Estrada JC, Terrados G, Blanco L, Samper E, Bernad A - PLoS Genet. (2009)

Bottom Line: In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development.Hematopoietic progenitors were reduced both in number and in expansion potential.Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

ABSTRACT
Polymerase micro (Polmicro) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development. In Polmicro(-/-) mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body gamma-irradiation revealed that Polmicro also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

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Polμ−/− hematopoietic progenitor cells have a reduced capacity for DSB repair.A. Number of γ-H2AX foci per nucleus in bone marrow and spleen cells from WT (solid bars) and Polμ−/− (open bars) γ-irradiated mice (5Gy); staining was performed 1 hour post-irradiation. B. Representative confocal images showing γ-H2AX foci (Red, Cy3; blue, DAPI) in WT and Polμ−/− bone marrow cells treated as in A. C. Western blot showing amounts of phosphorylated γ-H2AX protein in LPS stimulated wt and Polμ−/− splenocytes after γ-irradiation (8 Gy), and analyzed at different periods (1 h, 3 h, and 6 h) post-irradiation; NI: non-irradiated controls. Histone H3 was used as a loading control. D. Dot plot showing DNA comet tail moment of WT (closed squares) and Polμ−/− (open triangles) bone marrow cells retrieved from irradiated mice (5Gy) after 3 hours of in vivo DNA repair. Tail momentum was significantly increased in Polμ−/− cells. E. Representative images of irradiated WT and Polμ−/− bone marrow metaphase cells analyzed by telomere FISH 96 hours after irradiation. Telomeres were hybridized with a Cy-3 labeled PNA Probe (Red) and chromosomes counterstained with DAPI (blue). Arrows indicate structural chromosomal aberrations: Chd and Chr = chromatid and chromosome breaks, respectively; dic. = dicentric chromosomes. F. Number of breaks, radial configurations, end-to-end-fusions, and total aberrations per metaphase in bone marrow cells from WT (closed bars) and Polμ−/− (open bars) irradiated mice. The mice were γ-irradiated (5Gy) and maintained (6 h) to allow in vivo DNA repair; BM-cell chromosomal aberrations were detected 4 days post irradiation. All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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pgen-1000389-g006: Polμ−/− hematopoietic progenitor cells have a reduced capacity for DSB repair.A. Number of γ-H2AX foci per nucleus in bone marrow and spleen cells from WT (solid bars) and Polμ−/− (open bars) γ-irradiated mice (5Gy); staining was performed 1 hour post-irradiation. B. Representative confocal images showing γ-H2AX foci (Red, Cy3; blue, DAPI) in WT and Polμ−/− bone marrow cells treated as in A. C. Western blot showing amounts of phosphorylated γ-H2AX protein in LPS stimulated wt and Polμ−/− splenocytes after γ-irradiation (8 Gy), and analyzed at different periods (1 h, 3 h, and 6 h) post-irradiation; NI: non-irradiated controls. Histone H3 was used as a loading control. D. Dot plot showing DNA comet tail moment of WT (closed squares) and Polμ−/− (open triangles) bone marrow cells retrieved from irradiated mice (5Gy) after 3 hours of in vivo DNA repair. Tail momentum was significantly increased in Polμ−/− cells. E. Representative images of irradiated WT and Polμ−/− bone marrow metaphase cells analyzed by telomere FISH 96 hours after irradiation. Telomeres were hybridized with a Cy-3 labeled PNA Probe (Red) and chromosomes counterstained with DAPI (blue). Arrows indicate structural chromosomal aberrations: Chd and Chr = chromatid and chromosome breaks, respectively; dic. = dicentric chromosomes. F. Number of breaks, radial configurations, end-to-end-fusions, and total aberrations per metaphase in bone marrow cells from WT (closed bars) and Polμ−/− (open bars) irradiated mice. The mice were γ-irradiated (5Gy) and maintained (6 h) to allow in vivo DNA repair; BM-cell chromosomal aberrations were detected 4 days post irradiation. All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.

Mentions: To examine the effect of Polμ deficiency on DNA repair in the hematopoietic system more closely, we analyzed the frequency of phosphorylated γ-H2AX foci in bone marrow and spleen after whole animal γ-irradiation (5Gy). In both genotypes 91% of all BM cells were γ-H2AX+, showing that these cells are more susceptible to γ-irradiation. In contrast, the proportion of phospho-γ-H2AX positive cells in Polμ−/− splenocytes (95%) was significantly higher than in wt cells (27%; p<0.001). Further, the number of γ-H2AX foci per cell was significantly increased in Polμ−/− cells compared with wt (1.7-fold for BM cells and 4.8-fold for splenocytes, p<0.001; Figure 6A–B). Changes in γ-H2AX phosphorylation were confirmed by western blot of γ-irradiated (8 Gy) spleen B cells; as predicted, Polμ−/− cells show higher amounts of phospho-γ-H2AX, and these levels are sustained for longer (Figure 6C). We confirmed reduced DSB repair in Polμ−/− BM cells by comet assay. Before cell extraction, irradiated mice (5Gy) were left to recover for 3 hours to allow in vivo DNA repair (see Methods). BM cells from these mice were separated by single cell electrophoresis under alkaline conditions, to measure the relative levels of DNA breaks; comet tail moment was 2-fold higher in Polμ−/− cells compared with wt cells (p<0.001; Figure 6D), indicating reduced DNA repair in Polμ−/− cells.


Altered hematopoiesis in mice lacking DNA polymerase mu is due to inefficient double-strand break repair.

Lucas D, Escudero B, Ligos JM, Segovia JC, Estrada JC, Terrados G, Blanco L, Samper E, Bernad A - PLoS Genet. (2009)

Polμ−/− hematopoietic progenitor cells have a reduced capacity for DSB repair.A. Number of γ-H2AX foci per nucleus in bone marrow and spleen cells from WT (solid bars) and Polμ−/− (open bars) γ-irradiated mice (5Gy); staining was performed 1 hour post-irradiation. B. Representative confocal images showing γ-H2AX foci (Red, Cy3; blue, DAPI) in WT and Polμ−/− bone marrow cells treated as in A. C. Western blot showing amounts of phosphorylated γ-H2AX protein in LPS stimulated wt and Polμ−/− splenocytes after γ-irradiation (8 Gy), and analyzed at different periods (1 h, 3 h, and 6 h) post-irradiation; NI: non-irradiated controls. Histone H3 was used as a loading control. D. Dot plot showing DNA comet tail moment of WT (closed squares) and Polμ−/− (open triangles) bone marrow cells retrieved from irradiated mice (5Gy) after 3 hours of in vivo DNA repair. Tail momentum was significantly increased in Polμ−/− cells. E. Representative images of irradiated WT and Polμ−/− bone marrow metaphase cells analyzed by telomere FISH 96 hours after irradiation. Telomeres were hybridized with a Cy-3 labeled PNA Probe (Red) and chromosomes counterstained with DAPI (blue). Arrows indicate structural chromosomal aberrations: Chd and Chr = chromatid and chromosome breaks, respectively; dic. = dicentric chromosomes. F. Number of breaks, radial configurations, end-to-end-fusions, and total aberrations per metaphase in bone marrow cells from WT (closed bars) and Polμ−/− (open bars) irradiated mice. The mice were γ-irradiated (5Gy) and maintained (6 h) to allow in vivo DNA repair; BM-cell chromosomal aberrations were detected 4 days post irradiation. All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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pgen-1000389-g006: Polμ−/− hematopoietic progenitor cells have a reduced capacity for DSB repair.A. Number of γ-H2AX foci per nucleus in bone marrow and spleen cells from WT (solid bars) and Polμ−/− (open bars) γ-irradiated mice (5Gy); staining was performed 1 hour post-irradiation. B. Representative confocal images showing γ-H2AX foci (Red, Cy3; blue, DAPI) in WT and Polμ−/− bone marrow cells treated as in A. C. Western blot showing amounts of phosphorylated γ-H2AX protein in LPS stimulated wt and Polμ−/− splenocytes after γ-irradiation (8 Gy), and analyzed at different periods (1 h, 3 h, and 6 h) post-irradiation; NI: non-irradiated controls. Histone H3 was used as a loading control. D. Dot plot showing DNA comet tail moment of WT (closed squares) and Polμ−/− (open triangles) bone marrow cells retrieved from irradiated mice (5Gy) after 3 hours of in vivo DNA repair. Tail momentum was significantly increased in Polμ−/− cells. E. Representative images of irradiated WT and Polμ−/− bone marrow metaphase cells analyzed by telomere FISH 96 hours after irradiation. Telomeres were hybridized with a Cy-3 labeled PNA Probe (Red) and chromosomes counterstained with DAPI (blue). Arrows indicate structural chromosomal aberrations: Chd and Chr = chromatid and chromosome breaks, respectively; dic. = dicentric chromosomes. F. Number of breaks, radial configurations, end-to-end-fusions, and total aberrations per metaphase in bone marrow cells from WT (closed bars) and Polμ−/− (open bars) irradiated mice. The mice were γ-irradiated (5Gy) and maintained (6 h) to allow in vivo DNA repair; BM-cell chromosomal aberrations were detected 4 days post irradiation. All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
Mentions: To examine the effect of Polμ deficiency on DNA repair in the hematopoietic system more closely, we analyzed the frequency of phosphorylated γ-H2AX foci in bone marrow and spleen after whole animal γ-irradiation (5Gy). In both genotypes 91% of all BM cells were γ-H2AX+, showing that these cells are more susceptible to γ-irradiation. In contrast, the proportion of phospho-γ-H2AX positive cells in Polμ−/− splenocytes (95%) was significantly higher than in wt cells (27%; p<0.001). Further, the number of γ-H2AX foci per cell was significantly increased in Polμ−/− cells compared with wt (1.7-fold for BM cells and 4.8-fold for splenocytes, p<0.001; Figure 6A–B). Changes in γ-H2AX phosphorylation were confirmed by western blot of γ-irradiated (8 Gy) spleen B cells; as predicted, Polμ−/− cells show higher amounts of phospho-γ-H2AX, and these levels are sustained for longer (Figure 6C). We confirmed reduced DSB repair in Polμ−/− BM cells by comet assay. Before cell extraction, irradiated mice (5Gy) were left to recover for 3 hours to allow in vivo DNA repair (see Methods). BM cells from these mice were separated by single cell electrophoresis under alkaline conditions, to measure the relative levels of DNA breaks; comet tail moment was 2-fold higher in Polμ−/− cells compared with wt cells (p<0.001; Figure 6D), indicating reduced DNA repair in Polμ−/− cells.

Bottom Line: In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development.Hematopoietic progenitors were reduced both in number and in expansion potential.Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

ABSTRACT
Polymerase micro (Polmicro) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development. In Polmicro(-/-) mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body gamma-irradiation revealed that Polmicro also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

Show MeSH
Related in: MedlinePlus