Lysine methylation of FOXO3 regulates oxidative stress-induced neuronal cell death

Abstract

FOXO transcription factors have a critical role in oxidative stress-induced neuronal cell death. A variety of post-translational modifications of FOXO family proteins have been reported, including phosphorylation, acetylation, ubiqutination and recently arginine methylation. Here, we demonstrate that the methyltransferase Set9 methylates FOXO3 at lysine 270. Methylation of FOXO3 leads to the inhibition of its DNA-binding activity and transactivation. Accordingly, lysine methylation reduces oxidative stress-induced and FOXO3-mediated Bim expression and neuronal apoptosis in neurons. Collectively, these findings define a novel modification of FOXO3 and show that lysine methylation negatively regulates FOXO3-mediated transcription and neuronal apoptosis.

Figure 1

Set9 interacts with and methylates FOXO3 at K270. (A) Lysates of 293T cells transfected with plasmids encoding GFP-FOXO3 and FLAG-Set9 were immunoprecipitated with anti-FLAG antibody or IgG. Western blot analysis was performed using anti- GFP or FLAG antibody. (B) Anti-Set9 or IgG immunoprecipitates from CGNs were immunoblotted with FOXO3 antibody. (C) Right panel: In vitro methylation assays were performed by incubating GST-FOXO3, His-FOXO1 or GST with recombinant Set9 in the presence of ³H-SAM. The reaction products were subjected to SDS–PAGE, and the methylation of FOXO was detected by autoradiography. Left panel: BSA or bulk histone was used as the negative or positive substrate, respectively. Asterisks stand for the degraded species of FOXO1 or FOXO3 proteins. (D) Methylation assays were performed as in C, and the substrates are GST-fused proteins of the truncated fragments of FOXO3. Fig shows that the methylation mainly occurs in the fragment of GST-FOXO3 P2–3. Red asterisk indicates the methylated FOXO3 fragment; green asterisks stand for the GST-fused fragments of FOXO3. CB, Coomassie Blue. (E) The lysines in the fragment of GST-FOXO3 P2–3 (aa 154–409) are shown. (F) The fragmentation mass spectrum analysis of the FOXO3 peptide KKmeAALQAAPESADDSPSQLSK identified a mono-methylated residue at K270. (G) In vitro methylation assay was performed by incubating GST-FOXO3 P2–3 with recombinant WT-Set9 or H297A-Set9 in the presence of ³H-SAM. (H) Immunoblotting assay shows that FOXO3 K270 mono-methylated antibody recognizes methylated FOXO3 P2–3 by Set9. aa, amino acid; BSA, bovine serum albumin; CGN, cerebellar granule neuron; GFP, green fluorescent protein; GST, glutathione S-transferase; IP, immunoprecipitation; Me-FOXO3, methylated FOXO3; SAM, S-adenosyl-methionine; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; WT, wild type.

Figure 2

Set9 methylates FOXO3 at K270 in vivo. (A) Lysates of 293T cells transfected with FLAG-FOXO3 together with Myc-Set9 or control vector were immunoblotted with anti-K270 mono-methylated antibody. (B) Lysates of 293T cells transfected with plasmids encoding FLAG-FOXO3 WT or K270R together with Myc-Set9 WT or H297A were immunoblotted with the methylation (K270 mono-methylated) antibody. (C) Anti-Me-K270 FOXO3 immunoprecipitates from 293T cells transfected with Set9 shRNA plasmids or control vector were blotted with anti-FOXO3 antibody. (D) Anti-FOXO3 immunoprecipitates from 293T cells transfected with Set9 shRNA plasmids or control vector were blotted with anti-Me-K270 FOXO3 or anti-FOXO3 antibody. The input was immunoblotted with indicated antibodies. (E) Anti-Set9 immunoprecipitates from primary CGNs treated with 200 μM H₂O₂ from 0 to 2 h were immunoblotted with anti-FOXO3 antibody. (F) Anti-Me-K270 FOXO3 immunoprecipitates from CGNs and treated with 200 μM H₂O₂ from 0 to 2 h were immunoblotted with anti-FOXO3 antibody. CGN, cerebellar granule neuron; IP, immunoprecipitation; WT, wild type.

Figure 3

Methylation of Lys 270 inhibits the DNA-binding activity of FOXO3 and FOXO3-dependent transcription. (A) 293T cells were transfected with FKRE luciferase reporter gene and tk-renilla reporter together with plasmids encoding Set9 WT or H297A mutant, as well as FOXO3 or the control vectors. Shown is mean±s.e.m. firefly/renilla luciferase relative to control vectors (t-test, ^**P<0.01,^*P<0.05, n=3). (B) 293T cells were transfected with FKRE-luciferase reporter gene and tk-renilla reporter together with plasmids encoding FOXO3 WT or FOXO K270R, as well as Set9 or the vector control. Shown is mean±s.e.m. firefly/renilla luciferase relative to control vectors (t-test, ^**P<0.01, n=3). (C) The EMSA was performed using the indicated methylation products. 100X competitor indicates the amount of the cold probe. (D) MEF cells stably expressing inducible ER-FOXO3 TM (triple mutations on Akt phosphorylation sites) were transfected with Set9 together with FKRE-luciferase/tk-renilla reporter. At 24 h after transfection, cells were treated with or without 4-OHT for another 12 h before collecting. Set9 inhibits FOXO3 TM-dependent FKRE-luciferase reporter gene expression (t-test, ^***P<0.001, n=3). RLU, relative light unit. (E) Crosslinked chromatin was prepared from Neuro2A cells transfected with Set9 shRNA plasmid (#1 or #2) or the control plasmid (sh-Luc) and immunoprecipitated with anti-FOXO3 antibody or IgG alone, followed by real-time PCR using primers specific to Bim promoter. (F) Lysates of Neuro2A cells transfected with Set9 shRNA plasmid (#1 or #2) or the control plasmid (sh-Luc) were immunoblotted with the indicated antibodies. EMSA, electrophoretic mobility shift assay; FKRE, forkhead response element; SAM, S-adenosyl-methionine; shRNA, small hairpin RNA; TM, triple mutation; WT, wild type.

Figure 4

Set9 inhibits oxidative stress-induced neuronal cell death via methylation of FOXO3. (A) CGNs were transfected with the FOXO3, Set9 WT, Set9 H297A or control vector together with FKRE-luciferase reporter gene and tk-renilla reporter. Set9 significantly inhibits FOXO3's transcriptional activity in CGNs (t-test, P<0.01, n=3). (B) CGNs were transfected with the FOXO3 or the control vector with or without Set9 shRNA plasmid (#1 or #2), together with FKRE-luciferase/tk-renilla reporter. Knockdown of Set9 considerably increases the transcriptional activity of FOXO3 (t-test, P<0.01, n=3). (C) Total RNA was isolated from cultured Neuro2A cells and cDNA was synthesized by reverse transcription, followed by real-time PCR. Knockdown of Set9 considerably increases the expression of Bim (t-test, P<0.01, n=3). (D) CGNs transfected with the Set9 RNAi plasmid (sh-Set9 #1 and #2) or control vector (sh-Luc) together with GFP vector were treated with H₂O₂. Yellow arrowhead stands for the healthy cells and red arrowhead indicates apoptotic cells. Set9 knockdown increases cell apoptosis upon H₂O₂ treatment (ANOVA followed by Fisher's PLSD post hoc, P<0.01, n=4). (E) CGNs transfected with the Set9 RNAi plasmid (sh-Set9 #1) or control vector (sh-Luc), FOXO3 RNAi plasmid (U6/FOXO3) or its control vector U6 together with GFP vector were treated with H₂O₂. Set9 knockdown increases FOXO-dependent cell apoptosis upon H₂O₂ treatment (ANOVA followed by Fisher's PLSD post hoc, P<0.01, n=3). (F) Left panel: CGNs transfected with the FOXO3 RNAi plasmid (U6/FOXO3) together with indicated plasmids were treated with H₂O₂, and cell death was analysed as in E. Data are presented as mean±s.e.m. (ANOVA followed by Fisher's PLSD post hoc, P<0.01, n=3). Right panel: Lysates of 293T cells transfected with indicated plasmids were immunoblotted with anti-GFP or ERK1/2 antibody. ANOVA, analysis of variance; cDNA, complimentary DNA; CGN, cerebellar granule neuron; FKRE, forkhead response element; GFP, green fluorescent protein; mRNA, messenger RNA; RNAi, RNA-mediated interference; shRNA, small hairpin RNA.