Nrf2 is acetylated by p300/CBP in vivo and in vitro.
To determine if endogenous Nrf2 is acetylated, HCT116 cells cultured in 150-mm dishes were directly lysed under denaturing conditions to inactivate deacetylases and to disrupt protein-protein interactions. Diluted cell lysates were subjected to immunoprecipitation by normal immunoglobulin G (IgG) or anti-Nrf2 antibodies, followed by immunoblotting with antibodies specific for acetylated lysines. Acetylated Nrf2 was detectable under basal conditions (Fig. , lane 2). To assess whether acetylation is upregulated in response to oxidative stress, cells were treated for different times with 20 μM sodium arsenite [As(III)], an environmental carcinogen and a strong Nrf2 inducer. There was a steady increase of Nrf2 acetylation, along with an accumulation of Nrf2 proteins, after As(III) treatment (Fig. ). Since the accumulated Nrf2 protein level is always coupled with increased Nrf2 nuclear entry, it is conceivable that the observed enhancement in acetylation is due to prompt acetylation of Nrf2 by acetyltransferase(s) within the nucleus, which contribute to the quick activation of Nrf2 in response to oxidative stress.
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) (more ...)
To identify which acetyltransferase(s) acetylates Nrf2, HEK293T cells were cotransfected with vectors expressing HA-tagged Nrf2 and different HATs, including p300, CBP, and P/CAF. The cells were lysed under denaturing conditions to preserve the modification. The diluted cell lysates were subjected to immunoprecipitation with anti-HA antibodies, followed by immunoblotting with antibodies specific for acetylated lysine. Nrf2 was acetylated only by p300 and CBP, but not P/CAF (Fig. , lanes 2, 4, and 5). A DY point mutant of p300 failed to acetylate Nrf2 (Fig. , lane 3) (21
). p300 was the most potent in acetylating Nrf2 and therefore was chosen for the subsequent studies. Enhanced Nrf2 expression in the presence of p300/CBP was observed (Fig. , lanes 2 and 5). This was likely due to indirect effects of p300/CBP on the transcription of the Nrf2 transgene, since similar observations were made with other transgenes carried in the same expression vector. This notion is further supported by the fact that Nrf2 protein half-lives were not changed when p300 was overexpressed (see Fig. S4 in the supplemental material).
To determine if p300 is self-sufficient in acetylating Nrf2, an in vitro approach was utilized. Purified GST-tagged Nrf2 proteins were incubated with purified p300 proteins in the presence of 14C-labeled acetyl-CoA. Acetylation of Nrf2 was detected by autoradiography (Fig. , top, lane 2). The sample with GST alone (lane 1) did not give any positive signals, indicating that the acetylation reactions were specific for Nrf2. These results indicate that Nrf2 is a bona fide substrate of p300.
In the in vitro acetylation reaction, a series of Nrf2 deletion mutants were constructed in an effort to identify the major acetylation regions. The boundary of each domain is defined in Fig. , bottom. Deletion of the Neh1 DNA-binding domain almost completely abolished acetylation of Nrf2, suggesting that the Neh1 domain contains the major acetylation sites (Fig. , lane 8). Deletion of Neh4 and Neh5 also significantly decreased the acetylation levels of Nrf2 (Fig. , lanes 5 and 6), which is consistent with the finding that Neh5, in coordination with Neh4, mediates the binding of Nrf2 to p300 (Fig. , lanes 4 and 5). Deletion of other domains did not significantly alter the overall acetylation levels of Nrf2. Taken together, these results demonstrate that Nrf2 is acetylated by p300/CBP both in vivo and in vitro. The acetylation of Nrf2 is enhanced in response to arsenite exposure, and the Neh1 DNA-binding domain contains the majority of acetylated lysine residues.
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). (more ...) Nrf2 associates with p300.
ChIP analysis was performed to assess specific and coordinated recruitment of p300 and Nrf2 to the ARE in response to oxidative stress. HCT116 cells were either left untreated or treated with 20 μM As(III) for 4 h before being cross-linked and harvested. The cell lysates were subjected to immunoprecipitation with either anti-Nrf2 or anti-p300 antibody (with normal serum IgG as a negative control). The genomic DNA fragments bound to either Nrf2 or p300 proteins were recovered and quantified by qPCR using primer pairs specific for the NQO1-ARE region or the tubulin promoter region as negative controls. The amounts of NQO1-ARE bound to p300 or Nrf2 were increased nearly 10-fold compared to the mock-treated samples in response to As(III) treatment, while the amounts of tubulin promoter DNA bound to p300 or Nrf2 remained unchanged (Fig. ). This demonstrated that endogenous p300, along with Nrf2, was specifically recruited to the ARE-containing promoters in response to arsenite.
Interaction between Nrf2 and p300 was tested in HEK293T cells cotransfected with expression vectors for Flag-tagged p300 and HA-tagged Nrf2. Cell lysates were immunoprecipitated with anti-Flag M2 matrix. Both the immunoprecipitates and the total cell lysates were analyzed by immunoblotting with anti-HA antibodies. Nrf2 was detected in the Flag-p300 immunoprecipitates, while an unrelated protein, Cul4, was not detected (Fig. ), consistent with the previous finding for interactions between Nrf2 and CBP (29
Direct interactions between Nrf2 and p300 and interaction domains were assessed. p300 contains five major conserved domains, C/H1, KIX, BROMO, C/H2, and C/H3 (Fig. , top), whereas Nrf2 contains six major conserved domains, Neh2, Neh4, Neh5, Neh6, Neh1, and Neh3 (Fig. , bottom). The Neh2 domain is the “degron” that is bound and ubiquitinated by Keap1 (24
). The Neh4 and Neh5 domains are transactivation domains (29
). Neh1 contains a CNC (“cap'n'collar”)-type basic leucine zipper structure responsible for dimerization with Maf proteins and DNA binding (22
To identify the p300 binding domain(s) in Nrf2, a series of domain-specific deletion mutants of Nrf2 were used. GST-tagged Nrf2 proteins containing the deletions were purified. Equal amounts of GST-Nrf2 proteins were used for GST pull-down analysis with p300 proteins that were radiolabeled with [35
S]methionine. Consistent with a previous report (29
), the Neh4 and Neh5 domains of Nrf2, but mainly the Neh5 domain, are required for interaction with p300 (Fig. , lanes 4 and 5). In another set of experiments, a series of truncated forms of p300 were constructed and radiolabeled with [35
S]methionine (Fig. , top). These 35
S-labeled p300 proteins were subjected to GST pulled down with GST-tagged Nrf2 WT proteins, followed by SDS-PAGE and autoradiography (Fig. , bottom). Interactions with Nrf2 were mediated by p300 C-terminal positions 1725 to 2414 (Fig. , lanes 7). Radiolabeled luciferase served as a negative control to show the specificity of the in vitro interaction (Fig. , lanes 1). Collectively, these data suggest that the Neh4 and Neh5 domains of Nrf2 directly interact with the C/H3-containing C terminus of p300.
Identification of multiple acetylated lysines within the Neh1 DNA-binding domain of Nrf2.
Figure shows that the Neh1 domain (amino acids 434 to 561) of Nrf2 contains major acetylation sites. There are 18 lysines within this domain in human Nrf2 (see Fig. ). To further determine which lysine residues are major acetylation sites, GST-tagged Nrf2 with subdomain deletions within the Neh1 domain was used for the in vitro acetylation assay. None of subdomain deletion mutants were able to abolish Nrf2 acetylation to the same degree as the Neh1 deletion mutant, demonstrating that multiple lysine residues in Neh1 are acetylated (Fig. , compare lanes 4, 5, and 6 to lane 3).
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 (more ...)
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 (more ...)
Next, MS was used to identify exact acetylated lysine residues on Nrf2. HEK293T cells were transfected with expression vectors for HA-Nrf2 and p300. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies, followed by SDS-PAGE and Coomassie staining (see Fig. S1 in the supplemental material). The bands containing Nrf2 were isolated and analyzed by LC-MS/MS. Multiple acetylated lysines were detected (Fig. ; see Fig. ). Almost all acetylated lysines were within the Neh1 domain, which is consistent with the observation that deletion of Neh1 almost completely abolished acetylation of Nrf2 in the in vitro acetylation assay (Fig. and ).
Functional redundancy among different acetylation sites.
To elucidate the function of acetylation, lysine-to-arginine (K→R) substitutions were constructed in all lysine clusters within the Neh1 domain, and their effects on the overall Nrf2 acetylation levels (Fig. ) and Nrf2 transcriptional activity (Fig. ) were tested. As expected, none of the substitution constructs completely abolished acetylation of Nrf2, although a decrease in acetylation levels was visible for the K438R/K443R/K445R and K533R/K536R/K538R clusters (Fig. , top, lane 2 and lane 7). None of the cluster mutations had significant impacts on Nrf2 transcriptional activities, as measured with the luciferase reporter gene (Fig. ). This observation was confirmed by qRT-PCR analysis of NQO1 and HO1 mRNA levels from the Nrf2−/− MEFs expressing the cluster mutants of Nrf2 (see Fig. S2 in the supplemental material for one example). Since none of the K→R mutations within each lysine cluster alter the transcriptional activity of Nrf2, it is likely that there is intrinsic functional redundancy among acetylations on different sites. In other words, Nrf2 partial acetylation is sufficient for Nrf2 to reach its maximum transcriptional activity.
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 (more ...) Acetylation of the Neh1 domain does not regulate the stability of Nrf2 proteins.
To resolve the redundancy issue, a mutant with combined arginine replacement of all 18 lysines within the Neh1 domain was constructed and named 18KR. In addition, mutants with two other combined mutations of the six lysines within the CNC homology region and the 12 lysines within the bZIP (basic leucine zipper) region were also constructed and were named 6KR and 12KR, respectively (Fig. ).
The effects of the combined mutations on overall Nrf2 acetylation levels were tested. The 18KR mutation almost completely abolished acetylation of Nrf2 by p300 (Fig. , lane 3). The 6KR mutation seemed to decrease acetylation levels more than 12KR (Fig. , lanes 4 and 5), implying that the CNC homology region may be more heavily acetylated than the bZIP region. However, a conclusion about the relative acetylation levels of each site is difficult to draw because the acetylation-specific antibody may have a certain bias toward specific amino acid contexts of the acetylated residues when different acetylated lysine-specific antibodies are compared (see Fig. S3 in the supplemental material). Nevertheless, the 18 lysines in the Neh1 domain were established as the major acetylation sites by both in vivo and in vitro acetylation assays. Therefore, the combined 18KR mutant was chosen for the functional study of Nrf2 acetylation.
Because Nrf2 is mainly regulated by Keap1 at the levels of protein ubiquitination and degradation, it is important to know whether acetylation affects Nrf2 protein stability. To this end, the half-lives of Nrf2 proteins were measured under both basal and As(III)-treated conditions. MDA-MB-231 cells were chosen for this analysis because it is well established that the ubiquitination and stability of Nrf2 proteins are very sensitive to oxidative stress in this cell line (62
). MDA-MB-231 cells expressing Nrf2 WT or Nrf2 18KR, along with Keap1 and p300, were either left untreated or treated with 20 μM As(III) for 3 h, followed by the addition of 50 μM cycloheximide to block protein synthesis. Cells were then lysed at different time points, and the Nrf2 protein levels were determined by immunoblotting. There was no significant difference in Nrf2 half-lives between WT and 18KR under both basal and As(III)-treated conditions (Fig. ). Consistent with this result, overexpression of p300 did not cause an increase in the Nrf2 protein half-life (see Fig. S4 in the supplemental material). To assess if 18KR affects Nrf2 ubiquitination, MDA-MB-231 cells expressing the HA-Nrf2 WT or 18KR mutant were cotreated with 20 μM As(III) and 10 μM MG132 for 4 h and were then lysed under denaturing conditions. The diluted cell lysates were subjected to immunoprecipitation with anti-HA antibodies, followed by immunoblotting with antiubiquitin antibodies. Ubiquitin conjugation on Nrf2 WT or Nrf2 18KR was reduced to similar levels by As(III) (Fig. ). These results demonstrated that acetylation has no effect on Nrf2 ubiquitination or degradation.
Considering the importance of both Nrf2 nuclear import and Keap1-mediated Nrf2 nuclear export in turning the Nrf2 signaling pathway on and off (26
), the effects of 18KR on the subcellular localization of Nrf2 and on Keap1-Nrf2 interactions were tested. MDA-MB-231 cells expressing Nrf2 WT or 18KR and p300 were subjected to indirect immunofluorescent staining. Both Nrf2 WT and 18KR localized mainly in the nucleus with p300 (Fig. ). Furthermore, Nrf2 WT and 18KR bound to Keap1 equally well (Fig. ). Collectively, these results indicate that 18KR almost completely abolishes acetylation on Nrf2. Acetylation on the Nrf2 Neh1 domain does not regulate Nrf2 ubiquitination or protein stability, nor does it seem to contribute to the regulation of Nrf2 subcellular localization.
Acetylation plays a positive role in the transcriptional activity of Nrf2.
The transcriptional activity of the Nrf2 18KR mutant was measured in HEK293T cells cotransfected with expression vectors for either an NQO1-ARE-dependent (Fig. ) or GSTA1-ARE-dependent (see Fig. S5 in the supplemental material) firefly luciferase reporter gene, Nrf2 WT or 18KR, and p300. Luciferase reporter gene activities were measured. Nrf2 18KR showed a substantial decrease in its ability to drive the expression of both NQO1-ARE- and GSTA1-ARE-dependent luciferase, indicating that loss of acetylation at these 18 lysine residues impaired the transcriptional activity of Nrf2 (Fig. ). Although not as significant as the 18KR mutation, the 6KR mutation and the 12KR mutation also marginally decreased Nrf2-dependent transcription (Fig. ). An aliquot of cell lysates from the reporter gene assay was subjected to immunoblotting to ensure that the Nrf2 protein levels were equivalent among the different Nrf2 mutants (Fig. , top). Similar results were obtained in MDA-MB-231 cells, suggesting that the observation is not specific to HEK293T cells (see Fig. S6 in the supplemental material).
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 (more ...)
Next, the differential effects of oxidative stress on cells expressing Nrf2 WT versus Nrf2 18KR were analyzed in HEK293T cells overexpressing either Nrf2 WT or Nrf2 18KR, along with Keap1 and p300. Cells were treated with 20 μM As(III) for 12 h, and the NQO1-ARE-dependent luciferase reporter gene expression levels were measured (Fig. ). Compared to cells expressing Nrf2 WT, cells expressing Nrf2 18KR had an obviously dampened ARE-dependent transcription induction in response to arsenite challenge, although Keap1-mediated control of the total Nrf2 protein level was not affected (Fig. , bottom).
qRT-PCR was performed to confirm that acetylation positively regulates the transcriptional activity of Nrf2. mRNA was extracted from HEK293T cells expressing Nrf2 WT or Nrf2 18KR (Fig. , left). mRNA was also analyzed in cells that were either left untreated or treated with 20 μM As(III) for 12 h (Fig. , right). Overexpression of Nrf2 WT caused a two- to threefold increase in the mRNA levels of NQO1 and TXNRD1 compared to the mock-transfected control; the same increase in the mRNA levels of the two genes were observed upon As(III) treatment compared to the mock-treated control (Fig. , top two rows). This indicated that overexpression of Nrf2 mimicked the induction of endogenous Nrf2 by As(III) very well. However, As(III) caused a more dramatic induction of GCLM and HO-1, especially HO-1, compared to overexpression of Nrf2 WT (Fig. ). This is likely due to the fact that As(III) may also induce Nrf2-independent signal pathways that function synergistically with Nrf2 to transactivate these genes (1
). Consistent with the previous findings, As(III) did not change the mRNA levels of Nrf2 (Fig. ) (62
). In line with the results from the luciferase reporter gene assay, the 18KR mutant significantly compromised the induction of NQO1, TXNRD1, and GCLM (Fig. , left), demonstrating that acetylation plays a positive role in Nrf2-dependent transcription. Interestingly, the transcriptional activity of Nrf2 18KR on HO-1 was comparable to that of Nrf2 WT (Fig. , left), suggesting that acetylation of Nrf2 may preferentially regulate certain Nrf2 downstream genes. A parallel set of samples were subjected to immunoblot analysis to ensure that the cells were expressing equal amounts of Nrf2 WT and Nrf2 18KR (Fig. ).
To ensure that the observations made in HEK293T cells were not specific to the cell type, the mRNA levels of NQO1, TXNRD1, HO-1, GCLM, and Nrf2 were measured by qRT-PCR in Nrf2−/− MEFs expressing HA-Nrf2 WT or 18KR and p300. Consistent with the results obtained in HEK293T cells, acetylation of Nrf2 was found to be important in transactivating NQO1, TXNRD1, and GCLM, but not HO-1 (Fig. ).
The gene-specific effects of Nrf2 acetylation were further confirmed by comparing HCT116 p300−/− to p300+/+ cells and MEF CBP−/− to CBP+/+ cells (Fig. ). Loss of either p300 or CBP HAT activity led to a significant decrease in NQO1 induction in response to arsenite treatment, while induction of HO-1 remained unchanged (Fig. ). Nrf2 protein levels were elevated to similar extents upon arsenite exposure regardless of the p300/CBP status (Fig. ), which is consistent with the observation that p300/CBP does not regulate the protein stability of Nrf2. These results provide independent support for the notion that acetylation of Nrf2 regulates its transcriptional activity in a gene-specific manner.
Acetylation augments promoter-specific DNA binding of Nrf2.
Given the fact that acetylation mainly occurs in the DNA-binding domain, the effects of Nrf2 acetylation on ARE DNA binding were analyzed in vitro. Biotinylated NQO1-ARE DNA was incubated with whole-cell lysates from HEK293T cells expressing Nrf2 WT or the indicated mutant. ARE-bound Nrf2 proteins were pulled down by streptavidin beads and detected by immunoblotting them with anti-HA antibodies. Nrf2 18KR, but not 6KR or 12KR, had a marked reduction in ARE binding compared to the WT (Fig. , lanes 2 to 5). An ARE fragment with mutations in its core region was included as a control for binding specificity (Fig. , lane 1). These results demonstrate that acetylation of Nrf2 in the Neh1 DNA-binding domain promotes interaction between Nrf2 and the ARE.
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 (more ...)
Since the function of acetylation seems to be promoter specific, the binding of Nrf2 18KR to the AREs from GCLC and HO-1 was analyzed similarly. Consistent with the results of the qRT-PCR analysis (Fig. ), acetylation of Nrf2 promoted the binding of Nrf2 to the GCLC ARE, but not the HO-1 ARE, in HEK293T cells (Fig. ). The same finding was obtained when MDA-MB-231 cells were used (Fig. ). Next, purified Nrf2 proteins were subjected to electrophoretic mobility shift assays (Fig. ). GST-tagged Nrf2 WT and Nrf2 18KR proteins were purified from bacteria and incubated with purified Flag-tagged p300 proteins in an in vitro acetylation reaction, as shown in Fig. . Immunoblot analysis confirmed efficient conjugation of the acetyl group on Nrf2 only in the presence of acetyl-CoA, while the 18KR mutation almost completely abolished Nrf2 acetylation (Fig. ). In the mobility shift assay with the radiolabeled NQO1-ARE probe, the acetylated Nrf2 WT protein showed enhanced ability to form the ARE-binding heterodimer with in vitro-translated MafG, compared to unacetylated Nrf2 WT and Nrf2 18KR (Fig. , left, lanes 3 to 6). On the other hand, Nrf2 18KR retained the ability to bind the ARE, suggesting that the K→R mutation itself does not affect DNA recognition or dimerization between Nrf2 and small Maf proteins. When HO1-ARE was used, acetylated Nrf2 WT did not show increased DNA binding compared to unacetlyated Nrf2 WT (Fig. , right, lanes 3 and 4). These results demonstrate that acetylation of Nrf2 enhances its ARE binding in a promoter-specific manner.
To verify the above-mentioned findings obtained in vitro, the ARE-binding activity of 18KR was analyzed in vivo. ChIP analysis was performed in HEK293T cells either treated with 20 μM As(III) or overexpressing HA-Nrf2 WT or 18KR, as indicated, along with p300. Immunoprecipitation was performed with either IgG or anti-Nrf2 antibody. DNA fragments containing AREs from NQO1, TXNRD1, HO-1, and GCLM were amplified by PCR using specific primer sets and visualized on agarose gels (Fig. ). The tubulin promoter region was also amplified to serve as a negative control. Equal amounts of Nrf2 proteins and genomic DNA were used for each sample (Fig. ). The precipitated DNA from the ChIP assay was quantified by qPCR. The reading for the amount of DNA precipitated by anti-Nrf2 antibodies in the mock-treatment group was set as 1 (Fig. ). As(III) induced a seven- to ninefold increase in binding of endogenous Nrf2 to NQO1-ARE and TXNRD1-ARE; a comparable increase was observed in cells expressing exogenous WT Nrf2 (Fig. , left). This suggests that overexpression of Nrf2 mimics As(III)-induced DNA binding of endogenous Nrf2 to some degree. Abolishing acetylation in the DNA-binding domain significantly decreased binding of Nrf2 to the AREs from NQO1, TXNRD1, and GCLM, but not the HO-1 ARE (Fig. ). These results demonstrate that acetylation augments promoter-specific DNA binding of Nrf2 in vivo.