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SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress.

Kassner I, Andersson A, Fey M, Tomas M, Ferrando-May E, Hottiger MO - Open Biol (2013)

Bottom Line: Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo.Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner.Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

ABSTRACT
ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein-protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo. ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo. Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

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SET7/9-dependent methylation increases ARTD1 activity in cells. (a) After overexpression of Flag-SET7/9 WT or H297A, U2OS cells were treated with or without 1 mM H2O2 for 5 min and PAR formation was analysed by western blot. (b) H2O2-induced PAR formation was analysed by immunofluorescence in SET7/9 KO and WT MEFs. The intensity of the anti-PAR-stained cells was quantified. (c) H2O2-induced PAR formation in SET7/9 KO and WT MEFs was analysed as in (a). (d) U2OS cells were transfected with scrambled siRNA (scr) or siRNA targeting SET7/9. Three days after knockdown, H2O2-induced PAR formation was analysed as in (a). Short and long exposures (se and le, respectively) of the anti-PAR blot are shown. (e) ARTD1 activity was analysed in NEs from U2OS cells after knockdown of SET7/9 and ARTD1 for 3 days by radioactive ADP-ribosylation assays. All experiments were repeated at least twice, gave a similar result, and one representative blot is shown. Quantifications are shown in the electronic supplementary material, figures S3b and S4c,d.
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RSOB120173F3: SET7/9-dependent methylation increases ARTD1 activity in cells. (a) After overexpression of Flag-SET7/9 WT or H297A, U2OS cells were treated with or without 1 mM H2O2 for 5 min and PAR formation was analysed by western blot. (b) H2O2-induced PAR formation was analysed by immunofluorescence in SET7/9 KO and WT MEFs. The intensity of the anti-PAR-stained cells was quantified. (c) H2O2-induced PAR formation in SET7/9 KO and WT MEFs was analysed as in (a). (d) U2OS cells were transfected with scrambled siRNA (scr) or siRNA targeting SET7/9. Three days after knockdown, H2O2-induced PAR formation was analysed as in (a). Short and long exposures (se and le, respectively) of the anti-PAR blot are shown. (e) ARTD1 activity was analysed in NEs from U2OS cells after knockdown of SET7/9 and ARTD1 for 3 days by radioactive ADP-ribosylation assays. All experiments were repeated at least twice, gave a similar result, and one representative blot is shown. Quantifications are shown in the electronic supplementary material, figures S3b and S4c,d.

Mentions: In order to test the hypothesis that SET7/9 regulates the enzymatic activity of ARTD1 in vivo, we first confirmed that both enzymes are localized in the nucleus of U2OS cells. While ARTD1 was only present in the nucleus and enriched in the nucleoli, SET7/9 was localized throughout the cell except in the nucleoli (see electronic supplementary material, figure S3a). Next, Flag-tagged wild-type (WT) SET7/9 was overexpressed and PAR formation following oxidative stress by H2O2 was determined (see figure 3a and electronic supplementary material, figure S3b). PAR formation was indeed increased upon overexpression of WT SET7/9 (lanes 2 and 5), even in unstimulated cells (lane 2), while the enzymatically inactive SET7/9 mutant H297A did not cause this effect (lanes 3 and 6). To prove that SET7/9 stimulated PAR formation, we analysed mouse fibroblasts lacking SET7/9. Upon H2O2 stimulation, SET7/9-knockout MEFs showed significantly reduced PAR staining and lower PAR-synthesizing activity as compared with the WT control cells (see figure 3b,c and electronic supplementary material, figure S3c), suggesting that SET7/9 regulates PAR formation in vivo. This was also confirmed by SET7/9 knockdown in U2OS cells (see electronic supplementary material, figure S4a,b). Following oxidative stress by H2O2, siSET7/9-treated cells formed less PAR than cells transfected with a control siRNA (see figure 3d and electronic supplementary material, figure S4c). To further analyse the influence of SET7/9 on ARTD1 enzymatic activity in cells, nuclear extracts (NEs) from siRNA-treated U2OS cells (control siRNA or siRNA directed against SET7/9 or ARTD1) were prepared and auto-ADP-ribosylation of ARTD1 was tested in vitro in the presence or absence of exogenous DNA. Downregulation of SET7/9 reduced the basal ARTD1 activity to levels only slightly above those in siARTD1 cells (in the absence of exogenous DNA, figure 3e; electronic supplementary material, figure S4d). This effect was also seen, but to a lesser extent, when ARTD1 activity was stimulated by an excess of exogenous DNA, suggesting that SET7/9 methylation regulates ARTD1, especially in the absence of a strong stimulus.FigureĀ 3.


SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress.

Kassner I, Andersson A, Fey M, Tomas M, Ferrando-May E, Hottiger MO - Open Biol (2013)

SET7/9-dependent methylation increases ARTD1 activity in cells. (a) After overexpression of Flag-SET7/9 WT or H297A, U2OS cells were treated with or without 1 mM H2O2 for 5 min and PAR formation was analysed by western blot. (b) H2O2-induced PAR formation was analysed by immunofluorescence in SET7/9 KO and WT MEFs. The intensity of the anti-PAR-stained cells was quantified. (c) H2O2-induced PAR formation in SET7/9 KO and WT MEFs was analysed as in (a). (d) U2OS cells were transfected with scrambled siRNA (scr) or siRNA targeting SET7/9. Three days after knockdown, H2O2-induced PAR formation was analysed as in (a). Short and long exposures (se and le, respectively) of the anti-PAR blot are shown. (e) ARTD1 activity was analysed in NEs from U2OS cells after knockdown of SET7/9 and ARTD1 for 3 days by radioactive ADP-ribosylation assays. All experiments were repeated at least twice, gave a similar result, and one representative blot is shown. Quantifications are shown in the electronic supplementary material, figures S3b and S4c,d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814718&req=5

RSOB120173F3: SET7/9-dependent methylation increases ARTD1 activity in cells. (a) After overexpression of Flag-SET7/9 WT or H297A, U2OS cells were treated with or without 1 mM H2O2 for 5 min and PAR formation was analysed by western blot. (b) H2O2-induced PAR formation was analysed by immunofluorescence in SET7/9 KO and WT MEFs. The intensity of the anti-PAR-stained cells was quantified. (c) H2O2-induced PAR formation in SET7/9 KO and WT MEFs was analysed as in (a). (d) U2OS cells were transfected with scrambled siRNA (scr) or siRNA targeting SET7/9. Three days after knockdown, H2O2-induced PAR formation was analysed as in (a). Short and long exposures (se and le, respectively) of the anti-PAR blot are shown. (e) ARTD1 activity was analysed in NEs from U2OS cells after knockdown of SET7/9 and ARTD1 for 3 days by radioactive ADP-ribosylation assays. All experiments were repeated at least twice, gave a similar result, and one representative blot is shown. Quantifications are shown in the electronic supplementary material, figures S3b and S4c,d.
Mentions: In order to test the hypothesis that SET7/9 regulates the enzymatic activity of ARTD1 in vivo, we first confirmed that both enzymes are localized in the nucleus of U2OS cells. While ARTD1 was only present in the nucleus and enriched in the nucleoli, SET7/9 was localized throughout the cell except in the nucleoli (see electronic supplementary material, figure S3a). Next, Flag-tagged wild-type (WT) SET7/9 was overexpressed and PAR formation following oxidative stress by H2O2 was determined (see figure 3a and electronic supplementary material, figure S3b). PAR formation was indeed increased upon overexpression of WT SET7/9 (lanes 2 and 5), even in unstimulated cells (lane 2), while the enzymatically inactive SET7/9 mutant H297A did not cause this effect (lanes 3 and 6). To prove that SET7/9 stimulated PAR formation, we analysed mouse fibroblasts lacking SET7/9. Upon H2O2 stimulation, SET7/9-knockout MEFs showed significantly reduced PAR staining and lower PAR-synthesizing activity as compared with the WT control cells (see figure 3b,c and electronic supplementary material, figure S3c), suggesting that SET7/9 regulates PAR formation in vivo. This was also confirmed by SET7/9 knockdown in U2OS cells (see electronic supplementary material, figure S4a,b). Following oxidative stress by H2O2, siSET7/9-treated cells formed less PAR than cells transfected with a control siRNA (see figure 3d and electronic supplementary material, figure S4c). To further analyse the influence of SET7/9 on ARTD1 enzymatic activity in cells, nuclear extracts (NEs) from siRNA-treated U2OS cells (control siRNA or siRNA directed against SET7/9 or ARTD1) were prepared and auto-ADP-ribosylation of ARTD1 was tested in vitro in the presence or absence of exogenous DNA. Downregulation of SET7/9 reduced the basal ARTD1 activity to levels only slightly above those in siARTD1 cells (in the absence of exogenous DNA, figure 3e; electronic supplementary material, figure S4d). This effect was also seen, but to a lesser extent, when ARTD1 activity was stimulated by an excess of exogenous DNA, suggesting that SET7/9 methylation regulates ARTD1, especially in the absence of a strong stimulus.FigureĀ 3.

Bottom Line: Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo.Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner.Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

ABSTRACT
ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein-protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo. ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo. Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

Show MeSH
Related in: MedlinePlus