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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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ARTD1 is methylated at K508 by SET7/9 in vitro and in vivo. (a) GST, ARTD1, ARTD2 and H3 were incubated with SET7/9 and 14C-labelled SAM in an in vitro methylation assay, separated by SDS-PAGE and analysed by autoradiography (14C). Coomassie blue (CB) stained gels are shown below. (b) Full-length ARTD1 and fragments covering the whole protein were incubated in an in vitro methylation assay and analysed by autoradiography. (c) Decreasing amounts of WT ARTD1 and K508R ARTD1 were methylated by SET7/9 and analysed by autoradiography. (d) An antibody directed against a peptide carrying the methylated lysine residue of ARTD1 was generated and tested in a western blot with in vitro methylated ARTD1 WT and K508R. (e) U2OS cells were transfected with scrambled siRNA (scr) or siRNA directed against ARTD1. One day later, cells were transfected with an empty vector or with a plasmid containing WT SET7/9. Whole cell extracts were analysed by western blot on day 3 after knockdown using the same antibody as in (d). (f) U2OS cells were co-transfected with HA-ARTD1 (WT or K508R) and EGFP or Flag-HA-SET7/9 (WT or H297A). After immunoprecipitation with an anti-HA antibody, whole cell extracts and IP samples were analysed by western blotting with the indicated antibodies. All experiments were repeated at least twice, gave a similar result, and one representative blot is shown.
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RSOB120173F1: ARTD1 is methylated at K508 by SET7/9 in vitro and in vivo. (a) GST, ARTD1, ARTD2 and H3 were incubated with SET7/9 and 14C-labelled SAM in an in vitro methylation assay, separated by SDS-PAGE and analysed by autoradiography (14C). Coomassie blue (CB) stained gels are shown below. (b) Full-length ARTD1 and fragments covering the whole protein were incubated in an in vitro methylation assay and analysed by autoradiography. (c) Decreasing amounts of WT ARTD1 and K508R ARTD1 were methylated by SET7/9 and analysed by autoradiography. (d) An antibody directed against a peptide carrying the methylated lysine residue of ARTD1 was generated and tested in a western blot with in vitro methylated ARTD1 WT and K508R. (e) U2OS cells were transfected with scrambled siRNA (scr) or siRNA directed against ARTD1. One day later, cells were transfected with an empty vector or with a plasmid containing WT SET7/9. Whole cell extracts were analysed by western blot on day 3 after knockdown using the same antibody as in (d). (f) U2OS cells were co-transfected with HA-ARTD1 (WT or K508R) and EGFP or Flag-HA-SET7/9 (WT or H297A). After immunoprecipitation with an anti-HA antibody, whole cell extracts and IP samples were analysed by western blotting with the indicated antibodies. All experiments were repeated at least twice, gave a similar result, and one representative blot is shown.

Mentions: Based on methylation profile searches and preliminary experiments, it was hypothesized that SET7/9 directly methylates ARTD1. To determine whether SET7/9 indeed modifies ARTD1, biochemical in vitro methylation assays with purified proteins were performed. SET7/9 methylated the known substrate histone H3 as well as full-length ARTD1, while neither GST nor ARTD2, another member of the ARTD family, was modified (figure 1a). To localize the modification site, purified ARTD1 fragments covering the whole amino acid sequence were methylated by SET7/9 in vitro (figure 1b). The potential SET7/9 modification site(s) in ARTD1 could be narrowed down to the auto-modification domain (AD) consisting of amino acids 373–524, which was strongly methylated in vitro, while all other tested ARTD1 fragments (containing the DNA-binding (DBD), WGR or catalytic (CAT) domains) were not methylated (figure 1b). In silico analysis identified lysine 508 (K508) as the putative target site as it was the only lysine residue within this region matching the published [KR] [STA] [K(me)] consensus motif for SET7/9-dependent methylation [18]. Mutation of K508 to arginine (K508R) indeed abolished SET7/9-dependent methylation of full-length ARTD1 (figure 1c). ARTD1 K508 was confirmed as the target residue of SET7/9 by mass spectrometric analysis of recombinant ARTD1 (373–524) in vitro methylated by SET7/9 (see electronic supplementary material, figure S1a,b). To confirm methylation of ARTD1 K508 in cells, a polyclonal antibody against a synthetic human ARTD1 peptide containing monomethylated K508 was generated. The anti-meARTD1 antibody specifically recognized the monomethylated peptide (see electronic supplementary material, figure S1c) and full-length ARTD1 that was methylated by SET7/9 in vitro (see electronic supplementary material, figure S1d), while the methylation-deficient K508R mutant was not detected (figure 1d). In vivo, the same antibody specifically detected the methylation of ARTD1 in cells overexpressing SET7/9 (figure 1e,f). The antibody did not detect methylation of overexpressed mouse ARTD1 in mouse cells, which was most probably owing to sequence differences between human and mouse ARTD1 at the methylation site.Figure 1.


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)

ARTD1 is methylated at K508 by SET7/9 in vitro and in vivo. (a) GST, ARTD1, ARTD2 and H3 were incubated with SET7/9 and 14C-labelled SAM in an in vitro methylation assay, separated by SDS-PAGE and analysed by autoradiography (14C). Coomassie blue (CB) stained gels are shown below. (b) Full-length ARTD1 and fragments covering the whole protein were incubated in an in vitro methylation assay and analysed by autoradiography. (c) Decreasing amounts of WT ARTD1 and K508R ARTD1 were methylated by SET7/9 and analysed by autoradiography. (d) An antibody directed against a peptide carrying the methylated lysine residue of ARTD1 was generated and tested in a western blot with in vitro methylated ARTD1 WT and K508R. (e) U2OS cells were transfected with scrambled siRNA (scr) or siRNA directed against ARTD1. One day later, cells were transfected with an empty vector or with a plasmid containing WT SET7/9. Whole cell extracts were analysed by western blot on day 3 after knockdown using the same antibody as in (d). (f) U2OS cells were co-transfected with HA-ARTD1 (WT or K508R) and EGFP or Flag-HA-SET7/9 (WT or H297A). After immunoprecipitation with an anti-HA antibody, whole cell extracts and IP samples were analysed by western blotting with the indicated antibodies. All experiments were repeated at least twice, gave a similar result, and one representative blot is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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RSOB120173F1: ARTD1 is methylated at K508 by SET7/9 in vitro and in vivo. (a) GST, ARTD1, ARTD2 and H3 were incubated with SET7/9 and 14C-labelled SAM in an in vitro methylation assay, separated by SDS-PAGE and analysed by autoradiography (14C). Coomassie blue (CB) stained gels are shown below. (b) Full-length ARTD1 and fragments covering the whole protein were incubated in an in vitro methylation assay and analysed by autoradiography. (c) Decreasing amounts of WT ARTD1 and K508R ARTD1 were methylated by SET7/9 and analysed by autoradiography. (d) An antibody directed against a peptide carrying the methylated lysine residue of ARTD1 was generated and tested in a western blot with in vitro methylated ARTD1 WT and K508R. (e) U2OS cells were transfected with scrambled siRNA (scr) or siRNA directed against ARTD1. One day later, cells were transfected with an empty vector or with a plasmid containing WT SET7/9. Whole cell extracts were analysed by western blot on day 3 after knockdown using the same antibody as in (d). (f) U2OS cells were co-transfected with HA-ARTD1 (WT or K508R) and EGFP or Flag-HA-SET7/9 (WT or H297A). After immunoprecipitation with an anti-HA antibody, whole cell extracts and IP samples were analysed by western blotting with the indicated antibodies. All experiments were repeated at least twice, gave a similar result, and one representative blot is shown.
Mentions: Based on methylation profile searches and preliminary experiments, it was hypothesized that SET7/9 directly methylates ARTD1. To determine whether SET7/9 indeed modifies ARTD1, biochemical in vitro methylation assays with purified proteins were performed. SET7/9 methylated the known substrate histone H3 as well as full-length ARTD1, while neither GST nor ARTD2, another member of the ARTD family, was modified (figure 1a). To localize the modification site, purified ARTD1 fragments covering the whole amino acid sequence were methylated by SET7/9 in vitro (figure 1b). The potential SET7/9 modification site(s) in ARTD1 could be narrowed down to the auto-modification domain (AD) consisting of amino acids 373–524, which was strongly methylated in vitro, while all other tested ARTD1 fragments (containing the DNA-binding (DBD), WGR or catalytic (CAT) domains) were not methylated (figure 1b). In silico analysis identified lysine 508 (K508) as the putative target site as it was the only lysine residue within this region matching the published [KR] [STA] [K(me)] consensus motif for SET7/9-dependent methylation [18]. Mutation of K508 to arginine (K508R) indeed abolished SET7/9-dependent methylation of full-length ARTD1 (figure 1c). ARTD1 K508 was confirmed as the target residue of SET7/9 by mass spectrometric analysis of recombinant ARTD1 (373–524) in vitro methylated by SET7/9 (see electronic supplementary material, figure S1a,b). To confirm methylation of ARTD1 K508 in cells, a polyclonal antibody against a synthetic human ARTD1 peptide containing monomethylated K508 was generated. The anti-meARTD1 antibody specifically recognized the monomethylated peptide (see electronic supplementary material, figure S1c) and full-length ARTD1 that was methylated by SET7/9 in vitro (see electronic supplementary material, figure S1d), while the methylation-deficient K508R mutant was not detected (figure 1d). In vivo, the same antibody specifically detected the methylation of ARTD1 in cells overexpressing SET7/9 (figure 1e,f). The antibody did not detect methylation of overexpressed mouse ARTD1 in mouse cells, which was most probably owing to sequence differences between human and mouse ARTD1 at the methylation site.Figure 1.

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