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JWA regulates XRCC1 and functions as a novel base excision repair protein in oxidative-stress-induced DNA single-strand breaks.

Wang S, Gong Z, Chen R, Liu Y, Li A, Li G, Zhou J - Nucleic Acids Res. (2009)

Bottom Line: Our present studies demonstrated that a reduction in JWA protein levels in cells resulted in a decrease of SSB repair capacity and hypersensitivity to DNA-damaging agents such as methyl methanesulfonate and hydrogen peroxide.On the other hand, JWA via MAPK signaling pathway regulated nuclear factor E2F1, which further transcriptionally regulated XRCC1.In addition, JWA protected XRCC1 protein from ubiquitination and degradation by proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Toxicology, Cancer Centre, School of Public Health, Nanjing Medical University, Nanjing 210029, People's Republic of China.

ABSTRACT
JWA was recently demonstrated to be involved in cellular responses to environmental stress including oxidative stress. Although it was found that JWA protected cells from reactive oxygen species-induced DNA damage, upregulated base excision repair (BER) protein XRCC1 and downregulated PARP-1, the molecular mechanism of JWA in regulating the repair of DNA single-strand breaks (SSBs) is still unclear. Our present studies demonstrated that a reduction in JWA protein levels in cells resulted in a decrease of SSB repair capacity and hypersensitivity to DNA-damaging agents such as methyl methanesulfonate and hydrogen peroxide. JWA functioned as a repair protein by multi-interaction with XRCC1. On the one hand, JWA was translocated into the nucleus by the carrier protein XRCC1 and co-localized with XRCC1 foci after oxidative DNA damage. On the other hand, JWA via MAPK signaling pathway regulated nuclear factor E2F1, which further transcriptionally regulated XRCC1. In addition, JWA protected XRCC1 protein from ubiquitination and degradation by proteasome. These findings indicate that JWA may serve as a novel regulator of XRCC1 in the BER protein complex to facilitate the repair of DNA SSBs.

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JWA regulates XRCC1 transcription via the MAPK signaling pathway and E2F1. (A) JWA knockdown in NIH-3T3 cells significantly inhibits H2O2-induced transcription of XRCC1. NIH-3T3 cells were transfected with a control shRNA or a JWA shRNA plasmid, followed by treatment with or without 100 μM H2O2 for 30 min. Levels of JWA and XRCC1 transcription were detected by quantitative RT-PCR, and GAPDH was used as an endogenous control to normalize the differences in the amount of total RNA in each sample. (B) The E2F1-binding domain in the XRCC1 promoter is required for the JWA-mediated increase in XRCC1 expression after exposure to H2O2. NIH-3T3 cells were co-transfected with either control shRNA or JWA shRNA, together with the XRCC1 promoter-reporter (–881 to + 158, containing E2F1-binding domain) or an E2F1-binding site deleted XRCC1 promoter-reporter (ΔE2F1-XRCC1, –776 to + 158). After 24 h, the transfected cells were cultured with or without 100 μM H2O2 for 30 min, then the reporter activity was examined. The means ± SD of triplicate experiments are shown. *P < 0.05. (C) JWA is required for H2O2-induced E2F1 expression. NIH-3T3 cells were transfected with a control shRNA or JWA shRNA plasmid. Then 48 h after transfection, the cells were treated with or without 100 μM H2O2 for 30 min, and nuclear lysates were collected for detection of E2F1 by immunblotting. Histone H1 was used as the nuclear protein loading control. (D) JWA alters the affinity of E2F1 for the XRCC1 promoter, as detected by EMSA. The nuclear protein extracts of the NIH-3T3 cells (with or without treatment with 100 μM H2O2 for 30 min) were incubated with a biotin-labeled double-strand oligonucleotide probe of the XRCC1 promoter region, which contains an E2F1-binding domain (–826 to –797 bp). JWA shRNA transient transfection was used to knock down JWA expression in the NIH-3T3 cells. The DNA–protein complex (shift band) or DNA–protein–antibody complex (supershift band) is indicated by an arrow. Lane 1 contains no nuclear extracts. All other lanes contain 0.5-μg nuclear extracts except lanes 3 and 6 which contain 1-μg nuclear extracts. Lane 8 represents competition analysis using 100-fold unlabeled probes. The supershift band was observed when the E2F1 antibody was added (lane 9) and IgG was used as negative control for supershift (lane 10). (E) JWA regulates E2F1 expression via MAPK signaling cascades. JWA shRNA and control shRNA plasmids were transiently transfected into NIH-3T3 cells. After 46 h, the control shRNA vector transfected cells were incubated with 20 μM of PD98059 or 10 μM U0126 for another 2 h. All transfected cells were then cultured for another 30 min in the presence or absence of H2O2 (100 μM), and the whole-cell lysates were collected for western blotting.
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Figure 5: JWA regulates XRCC1 transcription via the MAPK signaling pathway and E2F1. (A) JWA knockdown in NIH-3T3 cells significantly inhibits H2O2-induced transcription of XRCC1. NIH-3T3 cells were transfected with a control shRNA or a JWA shRNA plasmid, followed by treatment with or without 100 μM H2O2 for 30 min. Levels of JWA and XRCC1 transcription were detected by quantitative RT-PCR, and GAPDH was used as an endogenous control to normalize the differences in the amount of total RNA in each sample. (B) The E2F1-binding domain in the XRCC1 promoter is required for the JWA-mediated increase in XRCC1 expression after exposure to H2O2. NIH-3T3 cells were co-transfected with either control shRNA or JWA shRNA, together with the XRCC1 promoter-reporter (–881 to + 158, containing E2F1-binding domain) or an E2F1-binding site deleted XRCC1 promoter-reporter (ΔE2F1-XRCC1, –776 to + 158). After 24 h, the transfected cells were cultured with or without 100 μM H2O2 for 30 min, then the reporter activity was examined. The means ± SD of triplicate experiments are shown. *P < 0.05. (C) JWA is required for H2O2-induced E2F1 expression. NIH-3T3 cells were transfected with a control shRNA or JWA shRNA plasmid. Then 48 h after transfection, the cells were treated with or without 100 μM H2O2 for 30 min, and nuclear lysates were collected for detection of E2F1 by immunblotting. Histone H1 was used as the nuclear protein loading control. (D) JWA alters the affinity of E2F1 for the XRCC1 promoter, as detected by EMSA. The nuclear protein extracts of the NIH-3T3 cells (with or without treatment with 100 μM H2O2 for 30 min) were incubated with a biotin-labeled double-strand oligonucleotide probe of the XRCC1 promoter region, which contains an E2F1-binding domain (–826 to –797 bp). JWA shRNA transient transfection was used to knock down JWA expression in the NIH-3T3 cells. The DNA–protein complex (shift band) or DNA–protein–antibody complex (supershift band) is indicated by an arrow. Lane 1 contains no nuclear extracts. All other lanes contain 0.5-μg nuclear extracts except lanes 3 and 6 which contain 1-μg nuclear extracts. Lane 8 represents competition analysis using 100-fold unlabeled probes. The supershift band was observed when the E2F1 antibody was added (lane 9) and IgG was used as negative control for supershift (lane 10). (E) JWA regulates E2F1 expression via MAPK signaling cascades. JWA shRNA and control shRNA plasmids were transiently transfected into NIH-3T3 cells. After 46 h, the control shRNA vector transfected cells were incubated with 20 μM of PD98059 or 10 μM U0126 for another 2 h. All transfected cells were then cultured for another 30 min in the presence or absence of H2O2 (100 μM), and the whole-cell lysates were collected for western blotting.

Mentions: To address how JWA downregulates XRCC1, the level of XRCC1 mRNA in JWA knockdown and control NIH-3T3 cells was determined. Quantitative RT-PCR results showed that the levels of JWA and XRCC1 mRNA were increased 2.3-fold and 1.5-fold in control cells in response of H2O2 treatment, respectively. In contrast, XRCC1 mRNA expression was reduced in JWA knockdown cells, and there was no significant increase in either JWA or XRCC1 mRNA expression following H2O2 treatment (Figure 5A).Figure 5.


JWA regulates XRCC1 and functions as a novel base excision repair protein in oxidative-stress-induced DNA single-strand breaks.

Wang S, Gong Z, Chen R, Liu Y, Li A, Li G, Zhou J - Nucleic Acids Res. (2009)

JWA regulates XRCC1 transcription via the MAPK signaling pathway and E2F1. (A) JWA knockdown in NIH-3T3 cells significantly inhibits H2O2-induced transcription of XRCC1. NIH-3T3 cells were transfected with a control shRNA or a JWA shRNA plasmid, followed by treatment with or without 100 μM H2O2 for 30 min. Levels of JWA and XRCC1 transcription were detected by quantitative RT-PCR, and GAPDH was used as an endogenous control to normalize the differences in the amount of total RNA in each sample. (B) The E2F1-binding domain in the XRCC1 promoter is required for the JWA-mediated increase in XRCC1 expression after exposure to H2O2. NIH-3T3 cells were co-transfected with either control shRNA or JWA shRNA, together with the XRCC1 promoter-reporter (–881 to + 158, containing E2F1-binding domain) or an E2F1-binding site deleted XRCC1 promoter-reporter (ΔE2F1-XRCC1, –776 to + 158). After 24 h, the transfected cells were cultured with or without 100 μM H2O2 for 30 min, then the reporter activity was examined. The means ± SD of triplicate experiments are shown. *P < 0.05. (C) JWA is required for H2O2-induced E2F1 expression. NIH-3T3 cells were transfected with a control shRNA or JWA shRNA plasmid. Then 48 h after transfection, the cells were treated with or without 100 μM H2O2 for 30 min, and nuclear lysates were collected for detection of E2F1 by immunblotting. Histone H1 was used as the nuclear protein loading control. (D) JWA alters the affinity of E2F1 for the XRCC1 promoter, as detected by EMSA. The nuclear protein extracts of the NIH-3T3 cells (with or without treatment with 100 μM H2O2 for 30 min) were incubated with a biotin-labeled double-strand oligonucleotide probe of the XRCC1 promoter region, which contains an E2F1-binding domain (–826 to –797 bp). JWA shRNA transient transfection was used to knock down JWA expression in the NIH-3T3 cells. The DNA–protein complex (shift band) or DNA–protein–antibody complex (supershift band) is indicated by an arrow. Lane 1 contains no nuclear extracts. All other lanes contain 0.5-μg nuclear extracts except lanes 3 and 6 which contain 1-μg nuclear extracts. Lane 8 represents competition analysis using 100-fold unlabeled probes. The supershift band was observed when the E2F1 antibody was added (lane 9) and IgG was used as negative control for supershift (lane 10). (E) JWA regulates E2F1 expression via MAPK signaling cascades. JWA shRNA and control shRNA plasmids were transiently transfected into NIH-3T3 cells. After 46 h, the control shRNA vector transfected cells were incubated with 20 μM of PD98059 or 10 μM U0126 for another 2 h. All transfected cells were then cultured for another 30 min in the presence or absence of H2O2 (100 μM), and the whole-cell lysates were collected for western blotting.
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Figure 5: JWA regulates XRCC1 transcription via the MAPK signaling pathway and E2F1. (A) JWA knockdown in NIH-3T3 cells significantly inhibits H2O2-induced transcription of XRCC1. NIH-3T3 cells were transfected with a control shRNA or a JWA shRNA plasmid, followed by treatment with or without 100 μM H2O2 for 30 min. Levels of JWA and XRCC1 transcription were detected by quantitative RT-PCR, and GAPDH was used as an endogenous control to normalize the differences in the amount of total RNA in each sample. (B) The E2F1-binding domain in the XRCC1 promoter is required for the JWA-mediated increase in XRCC1 expression after exposure to H2O2. NIH-3T3 cells were co-transfected with either control shRNA or JWA shRNA, together with the XRCC1 promoter-reporter (–881 to + 158, containing E2F1-binding domain) or an E2F1-binding site deleted XRCC1 promoter-reporter (ΔE2F1-XRCC1, –776 to + 158). After 24 h, the transfected cells were cultured with or without 100 μM H2O2 for 30 min, then the reporter activity was examined. The means ± SD of triplicate experiments are shown. *P < 0.05. (C) JWA is required for H2O2-induced E2F1 expression. NIH-3T3 cells were transfected with a control shRNA or JWA shRNA plasmid. Then 48 h after transfection, the cells were treated with or without 100 μM H2O2 for 30 min, and nuclear lysates were collected for detection of E2F1 by immunblotting. Histone H1 was used as the nuclear protein loading control. (D) JWA alters the affinity of E2F1 for the XRCC1 promoter, as detected by EMSA. The nuclear protein extracts of the NIH-3T3 cells (with or without treatment with 100 μM H2O2 for 30 min) were incubated with a biotin-labeled double-strand oligonucleotide probe of the XRCC1 promoter region, which contains an E2F1-binding domain (–826 to –797 bp). JWA shRNA transient transfection was used to knock down JWA expression in the NIH-3T3 cells. The DNA–protein complex (shift band) or DNA–protein–antibody complex (supershift band) is indicated by an arrow. Lane 1 contains no nuclear extracts. All other lanes contain 0.5-μg nuclear extracts except lanes 3 and 6 which contain 1-μg nuclear extracts. Lane 8 represents competition analysis using 100-fold unlabeled probes. The supershift band was observed when the E2F1 antibody was added (lane 9) and IgG was used as negative control for supershift (lane 10). (E) JWA regulates E2F1 expression via MAPK signaling cascades. JWA shRNA and control shRNA plasmids were transiently transfected into NIH-3T3 cells. After 46 h, the control shRNA vector transfected cells were incubated with 20 μM of PD98059 or 10 μM U0126 for another 2 h. All transfected cells were then cultured for another 30 min in the presence or absence of H2O2 (100 μM), and the whole-cell lysates were collected for western blotting.
Mentions: To address how JWA downregulates XRCC1, the level of XRCC1 mRNA in JWA knockdown and control NIH-3T3 cells was determined. Quantitative RT-PCR results showed that the levels of JWA and XRCC1 mRNA were increased 2.3-fold and 1.5-fold in control cells in response of H2O2 treatment, respectively. In contrast, XRCC1 mRNA expression was reduced in JWA knockdown cells, and there was no significant increase in either JWA or XRCC1 mRNA expression following H2O2 treatment (Figure 5A).Figure 5.

Bottom Line: Our present studies demonstrated that a reduction in JWA protein levels in cells resulted in a decrease of SSB repair capacity and hypersensitivity to DNA-damaging agents such as methyl methanesulfonate and hydrogen peroxide.On the other hand, JWA via MAPK signaling pathway regulated nuclear factor E2F1, which further transcriptionally regulated XRCC1.In addition, JWA protected XRCC1 protein from ubiquitination and degradation by proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Toxicology, Cancer Centre, School of Public Health, Nanjing Medical University, Nanjing 210029, People's Republic of China.

ABSTRACT
JWA was recently demonstrated to be involved in cellular responses to environmental stress including oxidative stress. Although it was found that JWA protected cells from reactive oxygen species-induced DNA damage, upregulated base excision repair (BER) protein XRCC1 and downregulated PARP-1, the molecular mechanism of JWA in regulating the repair of DNA single-strand breaks (SSBs) is still unclear. Our present studies demonstrated that a reduction in JWA protein levels in cells resulted in a decrease of SSB repair capacity and hypersensitivity to DNA-damaging agents such as methyl methanesulfonate and hydrogen peroxide. JWA functioned as a repair protein by multi-interaction with XRCC1. On the one hand, JWA was translocated into the nucleus by the carrier protein XRCC1 and co-localized with XRCC1 foci after oxidative DNA damage. On the other hand, JWA via MAPK signaling pathway regulated nuclear factor E2F1, which further transcriptionally regulated XRCC1. In addition, JWA protected XRCC1 protein from ubiquitination and degradation by proteasome. These findings indicate that JWA may serve as a novel regulator of XRCC1 in the BER protein complex to facilitate the repair of DNA SSBs.

Show MeSH
Related in: MedlinePlus