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Spartan deficiency causes genomic instability and progeroid phenotypes.

Maskey RS, Kim MS, Baker DJ, Childs B, Malureanu LA, Jeganathan KB, Machida Y, van Deursen JM, Machida YJ - Nat Commun (2014)

Bottom Line: However, the physiological relevance of Spartan has not been established.Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death.These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA.

ABSTRACT
Spartan (also known as DVC1 and C1orf124) is a PCNA-interacting protein implicated in translesion synthesis, a DNA damage tolerance process that allows the DNA replication machinery to replicate past nucleotide lesions. However, the physiological relevance of Spartan has not been established. Here we report that Spartan insufficiency in mice causes chromosomal instability, cellular senescence and early onset of age-related phenotypes. Whereas complete loss of Spartan causes early embryonic lethality, hypomorphic mice with low amounts of Spartan are viable. These mice are growth retarded and develop cataracts, lordokyphosis and cachexia at a young age. Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death. These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

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Genome instability in Sprtn KO MEFs.(a) Images showing chromatin bridges (arrows) and micronuclei (arrowheads) in SprtnF/− MEFs treated with MeOH or 4-OHT for 48 h. DNA was visualized by DAPI staining. (b) Quantitation of chromatin bridges. Experiments were performed as in a. At least 300 cells were scored for chromatin bridges and percentages of positive cells are shown. Three independent experiments were performed and mean±s.d. is shown. ****P<0.0001 (two-tailed unpaired t-test). (c) Quantitation of micronuclei-containing cells. Experiments were performed as in a. ****P<0.0001 (two-tailed unpaired t-test). (d) Formation of 53BP1 nuclear bodies. SprtnF/− MEFs were treated with MeOH or 4-OHT for 48 h. At least 300 cells were scored for 53BP1 nuclear bodies and percentages of nuclei with different number of 53BP1 nuclear bodies per nucleus are shown. (e) Chromosomal abnormalities in Sprtn KO cells. SprtnF/− MEFs were treated with MeOH or 4-OHT for 48 h. Twelve hours after completion of the treatments, mitotic spreads were prepared and examined by microscopy. Representative pictures of mitotic spreads are shown in the left panel. Arrows indicate some of the chromosome abnormalities. In the right panel, representative images of chromosome gaps in Sprtn KO MEFs are indicated by arrowheads. (f) Quantitation of abnormal chromosomes. Cells harbouring chromosomal abnormalities were scored in 50 mitotic spreads of SprtnF/− MEFs treated with MeOH or 4-OHT.
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f4: Genome instability in Sprtn KO MEFs.(a) Images showing chromatin bridges (arrows) and micronuclei (arrowheads) in SprtnF/− MEFs treated with MeOH or 4-OHT for 48 h. DNA was visualized by DAPI staining. (b) Quantitation of chromatin bridges. Experiments were performed as in a. At least 300 cells were scored for chromatin bridges and percentages of positive cells are shown. Three independent experiments were performed and mean±s.d. is shown. ****P<0.0001 (two-tailed unpaired t-test). (c) Quantitation of micronuclei-containing cells. Experiments were performed as in a. ****P<0.0001 (two-tailed unpaired t-test). (d) Formation of 53BP1 nuclear bodies. SprtnF/− MEFs were treated with MeOH or 4-OHT for 48 h. At least 300 cells were scored for 53BP1 nuclear bodies and percentages of nuclei with different number of 53BP1 nuclear bodies per nucleus are shown. (e) Chromosomal abnormalities in Sprtn KO cells. SprtnF/− MEFs were treated with MeOH or 4-OHT for 48 h. Twelve hours after completion of the treatments, mitotic spreads were prepared and examined by microscopy. Representative pictures of mitotic spreads are shown in the left panel. Arrows indicate some of the chromosome abnormalities. In the right panel, representative images of chromosome gaps in Sprtn KO MEFs are indicated by arrowheads. (f) Quantitation of abnormal chromosomes. Cells harbouring chromosomal abnormalities were scored in 50 mitotic spreads of SprtnF/− MEFs treated with MeOH or 4-OHT.

Mentions: The elevated levels of DNA damage accompanied by checkpoint activation in Sprtn−/− MEFs prompted us to assess whether loss of Spartan induces genomic instability. Microscopic analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained MEFs revealed that a substantial fraction of mitotic Sprtn−/− MEFs exhibited chromatin bridges and micronuclei, structures that have been associated with unresolved replication intermediates and unrepaired DNA breaks19 (Fig. 4a–c). In addition, these MEFs had increased numbers of 53BP1 nuclear bodies that signify incomplete DNA replication2021 (Fig. 4d). These data raise the possibility that the genome of Sprtn-deficient cells is subject to under-replication. Consistent with this interpretation, the vast majority of chromosomal abnormalities observed in the mitotic spreads of Sprtn−/− MEFs were chromosome gaps, which might represent under-replicated regions (Fig. 4e,f). In summary, these results suggest that Spartan is crucial for maintaining genome integrity.


Spartan deficiency causes genomic instability and progeroid phenotypes.

Maskey RS, Kim MS, Baker DJ, Childs B, Malureanu LA, Jeganathan KB, Machida Y, van Deursen JM, Machida YJ - Nat Commun (2014)

Genome instability in Sprtn KO MEFs.(a) Images showing chromatin bridges (arrows) and micronuclei (arrowheads) in SprtnF/− MEFs treated with MeOH or 4-OHT for 48 h. DNA was visualized by DAPI staining. (b) Quantitation of chromatin bridges. Experiments were performed as in a. At least 300 cells were scored for chromatin bridges and percentages of positive cells are shown. Three independent experiments were performed and mean±s.d. is shown. ****P<0.0001 (two-tailed unpaired t-test). (c) Quantitation of micronuclei-containing cells. Experiments were performed as in a. ****P<0.0001 (two-tailed unpaired t-test). (d) Formation of 53BP1 nuclear bodies. SprtnF/− MEFs were treated with MeOH or 4-OHT for 48 h. At least 300 cells were scored for 53BP1 nuclear bodies and percentages of nuclei with different number of 53BP1 nuclear bodies per nucleus are shown. (e) Chromosomal abnormalities in Sprtn KO cells. SprtnF/− MEFs were treated with MeOH or 4-OHT for 48 h. Twelve hours after completion of the treatments, mitotic spreads were prepared and examined by microscopy. Representative pictures of mitotic spreads are shown in the left panel. Arrows indicate some of the chromosome abnormalities. In the right panel, representative images of chromosome gaps in Sprtn KO MEFs are indicated by arrowheads. (f) Quantitation of abnormal chromosomes. Cells harbouring chromosomal abnormalities were scored in 50 mitotic spreads of SprtnF/− MEFs treated with MeOH or 4-OHT.
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Related In: Results  -  Collection

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f4: Genome instability in Sprtn KO MEFs.(a) Images showing chromatin bridges (arrows) and micronuclei (arrowheads) in SprtnF/− MEFs treated with MeOH or 4-OHT for 48 h. DNA was visualized by DAPI staining. (b) Quantitation of chromatin bridges. Experiments were performed as in a. At least 300 cells were scored for chromatin bridges and percentages of positive cells are shown. Three independent experiments were performed and mean±s.d. is shown. ****P<0.0001 (two-tailed unpaired t-test). (c) Quantitation of micronuclei-containing cells. Experiments were performed as in a. ****P<0.0001 (two-tailed unpaired t-test). (d) Formation of 53BP1 nuclear bodies. SprtnF/− MEFs were treated with MeOH or 4-OHT for 48 h. At least 300 cells were scored for 53BP1 nuclear bodies and percentages of nuclei with different number of 53BP1 nuclear bodies per nucleus are shown. (e) Chromosomal abnormalities in Sprtn KO cells. SprtnF/− MEFs were treated with MeOH or 4-OHT for 48 h. Twelve hours after completion of the treatments, mitotic spreads were prepared and examined by microscopy. Representative pictures of mitotic spreads are shown in the left panel. Arrows indicate some of the chromosome abnormalities. In the right panel, representative images of chromosome gaps in Sprtn KO MEFs are indicated by arrowheads. (f) Quantitation of abnormal chromosomes. Cells harbouring chromosomal abnormalities were scored in 50 mitotic spreads of SprtnF/− MEFs treated with MeOH or 4-OHT.
Mentions: The elevated levels of DNA damage accompanied by checkpoint activation in Sprtn−/− MEFs prompted us to assess whether loss of Spartan induces genomic instability. Microscopic analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained MEFs revealed that a substantial fraction of mitotic Sprtn−/− MEFs exhibited chromatin bridges and micronuclei, structures that have been associated with unresolved replication intermediates and unrepaired DNA breaks19 (Fig. 4a–c). In addition, these MEFs had increased numbers of 53BP1 nuclear bodies that signify incomplete DNA replication2021 (Fig. 4d). These data raise the possibility that the genome of Sprtn-deficient cells is subject to under-replication. Consistent with this interpretation, the vast majority of chromosomal abnormalities observed in the mitotic spreads of Sprtn−/− MEFs were chromosome gaps, which might represent under-replicated regions (Fig. 4e,f). In summary, these results suggest that Spartan is crucial for maintaining genome integrity.

Bottom Line: However, the physiological relevance of Spartan has not been established.Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death.These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA.

ABSTRACT
Spartan (also known as DVC1 and C1orf124) is a PCNA-interacting protein implicated in translesion synthesis, a DNA damage tolerance process that allows the DNA replication machinery to replicate past nucleotide lesions. However, the physiological relevance of Spartan has not been established. Here we report that Spartan insufficiency in mice causes chromosomal instability, cellular senescence and early onset of age-related phenotypes. Whereas complete loss of Spartan causes early embryonic lethality, hypomorphic mice with low amounts of Spartan are viable. These mice are growth retarded and develop cataracts, lordokyphosis and cachexia at a young age. Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death. These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

Show MeSH
Related in: MedlinePlus