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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

Hematopoietic progenitor and stem cell numbers are reduced in Polμ−/− mice.A. Number of myeloid progenitors (colony forming units) per bone marrow determined in clonogenic assays in WT (solid bars, n = 9–11) and Polμ−/− (open bars, n = 9–11) mice. CFU-GM (granulomonocytic cells), CFU-G (granulocytes), CFU-M (monocytes) and CFU-C (sum of CFU-GM, CFU-G and CFU-M cells). B. Number of CFU-PreB progenitors. C. Megakaryocyte colony-forming units (CFU-Mk). D. Number of erythroid burst-forming units (BFU-E). BFU-E were scored only if hemoglobin was evident within the colony. E. Flow cytometric determination of CLP (common lymphoid progenitors) in bone marrow of WT (closed circles, n = 8) or Polμ−/− (open circles, n = 8) C57BL/6 mice (left panel). F. Flow cytometric determination of bone marrow CMP (common myeloid progenitors), myelomonocytic progenitors (GMP) and megakaryocyte/erythroid progenitors (MEP) from WT (solid bar, n = 8) and Polμ−/− (open bar, n = 8) C57BL/6 mice (left panel). G. Flow cytometric determination of HSC in the bone marrow of the same mice analyzed in F; WT (solid bar, n = 8) and Polμ−/− (open bar, n = 8) C57BL/6 mice (left panel). Gating strategies for the analysis in E–G are indicated in the plots to the right. H. Experimental design for the competitive bone marrow transplantation assay. I. Frequency of competing repopulating units (RU) in the bone marrow of C57BL/6 wild-type (n = 4) or Polμ−/− mice (n = 4). J. Number of competing repopulating units (RU) per bone marrow of the mice analyzed in I. Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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pgen-1000389-g002: Hematopoietic progenitor and stem cell numbers are reduced in Polμ−/− mice.A. Number of myeloid progenitors (colony forming units) per bone marrow determined in clonogenic assays in WT (solid bars, n = 9–11) and Polμ−/− (open bars, n = 9–11) mice. CFU-GM (granulomonocytic cells), CFU-G (granulocytes), CFU-M (monocytes) and CFU-C (sum of CFU-GM, CFU-G and CFU-M cells). B. Number of CFU-PreB progenitors. C. Megakaryocyte colony-forming units (CFU-Mk). D. Number of erythroid burst-forming units (BFU-E). BFU-E were scored only if hemoglobin was evident within the colony. E. Flow cytometric determination of CLP (common lymphoid progenitors) in bone marrow of WT (closed circles, n = 8) or Polμ−/− (open circles, n = 8) C57BL/6 mice (left panel). F. Flow cytometric determination of bone marrow CMP (common myeloid progenitors), myelomonocytic progenitors (GMP) and megakaryocyte/erythroid progenitors (MEP) from WT (solid bar, n = 8) and Polμ−/− (open bar, n = 8) C57BL/6 mice (left panel). G. Flow cytometric determination of HSC in the bone marrow of the same mice analyzed in F; WT (solid bar, n = 8) and Polμ−/− (open bar, n = 8) C57BL/6 mice (left panel). Gating strategies for the analysis in E–G are indicated in the plots to the right. H. Experimental design for the competitive bone marrow transplantation assay. I. Frequency of competing repopulating units (RU) in the bone marrow of C57BL/6 wild-type (n = 4) or Polμ−/− mice (n = 4). J. Number of competing repopulating units (RU) per bone marrow of the mice analyzed in I. Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.

Mentions: Hematopoiesis is a clonal process in which immature progenitors give rise to more committed progenitors. We therefore used colony forming unit (CFU) assays to determine total progenitor numbers in wt and Polμ−/− BM (Figure 2A–D). The numbers of PreB and myeloid progenitors (CFU-PreB and CFU-C) in Polμ−/−mice were 2.2- and 1.3-fold lower than in wt (p<0.001, Figure 2A,B). Megakaryocytic and erythroid progenitors (CFU-Mk and BFU-E) were also affected, though to a lesser extent (Figure 2C,D). Importantly, whereas granulomonocyte and monocyte progenitors (CFU-GM and CFU-M) were reduced in Polμ−/− BM (correlating with the peripheral blood monocyte deficiency), granulocyte progenitors (CFU-G) were not, thus explaining the absence of neutropenia (Figure 1A). These results show that Polμ deficient mice have lowered numbers of committed progenitors and suggest that lack of Polμ differentially affects lineage-specific progenitors.


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)

Hematopoietic progenitor and stem cell numbers are reduced in Polμ−/− mice.A. Number of myeloid progenitors (colony forming units) per bone marrow determined in clonogenic assays in WT (solid bars, n = 9–11) and Polμ−/− (open bars, n = 9–11) mice. CFU-GM (granulomonocytic cells), CFU-G (granulocytes), CFU-M (monocytes) and CFU-C (sum of CFU-GM, CFU-G and CFU-M cells). B. Number of CFU-PreB progenitors. C. Megakaryocyte colony-forming units (CFU-Mk). D. Number of erythroid burst-forming units (BFU-E). BFU-E were scored only if hemoglobin was evident within the colony. E. Flow cytometric determination of CLP (common lymphoid progenitors) in bone marrow of WT (closed circles, n = 8) or Polμ−/− (open circles, n = 8) C57BL/6 mice (left panel). F. Flow cytometric determination of bone marrow CMP (common myeloid progenitors), myelomonocytic progenitors (GMP) and megakaryocyte/erythroid progenitors (MEP) from WT (solid bar, n = 8) and Polμ−/− (open bar, n = 8) C57BL/6 mice (left panel). G. Flow cytometric determination of HSC in the bone marrow of the same mice analyzed in F; WT (solid bar, n = 8) and Polμ−/− (open bar, n = 8) C57BL/6 mice (left panel). Gating strategies for the analysis in E–G are indicated in the plots to the right. H. Experimental design for the competitive bone marrow transplantation assay. I. Frequency of competing repopulating units (RU) in the bone marrow of C57BL/6 wild-type (n = 4) or Polμ−/− mice (n = 4). J. Number of competing repopulating units (RU) per bone marrow of the mice analyzed in I. 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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Show All Figures
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pgen-1000389-g002: Hematopoietic progenitor and stem cell numbers are reduced in Polμ−/− mice.A. Number of myeloid progenitors (colony forming units) per bone marrow determined in clonogenic assays in WT (solid bars, n = 9–11) and Polμ−/− (open bars, n = 9–11) mice. CFU-GM (granulomonocytic cells), CFU-G (granulocytes), CFU-M (monocytes) and CFU-C (sum of CFU-GM, CFU-G and CFU-M cells). B. Number of CFU-PreB progenitors. C. Megakaryocyte colony-forming units (CFU-Mk). D. Number of erythroid burst-forming units (BFU-E). BFU-E were scored only if hemoglobin was evident within the colony. E. Flow cytometric determination of CLP (common lymphoid progenitors) in bone marrow of WT (closed circles, n = 8) or Polμ−/− (open circles, n = 8) C57BL/6 mice (left panel). F. Flow cytometric determination of bone marrow CMP (common myeloid progenitors), myelomonocytic progenitors (GMP) and megakaryocyte/erythroid progenitors (MEP) from WT (solid bar, n = 8) and Polμ−/− (open bar, n = 8) C57BL/6 mice (left panel). G. Flow cytometric determination of HSC in the bone marrow of the same mice analyzed in F; WT (solid bar, n = 8) and Polμ−/− (open bar, n = 8) C57BL/6 mice (left panel). Gating strategies for the analysis in E–G are indicated in the plots to the right. H. Experimental design for the competitive bone marrow transplantation assay. I. Frequency of competing repopulating units (RU) in the bone marrow of C57BL/6 wild-type (n = 4) or Polμ−/− mice (n = 4). J. Number of competing repopulating units (RU) per bone marrow of the mice analyzed in I. Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
Mentions: Hematopoiesis is a clonal process in which immature progenitors give rise to more committed progenitors. We therefore used colony forming unit (CFU) assays to determine total progenitor numbers in wt and Polμ−/− BM (Figure 2A–D). The numbers of PreB and myeloid progenitors (CFU-PreB and CFU-C) in Polμ−/−mice were 2.2- and 1.3-fold lower than in wt (p<0.001, Figure 2A,B). Megakaryocytic and erythroid progenitors (CFU-Mk and BFU-E) were also affected, though to a lesser extent (Figure 2C,D). Importantly, whereas granulomonocyte and monocyte progenitors (CFU-GM and CFU-M) were reduced in Polμ−/− BM (correlating with the peripheral blood monocyte deficiency), granulocyte progenitors (CFU-G) were not, thus explaining the absence of neutropenia (Figure 1A). These results show that Polμ deficient mice have lowered numbers of committed progenitors and suggest that lack of Polμ differentially affects lineage-specific progenitors.

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