Fatty acid oxidation facilitates DNA double-strand break repair by promoting PARP1 acetylation

Abstract

DNA repair is a tightly coordinated stress response to DNA damage, which is critical for preserving genome integrity. Accruing evidence suggests that metabolic pathways have been correlated with cellular response to DNA damage. Here, we show that fatty acid oxidation (FAO) is a crucial regulator of DNA double-strand break repair, particularly homologous recombination repair. Mechanistically, FAO contributes to DNA repair by activating poly(ADP-ribose) polymerase 1 (PARP1), an enzyme that detects DNA breaks and promotes DNA repair pathway. Upon DNA damage, FAO facilitates PARP1 acetylation by providing acetyl-CoA, which is required for proper PARP1 activity. Indeed, cells reconstituted with PARP1 acetylation mutants display impaired DNA repair and enhanced sensitivity to DNA damage. Consequently, FAO inhibition reduces PARP1 activity, leading to increased genomic instability and decreased cell viability upon DNA damage. Finally, our data indicate that FAO serves as an important participant of cellular response to DNA damage, supporting DNA repair and genome stability.

Fig. 1. FAO supports DNA double-strand break repair.

A γH2AX protein levels in immortalized MEFs exposed to 3 Gy IR and treated with or without 200 μM ETO for the indicated times. β-actin was used as a loading control. B Immortalized MEFs were exposed to 3 Gy IR and then treated with or without ETO for the indicated times. Immunofluorescent staining was performed with a nuclear marker (DAPI) and anti-γH2AX (green) on the indicated cells. Scale bar represents 6 μm. C Number of γH2AX foci per nucleus in the indicated cells as shown in (B). The number of cells analyzed per condition (n) is indicated. Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons test. D Immortalized MEFs were transfected with siControl or two independent siRNAs against CPT1A and were exposed to 3 Gy IR. Representative images of immunofluorescence staining (γH2AX, Green; DAPI, Blue). Scale bar represents 6 μm. Number of γH2AX foci per nucleus as indicated (right). The number of cells analyzed is noted. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. E Immortalized MEFs were exposed to 3 Gy IR and then recovered for 24 h with or without ETO. DNA-damaged cells were measured using a neutral comet assay. Tail moment values are shown (right). The number of cells analyzed per condition (n) is indicated. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. Scale bar represents 2 mm. F Immortalized MEFs were exposed to 3 Gy IR and then treated with or without 200 μM ETO and/or 1 mM octanoate for the indicated times. Lysates were immunoblotted with anti-γH2AX antibody. C–E The number of cells was pooled from three independent experiments. All error bars ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig. 2. FAO affects HR repair by promoting BRCA1 recruitment.

A Experimental design for Fig. 2B and C. 293 cells were infected with DSB reporter and selected with G418. Selected cells were transfected with exogenous donor and I-SceI. After transfection, cells were treated with or without ETO for 48 h and analyzed using FACS. Quantification of (B) mCherry and (C) GFP expression in BFP positive cells. Three million cells per sample were analyzed. Statistical analysis was based on two-tailed Student’s t-test. D Immortalized MEFs were treated with 0.5 μM ETS, 200 μM ETO, or both as indicated. The Cell cycle was analyzed by BrdU staining. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. The indicated p-values (****) represent the comparative values between ETS-treated and ETS plus ETO-treated cells in G2/M phase. E γH2AX protein levels in immortalized MEFs. Cells, pre-treated with nocodazole (200 nM) for 20 h, were exposed to 3 Gy IR and then treated with or without 200 μM ETO for the indicated times. β-actin was used as loading control. F HeLa cells were exposed to 3 Gy IR and treated with or without ETO for 4 h. Representative images of immunofluorescence staining (53BP1, Red; γH2AX, Green; DAPI, Blue in upper panels and BRCA1, Green; γH2AX, Red; DAPI, Blue in lower panels). Scale bar represents 10 μm. Percentages of γH2AX positive cells with >10 Foci of BRCA1 or 53BP1 as indicated (right). Statistical analysis was performed using two-tailed Student’s t-test. All error bars ± SEM. *p < 0.05, **p < 0.01, and ****p < 0.0001.

Fig. 3. FAO participates in DNA repair by regulating PARP1 activity.

A Immortalized MEFs, transfected with siControl or siRNAs against CPT1A, were pre-treated with 10 μM PDD00017273 for 1 h and then exposed to 3 Gy IR. Immunofluorescent staining was performed with anti-PAR antibody and DAPI. Scale bar represents 20 μm. Relative fluorescence intensity of PAR as indicated (right). Statistical analysis was based on two-way ANOVA with Tukey’s multiple comparisons test. The number of cells analyzed per condition (n) is indicated. B 293 T cells were transfected with siControl or siRNA against CPT1A and were treated with 10 μM PDD00017273 for 1 h before irradiation (10 Gy). Lysates were subjected to immunoblotting analysis with anti-PAR and anti-PARP1 antibodies. C 293 T cells were transfected with pCMV-PARP1-3x Flag. After transfection, cells were treated with or without ETO for 4 h and then irradiated 10 Gy IR. Lysates were immunoprecipitated with Flag antibody, followed by immunoblotting with anti-PAR antibody. D 293 T cells were treated with or without ETO and/or 1 mM octanoate for 4 h and then incubated with 10 μM PDD00017273 for 1 h before 10 Gy IR. Lysates were immunoblotted with anti-PAR antibody. E Immortalized MEFs were treated with or without ETO and/or 1 mM octanoate for 4 h and then incubated with 10 μM PDD00017273 for 1 h before 3 Gy IR. Immunofluorescent staining was performed with a nuclear marker (DAPI) and anti-PAR antibody on the indicated cells. Scale bar represents 20 μm. Relative fluorescence intensity of PAR as indicated (right). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. The number of cells analyzed per condition (n) is noted. F Representative images of γH2AX foci per nucleus in Control or PARP1 knockdown MEF cells. Scale bar represents 6 μm. Number of γH2AX foci per nucleus as indicated (right). Statistical analysis was based on two-way ANOVA with Tukey’s multiple comparisons test. The number of cells analyzed per condition (n) is indicated. In (A), (E), and (F), the number of cells analyzed per condition was pooled from three independent experiments. All error bars ± SEM. *p < 0.05 and ****p < 0.0001.

Fig. 4. FAO adjusts PARP1 activity through acetyl-CoA upon DNA damage.

A Immunoblot for p-AMPK and AMPK in the indicated cells. Immortalized MEFs were exposed to 3 Gy IR and then treated with or without ETO for 4 h. β-actin was used as loading control. B γH2AX protein levels in cells treated with the indicated drugs. Immortalized MEFs were exposed to 3 Gy IR and treated with or without ETO and/or 4 mM NAC for 4 h. C The number of γH2AX foci per nucleus of the indicated cells. Immortalized MEFs were transfected with siControl or with siRNAs against CPT1A. After transfection, cells were exposed to 3 Gy IR and treated with or without 100 μM dNTP for 6 h. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. The number of cells analyzed per condition (n) is indicated. D Immunofluorescent staining was performed with a nuclear marker (DAPI) and anti-γH2AX antibody on cells. Immortalized MEFs were exposed to 3 Gy IR and then treated with or without 3 mM citrate for 4 h. Scale bar represents 6 μm. The number of γH2AX foci per nucleus as indicated (right). Statistical analysis was based on one-way ANOVA with Tukey’s multiple comparisons test. The number of cells analyzed is noted. E Representative images of immunofluorescence staining in control and CPT1A knockdown cells treated with the indicated drugs. Immunofluorescent staining was performed with nuclear marker (DAPI) and anti-PAR antibody on cells. Scale bar represents 20 μm. Relative fluorescence intensity of PAR as indicated (right). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. The number of cells analyzed is indicated. F, G 293 T cells were transfected with pCMV-PARP1-3x Flag. After transfection, cells were treated with ETO, 3 mM citrate, and/or 50 mM acetate for 4 h as indicated and then irradiated with 10 Gy IR. Lysates were immunoprecipitated with Flag antibody and immunoblotted with anti-PAR antibody. H Relative fluorescence intensity of PAR in the indicated cells. Immortalized MEFs were transfected with siControl or two independent siRNAs against ACLY. After transfection, cells were treated with or without ETO and/or 3 mM citrate for 4 h and then irradiated with 3 Gy IR. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. The number of cells analyzed per condition (n) is noted. In (C), (D), (E), and (H), the number of cells was pooled from three independent experiments. All error bars ± SEM. *p < 0.05, **p < 0.01 and ****p < 0.0001.

Fig. 5. DNA damage-induced PARP1 acetylation affects its activity.

A 293 T cells were transfected with pCMV-PARP1-3x Flag. Transfected cells were exposed to 10 Gy IR and treated with or without ETO for 4 h. Lysates were immunoprecipitated with Flag antibody and immunoblotted with anti-acetylated lysine (Lys-Ac) antibody. Relative PARP1 Lys-Ac levels as indicated (right). Statistical analysis was based on one-way ANOVA with Tukey’s multiple comparisons test. B 293 T cells were transfected with pCMV-PARP1-3x Flag. Transfected cells were treated with 0.3 μM TSA, 50 mM acetate, and/or ETO for 4 h before IR. Lysates were immunoprecipitated with Flag antibody and immunoblotted with anti-Lys-Ac antibody. C pCMV-PARP1-3x Flag transfected 293 T cells were treated with 0.3 μM TSA and/or ETO for 4 h and then irradiated 10 Gy IR. Lysates were immunoprecipitated with Flag antibody and immunoblotted with anti-PAR antibody. D Control or p300 knockdown 293 T cells, transfected with pCMV-PARP1-3x Flag, were exposed to 10 Gy IR. Lysates were immunoprecipitated with Flag antibody and immunoblotted with anti-PAR antibody. E, F 293 T cells were transfected with WT or two mutant forms of PARP1; 3 R (K498R/K521R/K524R) and 5 R (K498R/K505R/K508R/K521R/K524R). Transfected cells were exposed to 10 Gy IR. Lysates were immunoprecipitated with Flag antibody, followed by immunoblotting with anti-Lys-Ac and anti-PAR antibodies (upper panels). The immunoprecipitated Flag has been adjusted to be equal to make the levels of acetylated PARP1 (E) or PAR (F) comparable to those of immunoprecipitated Flag. Relative Lys-Ac and PARylation levels of PARP1 indicated (lower panels). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. G 293 T cells were transfected with WT or two mutant forms of PARP1. Transfected cells were treated with or without ETO and/or 1 mM octanoate for 4 h and then treated with 10 μM PDD00017273 for 1 h before IR. Lysates were immunoblotted with anti-PAR antibody. H PARP1 knockdown 293 T cells were transfected with WT or two mutant forms of PARP1 and then irradiated with 10 Gy IR. Lysates were subjected to immunoblotting analysis with anti-γH2AX antibody. All error bars ± SEM. *p < 0.05 and **p < 0.01.

Fig. 6. FAO is required to maintain genomic stability.

A Cell viability of immortalized MEFs treated with the indicated drugs. Cells were treated with or without 0.05 μM DOX, 200 μM ETO, 0.5 mM citrate, and/or 50 μM octanoate for 48 h. Statistical analysis was based on one-way ANOVA with Tukey’s multiple comparisons test. B Cell viability of the 293 T cells, reconstituted with WT or two mutant forms of PARP1, treated with DOX. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. C Immortalized MEFs were treated with or without 10 μM ETS, 200 μM ETO, 0.1 mM citrate, and/or 50 μM octanoate for 15 h. The following day, growth media was replaced with fresh media. Two days later, cells were stained with propidium iodide and analyzed by flow cytometry. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. D Percentages of multinucleated, binucleated, and mononucleated cells in the indicated cells. Immortalized MEFs were treated with ETS, ETO, citrate and/or octanoate as indicated. The number of cells pooled from three independent experiments is indicated. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. The indicated p-values represent comparison within multinuclei. Representative images of cells staining with DAPI and anti-phalloidin antibody as indicated (right). Scale bar represents 20 μm. E A proposed model for role of FAO in DNA repair. All error bars ± SEM. **p < 0.01, ***p < 0.001 and ****p < 0.0001.