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Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.

Li S, Li Z, Shu FJ, Xiong H, Phillips AC, Dynan WS - Nucleic Acids Res. (2014)

Bottom Line: We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes.Of 12 genes tested, none were downregulated, and several were upregulated.Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Emory University, Atlanta, GA 30322, USA Department of Biochemistry, Emory University, Atlanta, GA 30322, USA Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, GA 30912, USA.

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Delayed resolution of repair foci. Vectors encoding control or PSPC1 miRNA were introduced by electroporation into wild-type (+/0) or NONO-deficient (gt/0) MEFs. Cells were mock irradiated or exposed to 1 Gy of 137Cs γ-rays. At indicated times following irradiation, cells were fixed and analyzed by indirect immunofluorescence using anti-γ-H2AX primary and red secondary antibody and DAPI counterstain. (A, C, E) Representative fields showing γ-H2AX/DAPI channels (top row) or γ-H2AX/EmGFP/DAPI-merged images in NONO-deficient (gt/0) MEFs. Images were collected for non-irradiated control cells or for cells that were irradiated and allowed to recover for indicated times. Cell morphology and foci appearance were indistinguishable in wild-type (+/0) and NONO-deficient (gt/0) MEFs; only the latter are shown in the figure. Scale bar denotes 10 μm. (B, D, F) Foci per cell in populations corresponding to panels (A, C, E). A total of 30 nuclei were scored per experimental group. Blue symbols depict score for individual cells; red bar depicts mean. Treatment of NONO-deficient cells with PSPC1 RNA resulted in a significant increase in residual foci at 4 h post-irradiation (P < 0.01).
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Figure 5: Delayed resolution of repair foci. Vectors encoding control or PSPC1 miRNA were introduced by electroporation into wild-type (+/0) or NONO-deficient (gt/0) MEFs. Cells were mock irradiated or exposed to 1 Gy of 137Cs γ-rays. At indicated times following irradiation, cells were fixed and analyzed by indirect immunofluorescence using anti-γ-H2AX primary and red secondary antibody and DAPI counterstain. (A, C, E) Representative fields showing γ-H2AX/DAPI channels (top row) or γ-H2AX/EmGFP/DAPI-merged images in NONO-deficient (gt/0) MEFs. Images were collected for non-irradiated control cells or for cells that were irradiated and allowed to recover for indicated times. Cell morphology and foci appearance were indistinguishable in wild-type (+/0) and NONO-deficient (gt/0) MEFs; only the latter are shown in the figure. Scale bar denotes 10 μm. (B, D, F) Foci per cell in populations corresponding to panels (A, C, E). A total of 30 nuclei were scored per experimental group. Blue symbols depict score for individual cells; red bar depicts mean. Treatment of NONO-deficient cells with PSPC1 RNA resulted in a significant increase in residual foci at 4 h post-irradiation (P < 0.01).

Mentions: To determine whether the radiosensitivity arose from a deficit in DSB repair per se, we again used a single-cell assay. We transfected with miRNA, irradiated and scored γ-H2AX foci, a marker of unrepaired DSBs, in cells expressing the EmGFP transfection marker. We irradiated at 0 or 1 Gy, then scored cells in various treatment groups at 0.5 h post-irradiation, to examine the ability to form γ-H2AX foci, and at 4 h post-irradiation, to examine the ability to recover. There was a low background of spontaneous foci in the non-irradiated cells (Figure 5). This increased to about 30 foci/per cell at 30 min following treatment with 1 Gy of 137Cs γ-rays, consistent with the predicted number of DSBs at this dose assuming a diploid mouse genome. The increase was similar in all four experimental groups. After 4 hours, most of the foci resolved in the wild-type genetic background (with or without PSPC1 knockdown). Most of the foci also resolved in the NONO-deficient cells transfected with control miRNA. However, dual deficiency in NONO and PSPC1 resulted in near absence of recovery at the 4-h time point. Taken together, results indicate that deficiency in NONO/PSPC1 function had no effect on the formation of γ-H2AX foci, which is a very early step in the cascade of events involved in DSB repair. Deficiency did affect the resolution of γ-H2AX foci, which is a very late step.


Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.

Li S, Li Z, Shu FJ, Xiong H, Phillips AC, Dynan WS - Nucleic Acids Res. (2014)

Delayed resolution of repair foci. Vectors encoding control or PSPC1 miRNA were introduced by electroporation into wild-type (+/0) or NONO-deficient (gt/0) MEFs. Cells were mock irradiated or exposed to 1 Gy of 137Cs γ-rays. At indicated times following irradiation, cells were fixed and analyzed by indirect immunofluorescence using anti-γ-H2AX primary and red secondary antibody and DAPI counterstain. (A, C, E) Representative fields showing γ-H2AX/DAPI channels (top row) or γ-H2AX/EmGFP/DAPI-merged images in NONO-deficient (gt/0) MEFs. Images were collected for non-irradiated control cells or for cells that were irradiated and allowed to recover for indicated times. Cell morphology and foci appearance were indistinguishable in wild-type (+/0) and NONO-deficient (gt/0) MEFs; only the latter are shown in the figure. Scale bar denotes 10 μm. (B, D, F) Foci per cell in populations corresponding to panels (A, C, E). A total of 30 nuclei were scored per experimental group. Blue symbols depict score for individual cells; red bar depicts mean. Treatment of NONO-deficient cells with PSPC1 RNA resulted in a significant increase in residual foci at 4 h post-irradiation (P < 0.01).
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Related In: Results  -  Collection

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Figure 5: Delayed resolution of repair foci. Vectors encoding control or PSPC1 miRNA were introduced by electroporation into wild-type (+/0) or NONO-deficient (gt/0) MEFs. Cells were mock irradiated or exposed to 1 Gy of 137Cs γ-rays. At indicated times following irradiation, cells were fixed and analyzed by indirect immunofluorescence using anti-γ-H2AX primary and red secondary antibody and DAPI counterstain. (A, C, E) Representative fields showing γ-H2AX/DAPI channels (top row) or γ-H2AX/EmGFP/DAPI-merged images in NONO-deficient (gt/0) MEFs. Images were collected for non-irradiated control cells or for cells that were irradiated and allowed to recover for indicated times. Cell morphology and foci appearance were indistinguishable in wild-type (+/0) and NONO-deficient (gt/0) MEFs; only the latter are shown in the figure. Scale bar denotes 10 μm. (B, D, F) Foci per cell in populations corresponding to panels (A, C, E). A total of 30 nuclei were scored per experimental group. Blue symbols depict score for individual cells; red bar depicts mean. Treatment of NONO-deficient cells with PSPC1 RNA resulted in a significant increase in residual foci at 4 h post-irradiation (P < 0.01).
Mentions: To determine whether the radiosensitivity arose from a deficit in DSB repair per se, we again used a single-cell assay. We transfected with miRNA, irradiated and scored γ-H2AX foci, a marker of unrepaired DSBs, in cells expressing the EmGFP transfection marker. We irradiated at 0 or 1 Gy, then scored cells in various treatment groups at 0.5 h post-irradiation, to examine the ability to form γ-H2AX foci, and at 4 h post-irradiation, to examine the ability to recover. There was a low background of spontaneous foci in the non-irradiated cells (Figure 5). This increased to about 30 foci/per cell at 30 min following treatment with 1 Gy of 137Cs γ-rays, consistent with the predicted number of DSBs at this dose assuming a diploid mouse genome. The increase was similar in all four experimental groups. After 4 hours, most of the foci resolved in the wild-type genetic background (with or without PSPC1 knockdown). Most of the foci also resolved in the NONO-deficient cells transfected with control miRNA. However, dual deficiency in NONO and PSPC1 resulted in near absence of recovery at the 4-h time point. Taken together, results indicate that deficiency in NONO/PSPC1 function had no effect on the formation of γ-H2AX foci, which is a very early step in the cascade of events involved in DSB repair. Deficiency did affect the resolution of γ-H2AX foci, which is a very late step.

Bottom Line: We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes.Of 12 genes tested, none were downregulated, and several were upregulated.Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Emory University, Atlanta, GA 30322, USA Department of Biochemistry, Emory University, Atlanta, GA 30322, USA Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, GA 30912, USA.

Show MeSH
Related in: MedlinePlus