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The CENP-O complex requirement varies among different cell types.

Kagawa N, Hori T, Hoki Y, Hosoya O, Tsutsui K, Saga Y, Sado T, Fukagawa T - Chromosome Res. (2014)

Bottom Line: Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable.However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior.Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka, 411-8540, Japan.

ABSTRACT
CENP-U (CENP-50) is a component of the CENP-O complex, which includes CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized at kinetochores throughout the cell cycle in vertebrates. Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable. To examine the functional roles of CENP-U in an organism-dependent context, we generated CENP-U-deficient mice. The CENP-U-deficient mice died during early embryogenesis (approximately E7.5). Thus, conditional CENP-U-deficient mouse ES cells were generated to analyze CENP-U-deficient phenotypes at the cell level. When CENP-U was disrupted in the mouse ES cells, all CENP-O complex proteins disappeared from kinetochores. In contrast, other kinetochore proteins were recruited in CENP-U-deficient mouse ES cells as CENP-U-deficient DT40 cells. However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior. Although CENP-U was essential for cell viability during mouse early embryogenesis, CENP-U-deficient mouse embryonic fibroblast cells were viable, similar to the DT40 cells. Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types.

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CENP-U is essential for mouse embryogenesis a Genomic region of the mouse CENP-U locus and a gene disruption construct. Black boxes indicate the positions of exons. Sac I restriction sites are shown. The position of the probe used for Southern hybridization is indicated. A novel 12-kb Sac I fragment hybridized to the probe, if targeted integration of the construct occurred. b Restriction analysis of genomic DNAs with targeted integration of the CENP-U disruption construct. Genomic DNAs from wild-type ES cells and two clones (#13 and #19) after targeting (+/−) were analyzed by Southern hybridization using the probe indicated in (A). In #13 and #19, a novel 12-kb Sac I fragment was detected. c PCR genotyping of embryonic DNA from mice of CENP-U+/− heterozygous intercrosses. a Primer design, b PCR results with DNA from E3.5, and c PCR results with DNA from E6.5. d Genotyping of CENP-U+/− intercross mice at each stage. e Serial section analysis of E7.5 embryos from wild-type or CENP-U−/− mice. Sections were stained with toluidine blue. Scale bar, 100 μm. f DAPI (blue) and TUNEL (green) staining of E7.5 embryos from the wild-type or CENP-U−/− mice. Scale bar, 200 μm. An embryo (Em.) is outlined by a dashed line. TUNEL positive cells are enriched in the −/− embryo
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Fig1: CENP-U is essential for mouse embryogenesis a Genomic region of the mouse CENP-U locus and a gene disruption construct. Black boxes indicate the positions of exons. Sac I restriction sites are shown. The position of the probe used for Southern hybridization is indicated. A novel 12-kb Sac I fragment hybridized to the probe, if targeted integration of the construct occurred. b Restriction analysis of genomic DNAs with targeted integration of the CENP-U disruption construct. Genomic DNAs from wild-type ES cells and two clones (#13 and #19) after targeting (+/−) were analyzed by Southern hybridization using the probe indicated in (A). In #13 and #19, a novel 12-kb Sac I fragment was detected. c PCR genotyping of embryonic DNA from mice of CENP-U+/− heterozygous intercrosses. a Primer design, b PCR results with DNA from E3.5, and c PCR results with DNA from E6.5. d Genotyping of CENP-U+/− intercross mice at each stage. e Serial section analysis of E7.5 embryos from wild-type or CENP-U−/− mice. Sections were stained with toluidine blue. Scale bar, 100 μm. f DAPI (blue) and TUNEL (green) staining of E7.5 embryos from the wild-type or CENP-U−/− mice. Scale bar, 200 μm. An embryo (Em.) is outlined by a dashed line. TUNEL positive cells are enriched in the −/− embryo

Mentions: To disrupt the mouse CENP-U gene in ES cells, we used a promoter-less targeting construct to obtain a high level of homologous recombination. If a targeting reaction occurred, a neomycin resistance gene would be expressed under the control of the CENP-U promoter and exons 4 to 6 of the CENP-U gene would be deleted (Fig. 1a). We isolated several 129/Sv-derived ES cell clones with disrupted a CENP-U allele and confirmed targeted disruption by Southern blot analysis (Fig. 1b). ES cells with the CENP-U-disrupted allele were injected into C57BL/6 blastocysts, and the resulting chimerical mice were backcrossed with the wild-type C57BL/6 mice to generate CENP-U+/− heterozygous mice. We designed a PCR primer set to distinguish between wild-type and disrupted alleles and performed genotyping analysis of embryonic cells from CENP-U+/− heterozygous intercrosses (Fig. 1c).Fig. 1


The CENP-O complex requirement varies among different cell types.

Kagawa N, Hori T, Hoki Y, Hosoya O, Tsutsui K, Saga Y, Sado T, Fukagawa T - Chromosome Res. (2014)

CENP-U is essential for mouse embryogenesis a Genomic region of the mouse CENP-U locus and a gene disruption construct. Black boxes indicate the positions of exons. Sac I restriction sites are shown. The position of the probe used for Southern hybridization is indicated. A novel 12-kb Sac I fragment hybridized to the probe, if targeted integration of the construct occurred. b Restriction analysis of genomic DNAs with targeted integration of the CENP-U disruption construct. Genomic DNAs from wild-type ES cells and two clones (#13 and #19) after targeting (+/−) were analyzed by Southern hybridization using the probe indicated in (A). In #13 and #19, a novel 12-kb Sac I fragment was detected. c PCR genotyping of embryonic DNA from mice of CENP-U+/− heterozygous intercrosses. a Primer design, b PCR results with DNA from E3.5, and c PCR results with DNA from E6.5. d Genotyping of CENP-U+/− intercross mice at each stage. e Serial section analysis of E7.5 embryos from wild-type or CENP-U−/− mice. Sections were stained with toluidine blue. Scale bar, 100 μm. f DAPI (blue) and TUNEL (green) staining of E7.5 embryos from the wild-type or CENP-U−/− mice. Scale bar, 200 μm. An embryo (Em.) is outlined by a dashed line. TUNEL positive cells are enriched in the −/− embryo
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Related In: Results  -  Collection

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Fig1: CENP-U is essential for mouse embryogenesis a Genomic region of the mouse CENP-U locus and a gene disruption construct. Black boxes indicate the positions of exons. Sac I restriction sites are shown. The position of the probe used for Southern hybridization is indicated. A novel 12-kb Sac I fragment hybridized to the probe, if targeted integration of the construct occurred. b Restriction analysis of genomic DNAs with targeted integration of the CENP-U disruption construct. Genomic DNAs from wild-type ES cells and two clones (#13 and #19) after targeting (+/−) were analyzed by Southern hybridization using the probe indicated in (A). In #13 and #19, a novel 12-kb Sac I fragment was detected. c PCR genotyping of embryonic DNA from mice of CENP-U+/− heterozygous intercrosses. a Primer design, b PCR results with DNA from E3.5, and c PCR results with DNA from E6.5. d Genotyping of CENP-U+/− intercross mice at each stage. e Serial section analysis of E7.5 embryos from wild-type or CENP-U−/− mice. Sections were stained with toluidine blue. Scale bar, 100 μm. f DAPI (blue) and TUNEL (green) staining of E7.5 embryos from the wild-type or CENP-U−/− mice. Scale bar, 200 μm. An embryo (Em.) is outlined by a dashed line. TUNEL positive cells are enriched in the −/− embryo
Mentions: To disrupt the mouse CENP-U gene in ES cells, we used a promoter-less targeting construct to obtain a high level of homologous recombination. If a targeting reaction occurred, a neomycin resistance gene would be expressed under the control of the CENP-U promoter and exons 4 to 6 of the CENP-U gene would be deleted (Fig. 1a). We isolated several 129/Sv-derived ES cell clones with disrupted a CENP-U allele and confirmed targeted disruption by Southern blot analysis (Fig. 1b). ES cells with the CENP-U-disrupted allele were injected into C57BL/6 blastocysts, and the resulting chimerical mice were backcrossed with the wild-type C57BL/6 mice to generate CENP-U+/− heterozygous mice. We designed a PCR primer set to distinguish between wild-type and disrupted alleles and performed genotyping analysis of embryonic cells from CENP-U+/− heterozygous intercrosses (Fig. 1c).Fig. 1

Bottom Line: Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable.However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior.Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka, 411-8540, Japan.

ABSTRACT
CENP-U (CENP-50) is a component of the CENP-O complex, which includes CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized at kinetochores throughout the cell cycle in vertebrates. Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable. To examine the functional roles of CENP-U in an organism-dependent context, we generated CENP-U-deficient mice. The CENP-U-deficient mice died during early embryogenesis (approximately E7.5). Thus, conditional CENP-U-deficient mouse ES cells were generated to analyze CENP-U-deficient phenotypes at the cell level. When CENP-U was disrupted in the mouse ES cells, all CENP-O complex proteins disappeared from kinetochores. In contrast, other kinetochore proteins were recruited in CENP-U-deficient mouse ES cells as CENP-U-deficient DT40 cells. However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior. Although CENP-U was essential for cell viability during mouse early embryogenesis, CENP-U-deficient mouse embryonic fibroblast cells were viable, similar to the DT40 cells. Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types.

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