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Modification of RelA by O-linked N-acetylglucosamine links glucose to NF-κB and transcription

David F. Allisona, J. Jacob Wamsleya, Manish Kumara, Duo Lia, Lisa G. Graya, Gerald W. Hartb, David R. Jonesa,c, and Marty W. Mayoa,1

Departments of aBiochemistry and Molecular Genetics and cSurgery, University of Virginia, Charlottesville, VA 22908; and bDepartment of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205

Edited by George R. Stark, Lerner Research Institute, Cleveland, OH, and approved September 10, 2012 (received for review May 18, 2012) The molecular mechanisms linking glucose metabolism with active corepressor (NCoR) or silencing mediator for retinoid and thy- transcription remain undercharacterized in mammalian cells. Using roid-hormone receptor (SMRT) (18–21). The deacetylase ac- + nuclear factor-κB (NF-κB) as a glucose-responsive transcription fac- tivity of localized HDAC1/2/3 and NAD -dependent SIRT1/6 tor, we show that cells use the hexosamine pathway sustain the basal repression of these NF-κB–regulated promoters and O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) (20, 22–25). Our group has shown that -associated to potentiate in response to tumor necrosis factor recruitment of IKKα results in of SMRT, and (TNF) or etoposide. Chromatin immunoprecipitation assays dem- displacement of the SMRT/HDAC3 corepressor complex, en- onstrate that, upon induction, OGT localizes to NF-κB–regulated abling RelA/p50 dimers to activate NF-κB–regulated promoters promoters to enhance RelA acetylation. Knockdown of OGT abol- (21, 23). Once bound to NF-κB–regulated promoters, the acidic- ishes p300-mediated acetylation of RelA on K310, a posttransla- rich transactivation domain of RelA recruits com- tional mark required for full NF-κB transcription. Mapping studies plexes that include either p300 or the CREB-binding reveal T305 as an important residue required for attachment of the (CBP) (20, 26, 27). (HAT) activity of O-GlcNAc moiety on RelA. Furthermore, p300 fails to acetylate p300/CBP modifies local chromatin structure and facilitates re- a full-length RelA(T305A) mutant, linking O-GlcNAc and acetyla- cruitment of the transcriptional machinery, including the TFIID tion events on NF-κB. Reconstitution of RelA null cells with the complex and RNA polymerase II (28, 29). Complete NF-κB RelA(T305A) mutant illustrates the importance of this residue transcriptional activation requires specific acetylation of RelA for NF-κB–dependent gene expression and survival. Our work 310 (K310) by p300/CBP (23, 25, 30–32). provides evidence for a unique regulation where attachment of Because RelA has been shown to be modified by OGT (11, the O-GlcNAc moiety to RelA potentiates p300 acetylation and 15), we sought to determine whether attachment of an O-GlcNAc NF-κB transcription. modification impacts RelA acetylation. Data presented in this paper demonstrate that OGT inducibly localizes to chromatin p65 | NF kappa B | and drives p300-mediated acetylation of RelA(K310). Thus, attachment of the O-GlcNAc moiety to RelA is a prerequisite shunt of glycolysis known as the hexosamine biosynthesis for K310 acetylation, a molecular mechanism that links glucose Apathway (HBP) links cellular signaling and gene expression metabolism with NF-κB transcription. to glucose metabolism (1, 2). The HBP generates a metabolically expensive moiety used for , uridine diphosphate Results N-acetylglucosamine (UDP-GlcNAc) (1, 3, 4). The β-N-acetyl- Activation of NF-κB Is Glucose-Dependent. NF-κB is a glucose-re- glucosaminyltransferase (OGT) uses UDP-GlcNAc to sponsive (17). Using an NF-κB responsive covalently attach a single O-linked β-N-acetylglucosamine (O- luciferase reporter system, we demonstrate that HEK 293T cells GlcNAc) moiety to or (S/T) residues within tar- cultured in high glucose (25 mM) show elevated NF-κBtran- get (4). Conversely, O-GlcNAcase (OGA) removes the scriptional activity in response to TNF compared with cells modification. Both OGT and OGA are essential, ubiquitously cultured in 5 mM glucose (Fig. S1A). Treatment with lower expressed , making O-GlcNAcylation a highly dynamic concentrations of TNF (1 ng/mL), rather than 10 ng/mL, showed process (5, 6). O-GlcNAcylation regulates transcription through more sensitivity to change in glucose concentrations (Fig. S1A). OGT-associated chromatin-modifying complexes (4, 7–10) and For this reason, we used low concentrations of TNF throughout direct modification of transcription factors such as NF-κB (11–15). the rest of our studies. Quantitative real-time PCR (QRT-PCR) NF-κB is a glucose-responsive transcription factor that gov- demonstrates the induction of NF-κB–regulated genes (IL-8 and erns many biological processes including , sur- TNFAIP3) in cells incubated in high concentrations of glucose vival, and inflammation (16, 17). Five NF-κB family members compared with cells cultured in 5 mM glucose (Fig. 1A). This have been identified in humans: RelA/p65, RelB, cRel, p105/ effect was not limited to TNF because the topoisomerase II in- p50, and p100/p52. The most prevalent and best studied form of hibitor etoposide also stimulated NF-κB regulated genes (IL-8, NF-κB is the RelA/p50 heterodimer. Before stimulation, inhib- cIAP-2, and TNFAIP3) in a glucose-responsive manner (Fig. 1B). itor of κBalpha(IκBα) sequesters dormant RelA/p50 hetero- dimers in the cytosol. Canonical induction drives IκB (IKK) κ α complex-mediated phosphorylation of I B , resulting in K48- Author contributions: D.F.A., G.W.H., and M.W.M. designed research; D.F.A., J.J.W., M.K., linked polyubiquitination and subsequent degradation via the 26S D.L., and L.G.G. performed research; G.W.H. contributed new reagents/analytic tools; D.F.A., proteasome (16). The degradation of IκBα allows RelA/p50 het- D.R.J., and M.W.M. analyzed data; and D.F.A. and M.W.M. wrote the paper. erodimers to translocate to the nucleus where they displace re- The authors declare no conflict of interest. pressive p50 and p52 homodimers at NF-κB–regulated promoters. This article is a PNAS Direct Submission. In unstimulated cells, p50 or p52 homodimers bind to tar- 1To whom correspondence should be addressed. E-mail: [email protected]. getpromotersandsilencegeneexpression by recruiting his- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tone deacetylase (HDAC) activity tethered to nuclear receptor 1073/pnas.1208468109/-/DCSupplemental.

16888–16893 | PNAS | October 16, 2012 | vol. 109 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1208468109 Downloaded by guest on September 28, 2021 Fig. S1B. Experiments shown in Fig. 1 are consistent with previous reports (11), indicating that NF-κB responsive transcription is positively regulated by glucose flux through the HBP.

OGT Is Required for NF-κB Transcriptional Activity. To determine whether OGT positively regulates NF-κB transcription, we first confirmed that cells transfected with OGT siRNA displayed a significant knockdown of the OGT protein (Fig. 2A). HEK 293T cells transfected with siRNA targeting OGT displayed re- duced levels of IL-8 and BCL2A1 compared with cells trans- fected with control siRNA. Because the O-GlcNAc modification is dynamic and reversible, we tested whether ectopic expression of Myc-O-GlcNAcase (OGA) or V5-OGT altered NF-κB tran- scriptional activity. Exogenously expressed OGT increased tran- scription of IL-8 and BCL2A1 after TNF stimulation in HEK 293T cells (Fig. 2B). Additionally, ectopic expression of OGA effectively dampened the enhanced transcription of IL-8 and BCL2A1 observed from exogenous OGT expression. Immuno- blots confirm V5-tagged OGT and Myc-tagged OGA expression. These results indicate that OGT protein expression is required for endogenous NF-κB transcription, linking the attachment of the O-GlcNAc moiety with inducible NF-κB transcription.

Chromatin-Associated OGT Is Required for RelA Acetylation on NF-κB Regulated Promoters. Chromatin immunoprecipitation (ChIP) experiments were performed to ascertain whether OGT coloc- alizes with RelA on NF-κB regulated promoters. Similar to our previous report (23), TNF stimulation of DU145 cells results in the biphasic recruitment of RelA (20–50 and 90–120 min) to the IL-8 promoter. OGT was recruited to the IL-8 promoter in a stimulus-dependent manner and was present at the promoter over the same timeframe as both RelA and p300 (Fig. 3A). Re- ChIP experiments on the IL-8 promoter confirmed that RelA- bound complexes also contained increased levels of the O-GlcNAc modification. The relative IL-8 promoter occupancies of OGT and O-GlcNAcylated RelA were shown to have similar chromatin- associated patterns across the 120-min timeframe (Fig. 3A, Right). Next, experiments were undertaken to determine whether Fig. 1. Flux through the HBP enhances NF-κB transcription. (A) HEK 293T recruitment of OGT to chromatin corresponded with elevated cells were treated with TNF (1 ng/mL) after overnight incubation in serum- RelA(K310) acetylation. Consistent with Fig. 3A, RelA, p300, free DMEM containing either 5 mM or high (25 mM) glucose concentrations. and OGT colocalized to the IL-8 promoter with similar kinetics fi Expression of IL-8 (Left) and TNFAIP3 (Right) was quanti ed by using QRT- (Fig. 3B). Moreover, OGT and acetylated RelA(K310) appear PCR, and samples were normalized to GAPDH levels. Fold change values localized to the IL-8 promoter over the same timeframe. Spec- represent comparison with unstimulated cells incubated in 5 mM glucose. (B) fi fi HEK 293T cells were cultured overnight in either 5 mM or 25 mM glucose i city of the anti-RelA(K310Ac) antibody was con rmed in Fig. DMEM, and the next day, cells were treated with etoposide (100 μM) for 4 h. S2. The appearance of acetylated histone H3 Lys-14 () QRT-PCR demonstrates elevated NF-κB responsive transcripts in high glucose, on the IL-8 promoter indicates active transcription during OGT compared with cells cultured in 5 mM glucose. Fold change values represent and RelA(K310Ac) occupancy (Fig. 3B). Next, knockdown ex- comparison with untreated cells incubated in 5 mM glucose. (C) HEK 293T periments were used to confirm the role of OGT in p300-driven cells were cultured overnight in 5 mM glucose DMEM. Cells were then ex- acetylation of RelA on the promoter. As expected, knockdown posed to glucosamine (Glcn) for 2 h, stimulated with TNF (1 ng/mL) for 4 h, of OGT significantly reduced the level of OGT recruited to and QRT-PCR was performed. Fold change represents values obtained from chromatin after TNF stimulation (Fig. 3C). Although the loss of TNF-stumulated cells incubated in the absence of Glcn. (D) HEK 293T cells transfected with plasmids encoding Flag-RelA were incubated in 5 mM OGT expression had no effect on recruitment of either RelA or glucose media overnight and subsequently exposed to Glcn for 4 h. Flag- p300 to the cIAP-2 promoter, it ablated RelA(K310) acetylation. RelA was immunoprecipitated, and O-GlcNAc-modified RelA was analyzed In fact, in the absence of OGT, p300 remained loaded on the by immunoblotting. Total proteins were analyzed for the O-GlcNAc modi- cIAP-2 promoter with longer kinetics despite the lack of RelA fication by immunoblot. QRT-PCR results in A–C are a calculated mean ± SD, acetylation. Results shown in Fig. 3 suggest that the recruitment *P < 0.05, n = 3. Immunoblots are a representative example from at least of OGT to NF-κB–regulated promoters is required for p300- three independent experiments. mediated acetylation of RelA in response to TNF stimulation.

O-GlcNAc Modification Enhances RelA Acetylation. Because OGT Because glucose regulates numerous metabolic pathways, cells knockdown ablated RelA(K310Ac) occupancy to the IL-8 pro- cultured in 5 mM glucose were treated with D-glucosamine (Glcn) moter, we wanted to examine whether RelA acetylation was fi as a way to speci cally increase UDP-GlcNAc levels. The addi- sensitive to changes in glucose concentration. Expression of tion of Glcn increased IL-8 transcription in a dose-dependent p300 in HEK 293T cells effectively acetylates RelA as detected manner after TNF stimulation (Fig. 1C). RelA and total cellular by immunoprecipitation and immunoblotting with the anti- protein show increased O-GlcNAc modification through Glcn- RelA(K310Ac) antibody (Fig. 4A). Cells cultured in high glu-

induced upregulation of cytosolic UDP-GlcNAc (Fig. 1D). The cose showed more robust p300-directed acetylation of RelA BIOCHEMISTRY specificity of the pan anti–O-GlcNAc antibody was confirmed in compared with cells grown in 5 mM glucose. Moreover, cells

Allison et al. PNAS | October 16, 2012 | vol. 109 | no. 42 | 16889 Downloaded by guest on September 28, 2021 Fig. 2. NF-κB transcription requires OGT. (A) HEK 293T cells were transfected with siRNA targeting either OGT or nonspecific control. After knock- down, protein levels of OGT were analyzed by im- munoblot (Left), using α- as a loading control. HEK 293T cells were subjected to OGT knockdown, serum starved overnight, and treated with TNF (1 ng/mL) for 4 h. Expression of IL-8 or BCL2A1 was quantified by QRT-PCR (Right), and samples were normalized to GAPDH levels. Expres- sion is represented as fold change compared with the unstimulated control knockdown. (B)OGTand OGA were transfected into HEK 293T cells alone or in tandem. After transfection, cells were treated with TNF (1 ng/mL) for 4 h. Expression of IL-8 or BCL2A1 expression was quantified by QRT-PCR (Left). Rep- resented fold change values are compared with the unstimulated vector control. Immunoblots confirm the expression of V5-tagged OGT and Myc-tagged OGA (Right), compared with the α-tubulin loading control. Data are a calculated mean ± SD, *P < 0.05, n = 3.

cultured in high glucose displayed an overall increase in total immunoprecipitated RelA reveals that expression of ectopic V5- cellular O-GlcNAcylated protein. We then wanted to determine OGT robustly increased HA-p300–driven RelA(K310) acetylation whether OGT potentiated acetylation of RelA. Analysis of (Fig. 4B). Further, the increase in RelA acetylation corresponded with a concomitant elevation in O-GlcNAcylated RelA without altering expression of either RelA or p300. To clarify whether O-GlcNAc-modified RelA is a direct sub- strate for p300, in vitro acetylation assays were performed. Immu- noprecipitated protein, isolated from cells transfected with Flag- RelA alone or with both Flag-RelA and V5-OGT, was incubated with recombinant p300. Flag-RelA isolated from cells cotrans- fected with V5-OGT was acetylated by recombinant p300 more effectively than cells transfected with Flag-RelA alone (Fig. 4C). The expression of Myc-OGA or the knockdown of OGT conversely reduced levels of Flag-RelA K310 acetylation (Fig. 4 D and E). These results are consistent with our findings that O-GlcNAc– modified RelA is required for p300 to acetylate RelA(K310).

RelA(T305) Is Required for p300-Mediated Acetylation. Although a previous report describes multiple O-GlcNAc modifications on RelA, this event was not associated with stimulus-driven activa- tion of NF-κB (15). Therefore, we were interested in identifying O-GlcNAc–modified S/T residues within RelA associated with active transcription. Because RelA was shown to be modi- fied at T322 and T352 by O-GlcNAc (15), we focused on RelA sequences near K310 to exclude sequences beyond T322. The full-length RelA, as well as RelA(1-313) and RelA(1-317) pro- teins, displayed similar levels of the O-GlcNAc modification Fig. 3. NF-κB–regulated promoters require OGT for RelA acetylation. (A) relative to the amount of immunoprecipitated Flag-RelA (Fig. DU145 cells were treated with TNF (10 ng/mL) over a 2-h time course, and 5A). The reduced level of immunoprecipitated full-length RelA chromatin immunoprecipitation (ChIP) and Re-ChIP assays were performed. observed in Fig. 5A is consistent with a previously reported steric Control IgG antibody was used as a negative IP control, and inputs served as hindrance within the transactivation domain (33, 34). Compared a loading control. The relative IL-8 promoter occupancy was determined with RelA(1-313), the RelA(1-276) protein showed a significant after normalization to the maximum level of chromatin-bound OGT or RelA/ decrease in the O-GlcNAc modification, suggesting that the O-GlcNAc Re-ChIP complexes arbitrarily adjusted to one (Right). (B) DU145 cells were treated with TNF and subjected to ChIP and Re-ChIP with the in- major O-GlcNAc sites were between residues 276 and 313. Be- dicated antibodies. (C) DU145 cells transfected with siRNA targeting either cause this region encompasses part of the Rel homology domain OGT or control were treated with TNF and ChIP was performed. Data are (276-304) required for contact with p50 (33, 35, 36), we examined a representative example from at least three independent experiments. S/T residues outside of this region. We investigated T305 and

16890 | www.pnas.org/cgi/doi/10.1073/pnas.1208468109 Allison et al. Downloaded by guest on September 28, 2021 NF-κB prosurvival genes overcomes TNF- and etoposide-in- duced apoptosis (40, 41). To determine the importance of T305 − − for RelA-dependent transcription and cell survival, RelA / MEFs were stably reconstituted with wild-type or mutant Flag- RelA (T305A or K310R). Stable subclones expressed similar levels of wild-type and mutant RelA proteins (Fig. 6A). As − − expected, the introduction of wild-type RelA into MEFRelA / cells restored TNF-induced NF-κB expression of cIAP-2 and − − TNFIAP3 compared with MEFRelA / vector control cells (Fig. 6B). However, reintroduction of either RelA(T305A) or RelA − − (K310R) into MEFRelA / cellsfailedtorescueNF-κB–mediated transcription after TNF stimulation (Fig. 6B). These data suggest that the inability of RelA to be modified by O-GlcNAc at T305 significantly inhibits NF-κB transcription in a manner similar to cells expressing the nonacetylated mutant RelA(K310R). Ad- − − ditionally, MEFRelA / cells expressing either RelA(T305A) or RelA(K310R) remained sensitive to TNF- and etoposide-in- duced cell death as determined by elevated nucleosomal frag- mentation and a loss of cell viability (Fig. 6 C and D). Collectively, − − our data indicate that MEFRelA / cells expressing RelA(T305A) are deficient for NF-κB transcription and, as a result, remain more sensitive to TNF- and etoposide-induced apoptosis. Discussion Regulation of RelA Transcriptional Activity by the O-GlcNAc Modifi- cation. Previous studies provide evidence that the HBP pathway and OGT regulate NF-κB transcription through two distinct mechanisms: one in which OGT enhances the catalytic activity of IKKβ and another that regulates nuclear accumulation of RelA

Fig. 4. RelA acetylation is regulated by the O-GlcNAc modification. (A) The Flag-RelA expression plasmid was transfected into HEK 293T cells either alone or with HA-p300. After an overnight incubation in DMEM containing either 5 mM or 25 mM glucose, Flag-RelA was immunoprecipitated. O-GlcNAc modified RelA, and RelA(K310Ac) was subsequently analyzed by immunoblotting. (B) HEK 293T cells cotransfected with Flag-RelA, HA-p300, and V5-OGT were subjected to Flag immunoprecipitation. O-GlcNAc modi- fied RelA and acetylated RelA were detected by immunoblotting. (C) HEK 293T cells were transfected with Flag-RelA and V5-OGT or with Flag-RelA alone. After Flag IP, RelA was acetylated with recombinant (Rec) p300 in vitro. RelA(K310) acetylation levels were analyzed by immunoblot. (D) HEK 293T cells were cotransfected with Flag-RelA and HA-p300 in the presence or absence of Myc-OGA. O-GlcNAc modified and RelA(K310Ac) were detected by IP. (E) After knockdown of OGT, HEK 293T cells were transfected with Flag-RelA. Flag IPs were subjected to in vitro acetylation with recombinant p300. Immunoblots were analyzed with RelA(AcK310Ac) antibody. Data are a representative example from three independent experiments, and densi- tometry results indicate the relative change in RelA(K310) acetylation where values were normalized to Flag-RelA inputs.

T308 as possible residues for O-GlcNAc modification based on their relative location to K310. Immunoprecipitated Flag-RelA Fig. 5. O-GlcNAc modification of RelA is detected between residues 277– mutants (T305A and T308A) displayed reduced levels of the 313. (A) HEK 293T cells were transfected with the indicated Flag-RelA con- O-GlcNAc modification compared with wild-type RelA(1-313) struct (1–270, 1–313, 1–317, or 1–551) and incubated overnight with PugNAc μ fi (Fig. 5B). Next, acetylation assays were performed. Wild-type (100 M). O-GlcNAc modi ed RelA was subsequently detected by Flag IP. Arrows indicate the relative location of the Flag-RelA proteins. (B) HEK 293T RelA(1-317) and mutant RelA(T308A) proteins were robustly cells were transfected with a Flag-RelA 1–313 construct containing either acetylated by p300 at K310 (Fig. 5C). However, site-directed a T305A or T308A mutation. O-GlcNAc modified RelA was detected by Flag mutagenesis of T305 on Flag-RelA(1-317) and Flag-RelA(1-551) IP. (C) Flag-RelA 1–317 mutants were expressed in HEK 293T cells. After Flag abolished the ability of p300 to acetylate K310 without affecting IP, RelA was acetylated in vitro with recombinant p300. RelA(K310Ac) was RelA–p300 interaction. Thus, mutation of a single analyzed by immunoblot. (D) Wild-type and T305A Flag-RelA was expressed in within the full-length RelA(T305A) significantly reduces O- HEK 293T cells. HEK 293T cells were transfected with full-length Flag-RelA (WT or T305A). After Flag IP, RelA was acetylated in vitro by using recombinant GlcNAcylated protein and abolishes K310 acetylation. p300, and the samples were assayed for K310 acetylation by immunoblot. Data are a representative example from three independent experiments. κ – RelA(T305) Is Essential for NF- B Mediated Cell Survival. Cell sur- Band densities of O-GlcNAc modified RelA (A and B) and RelA(K310Ac) (C and

vival is one of the best characterized pathways regulated by D) are shown relative to the levels observed for the wild-type Flag-RelA BIOCHEMISTRY NF-κB gene products (37–39). The ability of stimuli to activate protein. Densitometry analysis is normalized to Flag-RelA inputs.

Allison et al. PNAS | October 16, 2012 | vol. 109 | no. 42 | 16891 Downloaded by guest on September 28, 2021 After TNF stimulation, we find that OGT localizes to chro- matin and p300-mediated acetylation of RelA K310 requires the O-GlcNAc modification. Moreover, our studies identify RelA (T305) as the major site required for O-GlcNAcylation. The proximity of RelA(T305) to the major activating acetylation site, K310, suggests coordinated regulation between OGT and p300. Interestingly, an alignment of with nonhistone p300 substrates (43) identifies several transcription factors that con- tain S/T five residues from known p300 acetylation sites, in- cluding c-Myb, , p73, and . Therefore, the O-GlcNAc modification may act as a prerequisite cue for p300/ CBP-dependent acetylation of multiple transcription factors.

RelA(T305) as a Site of O-GlcNAc Regulation. Site-directed muta- genesis of T305 reduced levels of O-GlcNAc–modified RelA. Although RelA possesses multiple sites of O-GlcNAcylation, T305 is evolutionally conserved from Homo sapiens to Xenopus (Fig. S4). RelA(T305) resides within α-helix four, a segment essential for high-affinity binding of IκBα to NF-κB (33, 35, 36). In unstimulated cells, IκBα binds to the RelA through α-helix four, creating a tight interaction that would prevent access to cytosolic OGT. Therefore, the unique location of T305 ensures that canonical signaling through the IKK complex is required to stimulate IκBα degradation, NF-κB nuclear translocation, and chromatin occupancy before OGT gains access to RelA—a model that is supported by our work. Thus, the degradation of IκBα not only liberates RelA for nuclear translocation, but also ensures chromatin-associated OGT access to T305. Together, our findings indicate that the O-GlcNAc modification plays a key role in NF- κB activation by promoting RelA acetylation on chromatin.

Relationship Between OGT and Transcriptional Regulation. To date, OGT has been shown to interact with at least 10 chromatin- associated complexes that either activate or repress target genes (3, 7, 8, 10, 44, 45). In terms of gene induction, OGT resides in the Set1/Ash1/MLLL histone H3K4 methyltransferase complex. The H3K4 modification is one of the most important histone Fig. 6. NF-κB–mediated cell survival depends on T305. (A) Expression levels marks associated with transcriptional activation (46, 47). − − of Flag-RelA were assessed in reconstituted RelA / MEFs by immunoblot. (B) Using RelA, as a HBP-responsive transcription factor, we −/− After overnight serum starvation, reconstituted RelA MEFs were treated provide evidence that OGT is required for p300-mediated with TNF (1 ng/mL) for 2 h. Expression of cIAP-2 and TNFAIP3 was analyzed acetylation of chromatin-associated RelA. Our study suggests by QRT-PCR and normalized to GAPDH levels. Fold change is displayed rel- − − fi fi ative to the untreated RelA / MEF control cell line. (C and D) Reconstituted that the O-GlcNAc modi cation on RelA speci es it as a pre- − − RelA / MEFs were serum starved overnight and treated with either TNF (10 ferred target for p300 acetylation. Moreover, the knockdown of ng/mL) for 8 h or etoposide (50 μM) for ten hours. After treatment, the cells OGT not only dampens p300-dependent acetylation of RelA were assayed for nucleosomal fragmentation by using the cell death ELISA K310, but it also down-regulates p300 autoacetylation at K1499 or viability using trypan blue exclusion. Nucleosomal activity and cell via- without impacting p300 interaction with RelA (Fig. S5). Thus, − − bilities are displayed relative to RelA / MEFs reconstituted with wild-type future work is needed to determine whether p300/CBP contain Flag-p65. QRT-PCR, apoptosis, and cell viability results are a calculated domains that specifically recognize O-GlcNAc–modified tran- ± fi < = mean SD; NS, not signi cant compared with controls; *P 0.05, n 3. scription factors and whether this event is required to further potentiate acetyltransferase activity. under basal conditions (12, 15). Additional reports demonstrate Materials and Methods that B and T cells use OGT to fully activate NF-κB transcription, Cell Culture and Reagents. HEK 293T and DU145 cells were obtained from which corresponds with increased O-GlcNAcylated RelA (11). −/− ATCC. RelA MEFs were kindly provided by Denis Guttridge (Ohio State Although these studies provide insight into the requirement of fi κ – University, Columbus, OH) with permission from Amer Beg (Mof tt Cancer OGT for activation of NF- B dependent gene expression, the Center, Tampa, FL). Cells were cultured as described (21, 23, 25). For 5 mM molecular mechanism by which O-GlcNAc drives RelA tran- glucose treatments, cells were incubated overnight in DMEM containing 1 g/ scription remains elusive. Here, we provide evidence that addition L glucose (CellGro; 10–014-CV), and dialyzed FBS (Gibco; 26400–036). Glcn of the O-GlcNAc modification on RelA promotes NF-κBtran- treatment lasted 4 h after overnight incubation in 5 mM glucose. Reagents scription by potentiating p300-dependent acetylation on K310 (Fig. purchased were as follows: TNF (Invitrogen, PHC3016), PugNAc (Toronto S3). Several laboratories including our own have demonstrated Research Chemicals), and Glcn and etoposide (Sigma-Aldrich). Antibodies α the importance of RelA(K310) acetylation for active NF-κBtran- were as follows: M2-Flag, M2-Flag Agarose, -tubulin, and OGT (Sigma; κ α scription (25, 30, 31). This observation is further supported by F1804, A2220, T9026, O6139); RelA, p105, I B , HDAC1, normal mouse IgG, and normal rabbit IgG (Santa Cruz; sc-372, sc-1191, sc-371, sc-7872, sc-2025, recent evidence that monomethylation of RelA(K310me1) acts – fi κ sc-2027); ChIP grade RelA and p300 (Millipore; AB1604, 05 257); RelA as an antagonistic modi cation to repress NF- B transcription (K310Ac) (Cell Signaling; 3045); H3K14Ac and RNA polymerase II (Active (42). Thus, posttranslational modifications that regulate the Motif; 39599, 39097); V5 (Invitrogen, R960-025); HA (Covance; MMS-101P); acetylation or status of RelA(K310) have a significant anti-mouse HRP and anti-rabbit HRP (Promega; W4021, W4011); anti-mouse impact on cellular processes regulated by NF-κB gene products. IgM (Bethyl, A90-101P). O-GlcNAc (CTD110.6) was provided by G.W.H.

16892 | www.pnas.org/cgi/doi/10.1073/pnas.1208468109 Allison et al. Downloaded by guest on September 28, 2021 Plasmid Constructs. Full-length cDNA OGT (Origene; TC124260) was used as Creation of Retrovirus and Stable Cell Lines. Retroviruses encoding pBabe- − − a template and subcloned into V5-His pCDNA3.1 (Invitrogen; V810-20). The Puro RelA and mutant RelA were generated. The RelA / MEFS were grown Myc-tagged OGA plasmid was provided by G.W.H. pBabe-Puro was kindly to subconfluency and infected as described (25). Forty-eight hours after in- provided by R. A. Weinberg (Whitehead Institute, Cambridge, MA). The Flag- fection, 1.5 μg/mL puromycin (Sigma) was added for selection. Colonies were RelA, HA-p300, β-Galactosidase, and the 3×-κB-luciferase reporter were de- isolated and expression of RelA was confirmed via immunoblotting. scribed (25). Plasmid mutagenesis was performed by using the QuikChange II − − XL Site-directed mutagenesis kit (Stratagene). Cell Death ELISA and Cell Viability Assay. The RelA / MEF cell lines stably expressing various constructs were treated for 8 h with 10 ng/mL TNF or for 10 h μ Transfections, Luciferase Assays, QRT-PCR, and ChIP Assays. Plasmid and siRNA with 50 M etoposide. After treatment, cell death ELISA (Roche; 11774425001) transfections and luciferase assays were performed as described (25). QRT- or cell viability assay was carried out by using trypan blue exclusion. PCR experiments were carried out as described (48). PCR primers are shown in Table S1. ChIP and Re-ChIP experiments were carried out as described (21). Statistics. Fold changes were log2 transformed, and a one-tailed Student t test was performed by using Microsoft Excel. Data for all experiments were fi < Immunoprecipitation, Acetylation Assays, and Immunoblotting. Immunopre- considered statistically signi cant when P 0.05. cipitation, acetylation assays, and immunoblotting were performed as de- scribed (21, 23, 25). In vitro acetylation assays were carried out by using ACKNOWLEDGMENTS. We thank Ms. A. Sherman for editorial assistance. Work was supported by National Institute of Health Grants R01CA132580, recombinant full-length p300 (ProteinOne; 2004–01) and the HAT assay kit R01CA104397 (to M.W.M.), R01CA136705 (to D.R.J.), R01DK61671, and – fi (Millipore; 17 289). To detect O-GlcNAc modi ed RelA, transfected HEK P01HL107153 (to G.W.H.). In addition, both M.W.M and D.R.J. were supported, 293T cells were incubated overnight with PugNAc (100 μM) and Flag IP RelA in part, by independent awards provided to the University of Virginia by Philip proteins were analyzed by immunoblotting. Morris USA through an external review process.

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