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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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Characterization of Sprtn hypomorphic cells and mice.(a) PCR-based genotyping of Sprtn+/+, Sprtn+/H and SprtnH/H mice at weaning. (b) Quantitative PCR analyses of Sprtn mRNA levels in Sprtn+/+ and SprtnH/H mice. Sprtn mRNA levels in the kidneys (left) and lungs (right) of two mice per genotype were measured three times by quantitative reverse transcription PCR and mean±s.d. is shown. Values were normalized to Gapdh and shown relative to Sprtn+/+ mouse #27. (c) Growth curves of Sprtn+/+, Sprtn+/H and SprtnH/H mice (n=5, mean±s.d.) of 4–16 weeks. (d,e) Quantitation of cells containing five or more γH2AX foci (d) and micronuclei (e) in the indicated Sprtn+/+ and SprtnH/H primary lung fibroblasts. At least 300 cells were scored in each experiment and percentages of positive cells are shown. Values are mean±s.d. of three independent experiments. For d,e, statistical significance is P<0.001 (H/H group versus +/+ group), two-tailed unpaired t-test. (f) Effect of ultraviolet irradiation on replication forks. DNA fiber assays were performed with Sprtn+/+ and SprtnH/H primary lung fibroblasts with or without ultraviolet irradiation (40 J m−2) between IdU and CldU labelling. Distribution of replication forks at different CldU/IdU ratios is shown. A horizontal red line indicates median value.
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f6: Characterization of Sprtn hypomorphic cells and mice.(a) PCR-based genotyping of Sprtn+/+, Sprtn+/H and SprtnH/H mice at weaning. (b) Quantitative PCR analyses of Sprtn mRNA levels in Sprtn+/+ and SprtnH/H mice. Sprtn mRNA levels in the kidneys (left) and lungs (right) of two mice per genotype were measured three times by quantitative reverse transcription PCR and mean±s.d. is shown. Values were normalized to Gapdh and shown relative to Sprtn+/+ mouse #27. (c) Growth curves of Sprtn+/+, Sprtn+/H and SprtnH/H mice (n=5, mean±s.d.) of 4–16 weeks. (d,e) Quantitation of cells containing five or more γH2AX foci (d) and micronuclei (e) in the indicated Sprtn+/+ and SprtnH/H primary lung fibroblasts. At least 300 cells were scored in each experiment and percentages of positive cells are shown. Values are mean±s.d. of three independent experiments. For d,e, statistical significance is P<0.001 (H/H group versus +/+ group), two-tailed unpaired t-test. (f) Effect of ultraviolet irradiation on replication forks. DNA fiber assays were performed with Sprtn+/+ and SprtnH/H primary lung fibroblasts with or without ultraviolet irradiation (40 J m−2) between IdU and CldU labelling. Distribution of replication forks at different CldU/IdU ratios is shown. A horizontal red line indicates median value.

Mentions: Although no viable SprtnH/− offspring were found in crossing of Sprtn+/− and Sprtn+/H mice, SprtnH/H mice were born at near Mendelian frequency from Sprtn+/H breeding pairs (Sprtn+/+, Sprtn+/H and SprtnH/H at 26.6, 53.3 and 20.1%, respectively), despite substantially reduced Sprtn expression (Fig. 6a,b). At weaning, SprtnH/H mice appeared healthy but were overtly smaller than control littermates. Dwarfism was observed in both sexes and persisted as SprtnH/H mice developed to adulthood (Fig. 6c). To investigate in more detail the effects of Sprtn insufficiency in SprtnH/H mice, we isolated primary lung fibroblasts and prostate epithelial cells from Sprtn+/+ and SprtnH/H mice. Sprtn expression was reduced to less than 10% in the SprtnH/H cells (Supplementary Fig. 5a,b). SprtnH/H lung fibroblasts showed elevated structural and numerical chromosomal instabilities, as evidenced by increased rates of γH2AX foci, aneuploidy and micronuclei (Fig. 6d,e; Table 3). In addition, SprtnH/H lung fibroblasts displayed defects in fork progression at ultraviolet-induced lesion, which was also confirmed in SprtnH/H prostate epithelial cells (Fig. 6f; Supplementary Fig. 5c). Taken together, these results indicate that cells from Sprtn hypomorphic mice exhibit genome instability and lesion bypass defects.


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)

Characterization of Sprtn hypomorphic cells and mice.(a) PCR-based genotyping of Sprtn+/+, Sprtn+/H and SprtnH/H mice at weaning. (b) Quantitative PCR analyses of Sprtn mRNA levels in Sprtn+/+ and SprtnH/H mice. Sprtn mRNA levels in the kidneys (left) and lungs (right) of two mice per genotype were measured three times by quantitative reverse transcription PCR and mean±s.d. is shown. Values were normalized to Gapdh and shown relative to Sprtn+/+ mouse #27. (c) Growth curves of Sprtn+/+, Sprtn+/H and SprtnH/H mice (n=5, mean±s.d.) of 4–16 weeks. (d,e) Quantitation of cells containing five or more γH2AX foci (d) and micronuclei (e) in the indicated Sprtn+/+ and SprtnH/H primary lung fibroblasts. At least 300 cells were scored in each experiment and percentages of positive cells are shown. Values are mean±s.d. of three independent experiments. For d,e, statistical significance is P<0.001 (H/H group versus +/+ group), two-tailed unpaired t-test. (f) Effect of ultraviolet irradiation on replication forks. DNA fiber assays were performed with Sprtn+/+ and SprtnH/H primary lung fibroblasts with or without ultraviolet irradiation (40 J m−2) between IdU and CldU labelling. Distribution of replication forks at different CldU/IdU ratios is shown. A horizontal red line indicates median value.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: Characterization of Sprtn hypomorphic cells and mice.(a) PCR-based genotyping of Sprtn+/+, Sprtn+/H and SprtnH/H mice at weaning. (b) Quantitative PCR analyses of Sprtn mRNA levels in Sprtn+/+ and SprtnH/H mice. Sprtn mRNA levels in the kidneys (left) and lungs (right) of two mice per genotype were measured three times by quantitative reverse transcription PCR and mean±s.d. is shown. Values were normalized to Gapdh and shown relative to Sprtn+/+ mouse #27. (c) Growth curves of Sprtn+/+, Sprtn+/H and SprtnH/H mice (n=5, mean±s.d.) of 4–16 weeks. (d,e) Quantitation of cells containing five or more γH2AX foci (d) and micronuclei (e) in the indicated Sprtn+/+ and SprtnH/H primary lung fibroblasts. At least 300 cells were scored in each experiment and percentages of positive cells are shown. Values are mean±s.d. of three independent experiments. For d,e, statistical significance is P<0.001 (H/H group versus +/+ group), two-tailed unpaired t-test. (f) Effect of ultraviolet irradiation on replication forks. DNA fiber assays were performed with Sprtn+/+ and SprtnH/H primary lung fibroblasts with or without ultraviolet irradiation (40 J m−2) between IdU and CldU labelling. Distribution of replication forks at different CldU/IdU ratios is shown. A horizontal red line indicates median value.
Mentions: Although no viable SprtnH/− offspring were found in crossing of Sprtn+/− and Sprtn+/H mice, SprtnH/H mice were born at near Mendelian frequency from Sprtn+/H breeding pairs (Sprtn+/+, Sprtn+/H and SprtnH/H at 26.6, 53.3 and 20.1%, respectively), despite substantially reduced Sprtn expression (Fig. 6a,b). At weaning, SprtnH/H mice appeared healthy but were overtly smaller than control littermates. Dwarfism was observed in both sexes and persisted as SprtnH/H mice developed to adulthood (Fig. 6c). To investigate in more detail the effects of Sprtn insufficiency in SprtnH/H mice, we isolated primary lung fibroblasts and prostate epithelial cells from Sprtn+/+ and SprtnH/H mice. Sprtn expression was reduced to less than 10% in the SprtnH/H cells (Supplementary Fig. 5a,b). SprtnH/H lung fibroblasts showed elevated structural and numerical chromosomal instabilities, as evidenced by increased rates of γH2AX foci, aneuploidy and micronuclei (Fig. 6d,e; Table 3). In addition, SprtnH/H lung fibroblasts displayed defects in fork progression at ultraviolet-induced lesion, which was also confirmed in SprtnH/H prostate epithelial cells (Fig. 6f; Supplementary Fig. 5c). Taken together, these results indicate that cells from Sprtn hypomorphic mice exhibit genome instability and lesion bypass defects.

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