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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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Methylation-deficient ARTD1 is less active and less efficiently recruited to sites of local DNA damage induced by femtosecond laser irradiation. (a) ARTD1 knockout MLFs were stably complemented with WT ARTD1 or two methylation-deficient mutants. Cells were then fractionated and CEs and NEs were analysed by western blot. (b) ARTD1 activity in NE from (a) was analysed by radioactive PAR assays in the absence or presence of 5 pmol-activating DNA. Experiments in A and B were repeated twice with a similar outcome and one representative blot is shown. Quantifications are shown in the electronic supplementary material, figure S5a,b. (c) Recruitment of WT and K508R ARTD1 to sites of local DNA damage induced by femtosecond laser irradiation at λ = 1050 nm. (d) Recruitment of macroH2A1.1-EGFP to sites of local DNA damage induced by femtosecond laser irradiation at λ = 1050 nm. (e) Recruitment of WT and K508R ARTD1 to sites of DNA damage by femtosecond laser irradiation at λ = 775 nm.
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RSOB120173F4: Methylation-deficient ARTD1 is less active and less efficiently recruited to sites of local DNA damage induced by femtosecond laser irradiation. (a) ARTD1 knockout MLFs were stably complemented with WT ARTD1 or two methylation-deficient mutants. Cells were then fractionated and CEs and NEs were analysed by western blot. (b) ARTD1 activity in NE from (a) was analysed by radioactive PAR assays in the absence or presence of 5 pmol-activating DNA. Experiments in A and B were repeated twice with a similar outcome and one representative blot is shown. Quantifications are shown in the electronic supplementary material, figure S5a,b. (c) Recruitment of WT and K508R ARTD1 to sites of local DNA damage induced by femtosecond laser irradiation at λ = 1050 nm. (d) Recruitment of macroH2A1.1-EGFP to sites of local DNA damage induced by femtosecond laser irradiation at λ = 1050 nm. (e) Recruitment of WT and K508R ARTD1 to sites of DNA damage by femtosecond laser irradiation at λ = 775 nm.

Mentions: To elucidate whether SET7/9-dependent methylation of ARTD1 at K508 is directly responsible for the observed influence of SET7/9 on ARTD1-dependent PAR formation in vivo, ARTD1−/− MLFs were stably genetically complemented with WT ARTD1 or with two methylation-deficient mutants (K508A and K508R). The WT and the mutant proteins were comparably expressed in the NEs, but not detectable in the cytoplasmic extracts (CEs) or the vector control (pRRL) (figure 4a). NEs containing WT or mutant ARTD1 were incubated with radioactively labelled NAD+, but without exogenous DNA, and ARTD1 auto-ADP-ribosylation was assessed. The methylation-deficient ARTD1 mutants K508A and K508R exhibited markedly reduced activity in comparison with the WT control (see figure 4b and electronic supplementary material, figure S5a). Upon addition of excess DNA, the methylation-deficient ARTD1 proteins K508A and K508R still exhibited reduced enzymatic activity, but the effect was less pronounced as compared with conditions without exogenous DNA (see figure 4b and electronic supplementary material, figure S5b), again pointing at a SET7/9 methylation effect on ARTD1 activation.Figure 4.


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)

Methylation-deficient ARTD1 is less active and less efficiently recruited to sites of local DNA damage induced by femtosecond laser irradiation. (a) ARTD1 knockout MLFs were stably complemented with WT ARTD1 or two methylation-deficient mutants. Cells were then fractionated and CEs and NEs were analysed by western blot. (b) ARTD1 activity in NE from (a) was analysed by radioactive PAR assays in the absence or presence of 5 pmol-activating DNA. Experiments in A and B were repeated twice with a similar outcome and one representative blot is shown. Quantifications are shown in the electronic supplementary material, figure S5a,b. (c) Recruitment of WT and K508R ARTD1 to sites of local DNA damage induced by femtosecond laser irradiation at λ = 1050 nm. (d) Recruitment of macroH2A1.1-EGFP to sites of local DNA damage induced by femtosecond laser irradiation at λ = 1050 nm. (e) Recruitment of WT and K508R ARTD1 to sites of DNA damage by femtosecond laser irradiation at λ = 775 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120173F4: Methylation-deficient ARTD1 is less active and less efficiently recruited to sites of local DNA damage induced by femtosecond laser irradiation. (a) ARTD1 knockout MLFs were stably complemented with WT ARTD1 or two methylation-deficient mutants. Cells were then fractionated and CEs and NEs were analysed by western blot. (b) ARTD1 activity in NE from (a) was analysed by radioactive PAR assays in the absence or presence of 5 pmol-activating DNA. Experiments in A and B were repeated twice with a similar outcome and one representative blot is shown. Quantifications are shown in the electronic supplementary material, figure S5a,b. (c) Recruitment of WT and K508R ARTD1 to sites of local DNA damage induced by femtosecond laser irradiation at λ = 1050 nm. (d) Recruitment of macroH2A1.1-EGFP to sites of local DNA damage induced by femtosecond laser irradiation at λ = 1050 nm. (e) Recruitment of WT and K508R ARTD1 to sites of DNA damage by femtosecond laser irradiation at λ = 775 nm.
Mentions: To elucidate whether SET7/9-dependent methylation of ARTD1 at K508 is directly responsible for the observed influence of SET7/9 on ARTD1-dependent PAR formation in vivo, ARTD1−/− MLFs were stably genetically complemented with WT ARTD1 or with two methylation-deficient mutants (K508A and K508R). The WT and the mutant proteins were comparably expressed in the NEs, but not detectable in the cytoplasmic extracts (CEs) or the vector control (pRRL) (figure 4a). NEs containing WT or mutant ARTD1 were incubated with radioactively labelled NAD+, but without exogenous DNA, and ARTD1 auto-ADP-ribosylation was assessed. The methylation-deficient ARTD1 mutants K508A and K508R exhibited markedly reduced activity in comparison with the WT control (see figure 4b and electronic supplementary material, figure S5a). Upon addition of excess DNA, the methylation-deficient ARTD1 proteins K508A and K508R still exhibited reduced enzymatic activity, but the effect was less pronounced as compared with conditions without exogenous DNA (see figure 4b and electronic supplementary material, figure S5b), again pointing at a SET7/9 methylation effect on ARTD1 activation.Figure 4.

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