Limits...
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.

Show MeSH

Related in: MedlinePlus

Sprtn KO causes DNA damage and checkpoint activation.(a) γH2AX focus formation. The indicated MEFs treated with MeOH or 4-OHT for 48 h were stained with anti-γH2AX. At least 300 cells were scored for γH2AX foci and percentages of cells with 5 or more foci are shown. Values are mean±s.d. of three independent experiments. NS, not significant; *P<0.05; ****P<0.0001 (two-tailed unpaired t-test). (b) Rad51 focus formation. The indicated MEFs were stained with anti-Rad51 after 48 h treatment with MeOH or 4-OHT. At least 300 cells were scored for Rad51 foci. Experiments were performed in triplicate and mean±s.d. is shown. ****P<0.0001 (two-tailed unpaired t-test). (c) Western blot analyses of phospho-Chk1 and Chk2. The indicated MEFs were treated with 4-OHT and harvested at various time points. SprtnF/− (H7) cells treated with ultraviolet (40 J m−2) or ionizing radiation (10 Gy) are shown as positive controls for checkpoint kinases activation. Chk1 is used as a loading control. P-Chk1, phospho-Chk1 (Ser345); P-Chk2, phospho-Chk2. Uncropped blots are shown in Supplementary Fig. 7.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4269170&req=5

f3: Sprtn KO causes DNA damage and checkpoint activation.(a) γH2AX focus formation. The indicated MEFs treated with MeOH or 4-OHT for 48 h were stained with anti-γH2AX. At least 300 cells were scored for γH2AX foci and percentages of cells with 5 or more foci are shown. Values are mean±s.d. of three independent experiments. NS, not significant; *P<0.05; ****P<0.0001 (two-tailed unpaired t-test). (b) Rad51 focus formation. The indicated MEFs were stained with anti-Rad51 after 48 h treatment with MeOH or 4-OHT. At least 300 cells were scored for Rad51 foci. Experiments were performed in triplicate and mean±s.d. is shown. ****P<0.0001 (two-tailed unpaired t-test). (c) Western blot analyses of phospho-Chk1 and Chk2. The indicated MEFs were treated with 4-OHT and harvested at various time points. SprtnF/− (H7) cells treated with ultraviolet (40 J m−2) or ionizing radiation (10 Gy) are shown as positive controls for checkpoint kinases activation. Chk1 is used as a loading control. P-Chk1, phospho-Chk1 (Ser345); P-Chk2, phospho-Chk2. Uncropped blots are shown in Supplementary Fig. 7.

Mentions: To determine the cause of the cell cycle defect in Sprtn−/− MEFs, we asked whether Spartan deficiency engages the DNA damage response pathway. γH2AX foci, a marker of DNA damage18, markedly increased in SprtnF/− MEFs after 4-OHT treatments, but not in Sprtn+/+ and SprtnF/+ MEFs (Fig. 3a). Consistent with increased DNA damage, Sprtn−/− MEFs showed high rates of double-strand DNA breaks as measured by immunostaining of the DNA recombination protein Rad51 (Fig. 3b) and profound activation of the checkpoint kinase Chk2 (Fig. 3c). Thus, loss of Sprtn in MEFs seemingly induces DNA damage, most likely DNA breaks, leading to activation of the DNA damage checkpoint.


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 DNA damage and checkpoint activation.(a) γH2AX focus formation. The indicated MEFs treated with MeOH or 4-OHT for 48 h were stained with anti-γH2AX. At least 300 cells were scored for γH2AX foci and percentages of cells with 5 or more foci are shown. Values are mean±s.d. of three independent experiments. NS, not significant; *P<0.05; ****P<0.0001 (two-tailed unpaired t-test). (b) Rad51 focus formation. The indicated MEFs were stained with anti-Rad51 after 48 h treatment with MeOH or 4-OHT. At least 300 cells were scored for Rad51 foci. Experiments were performed in triplicate and mean±s.d. is shown. ****P<0.0001 (two-tailed unpaired t-test). (c) Western blot analyses of phospho-Chk1 and Chk2. The indicated MEFs were treated with 4-OHT and harvested at various time points. SprtnF/− (H7) cells treated with ultraviolet (40 J m−2) or ionizing radiation (10 Gy) are shown as positive controls for checkpoint kinases activation. Chk1 is used as a loading control. P-Chk1, phospho-Chk1 (Ser345); P-Chk2, phospho-Chk2. Uncropped blots are shown in Supplementary Fig. 7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Sprtn KO causes DNA damage and checkpoint activation.(a) γH2AX focus formation. The indicated MEFs treated with MeOH or 4-OHT for 48 h were stained with anti-γH2AX. At least 300 cells were scored for γH2AX foci and percentages of cells with 5 or more foci are shown. Values are mean±s.d. of three independent experiments. NS, not significant; *P<0.05; ****P<0.0001 (two-tailed unpaired t-test). (b) Rad51 focus formation. The indicated MEFs were stained with anti-Rad51 after 48 h treatment with MeOH or 4-OHT. At least 300 cells were scored for Rad51 foci. Experiments were performed in triplicate and mean±s.d. is shown. ****P<0.0001 (two-tailed unpaired t-test). (c) Western blot analyses of phospho-Chk1 and Chk2. The indicated MEFs were treated with 4-OHT and harvested at various time points. SprtnF/− (H7) cells treated with ultraviolet (40 J m−2) or ionizing radiation (10 Gy) are shown as positive controls for checkpoint kinases activation. Chk1 is used as a loading control. P-Chk1, phospho-Chk1 (Ser345); P-Chk2, phospho-Chk2. Uncropped blots are shown in Supplementary Fig. 7.
Mentions: To determine the cause of the cell cycle defect in Sprtn−/− MEFs, we asked whether Spartan deficiency engages the DNA damage response pathway. γH2AX foci, a marker of DNA damage18, markedly increased in SprtnF/− MEFs after 4-OHT treatments, but not in Sprtn+/+ and SprtnF/+ MEFs (Fig. 3a). Consistent with increased DNA damage, Sprtn−/− MEFs showed high rates of double-strand DNA breaks as measured by immunostaining of the DNA recombination protein Rad51 (Fig. 3b) and profound activation of the checkpoint kinase Chk2 (Fig. 3c). Thus, loss of Sprtn in MEFs seemingly induces DNA damage, most likely DNA breaks, leading to activation of the DNA damage checkpoint.

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