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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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Sprtn KO causes impaired cell proliferation and cell death.(a) PCR-based genotyping of Sprtn alleles in the indicated MEF lines after 48 h treatment with MeOH or 2 μM 4-OHT. (b) Proliferation of Sprtn-targeted MEFs. Cells treated with MeOH or 4-OHT for 48 h were seeded and cell numbers were counted at the indicated time points. Values are mean±s.d. of three independent experiments. (c) Analyses of apoptosis in Sprtn-targeted MEFs. Two days after the completion of 48 h treatment with MeOH or 4-OHT, cells were stained with Annexin V and PI and analysed by flow cytometry. Values are mean±s.d. of three independent experiments. NS, not significant; ***P<0.001 (two-tailed unpaired t-test). (d) Cell cycle profiling of Sprtn-targeted MEFs. The indicated MEF lines were treated with MeOH or 4-OHT for 48 h. Two days later, cells were stained with PI and analysed by flow cytometry. (e) Cell cycle profiling of SprtnF/F; Cre-ERT2 MEFs (K3) expressing wild-type human Spartan or the E112A mutant after 48 h treatment with MeOH or 4-OHT.
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f2: Sprtn KO causes impaired cell proliferation and cell death.(a) PCR-based genotyping of Sprtn alleles in the indicated MEF lines after 48 h treatment with MeOH or 2 μM 4-OHT. (b) Proliferation of Sprtn-targeted MEFs. Cells treated with MeOH or 4-OHT for 48 h were seeded and cell numbers were counted at the indicated time points. Values are mean±s.d. of three independent experiments. (c) Analyses of apoptosis in Sprtn-targeted MEFs. Two days after the completion of 48 h treatment with MeOH or 4-OHT, cells were stained with Annexin V and PI and analysed by flow cytometry. Values are mean±s.d. of three independent experiments. NS, not significant; ***P<0.001 (two-tailed unpaired t-test). (d) Cell cycle profiling of Sprtn-targeted MEFs. The indicated MEF lines were treated with MeOH or 4-OHT for 48 h. Two days later, cells were stained with PI and analysed by flow cytometry. (e) Cell cycle profiling of SprtnF/F; Cre-ERT2 MEFs (K3) expressing wild-type human Spartan or the E112A mutant after 48 h treatment with MeOH or 4-OHT.

Mentions: To further investigate the role of Sprtn at the cellular level, we generated a conditional Sprtn KO system in mouse embryonic fibroblasts (MEFs). Sprtn+/+, SprtnF/+ and SprtnF/− MEFs were immortalized and transduced with retroviruses expressing Cre-ERT2. Treatment with 4-hydroxytamoxifen (4-OHT) resulted in excision of exon 2, efficiently converting the floxed allele to a KO allele (Fig. 2a). SprtnF/− MEFs, but not Sprtn+/+ and SprtnF/+ MEFs, showed markedly reduced proliferation and increased apoptosis after exposure to 4-OHT (Fig. 2b,c), suggesting that Sprtn is essential for cell proliferation and survival.


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)

Sprtn KO causes impaired cell proliferation and cell death.(a) PCR-based genotyping of Sprtn alleles in the indicated MEF lines after 48 h treatment with MeOH or 2 μM 4-OHT. (b) Proliferation of Sprtn-targeted MEFs. Cells treated with MeOH or 4-OHT for 48 h were seeded and cell numbers were counted at the indicated time points. Values are mean±s.d. of three independent experiments. (c) Analyses of apoptosis in Sprtn-targeted MEFs. Two days after the completion of 48 h treatment with MeOH or 4-OHT, cells were stained with Annexin V and PI and analysed by flow cytometry. Values are mean±s.d. of three independent experiments. NS, not significant; ***P<0.001 (two-tailed unpaired t-test). (d) Cell cycle profiling of Sprtn-targeted MEFs. The indicated MEF lines were treated with MeOH or 4-OHT for 48 h. Two days later, cells were stained with PI and analysed by flow cytometry. (e) Cell cycle profiling of SprtnF/F; Cre-ERT2 MEFs (K3) expressing wild-type human Spartan or the E112A mutant after 48 h treatment with MeOH or 4-OHT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269170&req=5

f2: Sprtn KO causes impaired cell proliferation and cell death.(a) PCR-based genotyping of Sprtn alleles in the indicated MEF lines after 48 h treatment with MeOH or 2 μM 4-OHT. (b) Proliferation of Sprtn-targeted MEFs. Cells treated with MeOH or 4-OHT for 48 h were seeded and cell numbers were counted at the indicated time points. Values are mean±s.d. of three independent experiments. (c) Analyses of apoptosis in Sprtn-targeted MEFs. Two days after the completion of 48 h treatment with MeOH or 4-OHT, cells were stained with Annexin V and PI and analysed by flow cytometry. Values are mean±s.d. of three independent experiments. NS, not significant; ***P<0.001 (two-tailed unpaired t-test). (d) Cell cycle profiling of Sprtn-targeted MEFs. The indicated MEF lines were treated with MeOH or 4-OHT for 48 h. Two days later, cells were stained with PI and analysed by flow cytometry. (e) Cell cycle profiling of SprtnF/F; Cre-ERT2 MEFs (K3) expressing wild-type human Spartan or the E112A mutant after 48 h treatment with MeOH or 4-OHT.
Mentions: To further investigate the role of Sprtn at the cellular level, we generated a conditional Sprtn KO system in mouse embryonic fibroblasts (MEFs). Sprtn+/+, SprtnF/+ and SprtnF/− MEFs were immortalized and transduced with retroviruses expressing Cre-ERT2. Treatment with 4-hydroxytamoxifen (4-OHT) resulted in excision of exon 2, efficiently converting the floxed allele to a KO allele (Fig. 2a). SprtnF/− MEFs, but not Sprtn+/+ and SprtnF/+ MEFs, showed markedly reduced proliferation and increased apoptosis after exposure to 4-OHT (Fig. 2b,c), suggesting that Sprtn is essential for cell proliferation and survival.

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