Acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 during the antioxidant response

Abstract

To maintain intracellular redox homeostasis, genes encoding many antioxidants and detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis antioxidant responsive elements (AREs) in their promoter regions. Nrf2 is the critical transcription factor responsible for ARE-dependent transcription. We and others have previously demonstrated that Nrf2 is targeted for ubiquitin-mediated degradation by Keap1 in a redox-sensitive manner through modifications of distinct cysteine residues of Keap1. Here, we report that p300/CBP directly acetylates Nrf2 in response to arsenite-induced stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.

FIG. 1.

Nrf2 is acetylated by p300/CBP in vivo and in vitro. (A) Endogenous Nrf2 is acetylated. HCT116 cells were lysed under denaturing conditions. The cell lysates were diluted and subjected to immunoprecipitation (IP) by normal IgG or anti-Nrf2 (α-Nrf2) antibodies, followed by immunoblotting (IB) with antibodies specific for acetylated lysine (AcK). Tub, tubulin. (B) Sodium arsenite enhances acetylation of Nrf2. HCT116 cells were treated with 20 μm As(III) for the indicated times. Nrf2 acetylation levels were measured as described for panel A. (C) Nrf2 is acetylated by p300/CBP, not P/CAF. HEK293T cells were cotransfected with vectors expressing HA-tagged Nrf2 and the indicated HATs. The cell lysates were immunoprecipitated with anti-HA antibodies, followed by immunoblotting with antibodies specific for acetylated lysine. (D) Nrf2 is a bona fide substrate of p300. (Top) Purified GST-tagged Nrf2 proteins were incubated in the presence of ¹⁴C-labeled acetyl-CoA with immunoprecipitated p300 proteins from COS-1 cells overexpressing Flag-p300. The reaction mixtures were resolved on SDS-PAGE, followed by autoradiography. (Bottom) The same amounts of GST-Nrf2 proteins as used in the in vitro acetylation assay described above were subjected to Coomassie staining.

FIG. 2.

Nrf2 associates with p300. (A) Endogenous Nrf2 and p300 are coordinately recruited to the ARE in response to As(III)-induced stress. ChIP analysis was performed in HCT116 cells with the indicated antibodies after 4 h of treatment of 20 μM As(III). (Top) The genomic DNA fragments bound to either Nrf2 or p300 protein were recovered and quantified by qPCR using primer pairs specific for the NQO1-ARE region or the tubulin promoter region as a negative control. IP, immunoprecipitation. (Bottom) The total DNAs were amplified and visualized as described above to ensure equal input between mock and As treatment groups. (B) The precipitated DNA fragments in panel A were quantified by qPCR. The error bars indicate standard deviations. Asterisks indicate significant differences from IgG controls and the untreated group. IP, immunoprecipitation; Tub, tubulin. (C) Nrf2 interacts with p300 in vivo. HEK293T cells expressing Flag-tagged p300 and HA-tagged Nrf2 were lysed. The cell lysates were immunoprecipitated with anti-Flag (α-Flag) M2 matrix, followed by immunoblotting (IB) with anti-HA antibodies. An unrelated protein, Cul4, was included as a negative control. (D) Schematic of conserved domains in p300 and Nrf2 proteins. p300 contains five conserved domains: C/H1, KIX, BROMO, C/H2, and C/H3. The catalytic region, also known as the “HAT domain,” is between amino acids 1195 and 1673. Nrf2 contains six conserved domains: Neh2, Neh4, Neh5, Neh6, Neh1, and Neh3. (E) Nrf2 transactivation domains Neh4 and Neh5, especially Neh5, directly bind p300. (Top) p300 proteins were labeled with [³⁵S]methionine and pulled down by GST-Nrf2 with different deletion mutants. The Nrf2-bound p300 was visualized by autoradiography. (Bottom) The same amounts of GST-Nrf2 proteins as used above were visualized by Coomassie blue staining. (F) The p300 C terminus directly interacts with Nrf2. (Top) Total inputs of different truncated forms of ³⁵S-labeled p300 proteins were visualized by autoradiography. (Bottom) ³⁵S-labeled p300 proteins were pulled down by GST-Nrf2 and visualized by autoradiography. Luciferase served as a negative control. FL, full length.

FIG. 3.

Identification of multiple acetylated lysine residues within the Neh1 DNA-binding domain of Nrf2. (A) The Neh1 domain contains multiple acetylation sites. GST-tagged Nrf2 proteins with the indicated deletions within the Neh1 domain were subjected to in vitro acetylation analysis as described in the legend to Fig. 1D. Ac, acetylated. (B) Multiple lysines in the Neh1 domain were identified as acetylation sites by LC-MS/MS. Immunoprecipitation was performed in HEK-293T cells expressing HA-Nrf2 and p300 with anti-HA antibodies. Immunoprecipitated Nrf2 proteins were visualized by Coomassie staining, isolated, and analyzed by LC-MS/MS.

FIG. 4.

Functional redundancy among different acetylation sites. (A) Arginine substitution for single or several adjacent lysine residue(s) does not change overall Nrf2 acetylation levels. Acetylation on Nrf2 was analyzed as described for HEK293T cells expressing the indicated HA-Nrf2 and p300. IP, immunoprecipitation; IB, immuno-blotting; α, anti. (B) HEK293T cells were cotransfected with vectors for the NQO1-ARE-dependent firefly luciferase reporter gene, TK Renilla luciferase gene, the indicated HA-Nrf2, and p300. Luciferase reporter gene activities were analyzed using the Promega dual-luciferase reporter gene assay system. Relative luciferase activities and standard deviations were calculated from three independent experiments. Inr, initiator. (C) Total cell lysates from the luciferase assay in panel B were subjected to immunoblot analysis with anti-HA antibodies. Tub, tubulin.

FIG. 5.

Acetylation on the Neh1 domain does not regulate the stability of Nrf2 proteins. (A) Distribution of lysines within the Neh1 domain of human Nrf2 protein. The asterisks indicate acetylated lysines as identified by MS. (B) Arginine substitution for all 18 lysines in the Neh1 domain abolishes acetylation of Nrf2 by p300. Acetylations on Nrf2 were analyzed as described for HEK293T cells expressing the indicated HA-Nrf2 and p300. IP, immunoprecipitation; IB, immunoblotting; α, anti; Tub, tubulin. (C) Mutation of 18KR in Nrf2 does not affect the half-lives of Nrf2 proteins under both basal and As-induced conditions. MDA-MB-231 cells expressing HA-Nrf2, Keap1, and p300, as indicated, were either left untreated or treated with 20 μM As(III) for 3 h, followed by cotreatment with 50 μM cycloheximide (CHX) for the indicated times. The cell lysates were analyzed by immunoblotting with anti-HA antibodies. The relative intensities of the Nrf2 bands were quantified, normalized to tubulin, and plotted on a semilog scale. The calculated half-lives (t_1/2) of Nrf2 in each group are shown. (D) Mutation of 18KR in Nrf2 does not affect overall Nrf2 ubiquitination levels. MDA-MB-231 cells expressing HA-Nrf2, CBD-tagged Keap1, and p300, as indicated, were cotreated with 20 μM As(III) and 10 μM proteasome inhibitor MG132 as indicated for 4 h and then lysed under denaturing conditions. The cell lysates were diluted and subjected to immunoprecipitation with anti-HA antibodies, followed by immunoblotting with antiubiqutin (α-Ub) antibodies. (E) Mutation of 18KR in Nrf2 does not affect colocalization of Nrf2 with p300 in the nucleus. MDA-MB-231 cells expressing the indicated HA-Nrf2 and Flag-tagged p300 were subjected to indirect immunofluorescence analysis using anti-HA and anti-Flag antibodies. (F) Mutation of 18KR in Nrf2 does not affect the interaction between Nrf2 and Keap1. HEK293T cells expressing the indicated HA-Nrf2, Keap1-CBD, and p300 were lysed. Keap1-containing protein complexes were pulled down with chitin beads and immunoblotted with anti-HA antibodies.

FIG. 6.

Acetylation plays a positive role in the transcriptional activity of Nrf2. (A) Nrf2 18KR has decreased transcriptional activity compared to Nrf2 WT. Luciferase reporter gene analysis was performed with NQO1 ARE reporters, as described in the legend to Fig. 4B, in HEK293T cells expressing HA-Nrf2 and p300, as indicated. The asterisk indicates a significant difference from Nrf2 WT. The error bars indicate standard deviations. Inr, initiator. (B) Arsenite has decreased induction effects on Nrf2 18KR compared to Nrf2 WT. HEK293T cells expressing HA-Nrf2, Keap1, and p300, as indicated, were treated with 10 μM As(III) for 12 h. NQO1-ARE luciferase (Luc) reporter gene expression was analyzed as for panel A. The asterisk indicates a significant difference from Nrf2 WT. (C) Total cell lysates from the two luciferase assays in panels A and B were subjected to immunoblot analysis with anti-HA (α-HA) antibodies. Tub, tubulin. (D) Nrf2 18KR has decreased activity in driving the transcription of NQO1, TXNRD1, and GCLM, but not HO-1. qRT-PCR was performed in HEK293T cells either expressing the indicated HA-Nrf2 and p300 or treated with 20 μM As(III) for 12 h. The error bars indicate the standard deviations from three experiments. The asterisks indicate significant differences from Nrf2 WT. (E) Total cell lysates from HEK293T cells prepared in parallel with cells for the qRT-PCR analysis in panel D were subjected to immunoblot analysis with anti-HA antibodies. (F and G) MEF Nrf2^−/− cells overexpressing HA-Nrf2 and p300, as indicated, were analyzed by qRT-PCR and immunoblotting. (H and J) HCT116 p300^+/+ and p300^−/− cells were treated with 20 μM As(III) for 12 h, followed by qRT-PCR and immunoblot analysis. The asterisk indicates a significant difference between p300^+/+ and p300^−/− cells within the same treatment group. (I and K) MEF CBP^+/+ and CBP^−/− cells were treated with 10 μM As(III) for 12 h, followed by qRT-PCR and immunoblot analysis. The asterisk indicates a significant difference between CBP^+/+ and CBP^−/− cells within the same treatment group.

FIG. 7.

Acetylation augments promoter-specific DNA binding of Nrf2. (A) Nrf2 18KR has decreased binding affinity to NQO1 ARE compared to Nrf2 WT. HEK293T cells expressing the indicated HA-Nrf2 and p300 were lysed. The whole-cell lysates were incubated with either WT (wt) or mutated (mu) biotinylated ARE from the NQO1 promoter region. The protein-DNA-binding complexes were pulled down by streptavidin beads and analyzed by immunoblotting with anti-HA (α-HA) antibodies. Tub, tubulin. (B) Nrf2 18KR has decreased binding affinity to the GCLC ARE, but not to the HO-1 ARE, compared to Nrf2 WT. HEK293T cells expressing the indicated HA-Nrf2 and p300 were subjected to DNA-binding assays with the AREs from GCLC and HO-1 as described for panel A. Tub, tubulin. (C) The same DNA-binding assay was repeated in MDA-MB-231 cells. (D) Protein input for the mobility shift assay. Two hundred nanograms of purified GST-tagged Nrf2 WT or Nrf2 18KR protein was incubated with purified p300 protein in the absence or presence of acetyl-CoA in an in vitro 50-μl acetylation reaction; 1/10 of the reaction products were analyzed by immunoblotting with acetylation-specific antibody and Nrf2 antibody. Ac, acetylated. (E) A mobility shift assay was performed with ³²P-labeled ARE probes in the presence of 2 μl of in vitro-translated MafG protein (Promega kit) and 4 μl of the indicated Nrf2 proteins from the in vitro acetylation reaction in panel D. (F) ChIP analysis was performed on HEK293T cells either treated with 20 μM As(III) or overexpressing the indicated HA-Nrf2 and p300. ChIP analysis was as described in the legend to Fig. 2A, with either IgG or anti-Nrf2 antibody. DNA fragments containing AREs of NQO1, TXNRD1, HO-1, and GCLM were amplified by PCR using specific primer sets and visualized on an agarose gel. The tubulin promoter region was also amplified to serve as a negative control. (G) Total cell lysates from HEK293T cells prepared in parallel with cells for the ChIP assay were subjected to immunoblot analysis with anti-HA antibodies. (H) Total DNA input was examined as for panel F. (I) The precipitated DNA fragments from the ChIP assay were quantified by qPCR. DNA precipitated by anti-Nrf2 antibodies in the mock-treatment group was set as 1. The error bars indicate the standard deviations from three experiments. The relative units of DNA amounts were plotted on the same scale for convenient comparison. The asterisks indicate significant differences from Nrf2 WT.