Human WDR23 regulates activation of NRF2 cytoprotection pathways

WDR-23 is the major regulator of SKN-1 activity, which is the C. elegans equivalent to mammalian NRF2/NFE2L2. Nematodes lack a KEAP1 homolog, but WDR-23 regulation of SKN-1 is mechanistically similar to KEAP1 regulation of NRF2, regulating turnover of the transcription factor by the ubiquitin proteasome system. Despite the evolution of the KEAP1 regulatory pathway, the WDR23 locus is exceptionally well conserved from worms to humans (Fig 1A, S1A Fig). Remarkably, a role for WDR23 in the regulation of the NRF2 cytoprotection pathway has yet to be described, and a general understanding of the role WDR23 plays in cell biology is lacking; there are two studies that have demonstrated a role for WDR23 in the regulation of SLBP[34, 35], and the only other published report describes altered expression of WDR23/DCAF11 in the mouse bladder epithelium in response to increased levels of urea and nitric oxide[36]. However, WDR23 has been identified in association with the CUL4-DDB1 E3 ligase complex, but like most E3 ligase receptors, specific target substrates remain elusive[37].

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Fig 1. WDR23 influences cytoprotective pathways via regulation of NRF2.

(A) Schematic of the domains found in human WDR23 isoform 1 (pink) and WDR23 isoform 2 (orange). (B,C) Representative images of HEK-293T cells overexpressing mCherry:LAMIN and either GFP:WDR23 isoform 1 (B) or GFP:WDR23 isoform 2 (C). Scale bar, 20μm. (D) Overexpression of WDR23 isoform 1 or WDR23 isoform 2 reduces the expression of an ARE-inducible luciferase reporter as compared to control (GFP expression) (Control n = 32, Iso 1 n = 24, Iso 2 n = 24). (E-H) Cells expressing WDR23 have reduced levels of the NRF2 target genes GSTA1 (Control n = 9, Iso 1 n = 6, Iso 2 n = 3) (E), CYP3A4 (Control n = 9, Iso 1 n = 6, Iso 2 n = 3) (F), ACADL (Control n = 12, Iso 1 n = 9, Iso 2 n = 9) (G), and ACADM (Control n = 12, Iso 1 n = 9, Iso 2 n = 9) (H) as compared to control (GFP expression). (I-M) Overexpression of WDR23 abrogates the effects of tBHQ-treatment on NRF2 targets GSR (Control n = 9, +tBHQ n = 9, Iso 1 n = 6, Iso 1 +tBHQ n = 6, Iso 2 n = 3, Iso 2 +tBHQ n = 3) (I), CYP1A1 (Control n = 9, +tBHQ n = 9, Iso 1 n = 6, Iso 1 +tBHQ n = 6, Iso 2 n = 3, Iso 2 +tBHQ n = 3) (J), CPT1A1 (Control n = 12, +tBHQ n = 12, Iso 1 n = 9, Iso 1 +tBHQ n = 9, Iso 2 n = 9, Iso 2 +tBHQ n = 9) (K), ACADS (Control n = 12, +tBHQ n = 12, Iso 1 n = 9, Iso 1 +tBHQ n = 9, Iso 2 n = 9, Iso 2 +tBHQ n = 9) (L), and ACADL (Control n = 12, +tBHQ n = 12, Iso 1 n = 9, Iso 1 +tBHQ n = 9, Iso 2 n = 9, Iso 2 +tBHQ n = 9) (M) as compared to control (GFP overexpression). Wdr23 knockout MEF cells have increased levels of the NRF2 target genes Gclm (Control n = 5, KO n = 12) (N), Nqo1 (Control n = 5, KO n = 12) (O), and Gsr (Control n = 5, KO n = 12) (P). Data are mean ± s.e.m.; (D) two-tailed t-test relative to control samples; (E-P) one-tailed t-test relative to control samples. *P<0.05, **P<0.01, ***P<0.001.

https://doi.org/10.1371/journal.pgen.1006762.g001

Two major isoforms (iso) of WDR23 are expressed in mammals (Fig 1A). WDR23 isoform 1 (UniProtKB/Swiss-Pro Accession: Q8TEB1-2) encodes a 546 amino acid polypeptide with a predicted molecular mass of 61.7 kDa, while the second isoform, WDR23 isoform 2 (UniProtKB/Swiss-Prot Accession: Q8TEB1-1), encodes a 520 amino acid polypeptide with a predicted molecular mass of 58.8 kDa. GFP tagged WDR23 isoform 1 is localized primarily to the cytoplasm (Fig 1B), while GFP:WDR23 isoform 2 is enriched in the nucleus, but can be found in the cytoplasm when overexpressed in HEK-293T (Fig 1C) or HepG2 (S1B and S1C Fig) cells. The cellular distribution of the two isoforms in human cell culture is consistent with the localization of the two predominant CeWDR-23 isoforms in worms (S1D and S1E Fig)[38]. Although NRF2 activation by xenobiotic electrophiles leads to NRF2 accumulation in the nucleus[39], the subcellular localization of WDR23 does not change with stress (S1F–S1I Fig). The localization of WDR23 isoform 2 in the nucleus is intriguing, as KEAP1 regulation of NRF2 is thought to be restricted to the cytoplasm[40–42]. As such, KEAP1 and WDR23 may coordinately regulate NRF2 in either compartment.

To mount an appropriate response to cellular stress, NRF2 regulates the expression of several classes of xenobiotic response genes, including: glutathione homeostasis, drug metabolism, iron metabolism, multidrug resistance transporters, cellular energy metabolism, biogenesis of circulatory signaling molecules and receptors, and calcium homeostasis[43]. These genes all contain an antioxidant response element (ARE) and are positively regulated by NRF2. To assess whether WDR23 is a functional regulator of NRF2 cytoprotection pathways, we measured NRF2-dependent activation of an ARE-luciferase reporter co-transfected with a renilla control plasmid in HEK-293T cells that were overexpressing GFP tagged WDR23 (Fig 1D). ARE-luciferase activity was inversely related to WDR23 expression levels, supporting a model where WDR23 functions as a negative regulator of NRF2. Surprisingly, expression of CeWDR-23 did not impact ARE-luciferase expression in unstressed cells or in KEAP1 siRNA treated cells. Thus, although WDR23 is an ancient regulator of cytoprotection, its functionality in the SKN-1 and NRF2 pathways is species specific (S2A and S2B Fig).

We were intrigued by the ability of WDR23 to influence the expression of cellular antioxidant responses via the ARE. We next determined if WDR23 could repress the expression of specific NRF2 targets that are responsible for the diversity in NRF2 cellular stress response [27, 44–50]. Increased expression of WDR23 resulted in reduced steady state expression of several NRF2 targets, including: GSTA1 (Fig 1E), CYP3A4 (Fig 1F), ACADL (Fig 1G), and ACADM (Fig 1H); however, not all NRF2 targets were altered (S2C–S2E Fig). Turning off the NRF2 response is equally important, particularly in the context of cancer cells where NRF2 is deregulated. Treatment of cells with tert-butylhydroquinone (tBHQ) activates NRF2-dependent transcription of cytoprotection genes[39], but when combined with WDR23 overexpression, the induction of electrophile induced NRF2 targets, including: GSR (Fig 1I), CYP1A1 (Fig 1J), CPT1A1 (Fig 1K), ACADS (Fig 1L), and ACADL (Fig 1M) were attenuated, while other NRF2-dependent transcripts were unaffected (S2F–S2H Fig). The fact that not all NRF2 targets were influenced by WDR23 may be indicative of the WDR23-regulatory pathway to direct a specific subset of NRF2 targets, of the differential impact the WDR23-NRF2 pathway plays in NRF2-cytoprotection in a cell-type dependent manner, or perhaps one function of the WDR23 control is to turn off NRF2 following transcriptional activation, which is more important for some targets.

In order to determine the effects of loss of WDR23, we derived MEF cells from the Wdr23 knockout (KO) mouse that we generated. Wdr23 KO MEF cells behave similarly to wildtype MEF cells, and we have not observed any differences in cellular fitness between the two genotypes. In line with the overexpression data that we had observed, the MEF KO cells show the opposite effect and have increased expression of NRF2 target genes, including: Gclm (Fig 1N), Nqo1 (Fig 1O), and Gsr (Fig 1P). Notably, these cells have functional KEAP1; in fact, there is an increase in Keap1 transcript levels (S2J Fig), consistent with the model of KEAP1 and WDR23 behaving complementary to each other. Additionally, the changes in NRF2 activity from modulation of WDR23 levels are independent of the phenotypes associated with WDR23’s role in SLBP regulation, as we do not observe an increase in NRF2 target expression when cells are depleted of Slbp (S2K–S2Q Fig). Unlike the WDR23 studies about SLBP by Brodersen et. al., reducing WDR23 levels in this context does not appear to be pleiotropic, likely due to compensation from KEAP1. Together with the overexpression data, these results demonstrate WDR23’s role as a negative regulator of NRF2.

CUL4-DDB-1-WDR23 regulates NRF2 stability

The CUL4-DDB1 E3 ligase complex licenses WDR proteins as receptors for substrate recognition; however, very few receptor-substrate pairs are defined. CeWDR-23 is thought to physically bind SKN-1[21] to regulate its abundance in the cell, but an interaction between WDR23 and NRF2 in humans has not been shown. To reveal the ability of WDR23 to interact with NRF2, we transfected cells with GFP:WDR23 and HA-NRF2 and tested for an interaction biochemically. We immunoprecipitated (IP) GFP:WDR23 isoform 1 (Fig 2A) or GFP:WDR23 isoform 2 (Fig 2B) and found that HA-NRF2 was efficiently co-immunoprecipitated with either isoform of WDR23, which indicates the ability of these two proteins to complex. Importantly, overexpression of GFP alone however did not sequester NRF2 (S3A Fig).

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Fig 2. CUL4-DDB1-WDR23 regulates NRF2 stability.

(A-D) In cells overexpressing tagged versions of WDR23 and NRF2, NRF2 immunoprecipitates (IP) with WDR23 isoform 1 (A) or WDR23 isoform 2 (B) along with DDB1 and CUL4A, but not KEAP1. NRF1 does not IP with the WDR23-DDB1 complex in cells overexpressing tagged versions of WDR23 and NRF1 (C); as diagramed in (D). (E-H) overexpressing WDR23 reduces the abundance of co-expressed FLAG:NRF2 (E) in a proteasome dependent manner (F), increases the abundance of poly-ubiquitinated-NRF2 in a time dependent manner (G), and can also reduce endogenous NRF2 in total cell lysate (H) as compared to control (GFP expression). Control n = 3, Iso 1 n = 6, Iso 2 n = 5. (I) WDR23 interacts with NRF2 in cells treated with H2O2 to induce oxidative stress. (J) The increased stability of NRF2 following H2O2 exposure is abrogated when WDR23 is overexpressed. Oxidative stress also destabilizes WDR23. *P<0.05.

https://doi.org/10.1371/journal.pgen.1006762.g002

We were able to co-IP the several components of the CUL4 complex. IP of WDR23 efficiently pulled down both DDB1 and CUL4A, but not KEAP1 (Fig 2A and 2B), which defines NRF2 as a novel substrate of the CUL4A E3 ligase complex that operates independently of the established CUL3-KEAP1 E3 ligase machinery. We next examined the specificity of the role that WDR23 plays in the maintenance of the NRF family of transcription factors. In addition to NRF2, mammals express NRF1, NRF3, and NF-E2, which all contribute to ARE activation[51–53]. NRF1 is ubiquitously expressed, similar to NRF2, while NRF3 expression is restricted to the placenta and liver tissues, and NF-E2 is only expressed in erythrocytes. Moreover, NRF1 and NRF2 have distinct cellular roles[54, 55]. The interaction of WDR23 with NRF2 was specific, as we were unable to detect an interaction with NRF1 in cells overexpressing tagged versions of WDR23 and NRF1 (Fig 2C). As such, the WDR23-DDB1-CUL4 E3 ligase complex is specific to NRF2-dependent cytoprotection (Fig 2D).

Our experiments follow previous studies[24, 56, 57] that demonstrate that WDR23 is a component of the CUL4A-DDB1 E3 ligase complex (Fig 2A and 2B) and predict that the underlying mechanism of NRF2 regulation would be at the level of protein turnover and stability. As such, we examined whether modulating WDR23 levels could alter the abundance of NRF2 protein. The increased expression of WDR23 in HEK-293T cells decreased the abundance of co-transfected NRF2 in a dose dependent manner (Fig 2E). The reduction of NRF2 was dependent on the increase in WDR23 expression since co-transfection of a WDR23 siRNA restored NRF2 levels (S4A–S4C Fig). As predicted, the WDR23-mediated degradation of NRF2 was dependent on the ubiquitin proteasome system, as the WDR23-mediated reduction of NRF2 was attenuated when cells were treated with the proteasome inhibitor peptide MG-132 (Fig 2F). Although we are unable to detect a significant change in the rate of turnover of NRF2 when co-expressed with WDR23 (S4D Fig), this is likely complicated by the already significant reduction of NRF2 levels prior to treatment with cyclohexamide. Additionally, poly-ubiquitination signals proteins for degradation via the ubiquitin proteasome system, which led us to examine levels of ubiquinated-NRF2. To further test the functionality of WDR23 in NRF2 proteostasis, we purified the WDR23-DDB1-CUL4 complex (Fig 2A) and discovered that it could efficiently add ubiquitin chains to purified NRF2 in vitro (Fig 2G, S4E Fig). Taken together these data suggests that WDR23 drives NRF2 turnover by the ubiquitin proteasome system. As observed by others, we found endogenous NRF2 levels to be relatively low under basal conditions. However, overexpression of isoform 2 of WDR23 reduced endogenous NRF2 protein levels (Fig 2H). These data indicate that the regulation of NRF2 by WDR23 is in part at the level of NRF2 stability.

KEAP1 function is primarily restricted to the cytoplasm, but KEAP1-independent regulation of NRF2, perhaps in the nucleus, has long been hypothesized[58]. Between the two isoforms, there is expression of WDR23 in both the cytoplasm and nucleus, and the functional capacity of both isoforms of WDR23 suggests that its role in NRF2 regulation contributes to the unknown of KEAP1-independent mechanisms. The stable localization of WDR23 in the presence or absence of xenobiotic stress predicts that this regulation can occur regardless of the redox state of the cell. During electrophilic stress, such as treatment with oxidizing agents, the physical association of NRF2 with KEAP1 is disrupted, which stabilizes NRF2, allowing its accumulation in the nucleus[59]. We find that the interaction of WDR23 with NRF2 also occurs in cells treated with H2O2 (Fig 2I and S5A Fig), which is consistent with the idea that WDR23 regulates NRF2 independent of KEAP1. We next challenged the WDR23 regulatory system to turn over activated NRF2 following oxidative stress. In line with our studies in non-stressed cells, overexpression of WDR23 was sufficient to abrogate the increased accumulation of NRF2 following exposure to hydrogen peroxide (Fig 2J).

The conserved capacity of C. elegans WDR-23 to regulate similar cellular cytoprotection responses, albeit mediated by SKN-1, suggested we could exploit our C. elegans genetic system to identify the domains of WDR-23 that would be of functional significance for regulation of the mammalian NRF2 pathway. In C. elegans, WDR-23 is a direct regulator of SKN-1; WDR-23 delivers SKN-1 to the proteasome to regulate the abundance of the transcription factor. Therefore, we utilized the worm as a genetic tool to dissect the mechanisms behind this conserved pathway. To that end, we performed an ethyl methanesulfonate (EMS) mutagenesis screen to identify wdr-23 mutants (S6A Fig), which we predicted would be enriched, as WDR-23 is the canonical negative regulator of SKN-1 activity in worms. We sequenced the wdr-23 locus in all isolated mutants that mapped to linkage group I[60] and identified eight novel alleles of wdr-23 (Fig 3A and S2 Table) that map to conserved regions of the WDR23 protein (S6B Fig)[61]. Each of these mutations is fully recessive and although variable in strength, can enhance animal survival during xenobiotic stress (Fig 3B) and activate the transcription of cytoprotection genes (Fig 3C–3E, S6C Fig) in a skn-1-dependent manner (S6D–S6S Fig). The mutations in wdr-23 cluster around WD40 repeats 4 and 5, which are near the conserved DWD-box found in WDR23 across species (S3 Table). Notably, many of these mutations are in residues that are conserved from worm to man.

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Fig 3. A central highly conserved domain of WDR23 facilitates binding to NRF2.

(A) Schematic of C. elegans WDR-23 and the identity of eight recessive loss of function alleles. Conserved residues between worm and human WDR23 that are mutated for structure function analysis are in pink. (B) Most mutations in WDR-23 result in an increase in cytoprotection from heavy metals. WT n = 6, Q80Stop n = 5, D387N n = 6, T400I n = 6, W339Stop n = 3, frameshift n = 6, H310Y n = 6, D313N n = 6, G460R n = 6. (C-E) Strains harboring mutant versions of WDR-23 display increased expression of the SKN-1/NRF2 cytoprotection targets gcs-1 (WT n = 3, Q80Stop n = 3, D387N n = 3, T400I n = 3, W339Stop n = 3, frameshift n = 3, H310Y n = 3, D313N n = 3, G460R n = 3) (C), gst-4 (WT n = 3, Q80Stop n = 3, D387N n = 3, T400I n = 3, W339Stop n = 3, frameshift n = 3, H310Y n = 3, D313N n = 3, G460R n = 3) (D), ugt-11 (WT n = 3, Q80Stop n = 3, D387N n = 3, T400I n = 3, W339Stop n = 3, frameshift n = 3, H310Y n = 3, D313N n = 3, G460R n = 3) (E). (F-H) As compared to cells over expressing wild type WDR23 and NRF2 (F), the WDR23(W335Stop) mutation (G) impairs binding of NRF2 and disrupts association with the DDB1-CUL4A complex, while the H306Y mutation modestly reduces binding of both (H). Data are mean ± s.e.m.; one-tailed t-test relative to control samples. *P<0.05,**P<0.01, ***P<0.001.

https://doi.org/10.1371/journal.pgen.1006762.g003

Our studies identify NRF2 as a substrate for the CUL4 adapter protein WDR23. Although WDR23 has previously been shown to bind to the CUL4-DDB1 complex[37, 62], the biochemical mechanism underlying this interaction is unknown. Informed by our worm mutants, we used site-directed mutagenesis to generate orthologous mutations in highly conserved residues in WDR23: H306Y and W335Stop (Fig 3A and S6B Fig). These mutant versions of WDR23 did not alter the subcellular localization of WDR23 isoform 1, but we often observed non-nuclear localized WDR23 isoform 2 harboring these mutations, which might impact their functionality (S7A–S7D Fig). Both mutations were stably expressed and could be enriched by our IP strategy, but each mutation weakened the WDR23 interaction with NRF2. The W335Stop mutation disrupted the association of both WDR23 isoforms with NRF2, DDB1 and CUL4A (Fig 3F and 3G, S7E Fig), and the H306Y mutation reduced binding to DDB1 and CUL4A and also modestly reduced NRF2 binding (Fig 3F and 3H, S7F Fig). Informed by our invertebrate studies, these results implicate a potential function of the C-terminus of WDR23 for substrate binding and recruitment of the CUL4 E3 ligase complex. Further dissection of this region will allow us to pinpoint the required residues for this interaction.

WDR23 regulates NRF2 independently of KEAP1

The regulation of NRF2 by KEAP1 is thought to occur in the cytoplasm[40–42]. Our immunoprecipitation studies of WDR23 did not pull down KEAP1, supporting the formation of a CUL3-KEAP1-independent regulatory complex. Six NRF2-ECH homology (Neh) domains have been defined within NRF2 that are key determinants of NRF2 regulation and activity[59, 63–66] (Fig 4A). We systematically examined a panel of NRF2 mutants, each with a different Neh domain deleted[12], and measured the capacity of WDR23 to bind the truncated protein. After transfecting tagged versions of WDR23 and the NRF2 mutants, we observed that NRF2(ΔNeh2) failed to co-IP with either WDR23 isoform (Fig 4B, S5A and S8A Figs), while binding still occurred with all other truncated versions of NRF2 (S8B–S8I Fig). This result indicates the absolute requirement of the Neh2 domain to facilitate the interaction of WDR23 with NRF2.

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Fig 4. WDR23 regulates NRF2 independently of KEAP1.

(A) Schematic of the NRF2 protein, the location of each Neh domain, and the amino acid sequence of the DIDLID, DLG, and ETGE motifs. (B) WDR23 requires the Neh2 domain of NRF2 for binding. Wildtype control IP in Fig 2A. (C,D) Reduction of KEAP1 by RNAi induces the activation of a NRF2-dependent ARE-luciferase reporter (Control n = 32, KEAP1 RNAi n = 32, Iso 1 n = 24, Iso 1 + Keap1 RNAi n = 24, Iso 2 n = 24, Iso 2 + KEAP1 RNAi n = 24) (C) and the increased expression of the NRF2 target GCLC (Control n = 6, KEAP1 RNAi n = 6, Iso 1 n = 6, Iso 1 + KEAP1 RNAi n = 6, Iso 2 n = 6, Iso 2 + KEAP1 RNAi n = 6) (D). This induction is attenuated when WDR23 is overexpressed as compared to control (GFP expression). (E-G) Despite shared use of the Neh2 domain by KEAP1 and WDR23 for binding, WDR23 does not require the DLG (E) or ETGE (F) motifs utilized by KEAP1, but instead requires the conserved DIDLID motif (G). Data are mean ± s.e.m.; one-tailed t-test relative to control samples. *P<0.05, ***P<0.001.

https://doi.org/10.1371/journal.pgen.1006762.g004

KEAP1 also regulates NRF2 via the Neh2 domain[17], which might suggest a common mechanism of WDR23 and KEAP1 regulation of NRF2, despite lack of a detectable interaction between WDR23 and KEAP1. To confirm a KEAP1-independent axis of NRF2 regulation by WDR23, we assessed the capacity of WDR23 to suppress the activation of NRF2 when KEAP1 is inhibited. Overexpression of WDR23 reduced ARE-luciferase activation in cells transfected with KEAP1 siRNA (Fig 4C, S1 Table). Specifically, overexpression of WDR23 suppressed the induction of the canonical KEAP1-NRF2 pathway targets GCLC (Fig 4D), but not NQO1 (S9A Fig).

The Neh2 domain contains 86 amino acids (Fig 4A). To better define the location where WDR23 regulates NRF2 we tested WDR23 binding to NRF2 mutants where either the first or second 43 amino acids were removed, Neh2A(Δ2–43) and Neh2B(Δ44–86). In cells overexpressing tagged version of WDR23 and Neh2 mutants, WDR23 was still able to bind Neh2B(Δ44–86), but not Neh2A(Δ2–43), indicating the binding site is the N-terminal portion of the domain (S9B–S9D Fig). This region of Neh2 contains three identifiable motifs: DIDLID, DLG, and ETGE (Fig 4A). The shared use of the Neh2 domain for binding of NRF2 by WDR23 and KEAP1 may reflect a competition between the CUL4 and CUL3 E3 ligases for NRF2 regulation. To determine whether this model was correct, we tested if WDR23 could associate with NRF2 when the motifs utilized by KEAP1 for binding were mutated[67]. Mutation of the DLG (Fig 4E and S9E Fig) or ETGE (Fig 4F, S9F Fig) motifs did not abolish binding. These findings further support the model where WDR23 can restore regulatory control of NRF2 independent of KEAP1 function (Fig 2D).

In worms, WDR-23 regulates the activity of the cytoprotective transcription factor SKN-1. SKN-1 contains a DIDLID motif that is critical for SKN-1 activity[68]. NRF2 also contains a DIDLID motif, which is found in the Neh2 domain (Fig 4A). Deletion of the DIDLID motif in NRF2 impaired WDR23 binding (Fig 4G, S9D Fig), revealing that the conserved DIDLID motif has been maintained over evolution as a mechanism of regulation. Moreover, this finding defines the DIDLID and the DLG/ETGE motifs are two independent sequences in the Neh2 domain that cooperatively regulate NRF2 by WDR23 and KEAP1, respectively.

WDR23 impacts chemotherapy sensitivity and restores NRF2 homeostasis in cancer cells

KEAP1 function is perturbed in several aggressive cancers that are resistant to chemo- and radiation-based therapies due to enhanced NRF2 activity, making them particularly hard to treat[69–71]. NRF2 stability leads to enhanced resistance to the cytotoxic drugs etoposide, doxorubicin, and cisplatin[72]. As such, we challenged HEK-293T cells overexpressing either GFP (control), GFP:WDR23 isoform 1, or GFP:WDR23 isoform 2 to increasing concentrations of these anti-cancer molecules. Overexpression of WDR23 isoform 1 or WDR23 isoform 2 resulted in increased sensitivity to each cytotoxic drug tested as compared to cells expressing GFP alone (Fig 5A–5C, S10A–S10D Fig and S4 Table). Cells overexpressing either isoform of WDR23 displayed a significant increase in DNA damage, as measured by dual phospho-ATM and phospho-H2Ax staining, which is indicative of DNA double strand breaks (DSB) (Fig 5D and 5E, S10E–S10J Fig). Similarly, the increased toxicity of etoposide treatment for cells overexpressing WDR23 isoform 1 was correlated with an increased DSB in those cells. Lastly, we observed enhanced apoptosis, as measured by Annexin V staining in cells overexpressing either isoform of WDR23 (Fig 5F, S10K–S10M Fig).

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Fig 5. WDR23 overexpression enhances chemotherapy toxicity.

(A-C) Cells overexpressing GFP:WDR23 isoform 1 (pink) or GFP:WDR23 isoform 2 (orange) are more sensitive to increasing concentrations of (A) etoposide (25uM n = 12 each, 50um n = 8 each, 75um n = 8 each), (B) doxorubicin (1uM n = 12 each, 2uM n = 8 each), or (C) cisplatin (50uM n = 12 each, 75uM n = 8 each, 100uM n = 8 each) as compared to cells expressing GFP alone (black). (D,E) The percentage of cells carrying DNA double strand breaks (DSB), as indicated by dual phospho-H2Ax and phospho-ATM staining, is increased when WDR23 isoform 1 or WDR23 isoform 2 is overexpressed (Control n = 3, Iso 1 n = 3, Iso 2 n = 3) and (E) enhances DSB incidence following etoposide treatment (Control n = 3, Iso 1 n = 3, Iso 2 n = 3). (F) Cellular apoptosis (Annexin V positive) is increased in cells overexpressing WDR23 isoform 1 or WDR23 isoform 2 as compared to control cells overexpressing GFP alone (Control n = 3, Iso 1 n = 3, Iso 2 n = 3). Data are mean ± s.e.m.; one-tailed t-test relative to control GFP overexpression for each treatment condition. *P<0.05, **P<0.01, ***P<0.001.

https://doi.org/10.1371/journal.pgen.1006762.g005

In light of the impact WDR23 had on the NRF2 activity in untransformed cells, we predicted that WDR23 could compensate for KEAP1 loss in cancer cell lines derived from human tumors. To that end, we overexpressed either WDR23 isoform 1 or WDR23 isoform 2 in A549 lung carcinoma cells, where loss of KEAP1 results in NRF2 nuclear accumulation. We exploited the transient transfection system, which advantageously facilitated a side-by-side comparison of cell expressing WDR23 to those without. In support of our hypothesis, cells transfected with either isoform of WDR23 had reduced endogenous nuclear NRF2, while NRF2 in non-transfected cells remained nuclear (Fig 6A–6C). Moreover, when looking at the immunostaining of individual cells, the reduction of nuclear NRF2 was more pronounced when WDR23 isoform 2 was overexpressed, and is consistent with the idea that the WDR23 is the nuclear complement to the cytoplasmic KEAP1 system. Additionally, we also examined the effect of WDR23 overexpression in H460 human non-small-cell lung carcinoma cells, which also harbor a KEAP1 mutation (different from that of A549) that results in nuclear NRF2[73]. H460 cells that overexpress either isoform of WDR23 also had reduced endogenous nuclear NRF2, (S11F and S11G Fig), in line with what we observed in A549 cells. Quantification of total NRF2 protein levels in A549 cells overexpressing WDR23 isoform 1 or WDR23 isoform 2 reveal a reduction of approximately 20%, although based on the difficulty in transfection of these cells, and the resulting mosaic nature of the population, this is likely an underestimate of the effect WDR23 has on NRF2 stability (S11D and S11E Fig). Lastly, A549 lung carcinoma cells that overexpress either isoform of WDR23 have reduced expression of the NRF2 targets GSTA (Fig 6D) and PRDX1 (Fig 6E), which are often induced in cancer cells and have been identified as potential targets for directed therapy[74, 75]. Collectively, our studies provide new mechanistic insight underlying the complex regulation of NRF2-dependent cytoprotection (Fig 7). Additionally, these findings are of particular medical relevance as the ability to shutdown NRF2 activity, independently from KEAP1, is of particular clinical interest for cancers where activated NRF2 contributes to both the severity and resistance to treatment by radiation- or chemo-based therapies.

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Fig 6. WDR23 restores NRF2 regulation in cancer cells.

(A,B) Overexpression of GFP:WDR23 isoform 1 (A) or GFP:WDR23 isoform 2 (B) can deplete activated endogenous NRF2 (Alexa Fluor 594, red) from the nucleus (DAPI, blue) in A549 lung cancer cells. Scale bar, 20μm. Arrows denote transfected cells. (C) Quantification of nuclear or diffuse localization of endogenous NRF2 in cells over expressing GFP:WDR23 isoform 1 (n = 74), GFP:WDR23 isoform 2 (n = 84) or non-transfected cells from the same experiment (n = 490). Each bar represents an independent immunostaining experiment. Fisher’s exact test; **P<0.01, ***P<0.001. (D,E) A549 cancer cells overexpressing WDR23 have reduced levels of the NRF2 target genes GSTA1 (Control n = 12, Iso 1 n = 6, Iso 2 n = 6) (D) and PRDX1 (Control n = 12, Iso 1 n = 6, Iso 2 n = 6) (E). Data are mean ± s.e.m.; one-tailed t-test relative to control samples. *P<0.05.

https://doi.org/10.1371/journal.pgen.1006762.g006

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Fig 7. WDR23 and KEAP1 pathways coordinate regulation of NRF2.

Model for the shared regulation of NRF2 by the cytoplasmic Cullin3 E3-ubiquitin ligase complex (CUL3-KEAP1) and the nuclear and the cytoplasmic Cullin4 E3-ubiquitin ligase complexes (CUL4-DDB1-WDR23).

https://doi.org/10.1371/journal.pgen.1006762.g007

Cell cultures, transfections, and chemicals

Cell cultures were maintained as previously described[85]. HEK-293T and HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic (Thermo Fisher) at 37°C, 5% CO2. A549 (ATCC) cells were maintained in Ham’s F-12K (Kaighn’s) medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic at 37°C, 5% CO2. H460 (ATCC) cells were maintained in RMPI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic at 37°C, 5% CO2. Transfections were performed with Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s protocol. siRNAs (Thermo Fisher) used include: KEAP1 (HSS114799, HSS114800, HSS190639), WDR23 (HSS129631, HSS129632, HSS129633), NRF2 (s9492). For genes with more than one siRNA listed, a cocktail mixture of the previously mentioned siRNAs is used for efficient knockdown. Chemical treatments include: 50μM tert-Butylhydroquinone (Sigma) and 250μM H2O2 (Sigma). 50μM tert-Butylhydroquinone (Sigma), 250μM H2O2 (Sigma), and 10μM MG-132 (Sigma).

Recombinant DNA

Full-length cDNA sequence of Hs Wdr23 Isoforms 1 and 2 and Ce wdr-23 Isoforms A and B were cloned into pcDNA 6.2/N-EmGFP/TOPO (Thermo Fisher). 3xFLAG:Nrf2 and 3xFLAG:Nrf1 were purchased from GeneCopeia. mCherry-LaminA-C-18 was a gift from Michael Davidson (Addgene plasmid # 55068). Nrf2(ΔNeh) plasmids were a generous gift from Donna Zhang (University of Arizona). Additional mutants were generated from existing plasmids using Q5 Site-Directed Mutagenesis (NEB).

C. elegans strains utilized and culture methods

C. elegans were cultured using standard techniques[86]. The following strains were used: wild-type N2 Bristol, CL2166[gst-4p::gfp], SPC296[wdr-23(lax101;Q80Stop)], SPC318[wdr-23(lax123;D387N)], SPC302[wdr-23(lax124;T400I)], SPC306[wdr-23(lax126;W339Stop)], SPC299[wdr-23(lax129;frameshift)], SPC315[wdr-23(lax134;H310Y)], SPC303[wdr-23(lax211;D313N)], and SPC317[wdr-23(lax213;G460R)]. Double mutants were generated by standard genetic techniques. For RNAi experiments, NGM plates containing 5 mM IPTG and 100 μg ml^-1 carbencillin were seeded with overnight cultures of double-stranded RNAi-expressing HT115 bacteria. Plates were allowed to induce overnight followed by transfer of age-synchronous populations of C. elegans. For arsenite survival, L4 worms of indicated genotype were transferred to plates containing 5mM arsenite (J.T.Baker) and counted for survival after 24 hours.

Isolation of wdr-23 mutants

Ethyl methanesulfonate (EMS) mutagenesis was performed as previously described[25]. Briefly, a C. elegans strain harboring the SKN-1 transcriptional reporter gst-4p::gfp was mutagenized with EMS, and F1 worms with high GFP expression (indicating SKN-1 activation) were selected. A complementation group of eight recessive alleles were isolated and mapped to chromosome I. The wdr-23 gene was sequenced in each mutant isolated to determine the specific mutation in each strain.

Fluorescent imaging

Cells were grown on coverslips coated with poly-D-lysine (Corning) and transiently transfected with indicated plasmids. Twenty-four hours post-transfection, coverslips were mounted on cover slides and imaged with a Zeiss Axio Imager.M2m microscope, Axio Cam MRm camera, and Zen Blue software.

RNA extraction and quantitative PCR

Quantitative PCR was performed as previously described[27]. Briefly, either human cells or worms of the indicated genotypes and treatments were collected and lysed in Tri reagent (Zymo Research). RNA was extracted according to the manufacturer’s protocol. DNA contamination was digested with DNase I and subsequently, RNA was reverse-transcribed to complementary DNA using qScript cDNA SuperMix (Quanta Biosciences). Quantitative PCR was performed by using SYBR Green (BioRad). The expression levels of ​snb-1 and B2M were used to normalize samples in worms and human cells, respectively. Primer sequences are listed in Supplemental S6 Table and raw data in S7 Table.

Luciferase reporter gene assay

HEK-293T cells were transiently transfected with the indicated plasmids and/or siRNA and Cignal antioxidant response luciferase reporter (Qiagen). Forty-eight hours post-transfection, cells were assayed using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to renilla luciferase activity (S8 Table).

Co-immunoprecipitation

HEK-293T cells were transiently transfected with indicated plasmids. Twenty-four hours post-transfection and without treatment of the proteasome inhibitor MG-132, cells were lysed in 0.5% CHAPS buffer (10mM Tris/Cl pH 7.5, 150mM NaCl, 0.5mM EDTA, 0.5% CHAPS) containing Halt Protease Inhibitor (Thermo Fisher). Immunoprecipitation of GFP:WDR23 was performed according to the manufacturer’s protocol (ChromoTek). Briefly, cell lysates were precleared with blocked magnetic agarose GFP Trap beads for 1 hour at 4°C, followed by incubation with magnetic agarose GFP Trap beads for 1 hour at 4°C. After three washes (1mM Tris/Cl pH 7.5, 150mM NaCl, 0.5mM EDTA) post-immunoprecipitation, immunoprecipitated protein complexes were eluted in 2X sample buffer (0.1M Tris/Cl pH 6.8, 4% SDS, 20% glycerol, 0.2M DTT, 0.1% bromophenol blue) by boiling for 10 minutes at 95°C. Samples were analyzed by Western blot.

Western blot analysis and antibodies

For detection of protein expression in total cell lysates, cells were lysed in RIPA buffer (50mM Tris/Cl pH 8, 150mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS with Halt Protease inhibitor (Thermo Fisher)). Protein concentrations were measured with Bradford (Amaresco), then prepared with 5X sample buffer (0.25M Tris/Cl pH 6.8, 10% SDS, 50% glycerol, 0.5M DTT, 0.25% bromophenol blue), electrophoresed through Bolt 4–12% bis-tris polyacrylamide gels in MOPS running buffer (Thermo Fisher), transferred to nitrocellulose membranes, and subjected to immunoblot analysis. Antibodies used include: GFP GF28R (Thermo Fisher), FLAG M2 (Sigma), NRF2 H-300 (Santa Cruz), NRF2 C-20 (Santa Cruz), DDB1 A300-462 (Bethyl), CUL4A 113876 (GeneTex), KEAP1 ab66620 (Abcam), Actin A5441 (Sigma), Tubulin 21485 (CST), Ubiquitin 1859660 (Thermo Fisher).

Immunocytochemistry

A549 cells were grown on coverslips coated with poly-D-lysine (Corning) and transiently transfected with indicated plasmids. Forty-eight hours post-transfection, cells were fixed in 100% methanol in -20°C for 5 minutes, blocked in 10% normal goat serum/PBS for 20 minutes, incubated in primary antibody for 1 hour each, incubated in secondary Alexa Fluor antibody (Abcam) for 1 hour each, and mounted with Vectashield with DAPI (Vector Labs). Images were taken with a Zeiss Axio Imager.M2m microscope, Axio Cam MRm camera, and Zen Blue software (S9 Table).

Chemotherapy treatment and cytology

HEK-293T cells were transiently transfected with indicated plasmids. Forty-eight hours post-transfection, cells were treated with the indicated chemical: Etoposide (Cayman), Doxorubicin (Cayman), or Cisplatin (Cayman). For viability assays, after forty-eight hours of treatment, cells were assayed using the Vybrant MTT Cell Proliferation Assay Kit (Thermo Scientific), performed according to the manufacturer’s protocol. Viability was calculated relative to the vehicle treatment for each transfection condition. For DNA damage analysis, after twenty-four hours of treatment, cells were assayed using the Muse Multi-Color DNA Damage Kit (EMD Millipore) and Muse Cell Analyzer (EMD Millipore), performed according to the manufacturer’s protocol. For apoptotic cell analysis, cells were assayed using the Muse Annexin V and Dead Cell Assay Kit (EMD Millipore) and Muse Cell Analyzer (EMD Millipore), performed according to the manufacturer’s protocol.

In vitro ubiquitylation assay

Reactions were performed as described in Broderson M. M. L. et al. (2016), with the exception of NRF2 eluate from a PURExpress In Vitro Protein Synthesis Kit (NEB). Briefly, Flag tagged NRF2 was purified from HEK-293T cells with Flag-M2 affinity resin (Sigma) for Fig 2G or 250 ng of HALO-NRF2 was incubated with PURExpress components in a 25 μl reaction for 3 hours at 37°C for S4E Fig and 5 μl was used for each in vitro ubiquitylation reaction. CUL4 ligase (DDB1, CUL4A, WDR23) purification is described above.

Statistics

Statistical analyses were performed with GraphPad Prism 6 software. Data are presented as mean ± s.e.m. Data were analyzed by using unpaired Student’s t-test, one-way ANOVA, and Fisher’s exact test, where indicated. P<0.05 was considered as significant.