Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

Inhibition of the proteasome leads to proteotoxic stress, which is characterized by the buildup of ubiquitinated proteins that cannot be degraded properly. The transcription factor Nrf1 (also called NFE2L1) counteracts proteotoxic stress by inducing transcription of proteasome subunit genes, resulting in the restoration of proteasome activity. Further understanding of the Nrf1 pathway is therefore of interest in both neurodegeneration, where proteasome activity could be enhanced, and cancer, where suppression of this pathway could potentiate the cell-killing effect mediated by proteasome inhibitor drugs. Here, to identify novel regulators of Nrf1, we performed an RNAi screen in an engineered cell line, reporting on Nrf1 transcriptional activity. In addition to validating known regulators, we discovered that the AAA+ ATPase RUVBL1 is necessary for Nrf1's transcriptional activity. Given that RUVBL1 is part of different multisubunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin-regulatory complex is essential for Nrf1-dependent transcription of proteasome genes. Consistent with these observations, Nrf1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Overall, our study provides a framework for manipulating the TIP60–Nrf1 axis to alter proteasome function in various human diseases, including cancer.

Inhibition of the proteasome leads to proteotoxic stress, which is characterized by the buildup of ubiquitinated proteins that cannot be degraded properly. The transcription factor Nrf1 (also called NFE2L1) counteracts proteotoxic stress by inducing transcription of proteasome subunit genes, resulting in the restoration of proteasome activity. Further understanding of the Nrf1 pathway is therefore of interest in both neurodegeneration, where proteasome activity could be enhanced, and cancer, where suppression of this pathway could potentiate the cellkilling effect mediated by proteasome inhibitor drugs. Here, to identify novel regulators of Nrf1, we performed an RNAi screen in an engineered cell line, reporting on Nrf1 transcriptional activity. In addition to validating known regulators, we discovered that the AAA؉ ATPase RUVBL1 is necessary for Nrf1's transcriptional activity. Given that RUVBL1 is part of different multisubunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin-regulatory complex is essential for Nrf1-dependent transcription of proteasome genes. Consistent with these observations, Nrf1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Overall, our study provides a framework for manipulating the TIP60 -Nrf1 axis to alter proteasome function in various human diseases, including cancer.
Protein homeostasis or proteostasis refers to the sum total of highly regulated and often interconnected cellular processes that include synthesis, folding, and destruction of proteins (1). The ubiquitin-proteasome system plays a pivotal role in proteostasis by orchestrating protein degradation. A central component of the ubiquitin-proteasome system is the 26S proteasome, a multisubunit molecular machine with proteolytic activity that is directly responsible for degrading its protein substrates, which are often tagged with polyubiquitin signals. The 26S proteasome, in turn, is composed of a 20S core that serves as the main proteolytic chamber and is capped on one or both ends by the 19S regulatory particle (2). Cellular proteasome activity is regulated at multiple levels, including transcription, translation, assembly, and posttranslational modifications of subunits (3).
We and others have demonstrated previously that the transcription factor nuclear factor erythroid-derived 2-related factor 1 (Nrf1, also called NFE2L1) functions as a master regulator of proteasome subunit genes (4 -7). Nrf1 belongs to the cap 'n' collar basic region leucine zipper family of transcription factors, which also includes p45 Nfe2, Nrf2, and Nrf3 (8,9). Although Nrf2, the most studied of the cap 'n' collar basic region leucine zipper factors, responds to oxidative stress (10), Nrf1 takes center stage when cells experience proteotoxic stress (4,6). In response to proteasome inhibition, the Nrf1 pathway is activated, leading to de novo synthesis of proteasome subunit genes, resulting in "bounce back" or recovery of proteasome activity (4). Apart from responding to proteotoxic stress, Nrf1 is also responsible for basal transcription of proteasome genes in a tissue-specific manner. Nrf1-deficient mouse neuronal cells display accumulation of ubiquitinated proteins and decreased proteasome activity concomitant with a reduction in proteasome gene expression (11). A similar phenomenon is also evident in hepatocytes from mice that have a liver-specific knockout of Nrf1 (12).
From a molecular perspective, Nrf1 is co-translationally inserted into the endoplasmic reticulum (ER) 2 membrane in such a way that only a small portion of its N terminus protrudes into the cytosol, whereas the bulk of its polypeptide, including the transcriptional activation and DNA-binding domains, is embedded in the ER lumen. Thus, Nrf1 depends on the action of the ATPase p97 to be retrotranslocated to the cytosolic side either to be constitutively degraded in the absence of proteotoxic stress or activated and mobilized to the nucleus when cells are subjected to proteasome inhibition (13). Activation of Nrf1 also requires its deglycosylation, which is mediated by a p97associated N-glycanase enzyme, NGLY1 (14), and a proteolytic cleavage step that serves to trim the N terminus of Nrf1, which harbors the transmembrane domain. The protease involved in this process was recently discovered to be DDI2, a member of the aspartic family of proteases (15,16).
Proteasome inhibitors are currently being used in the clinic against multiple myeloma and mantle cell lymphoma (17). One way to make this therapy more effective would be to devise strategies to block the Nrf1 pathway, thus impairing the ability of the cancer cells to recover their proteasome activity in response to inhibition of the proteasome. In support of this notion, a recent study demonstrated that attenuation of Nrf1 activation via p97 inhibition increased the efficacy of proteasome inhibitor drugs in multiple myeloma models (18). On the flip side, in certain proteinopathies, especially neurodegenerative diseases where the neurons display ubiquitinated protein aggregates, indicative of proteasome dysfunction, there could be interest in enhancing the Nrf1 pathway to directly increase proteasomal capacity (3). Thus, further understanding the Nrf1-proteasome axis could help pave the way for devising novel therapeutic agents targeted at different human diseases. In this study, we identified a requirement for the TIP60 chromatin-modifying complex in enabling Nrf1-mediated proteasomal gene expression in response to inhibition of the proteasome. This expands our view of the inner workings of Nrf1mediated transcription and could offer strategies to manipulate this pathway in human diseases.

RUVBL1 is necessary for Nrf1-mediated transcriptional response during proteotoxic stress
To identify factors that regulate Nrf1 activity under conditions of proteasome inhibition, we constructed a cell-based screening system in WT NIH-3T3 mouse fibroblasts. This cell line was engineered to stably express firefly luciferase under the control of 8x antioxidant response element (ARE; the sequence to which Nrf1 is known to bind (8)) repeats coupled to a minimal promoter. In addition, as a control, these cells also expressed Renilla luciferase driven by the human phosphoglycerate kinase (hPGK) promoter (Fig. 1A). The resultant screening system is referred to as WT 8xARE-Luc, whereas a similar system in an NIH-3T3 Nrf1-deficient background (19) is referred to as Nrf1 Ϫ/Ϫ 8xARE-Luc. We then confirmed the status of Nrf1 in these two cell lines. Treatment with the proteasome inhibitor carfilzomib (CFZ) resulted in the accumulation of Nrf1 p120 (precursor) and p110 (processed form; transcriptionally active) in the WT 8xARE-Luc but not in Nrf1 Ϫ/Ϫ 8xARE-Luc cell line, as expected (Fig. 1B). More importantly, although the WT 8xARE-Luc cells showed a dose-dependent increase in normalized luciferase activity (ratio of firefly to Renilla luciferase values) in response to CFZ, the Nrf1 Ϫ/Ϫ 8xARE-Luc cell line showed no such increase (Fig. 1C), implying that this effect is Nrf1-dependent. In light of our previous study, which demonstrated a strict reliance of Nrf1 function on p97 ATPase activity (13), we used NMS-873, an inhibitor of p97 (20), to further test our screening system. NMS-873 was able to effectively attenuate CFZ-induced increased luciferase activity in the WT 8xARE-Luc cell line (Fig. 1D), additionally validating our screening system.
Next, we used our WT 8xARE-Luc cell system in an RNAi screen where we used a focused library of siRNAs that target various epigenetic regulators and possible Nrf1 pathway influencers gleaned from public databases and the literature (8,9,21,22). As positive controls, we used Nrf1-specific siRNAs and also siRNAs targeting known Nrf1 regulators such as p97 and the protease DDI2. In the screen, we found that depletion of any of the controls (Nrf1, p97, or DDI2) strongly attenuated the CFZinduced increase in luciferase activity (Fig. 1E). Of the other test genes in the library, we observed that knockdown of RUVBL1 elicited an effect that was quite similar to the ones produced by the three positive control siRNAs (Fig. 1E). The results from the entire screen are listed in Table S1. RUVBL1 (also called Pontin/TIP49) and the related protein RUVBL2 (also called Reptin/TIP48) are AAAϩ ATPases (ATPases associated with diverse cellular activities) that most often occur together and participate in various cellular functions, including transcriptional regulation (23). Given that we identified RUVBL1 as a "hit" in our screen, it most likely is involved in Nrf1-mediated transcriptional activation of its target genes. To further confirm the involvement of RUVBL1 in the Nrf1 pathway, we examined the changes in proteasome gene transcription in response to CFZ treatment in control and siRUVBL1-treated NIH3T3 cells. Although the control cells displayed a functional Nrf1 pathway by up-regulating representative proteasome subunit genes as expected, cells with RUVBL1 depletion were profoundly defective in this response (Fig. 1F). Under these circumstances, we did not see a significant difference in Nrf1 protein levels or its ability to be processed into the p110 form after RUVBL1 knockdown (Fig. 1G). Taken together, these results reinforce a model where RUVBL1 could regulate the transcriptional activity of Nrf1, as seen for certain other transcription factors such as c-Myc, ␤-catenin, E2F1, NF-B, and hypoxia-inducible factor 1␣ (HIF1␣) (23). Interestingly, in the control immunoblots, to verify knockdown efficiency in this experiment, we noticed that, apart from RUVBL1, the protein levels of RUVBL2 were also attenuated in lysates derived from siRUVBL1-treated samples (Fig. 1G). This is consistent with previous observations where depletion of either factor results in a concomitant decrease in the other, in line with the notion that RUVBL1 and RUVBL2 together need to be incorporated into a hexameric complex to attain stability (24 -26).
To rule out any cell type-specific bias, we tested other cell lines of various origin: HCT116 colon cancer, MDA-MB-231 breast cancer, and MIA-PaCa2 pancreatic cancer cells. Regardless of the cell type, we observed that, although proteasome inhibition up-regulated the transcript levels of the tested proteasome genes, depletion of RUVBL1 (Fig. S1) severely compromised this effect ( Fig. 2A). Similar to our observation with NIH-3T3 cells, in all of these cancer cell lines, depletion of RUVBL1 resulted in decreases in the protein levels of both RUVBL1 and RUVBL2 (Fig. 2B).

The TIP60 complex is required for Nrf1-dependent transcription
Although, in some instances, RUVBL1 and RUVBL2 act as a heterohexamer to regulate gene transcription (27), in most cases these proteins are part of multisubunit complexes with roles in transcription (23). The most prominent RUVBL1/2containing complexes are INO80, SRCAP, TIP60, and R2TP (Fig. 3A). To test whether one or more of these complexes could be involved in mediating Nrf1-dependent transcription under

Nrf1-dependent transcription requires TIP60
proteotoxic stress, we proceeded to deplete a key component in each complex (the INO80 subunit in the INO80 complex, the SRCAP subunit in the SRCAP complex, the TIP60 subunit in the TIP60 complex, and the PIH1 subunit in the R2TP complex, all of which are indicated by arrows in Fig. 3A) in the WT 8xARE-Luc cell line. We observed that depletion of TIP60, but not any of the other subunits tested (Fig. S2), recapitulated the effect of RUVBL1 knockdown in blocking the proteasome inhibitor-mediated increase in luciferase activity (Fig. 3B). Likewise, when we measured transcriptional up-regulation of proteasome genes in response to proteasome inhibition, depletion of TIP60 attenuated this response, similar to what was  (n ϭ 3). G, NIH-3T3 cells treated as described in F were used for immunoblotting with antibodies against Nrf1, RUVBL1, RUVBL2, ubiquitin, and ␤-actin as indicated. The experiments were performed three independent times, and a representative blot is shown.

Nrf1-dependent transcription requires TIP60
observed with RUVBL1 depletion (Fig. 3C). Taken together, our results attribute a critical role of the TIP60 complex in Nrf1-dependent transcriptional response to proteotoxic stress. Interestingly, in our control immunoblots, we observed that depletion of RUVBL1 also decreased TIP60 and, to a certain extent, INO80 and PIH1 protein levels (Fig. 3D). This is consistent with an earlier study that indicated that the RUVBL1/2 complex is essential for assembly of a functional TIP60 complex (28).

Interaction between Nrf1 and the TIP60 complex
Based on the known biology of TIP60 as a transcriptional co-activator (29 -33), it is possible that this complex interacts with Nrf1 on the chromatin to aid in its transcription function. To verify this hypothesis, we compared the ARE-containing promoter regions of the Nrf1 target genes PSMA7, PSMB7, and PSMD12 in WT and Nrf1 Ϫ/Ϫ NIH3T3 cells using ChIP assays. Under the condition of proteasome inhibition with CFZ, we could observe recruitment of Nrf1 in the promoter regions of proteasome genes in WT but not Nrf1 Ϫ/Ϫ cells, as expected (Fig. 4A). Importantly, we observed a similar trend for RUVBL1 and TIP60 subunits, implying Nrf1-dependent recruitment of these factors to the proteasome gene promoters. Consistent with this notion, we were also able to detect interaction between Nrf1 and RUVBL1/2 using co-immunoprecipitation assays (Fig. 4B).

Functional consequences of depletion of the TIP60 complex during proteotoxic stress
Our previous work conclusively demonstrated a cellular requirement for Nrf1 in recovering from inhibition of the proteasome (4). To verify whether this effect is recapitulated in cells deficient in RUVBL1 or TIP60, we performed proteasome recovery assays (Fig. 5A). Here we exploited the differences in the nature of binding of proteasome inhibitors to the active site of the proteasome. Although MG132 is a completely reversible proteasome inhibitor, CFZ binds to the proteasome active site in a covalent/irreversible manner. When cells are treated with MG132 briefly and then washed off, recovery of the proteasome activity is achieved by simple dissociation of the drug from the proteasome active site as well as via the action of the Nrf1 pathway during the time when the proteasome was inhibited. In the case of CFZ, given its covalent binding to the proteasome, the only way for the cells to recover after drug washout is by invoking the Nrf1 pathway to synthesize new proteasomes. Thus, impairing Nrf1 function in this context will severely undermine the ability of the cells to recover proteasome activity (4). To test  (n ϭ 3). B, the cell lines above were treated similarly as described in A and subjected to immunoblotting with antibodies specific for RUVBL1, RUVBL2, Nrf1, ubiquitin, and ␤-actin. The experiments were performed three independent times, and a representative blot is shown.

Nrf1-dependent transcription requires TIP60
these predictions in our study, we compared WT NIH-3T3 cells with siRUVBL1-or siTIP60-transfected cells for their relative ability to recover proteasome activity after pulse treatment with MG132 or CFZ at appropriate concentrations to achieve about 90% inhibition of proteasome activity in 1 h. Following drug washout, we found that cells depleted of RUVBL1 or TIP60 were able to recover their proteasome activity after MG132 pulse treatment but were partially impaired in their ability to do so when CFZ was employed (Fig. 5B). These results underscore the importance of RUVBL1 and TIP60 in mediating the recovery of proteasome activity after inhibition of the proteasome.
Irresolvable proteotoxic stress could lead to cell death, particularly in cancer cells, which are thought to be over-reliant on proteasome function (34,35). Our previous studies have demonstrated potentiation of apoptosis in Nrf1-impaired cancer cells that were treated with proteasome inhibitors (4,14). To evaluate the contribution of the TIP60 complex in this context in cancer cells, we first exposed control and siRUVBL1-or siTIP60-treated cells (HCT116, MDA-MB-231, and MIA-PaCa2) to different concentrations of CFZ. Although CFZ by itself caused a dose-dependent decrease in cell viability, this effect was exacerbated in siRUVBL1-and siTIP60-treated cells  (n ϭ 3). D, WT NIH-3T3 cells were treated as described in C, and the lysates were used for immunoblotting to measure the levels of different proteins as indicated. ␤-Actin was used as a loading control. The experiments were performed three independent times, and a representative blot is shown.

Nrf1-dependent transcription requires TIP60
( Fig. 5C). Consistent with these results, immunoblotting experiments revealed that, compared with the control, the levels of cleaved caspase-3 (a marker for apoptosis) were markedly elevated in RUVBL1-or TIP60-depleted cells that were further treated with CFZ (Fig. 5D). Taken together, our results indicate that modulating the activity of the TIP60 complex could enable manipulation of the functional output from the Nrf1 pathway in cells experiencing proteotoxic stress.

Discussion
Nrf1 has emerged as a critical transcription factor in the cellular arsenal to combat proteotoxic stress or proteasome insufficiency. In response to inhibition of the proteasome, Nrf1 is mobilized to enable increased transcription of proteasome subunit genes, culminating in the formation of new proteasomes (4,6,13,19). Several previous studies, including ours, have dissected the regulation of Nrf1 function that is achieved via multiple mechanisms in the cell: regulation of its abundance by the ubiquitin ligases HRD1, FBXW7, and ␤-TRCP; regulation of its extraction from the ER by p97; regulation of its proteolytic processing by DDI2; regulation of its deglycosylation by NGLY1; and regulation of its phosphorylation and, thereby, its transcriptional activity by casein kinase 2, to name a few (9,14,15). Our current study adds the TIP60 chromatin-modifying complex to the growing list of factors that impact Nrf1 activity. To our knowledge, this is the first example of an epigenetic regulator that appears to modulate Nrf1 function.
Although we have demonstrated that RUVBL1 and TIP60, both of which are subunits of the TIP60 complex, are necessary for Nrf1-mediated transcription after proteasome inhibition, the exact nature of their requirement remains to be uncovered. Based on the well-established role of RUVBL1/2 in chromatin remodeling, it could be that these factors serve to open up the chromatin at Nrf1 target gene promoters, as seen previously in the case of the E2F1 transcription factor (36). Also, TIP60, which harbors acetyl transferase activity, is known to acetylate histones H4 and H2A, thereby relaxing the chromatin in promoter regions and providing access to the transcriptional machinery (37). This is exemplified in the case of the hypoxia response, where HIF1␣ recruits TIP60, which enables acetylation of histones and subsequent recruitment and activation of RNA polymerase II at HIF1␣ target gene promoters (30). In our case, it could be that TIP60 provides such a function in Nrf1dependent transcription. Given that TIP60 can also directly acetylate some transcription factors, such as c-myc and the androgen receptor, and modulate their function (38,39), it could also be that Nrf1 is subject to such modification. It is important to note that all of these mechanisms may also occur together and are not necessarily mutually exclusive.
Mechanism aside, our findings could have important therapeutic implications, especially in cancer. RUVBL1 and RUVBL2 have been shown to be overexpressed in a number of cancer types, including colorectal, liver, breast, lung, gastric, esophageal, pancreatic, and kidney cancer and leukemia (23). More importantly, depletion of RUVBL1/2 seemed to delay or even shrink the tumors in preclinical models of some of the cancer types mentioned above. Accordingly, there is intense interest in developing small-molecule inhibitors of the ATPase activity of RUVBL1/2 (40, 41). These early-stage inhibitors already show promise in cell culture and xenograft studies in mice. Likewise, TIP60 has also been regarded as a viable target in cancer, and there have been efforts directed to identify inhibitors of its acetyl transferase activity (42). Given that in our study, depletion of RUVBL1 or TIP60 enhances cell killing by carfilzomib, this provides a framework for testing RUVBL1 or TIP60 inhibitors in combination with proteasome inhibitors in various cancer types. This combination would also make sense from the point of view of proteasome inhibitors, which, apart from their current use in the clinic against multiple myeloma and mantle cell lymphoma, are now being evaluated in combination with other chemotherapeutic agents in several types of solid tumors (43). Our findings, which demonstrates a strong reliance of the Nrf1-mediated proteasome recovery pathway on the functional TIP60 complex, prompt further consideration of combination trials with proteasome and TIP60 complex inhibitors.

Screening system
The lentiviral construct PLKO-luc-mcherry-puro-Renilla (Addgene plasmid 29783) was a gift from Dr. Carl Novina and has been described previously (44). This construct was digested with AgeI and EcoRI to generate PLKO-luc-mcherry-puro. The The cells were then subjected to ChIP with IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for ARE-containing promoter regions of the proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote S.D. (n ϭ 3). B, HEK293 cells stably expressing tagged Nrf1 (Nrf1 3xFLAG ) were treated with 200 nM CFZ for 8 h or left untreated. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times, and a representative blot is shown.

Cell culture and siRNA transfections
All cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Atlanta Biologicals) and penicillin and streptomycin (Invitrogen) at 37°C in a humidified incubator with 5% CO 2 . All siRNAs (sequences are shown in Tables S2 and S3) were purchased from Dharmacon and were transfected into the cell lines, typically at 40 nM concentration, using DharmaFECT-1 reagent (Dharmacon) according to the manufacturer's recommenda-

Nrf1-dependent transcription requires TIP60
tions. Forty-eight hours after transfection, the cells were used for downstream experiments.

Quantitative RT-PCR
TRIzol reagent (Life Technologies) was added directly to the cell culture plates, and total RNA was isolated according to the manufacturer's instructions. Complementary DNA was then prepared from 1000 ng of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was carried out with iTaq universal SYBR Green Supermix (Bio-Rad) using the C1000 Touch Thermal cycler (Bio-Rad). Data were analyzed using CFX Manager 3.1 (Bio-Rad). The levels of 18S rRNA were used for normalization. The primers used for the assays are listed in Table S4.

Chromatin immunoprecipitation
Ten million cells were fixed with freshly prepared formaldehyde solution (1% final volume) and incubated at room temperature for 10 min, followed by addition of glycine to quench the formaldehyde. Plates were then washed twice with ice-cold PBS and collected in PBS supplemented with a protease inhibitor mixture. Pellets were collected after centrifugation at 800 ϫ g at 4°C. The EZ-Magna ChIP A/G (Millipore) kit was used for further steps according to the manufacturer's protocol. Briefly, cells were lysed in cell lysis buffer, and the nuclei were pelleted. These pellets were suspended in nuclear lysis buffer and used for shearing chromatin with Covaris M220. Shearing was done at 10% duty factor (df) for 16 min. Sheared chromatin was centrifuged at 10,000 ϫ g for 10 min, and the supernatant was collected. 20 l of protein A/G magnetic beads was used for preclearing the sheared chromatin for 1 h at 4°C on a rotor. 50 l of precleared chromatin was used for each immunoprecipitation with 5 g of specific antibody overnight at 4°C. Beads were then washed with low salt buffer followed by high salt buffer, LiCl wash buffer, and TE buffer (20 mM Tris (pH 8.0) and 5 mM EDTA). Elution was done with elution buffer at 65°C for 4 h, followed by column purification (Qiagen) to obtain purified chromatin. Quantitative PCR was used to analyze the chromatin. Primers used for analysis are shown in Table S4.

Cell viability assays
Cells were typically grown in 96-well plates and treated with drugs as necessary. For quantifying cell viability, the Cell-Titer Glo kit (Promega) was used. This kit measures the level of ATP, which, in turn, is proportional to the number of viable cells. Qualitative measure of cell viability was assessed using immunoblots for cleaved caspase-3.

Proteasome activity recovery assays
Cells grown in 96-well plates were treated for an hour with 500 nM MG132 or 50 nM carfilzomib, doses that were determined to inhibit proteasome activity by 90%. The cells were then washed with PBS three times and allowed to recover in fresh medium. The cells were frozen in TE buffer at different time points. At the time of the assays, the cells were thawed and used for measuring proteasome activity as described previously (4). Briefly, the cell lysates obtained by freeze-thaw lysis were mixed with the fluorogenic substrate succinyl-Leu-Leu-Val-Tyr-amino-4-methylcoumarin (specific for measuring the chymotrypsin-like activity of the proteasome), and the resulting fluorescence was measured at 360/460 nm excitation/emission. The fluorescence values were then normalized by cell number, as determined using the Cell-Titer Glo kit (Promega), which measures cellular ATP levels.

Co-immunoprecipitation assays
Human embryonic kidney 293 cells stably expressing C-terminally tagged Nrf1 (Nrf1 3xFLAG ) after appropriate treatments were pelleted and resuspended in IP buffer (50 mM Tris (pH 7.4), 0.5 M NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with protease and phosphatase inhibitor mixture (Pierce) and incubated on ice for 30 min. These were then centrifuged at 13,000 rpm in a refrigerated tabletop centrifuge for 20 min. The lysate supernatants were then used for immunoprecipitation with anti-FLAG beads (Sigma-Aldrich) according to the manufacturer's recommendations. The beads (20 l of the 50% slurry for each pulldown) were usually blocked with 5% BSA for 2 h before use. Typically, 500 g of total protein (as determined by Bradford assay) was used for each immunoprecipitation that was allowed to proceed overnight. The beads were then washed three times with IP buffer, eluted in Laemmli buffer, and analyzed by SDS-PAGE, followed by immunoblotting.
Author contributions-J. R. V. and S. K. R. conceptualization; J. R. V. and S. K. R. formal analysis; J. R. V. and S. K. R. methodology; J. R. V. and S. K. R. writing-original draft; J. R. V. and S. K. R. writingreview and editing; S. K. R. supervision; S. K. R. funding acquisition.