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SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response.

Mund A, Schubert T, Staege H, Kinkley S, Reumann K, Kriegs M, Fritsch L, Battisti V, Ait-Si-Ali S, Hoffbeck AS, Soutoglou E, Will H - Nucleic Acids Res. (2012)

Bottom Line: Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner.SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1.In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation.

View Article: PubMed Central - PubMed

Affiliation: Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Department of General Virology, Martinistrasse 52, 20251 Hamburg, Germany.

ABSTRACT
Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.

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SPOC1-expression levels modulate NHEJ activity. (A) siRNA-mediated SPOC1 depletion increases NHEJ activity in H1299.EJ reporter cells compared to control siRNA-transfected cells as quantified by flow cytometry detecting EGFP expression. NHEJ activity is only evident after I-Scel-mediated cleavage in cells transfected with a plasmid expressing cytoplasmic SceI fused to glucocorticoid receptor ligand-binding domain (SceI-GR), plus TA to translocate the fusion protein into the nucleus. (B) Enhanced expression of SPOC1wt and a SPOC1 N-terminal domain deletion (deltaN), but not a PHD-deficient derivative (PHDmt) strongly reduced NHEJ activity when analyzed and quantified as in (A). (C) DNA repair activity by HR in H1299.GC reporter cells after SPOC1 siRNA or control siRNA transfection, quantified as in (A), were similar. (D) HR-mediated DNA repair activity in U2OS-DR-GFP reporter cells was reduced by >60% after siRNA-induced SPOC1 knockdown compared to control siRNA treated cells. Knockdown of CtIP, a protein critical for the initial steps of HR, reduced HR activity by ∼90% compared to the control siRNA transfected cells. All data are from triplicates derived from three independent experiments (±SEM) ****P ≤ 0.0001. (A–D) Successful SPOC1 and CtIP knockdown as well as equivalent expression levels of SPOC1 wt and mutant proteins were confirmed by immunoblotting of cell extracts using anti-SPOC1, anti-CtIP and anti-GAPDH antibodies.
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gks868-F4: SPOC1-expression levels modulate NHEJ activity. (A) siRNA-mediated SPOC1 depletion increases NHEJ activity in H1299.EJ reporter cells compared to control siRNA-transfected cells as quantified by flow cytometry detecting EGFP expression. NHEJ activity is only evident after I-Scel-mediated cleavage in cells transfected with a plasmid expressing cytoplasmic SceI fused to glucocorticoid receptor ligand-binding domain (SceI-GR), plus TA to translocate the fusion protein into the nucleus. (B) Enhanced expression of SPOC1wt and a SPOC1 N-terminal domain deletion (deltaN), but not a PHD-deficient derivative (PHDmt) strongly reduced NHEJ activity when analyzed and quantified as in (A). (C) DNA repair activity by HR in H1299.GC reporter cells after SPOC1 siRNA or control siRNA transfection, quantified as in (A), were similar. (D) HR-mediated DNA repair activity in U2OS-DR-GFP reporter cells was reduced by >60% after siRNA-induced SPOC1 knockdown compared to control siRNA treated cells. Knockdown of CtIP, a protein critical for the initial steps of HR, reduced HR activity by ∼90% compared to the control siRNA transfected cells. All data are from triplicates derived from three independent experiments (±SEM) ****P ≤ 0.0001. (A–D) Successful SPOC1 and CtIP knockdown as well as equivalent expression levels of SPOC1 wt and mutant proteins were confirmed by immunoblotting of cell extracts using anti-SPOC1, anti-CtIP and anti-GAPDH antibodies.

Mentions: To evaluate which DNA DSB repair pathways SPOC1 influences we used the well-established H1299.EJ (NHEJ) and H1299.GC (HR) reporter cell lines (39,41). Both cell lines contain chromosomally integrated EGFP that is only expressed after I-Scel-mediated cleavage and repair (Supplementary Figure S2). By quantifying in vivo EGFP expression, we observed ∼20% more NHEJ activity after SPOC1 knockdown, but no change in HR activity (Figure 4A and C). In contrast, SPOC1 overexpression in H1299.EJ cells decreased NHEJ activity by 40% compared to control cells, as did a SPOC1 N-terminal domain deletion (deltaN) (Figure 4B). Interestingly, this inhibitory effect was not observed with SPOC1 containing two critical amino acid mutations in the H3K4me3-binding pocket (PHDmt). The effect of SPOC1 on NHEJ is specific since EGFP-reporter expression was not significantly changed by transfection of H1299.EJ cells with SPOC1-expression vectors or siRNA (Supplementary Figure S3). These data indicate that NHEJ DNA repair activity is modulated by SPOC1 in a dose- and PHD-dependent mannerFigure 4.


SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response.

Mund A, Schubert T, Staege H, Kinkley S, Reumann K, Kriegs M, Fritsch L, Battisti V, Ait-Si-Ali S, Hoffbeck AS, Soutoglou E, Will H - Nucleic Acids Res. (2012)

SPOC1-expression levels modulate NHEJ activity. (A) siRNA-mediated SPOC1 depletion increases NHEJ activity in H1299.EJ reporter cells compared to control siRNA-transfected cells as quantified by flow cytometry detecting EGFP expression. NHEJ activity is only evident after I-Scel-mediated cleavage in cells transfected with a plasmid expressing cytoplasmic SceI fused to glucocorticoid receptor ligand-binding domain (SceI-GR), plus TA to translocate the fusion protein into the nucleus. (B) Enhanced expression of SPOC1wt and a SPOC1 N-terminal domain deletion (deltaN), but not a PHD-deficient derivative (PHDmt) strongly reduced NHEJ activity when analyzed and quantified as in (A). (C) DNA repair activity by HR in H1299.GC reporter cells after SPOC1 siRNA or control siRNA transfection, quantified as in (A), were similar. (D) HR-mediated DNA repair activity in U2OS-DR-GFP reporter cells was reduced by >60% after siRNA-induced SPOC1 knockdown compared to control siRNA treated cells. Knockdown of CtIP, a protein critical for the initial steps of HR, reduced HR activity by ∼90% compared to the control siRNA transfected cells. All data are from triplicates derived from three independent experiments (±SEM) ****P ≤ 0.0001. (A–D) Successful SPOC1 and CtIP knockdown as well as equivalent expression levels of SPOC1 wt and mutant proteins were confirmed by immunoblotting of cell extracts using anti-SPOC1, anti-CtIP and anti-GAPDH antibodies.
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Related In: Results  -  Collection

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gks868-F4: SPOC1-expression levels modulate NHEJ activity. (A) siRNA-mediated SPOC1 depletion increases NHEJ activity in H1299.EJ reporter cells compared to control siRNA-transfected cells as quantified by flow cytometry detecting EGFP expression. NHEJ activity is only evident after I-Scel-mediated cleavage in cells transfected with a plasmid expressing cytoplasmic SceI fused to glucocorticoid receptor ligand-binding domain (SceI-GR), plus TA to translocate the fusion protein into the nucleus. (B) Enhanced expression of SPOC1wt and a SPOC1 N-terminal domain deletion (deltaN), but not a PHD-deficient derivative (PHDmt) strongly reduced NHEJ activity when analyzed and quantified as in (A). (C) DNA repair activity by HR in H1299.GC reporter cells after SPOC1 siRNA or control siRNA transfection, quantified as in (A), were similar. (D) HR-mediated DNA repair activity in U2OS-DR-GFP reporter cells was reduced by >60% after siRNA-induced SPOC1 knockdown compared to control siRNA treated cells. Knockdown of CtIP, a protein critical for the initial steps of HR, reduced HR activity by ∼90% compared to the control siRNA transfected cells. All data are from triplicates derived from three independent experiments (±SEM) ****P ≤ 0.0001. (A–D) Successful SPOC1 and CtIP knockdown as well as equivalent expression levels of SPOC1 wt and mutant proteins were confirmed by immunoblotting of cell extracts using anti-SPOC1, anti-CtIP and anti-GAPDH antibodies.
Mentions: To evaluate which DNA DSB repair pathways SPOC1 influences we used the well-established H1299.EJ (NHEJ) and H1299.GC (HR) reporter cell lines (39,41). Both cell lines contain chromosomally integrated EGFP that is only expressed after I-Scel-mediated cleavage and repair (Supplementary Figure S2). By quantifying in vivo EGFP expression, we observed ∼20% more NHEJ activity after SPOC1 knockdown, but no change in HR activity (Figure 4A and C). In contrast, SPOC1 overexpression in H1299.EJ cells decreased NHEJ activity by 40% compared to control cells, as did a SPOC1 N-terminal domain deletion (deltaN) (Figure 4B). Interestingly, this inhibitory effect was not observed with SPOC1 containing two critical amino acid mutations in the H3K4me3-binding pocket (PHDmt). The effect of SPOC1 on NHEJ is specific since EGFP-reporter expression was not significantly changed by transfection of H1299.EJ cells with SPOC1-expression vectors or siRNA (Supplementary Figure S3). These data indicate that NHEJ DNA repair activity is modulated by SPOC1 in a dose- and PHD-dependent mannerFigure 4.

Bottom Line: Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner.SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1.In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation.

View Article: PubMed Central - PubMed

Affiliation: Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Department of General Virology, Martinistrasse 52, 20251 Hamburg, Germany.

ABSTRACT
Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.

Show MeSH
Related in: MedlinePlus