Modification of RelA by O-linked N-acetylglucosamine links glucose metabolism to NF-κB acetylation 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 biosynthesis 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 chromatin-associated to potentiate gene expression in response to tumor necrosis factor recruitment of IKKα results in phosphorylation 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 coactivator com- tional mark required for full NF-κB transcription. Mapping studies plexes that include either p300 or the CREB-binding protein reveal T305 as an important residue required for attachment of the (CBP) (20, 26, 27). Histone acetyltransferase (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 cell survival. Our work lysine 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 | apoptosis 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 glycosylation, 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) enzyme uses UDP-GlcNAc to sponsive transcription factor (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 serine or threonine (S/T) residues within tar- cultured in high glucose (25 mM) show elevated NF-κBtran- get proteins (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 enzymes, 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 cell proliferation, 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 kinase (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 (H3K14Ac) 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.