JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

Abstract

The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6), promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB) repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

Figure 1. Inhibition of JNK Signaling Abrogates the Ability of SIRT6 to Stimulate DSB Repair in Response to Oxidative Stress

(A) Immortalized normal diploid human fibroblast cells containing a chromosomally integrated NHEJ reporter cassette (see Figure S1A) were co-transfected with I-SceI and DsRed expression vectors as well as either an SIRT6-encoding plasmid or a control plasmid in the presence or absence of paraquat and a JNK inhibitor (SP600125). SIRT6 expression stimulated NHEJ 2.3-fold relative to control; when cells were pretreated with 1 mM paraquat, SIRT6 expression stimulated NHEJ 9.4-fold relative to control. Pretreating cells with 10 µMJNK inhibitor did not affect the ability of SIRT6 to stimulate NHEJ under basal conditions; when cells were pretreated with both paraquat and JNK inhibitor, however, SIRT6 failed to stimulate NHEJ. Western blots indicate activation of JNK signaling in response to paraquat, as indicated by phosphorylation of c-JUN (p-cJUN); treatment with the JNK inhibitor SP600125 effectively abrogated JNK signaling, but did not affect the paraquat-induced increase in the levels of SIRT6 protein (bottom panel). Error bars indicate SD (n = 6). See also Figure S1. (B) The requirement of JNK signaling for SIRT6 expression to stimulate NHEJ in response to stress was confirmed using siRNAs. HCA2-hTERT-NHEJ cells were transfected as in (A), but, instead of exposure to a chemical inhibitor, the cells were co-transfected with siRNAs specific to JNK1/2 or a scrambled, control siRNA. SIRT6 expression massively stimulated NHEJ in cells pretreated with paraquat, but failed to do so when the cells also had been transfected with JNK siRNAs. Western blots indicate activation of JNK signaling in response to paraquat, as indicated by phosphorylation of c-JUN (p-cJUN); treatment siRNAs targeting JNK effectively abrogated JNK signaling. Error bars indicate SD (n = 5). (C) Immortalized normal diploid human fibroblast cells containing a chromosomally integrated HR reporter cassette (see Figure S1B) were co-transfected with I-SceI and DsRed expression vectors as well as either an SIRT6-encoding plasmid or a control plasmid in the presence or absence of paraquat and a JNK inhibitor (SP600125). SIRT6 expression stimulated HR 3.1-fold relative to control; when cells were pretreated with 1 mM paraquat, SIRT6 expression stimulated NHEJ 10.4-fold relative to control. Pretreating cells with 10 µM JNK inhibitor did not affect the ability of SIRT6 to stimulate HR under basal conditions; when cells were pretreated with both paraquat and JNK inhibitor, however, SIRT6 failed to stimulate HR. Error bars indicate SD (n = 4). (D) SIRT6 expression accelerates the clearance of the DNA DSB marker γH2AX in HCA2-hTERT cells that had been pretreated with 1 mM paraquat for 16 hr. Inhibition of JNK signaling with SP600125 or JNK siRNA abrogates the effect of SIRT6 overexpression. Data represent the average number of γH2AX foci per nucleus. At least 50 nuclei were scored for each time point. Error bars indicate SEM. (E) Human fibroblasts, transfected with a plasmid encoding either SIRT6 or a control vector, were treated with 1 mM paraquat for 16 hr. Repair was measured 3 hr after the treatment using a comet assay kit (Trevigen) according to the manufacturer’s instructions. Tail moments were determined using CometScore software. One hundred cells were scored for each independent experiment. Error bars indicate SD (n = 3; *p < 0.05 and **p < 0.01). See also Figure S1 for inhibitors of other kinases.

Figure 2. JNK Phosphorylates SIRT6 at Residue S10 to Promote DSB Repair

(A) CoIP reveals that SIRT6 interacts with JNK in HCA2-hTERT cells only when the cells have been exposed to oxidative stress (1 mM paraquat for 16hr). The experiment was repeated at least four times. (B) In vitro phosphorylation assay demonstrating that JNK can phosphorylate SIRT6 in vitro. Anisomysin-activated JNK, purified from HEK293 cells, was incubated with BSA and bacterially purified SIRT6 in the presence of ³²P-ATP and a kinase reaction buffer. SIRT6 specifically incorporated the radiolabel in these reactions, indicating that it was phosphorylated by JNK. The experiment was repeated three times and a representative gel is shown. See also Figure S2. (C) SIRT6 plasmids encoding mutations at the indicated putative phosphorylation sites were overexpressed in HCA2-hTERT-NHEJ cells to measure their ability to stimulate NHEJ. SIRT6 T294A, S303A, S330A, and S338A all stimulated NHEJ similarly to WT SIRT6. SIRT6 S10A, however, failed to stimulate NHEJ in response to stress. Expression of an SIRT6 plasmid encoding an S10E phospho-mimetic mutation was able to powerfully stimulate NHEJ in the absence of oxidative stress. The effect of S10E mutation on DNA repair was resistant to JNK inhibition with SP600125. Error bars indicate SD (n = 4). Immunoblot (above) demonstrates that all of the indicated SIRT6 vectors were expressed stably and at comparable levels (*p < 0.05 and **p < 0.01). (D) NHEJ reporter construct was integrated into SIRT6^−/− MEF to measure the SIRT6 S10A and S10E activity in DNA repair in the absence of endogenous SIRT6. Cells expressing SIRT6 S10A mutant showed no stimulation of NHEJ repair in response to paraquat-induced oxidative stress. SIRT6 S10E phospho-mimetic mutant stimulated NHEJ under basal conditions, which could be further stimulated by stress; however, this additional stimulation of NHEJ was not affected by JNK inhibitor SP600125. Error bars indicate SD (n = 3; *p < 0.05 and **p < 0.01). (E) In vitro phosphorylation assay demonstrates that, while JNK can phosphorylate WT SIRT6, it cannot phosphorylate SIRT6 S10A. Anisomysin-activated JNK, purified fromHEK293 cells, was incubated with BSA and bacterially purified WT SIRT6 of SIRT6 S10A in the presence of ³²P-ATP and a kinase reaction buffer. The experiment was repeated three times and a representative gel is shown. (F) SIRT6 is phosphorylated on S10 in vivo after oxidative stress and the phosphorylation is diminished by JNK inhibitor (SP600125). Custom rabbit polyclonal antibodies (Rb5159 and Rb5160) were generated by immunizing rabbits with YAAGLpSPYADKGKC peptide (see Figure S2D for antibody specificity assays). The hTERT-immortalized human fibroblasts HCA2 were transfected with WT SIRT6-expressing plasmid, then treated with paraquat and/or JNK inhibitor and SIRT6 S10-P, and total SIRT6 levels were assessed by western blot (Rb5159 is pictured; both antibodies gave comparable results). To further confirm the specificity of S10-P antibodies, replicate samples were run and treated with Lambda Protein Phosphatase (LPP) for 1 hr, prior to antibody staining. The experiment was repeated three times and a representative blot is shown.

Figure 3. Phosphorylation at SIRT6 Residue S10 Stimulates Recruitment of SIRT6 to DSB Sites

(A) Chromatin-enriched fractions from WT MEFs indicate that SIRT6 is rapidly recruited to chromatin following exposure of cells to paraquat. Cells were treated with 0.5 mM paraquat, and chromatin-enriched extracts were prepared at the indicated time points (n = 3). A representative blot is shown. (B) Recruitment of SIRT6-GFP to sites of laser-induced DNA damage was monitored in U2OS cells transfected with SIRT6-GFP in the presence or absence of a JNK inhibitor (SP600125). Cells pretreated with 20 µM JNK inhibitor for 2 hr exhibited severe defects in their ability to recruit SIRT6 to DSB sites. Representative images are shown (n > 8 for each sample). Error bars indicate SEM. See also Figure S3. (C) Recruitment of WT, S10A, or S10E SIRT6-GFP to sites of laser-induced DNA damage was monitored in U2OS cells. S10E SIRT6-GFP exhibited enhanced recruitment efficiency to sites of DSBs, whereas S10A SIRT6-GFP exhibited diminished recruitment efficiency to DSB sites. Representative images are shown (n > 8 for each sample). Error bars indicate SEM.

Figure 4. Phosphorylation of SIRT6 by JNK Stimulates Its Ability to Activate PARP1

(A) In vitro mono-ADP ribosylation reaction. Bacterially purified recombinant WT and S10A and S10E SIRT6 proteins were incubated with catalytically inactive recombinant PARP1 (C-terminal truncation, containing only aa 1–655) for 2 hr. S10E SIRT6 was able to more robustly mono-ADP ribosylate the PARP1 substrate than either WT or S10A SIRT6 (n = 3). A representative reaction is shown. (B) In vitro ribosylation assay demonstrating S10E SIRT6 more robustly stimulates PARP1 activity. Bacterially purified WT, S10A, or S10E SIRT6 was incubated with PARP1. PARP1 activity was measured by quantifying the amount of auto-poly-ADP ribosylation of the protein by immunoblotting with antibodies targeting poly-ADP ribose. The experiment was independently repeated three times; the right panel shows quantification; error bars indicate SD (*p < 0.05 and **p < 0.01).

Figure 5. Phosphorylation of SIRT6 by JNK Accelerates the Recruitment of PARP1 to DSB Sites

(A) Recruitment of PARP1-GFP to sites of laser-induced DNA damage was measured in WT and SIRT6 KO MEFs. In the absence of SIRT6, PARP1 exhibited a striking failure to fully recruit to DNA break sites. Representative images are shown (n > 8 for each condition); error bars indicate SEM. (B) Recruitment of SIRT6-GFP to sites of laser-induced DNA damage was measured in WT and PARP1 KO MEFs. In the absence of PARP1, SIRT6 was able to be fully recruited to DNA break sites. Representative images are shown (n > 8 for each condition). Error bars represent SEM (n.s., not significant). (C) Recruitment of PARP1-GFP to sites of laser-induced DNA damage in MEFs overexpressing WT, S10A, or S10E SIRT6. Cells overexpressing WT SIRT6 and S10E SIRT6 were able to more robustly recruit PARP1-GFP to sites of DNA damage. By contrast, cells overexpressing S10A SIRT6 failed to stimulate PARP1 recruitment to DNA damage sites. Error bars indicate SEM (n > 8). (D) Modification of K521A PARP1 is required for the efficient recruitment of PARP1 to DSB sites. Recruitment of PARP-GFP1 or K521A PARP1-GFP to sites of laser-induced DNA damage was measured in MEFs. K521A PARP1-GFP exhibited a failure to efficiently recruit to DNA damage sites. Representative images are shown (n > 8 for each condition). Error bars indicate SEM (n > 8). (E) JNK inhibition abrogates PARP1 recruitment to DNA damage sites. Recruitment of PARP1-GFP to sites of laser-induced DNA damage was measured in WT MEFs in the presence or absence of a JNK inhibitor. JNK inhibition with SP600125 resulted in failure to recruit PARP1 to DNA damage sites. Error bars indicate SEM (n > 8).

Figure 6. Model for JNK-Mediated Activation of DSB Repair

Upon oxidative stress JNK phosphorylates SIRT6 on Serine 10. This results in rapid recruitment of SIRT6 to the DSB site and simultaneously stimulates SIRT6 mono-ADP ribosylation of PARP1. PARP1 mono-ADP ribosylation leads to recruitment of PARP1 to DSB site and activates PARP1 poly-ADP ribosylation activity. This sequence of events represents the initial steps in the assembly of repair machinery on a DSB, and it is required for efficient DSB repair under oxidative stress conditions.