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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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Related in: MedlinePlus

Blood and bone marrow cell profiles are altered and bone marrow cell numbers reduced in Polμ−/− mice.A. Blood cell populations in wildtype (WT: n = 6–12; solid bars) and Polμ−/− mice (n = 7–13; open bars). CD19, B lymphocytes; CD3, T lymphocytes; Mac1+Gr1−, monocytes; and Mac1+Gr1+, neutrophils. B. Representative experiment showing platelet numbers in WT (solid bar; n = 6) and Polμ−/− mice (open bar; n = 16). C. Representative experiment showing distribution of bone marrow (BM) populations (two femurs per mouse) in WT (closed circles; n = 4–7) and Polμ−/− mice (open circles; n = 4–8). D. Histological sections of Polμ−/− and WT BM; endothelial sinusoids are delineated by a dashed line (blue) and marked by blue arrowheads. E. BM cell population analysis by flow cytometry (WT, solid bars; n = 7; Polμ−/−, open bars; n = 8), showing B220 (B cell) and CD3 (T cell), Mac1Gr1 (myelomonocytic), Ter119 (erythroid), and CD41 (megakaryocytic) lineages. F. Representative flow cytometry plots of B cell differentiation analysis in the bone marrow of WT (red plots) or Polμ−/− (blue plots) C57BL/6 mice. PreB+ProB cells were further analyzed according to CD25 and CD43 expression to distinguish between PreB and ProB cells. G. Cell number per bone marrow (2 tibias, 2 femur) of the B cell subsets analyzed in F; n = 8. H. Frequency (percentage of total B cell population) of the B cell subsets analyzed in F; n = 8. Results are pooled data from two independent experiments. Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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pgen-1000389-g001: Blood and bone marrow cell profiles are altered and bone marrow cell numbers reduced in Polμ−/− mice.A. Blood cell populations in wildtype (WT: n = 6–12; solid bars) and Polμ−/− mice (n = 7–13; open bars). CD19, B lymphocytes; CD3, T lymphocytes; Mac1+Gr1−, monocytes; and Mac1+Gr1+, neutrophils. B. Representative experiment showing platelet numbers in WT (solid bar; n = 6) and Polμ−/− mice (open bar; n = 16). C. Representative experiment showing distribution of bone marrow (BM) populations (two femurs per mouse) in WT (closed circles; n = 4–7) and Polμ−/− mice (open circles; n = 4–8). D. Histological sections of Polμ−/− and WT BM; endothelial sinusoids are delineated by a dashed line (blue) and marked by blue arrowheads. E. BM cell population analysis by flow cytometry (WT, solid bars; n = 7; Polμ−/−, open bars; n = 8), showing B220 (B cell) and CD3 (T cell), Mac1Gr1 (myelomonocytic), Ter119 (erythroid), and CD41 (megakaryocytic) lineages. F. Representative flow cytometry plots of B cell differentiation analysis in the bone marrow of WT (red plots) or Polμ−/− (blue plots) C57BL/6 mice. PreB+ProB cells were further analyzed according to CD25 and CD43 expression to distinguish between PreB and ProB cells. G. Cell number per bone marrow (2 tibias, 2 femur) of the B cell subsets analyzed in F; n = 8. H. Frequency (percentage of total B cell population) of the B cell subsets analyzed in F; n = 8. Results are pooled data from two independent experiments. Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.

Mentions: Polμ is widely expressed, but is especially abundant in lympohematopoietic tissues and cells [11],[14]. Polμ is known to participate in DSB repair reactions in vitro[15]–[17], but the only in vivo role assigned so far is in Vκ-Jκ recombination during B cell development [18]. To investigate whether Polμ has a function in other hematopoietic lineages, we first studied peripheral blood (PB) cell populations in Polμ−/− mice. Concurring with the reduction in B cell numbers associated with abnormal Vκ-Jκ recombination, [18], the B cell count in Polμ−/− mice was 4.9-fold (p<0.001) lower than in wt controls (Figure 1A; CD19+ cells). Monocyte numbers were also reduced (2.2-fold, p<0.01, Figure 1A; Mac1+ Gr1− cells), as were neutrophil numbers, though here the difference was not statistically significant (p<0.09, Figure 1A; Mac1+Gr1+ cells). The only cell type analyzed that was apparently unaffected was the T cell lineage (CD3+ cells). Hematological analysis revealed moderate thrombocytopenia, with 403×109 platelets/ml in Polμ−/− mice, compared with 663×109 platelets/ml in wt counterparts (p<0.01, Figure 1B); this was associated with increased bleeding times (Figure S1) Thus peripheral blood populations other than B lymphocytes are altered in Polμ−/− mice. To assess whether the defective blood population profile originated in bone marrow (BM), we compared BM cell numbers between wt and Polμ−/− mice. Our results show that BM cellularity was 1.5-fold lower in Polμ−/− mice (p<0.001, Figure 1C). Immunohistochemistry confirmed that Polμ−/− BM contained fewer cells than wt BM, as shown by the dilated endothelial bone marrow sinusoids (Figure 1D). Therefore the most likely cause of the thrombocytopenia and reduced myeloid cell numbers in peripheral blood in Polμ−/− mice is a defect in BM hematopoiesis.


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)

Blood and bone marrow cell profiles are altered and bone marrow cell numbers reduced in Polμ−/− mice.A. Blood cell populations in wildtype (WT: n = 6–12; solid bars) and Polμ−/− mice (n = 7–13; open bars). CD19, B lymphocytes; CD3, T lymphocytes; Mac1+Gr1−, monocytes; and Mac1+Gr1+, neutrophils. B. Representative experiment showing platelet numbers in WT (solid bar; n = 6) and Polμ−/− mice (open bar; n = 16). C. Representative experiment showing distribution of bone marrow (BM) populations (two femurs per mouse) in WT (closed circles; n = 4–7) and Polμ−/− mice (open circles; n = 4–8). D. Histological sections of Polμ−/− and WT BM; endothelial sinusoids are delineated by a dashed line (blue) and marked by blue arrowheads. E. BM cell population analysis by flow cytometry (WT, solid bars; n = 7; Polμ−/−, open bars; n = 8), showing B220 (B cell) and CD3 (T cell), Mac1Gr1 (myelomonocytic), Ter119 (erythroid), and CD41 (megakaryocytic) lineages. F. Representative flow cytometry plots of B cell differentiation analysis in the bone marrow of WT (red plots) or Polμ−/− (blue plots) C57BL/6 mice. PreB+ProB cells were further analyzed according to CD25 and CD43 expression to distinguish between PreB and ProB cells. G. Cell number per bone marrow (2 tibias, 2 femur) of the B cell subsets analyzed in F; n = 8. H. Frequency (percentage of total B cell population) of the B cell subsets analyzed in F; n = 8. Results are pooled data from two independent experiments. Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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Related In: Results  -  Collection

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pgen-1000389-g001: Blood and bone marrow cell profiles are altered and bone marrow cell numbers reduced in Polμ−/− mice.A. Blood cell populations in wildtype (WT: n = 6–12; solid bars) and Polμ−/− mice (n = 7–13; open bars). CD19, B lymphocytes; CD3, T lymphocytes; Mac1+Gr1−, monocytes; and Mac1+Gr1+, neutrophils. B. Representative experiment showing platelet numbers in WT (solid bar; n = 6) and Polμ−/− mice (open bar; n = 16). C. Representative experiment showing distribution of bone marrow (BM) populations (two femurs per mouse) in WT (closed circles; n = 4–7) and Polμ−/− mice (open circles; n = 4–8). D. Histological sections of Polμ−/− and WT BM; endothelial sinusoids are delineated by a dashed line (blue) and marked by blue arrowheads. E. BM cell population analysis by flow cytometry (WT, solid bars; n = 7; Polμ−/−, open bars; n = 8), showing B220 (B cell) and CD3 (T cell), Mac1Gr1 (myelomonocytic), Ter119 (erythroid), and CD41 (megakaryocytic) lineages. F. Representative flow cytometry plots of B cell differentiation analysis in the bone marrow of WT (red plots) or Polμ−/− (blue plots) C57BL/6 mice. PreB+ProB cells were further analyzed according to CD25 and CD43 expression to distinguish between PreB and ProB cells. G. Cell number per bone marrow (2 tibias, 2 femur) of the B cell subsets analyzed in F; n = 8. H. Frequency (percentage of total B cell population) of the B cell subsets analyzed in F; n = 8. Results are pooled data from two independent experiments. Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
Mentions: Polμ is widely expressed, but is especially abundant in lympohematopoietic tissues and cells [11],[14]. Polμ is known to participate in DSB repair reactions in vitro[15]–[17], but the only in vivo role assigned so far is in Vκ-Jκ recombination during B cell development [18]. To investigate whether Polμ has a function in other hematopoietic lineages, we first studied peripheral blood (PB) cell populations in Polμ−/− mice. Concurring with the reduction in B cell numbers associated with abnormal Vκ-Jκ recombination, [18], the B cell count in Polμ−/− mice was 4.9-fold (p<0.001) lower than in wt controls (Figure 1A; CD19+ cells). Monocyte numbers were also reduced (2.2-fold, p<0.01, Figure 1A; Mac1+ Gr1− cells), as were neutrophil numbers, though here the difference was not statistically significant (p<0.09, Figure 1A; Mac1+Gr1+ cells). The only cell type analyzed that was apparently unaffected was the T cell lineage (CD3+ cells). Hematological analysis revealed moderate thrombocytopenia, with 403×109 platelets/ml in Polμ−/− mice, compared with 663×109 platelets/ml in wt counterparts (p<0.01, Figure 1B); this was associated with increased bleeding times (Figure S1) Thus peripheral blood populations other than B lymphocytes are altered in Polμ−/− mice. To assess whether the defective blood population profile originated in bone marrow (BM), we compared BM cell numbers between wt and Polμ−/− mice. Our results show that BM cellularity was 1.5-fold lower in Polμ−/− mice (p<0.001, Figure 1C). Immunohistochemistry confirmed that Polμ−/− BM contained fewer cells than wt BM, as shown by the dilated endothelial bone marrow sinusoids (Figure 1D). Therefore the most likely cause of the thrombocytopenia and reduced myeloid cell numbers in peripheral blood in Polμ−/− mice is a defect in BM hematopoiesis.

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