Metabolic Reprogramming Induces Germinal Center B Cell Differentiation through Bcl6 Locus Remodeling

•IL-4-signaling induces Bcl6 expression and GC B cell differentiation•IL-4 alters TCA cycle to accumulate αKG, a cofactor for H3K27-demethylase•STAT6 recruits H3K27-demethylase UTX, leading to activation of the Bcl6 locus•GC B cell development requires αKG and enzymes regulating αKG level The germinal center (GC) reaction is essential for long-lived humoral immunity. However, molecular requirements for the induction of Bcl6, the master regulator for GC B cell differentiation, remain unclear. Through screening for cytokines and other stimuli that regulate Bcl6 expression, we identify IL-4 as the strongest inducer. IL-4 signaling alters the metabolomic profile in activated B cells and induces accumulation of the TCA cycle intermediate α-ketoglutarate (αKG), which is required for activation of the Bcl6 gene locus. Mechanistically, after IL-4 treatment, STAT6 bound to the known enhancers in the Bcl6 locus recruits UTX, a demethylase for the repressive histone mark H3K27me3 that requires αKG as a cofactor. In turn, the H3K27me3 demethylation activates the enhancers and transcription of the Bcl6 gene. We propose that IL-4-mediated metabolic reprogramming in B cells is pivotal for epigenomic activation of Bcl6 expression to promote GC B cell differentiation. The germinal center (GC) reaction is essential for long-lived humoral immunity. However, molecular requirements for the induction of Bcl6, the master regulator for GC B cell differentiation, remain unclear. Through screening for cytokines and other stimuli that regulate Bcl6 expression, we identify IL-4 as the strongest inducer. IL-4 signaling alters the metabolomic profile in activated B cells and induces accumulation of the TCA cycle intermediate α-ketoglutarate (αKG), which is required for activation of the Bcl6 gene locus. Mechanistically, after IL-4 treatment, STAT6 bound to the known enhancers in the Bcl6 locus recruits UTX, a demethylase for the repressive histone mark H3K27me3 that requires αKG as a cofactor. In turn, the H3K27me3 demethylation activates the enhancers and transcription of the Bcl6 gene. We propose that IL-4-mediated metabolic reprogramming in B cells is pivotal for epigenomic activation of Bcl6 expression to promote GC B cell differentiation. One of the hallmarks of the adaptive immune response is the generation of immunological memory. Long-lived memory B cells and plasma cells (PCs) with high affinity for antigen are formed in the germinal center (GC) through B cell proliferation, somatic hypermutation (SHM) and affinity-based selection (Victora and Nussenzweig, 2012Victora G.D. Nussenzweig M.C. Germinal centers.Annu. Rev. Immunol. 2012; 30: 429-457Crossref PubMed Scopus (1354) Google Scholar). Thus, GC formation is essential for efficient adaptive immune responses. Upon antigen recognition by their B cell receptor (BCR), B cells interact with cognate T cells and are activated by signals through CD40 and cytokine receptors, determining their cell fate. Only B cells expressing the transcriptional repressor Bcl6 can differentiate into GC B cells. Bcl6 is the master regulator for GC B cell differentiation; it directly suppresses the expression of many genes involved in signal transduction through BCR and CD40, PC differentiation, cell-cycle arrest, and DNA damage responses (Basso and Dalla-Favera, 2012Basso K. Dalla-Favera R. Roles of BCL6 in normal and transformed germinal center B cells.Immunol. Rev. 2012; 247: 172-183Crossref PubMed Scopus (290) Google Scholar). Therefore, Bcl6 expression prevents premature activation and terminal differentiation, and makes it possible for GC B cells to undergo massive proliferation, SHM, and the consequent affinity maturation (Basso and Dalla-Favera, 2012Basso K. Dalla-Favera R. Roles of BCL6 in normal and transformed germinal center B cells.Immunol. Rev. 2012; 247: 172-183Crossref PubMed Scopus (290) Google Scholar). Although the function of Bcl6 has been studied in detail, the molecular mechanisms underlying the induction of Bcl6 expression remain elusive. This is mainly due to the lack of an in vitro system in which Bcl6 expression can be reliably induced. Immune cell activation is accompanied by metabolic reprogramming to meet increased bioenergetic and biosynthetic demands for cellular growth and proliferation (Pearce et al., 2013Pearce E.L. Poffenberger M.C. Chang C.-H. Jones R.G. Fueling immunity: insights into metabolism and lymphocyte function.Science. 2013; 342: 1242454Crossref PubMed Scopus (833) Google Scholar). Once activated, lymphocytes engage in aerobic glycolysis, producing lactate, rather than in mitochondrial oxidative metabolism, even in the presence of oxygen. This process is known as the Warburg effect, a common feature of actively proliferating cells, which allows cells to produce ATP quickly and to generate the metabolic intermediates required for biosynthesis (Vander Heiden et al., 2009Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science. 2009; 324: 1029-1033Crossref PubMed Scopus (10206) Google Scholar). Thus, after BCR or CD40 stimulation, B cells dramatically increase glycolytic activity (Woodland et al., 2008Woodland R.T. Fox C.J. Schmidt M.R. Hammerman P.S. Opferman J.T. Korsmeyer S.J. Hilbert D.M. Thompson C.B. Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival.Blood. 2008; 111: 750-760Crossref PubMed Scopus (153) Google Scholar). Furthermore, among highly proliferative GC B cells, a fraction of light-zone B cells that can receive T cell help activate mammalian target of rapamycin complex 1 (mTORC1) signaling and express c-Myc, both of which positively regulate glycolysis and anabolic metabolism and are required for GC development (Calado et al., 2012Calado D.P. Sasaki Y. Godinho S.A. Pellerin A. Köchert K. Sleckman B.P. de Alborán I.M. Janz M. Rodig S. Rajewsky K. The cell-cycle regulator c-Myc is essential for the formation and maintenance of germinal centers.Nat. Immunol. 2012; 13: 1092-1100Crossref PubMed Scopus (268) Google Scholar; Dominguez-Sola et al., 2012Dominguez-Sola D. Victora G.D. Ying C.Y. Phan R.T. Saito M. Nussenzweig M.C. Dalla-Favera R. The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry.Nat. Immunol. 2012; 13: 1083-1091Crossref PubMed Scopus (293) Google Scholar; Ersching et al., 2017Ersching J. Efeyan A. Mesin L. Jacobsen J.T. Pasqual G. Grabiner B.C. Dominguez-Sola D. Sabatini D.M. Victora G.D. Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase.Immunity. 2017; 46: 1045-1058.e6Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). A growing body of evidence suggests that some metabolic pathways influence epigenetic gene regulation by providing donors and cofactors for epigenetic modifiers. For example, the threonine-fueled metabolism in embryonic stem cells (ESCs) provides S-adenosylmethionine, the methyl-group donor for methylation, to maintain the histone methylation required for pluripotency (Shyh-Chang et al., 2013Shyh-Chang N. Locasale J.W. Lyssiotis C.A. Zheng Y. Teo R.Y. Ratanasirintrawoot S. Zhang J. Onder T. Unternaehrer J.J. Zhu H. et al.Influence of threonine metabolism on S-adenosylmethionine and histone methylation.Science. 2013; 339: 222-226Crossref PubMed Scopus (453) Google Scholar). Another example is that enhanced glycolysis elevates cytosolic acetyl-coenzyme A (CoA), a universal donor for acetylation, to enhance histone acetylation and expression of Ifng in activated T cells (Peng et al., 2016Peng M. Yin N. Chhangawala S. Xu K. Leslie C.S. Li M.O. Aerobic glycolysis promotes T helper 1 cell differentiation through an epigenetic mechanism.Science. 2016; 354: 481-484Crossref PubMed Scopus (428) Google Scholar). Furthermore, the accumulation of the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate (αKG), a required cofactor for Jumonji C domain-containing (JmjC) histone demethylases and ten-eleven translocation (TET) DNA dioxygenases, has been reported to be required for maintaining the pluripotency of ESCs (Carey et al., 2015Carey B.W. Finley L.W.S. Cross J.R. Allis C.D. Thompson C.B. Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells.Nature. 2015; 518: 413-416Crossref PubMed Scopus (597) Google Scholar) and primordial germ cells (Tischler et al., 2019Tischler J. Gruhn W.H. Reid J. Allgeyer E. Buettner F. Marr C. Theis F. Simons B.D. Wernisch L. Surani M.A. Metabolic regulation of pluripotency and germ cell fate through α-ketoglutarate.EMBO J. 2019; 38: e99518Crossref PubMed Scopus (57) Google Scholar) and for activating effector gene programs in T cells (Chisolm et al., 2017Chisolm D.A. Savic D. Moore A.J. Ballesteros-Tato A. León B. Crossman D.K. Murre C. Myers R.M. Weinmann A.S. CCCTC-Binding Factor Translates Interleukin 2- and α-Ketoglutarate-Sensitive Metabolic Changes in T Cells into Context-Dependent Gene Programs.Immunity. 2017; 47: 251-267.e7Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To date, it is unknown whether B cell metabolism can control differentiation gene programs beyond meeting their bioenergetic and biosynthetic demands. Here, we demonstrated that IL-4-mediated reprogramming of TCA cycle metabolism drives the accumulation of αKG that integrates epigenetic activation of the Bcl6 gene to induce GC B cell differentiation. To elucidate the mechanisms upregulating Bcl6 expression in B cells at a molecular level, we used our previously reported B cell culture system called “iGB culture” (Nojima et al., 2011Nojima T. Haniuda K. Moutai T. Matsudaira M. Mizokawa S. Shiratori I. Azuma T. Kitamura D. In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo.Nat. Commun. 2011; 2: 465Crossref PubMed Scopus (174) Google Scholar). In this system, splenic B cells are cultured with interleukin-4 (IL-4) on feeder cells expressing CD40L and B-cell activating factor BAFF (40LB) and extensively proliferate and efficiently undergo class switching to immunoglobulin G1 (IgG1) and IgE. These B cells (called iGB cells) express modest levels of Bcl6 mRNA (Nojima et al., 2011Nojima T. Haniuda K. Moutai T. Matsudaira M. Mizokawa S. Shiratori I. Azuma T. Kitamura D. In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo.Nat. Commun. 2011; 2: 465Crossref PubMed Scopus (174) Google Scholar) and protein (Figure 1A), probably because chronic CD40 stimulation suppresses Bcl6 expression (Saito et al., 2007Saito M. Gao J. Basso K. Kitagawa Y. Smith P.M. Bhagat G. Pernis A. Pasqualucci L. Dalla-Favera R. A signaling pathway mediating downregulation of BCL6 in germinal center B cells is blocked by BCL6 gene alterations in B cell lymphoma.Cancer Cell. 2007; 12: 280-292Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar; Zhang et al., 2017Zhang T.T. Gonzalez D.G. Cote C.M. Kerfoot S.M. Deng S. Cheng Y. Magari M. Haberman A.M. Germinal center B cell development has distinctly regulated stages completed by disengagement from T cell help.eLife. 2017; 6: e19552Crossref PubMed Scopus (42) Google Scholar). “Plain culture” with medium alone for 1 day after removal of the feeder cells at day 3 of the iGB culture produced a distinct population of IgG1+ B cells highly expressing Bcl6 (Figure 1A). However, 60%–70% of the IgG1+ B cells remained Bcl6− after the plain culture, despite the preceding stimulation. Thus, we sought to find stimuli that induce Bcl6 expression in activated B cells by screening cytokines and other stimuli that could convert these Bcl6− cells into Bcl6+ cells. We treated the iGB cells with a panel of cytokines and antibodies (Abs) during the plain culture period for 1 day and then analyzed them by flow cytometry. We found that IL-4, IL-6, IL-13, and IL-21 increased the frequency of Bcl6+ cells among IgG1+ cells, and that IL-4 was the most potent inducer (Figures 1B, 1C, and S1A). In contrast, BCR stimulation by anti-IgG Ab strongly reduced the frequency of Bcl6+ cells, but instead increased the CD138+ PC (Figures 1B, 1C, and S1A). In line with these data, IL-4 upregulated Bcl6 mRNA expression, whereas anti-IgG Ab downregulated it and upregulated Irf4 expression (Figure 1D). To assess the impact of IL-4 on GC development, we administered IL-4 complexed with an anti-IL4 Ab (IL-4c), which extends the bioactive half-life of the cytokine (Finkelman et al., 1993Finkelman F.D. Madden K.B. Morris S.C. Holmes J.M. Boiani N. Katona I.M. Maliszewski C.R. Anti-cytokine antibodies as carrier proteins. Prolongation of in vivo effects of exogenous cytokines by injection of cytokine-anti-cytokine antibody complexes.J. Immunol. 1993; 151: 1235-1244Crossref PubMed Google Scholar) into immunized mice during the early stage of GC formation. IL-4c treatment significantly increased the number of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific GC B cells (Figure S1D). Given that cellular metabolism has been reported to be linked to gene expression and differentiation (Pearce et al., 2013Pearce E.L. Poffenberger M.C. Chang C.-H. Jones R.G. Fueling immunity: insights into metabolism and lymphocyte function.Science. 2013; 342: 1242454Crossref PubMed Scopus (833) Google Scholar), we next assessed the metabolic properties of the iGB cells after stimulation during the plain culture period. We measured mitochondrial membrane potential (ΔΨm) intimately linked to electron transport chain (ETC) activity by staining with the fluorescent dye TMRM (tetramethylrhodamine, methyl ester, perchlorate). We found that stimulation with IL-4 increased the ΔΨm of iGB cells, whereas anti-IgG stimulation decreased it, as compared to non-stimulated cultures (Figure 1E). Next, we measured lactate production 4 h after stimulation and found that anti-IgG but not IL-4 facilitated its production (Figure 1F). These data indicated that IL-4-receptor (IL-4R) or BCR signaling activates mitochondrial or glycolytic metabolism, respectively. The inhibition of mitochondrial oxidative metabolism by treatment with oligomycin, an inhibitor of the mitochondrial ETC (Figure S1E), abolished Bcl6 upregulation after IL-4 stimulation, but not PC differentiation by anti-IgG (Figure 1G). These findings suggest that IL-4 signaling upregulates Bcl6 expression through potentiating mitochondrial oxidative metabolism. Given the unique dependency of Bcl6 expression on mitochondrial oxidative metabolism, we decided to characterize the metabolic status of B cell subsets that develop after antigen challenge. To identify antigen-specific precursors of GC B cells (pre-GC B cell) and GC B cells, we transferred naive B (NB) cells from CD45.1 mice carrying a knockin allele encoding a hapten NP-specific VH region (B1-8hi) into C57BL/6 (B6) mice, which were immunized with a conjugate of NP and chicken γ-globulin (CGG) in alum the next day. Among the donor-derived B220+ NP-binding B cells, pre-GC B cells were defined as GL7+ CD38hi cells on day 3.5 after immunization and GC B cells as GL7+ CD38lo cells on day 7. We analyzed these cells, as well as PCs on day 7 and NB cells from unimmunized mice (Figures 2A and S2A), for mitochondrial mass, ΔΨm, and mitochondrial reactive oxygen species (ROS) by staining with MitoTracker, TMRM, or MitoSOX, respectively. Mitochondrial mass, ΔΨm, and mitochondrial ROS were higher in pre-GC and GC B cells than in NB cells or PCs (Figure 2B), suggesting activated mitochondrial metabolism in pre-GC and GC B cells. Glycolytic activity, assessed by the uptake of 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; a fluorescent analog of glucose) and production of lactate (an end product of glycolysis) was markedly high in pre-GC B cells and PCs and modest but higher than in NB cells in GC B cells (Figures 2B and 2C). The enzymatic activity of pyruvate dehydrogenase (PDH), which promotes pyruvate entry into the TCA cycle at the expense of lactate production (Figure S1E), was increased in GC B cells compared with NB cells or PCs (Figures 2D and S2C), with a concomitant decrease in the inhibitory PDH phosphorylation in GC B cells (Figure S2D). These data indicate that GC B cells have activated mitochondrial oxidative metabolism and restrained aerobic glycolysis, suggesting that the Warburg effect may not fit overall GC B cells, even though GC B cells display an increased cellular size, intimately linked to biosynthesis, and the highest proliferative capacity of any of the B cell subsets (Figures 2B and 2E). To address the requirement for mitochondrial oxidative metabolism in each B cell subset, we treated immunized mice with oligomycin for 1 day before analysis (Figure 2F). The administration of oligomycin partially but significantly reduced the number of GC B cells, but not of pre-GC B cells, PCs, or follicular helper T (Tfh) cells (known to be required for GC formation) (Victora and Nussenzweig, 2012Victora G.D. Nussenzweig M.C. Germinal centers.Annu. Rev. Immunol. 2012; 30: 429-457Crossref PubMed Scopus (1354) Google Scholar), and attenuated the expression of Bcl6 in GC B cells but not in Tfh cells (Figures 2G, 2H, and S2E–S2G). In addition, inhibition of the first step of glycolysis by administrating 2-deoxyglucose at the same time point also diminished GC B cell numbers (data not shown), as reported previously (Jellusova et al., 2017Jellusova J. Cato M.H. Apgar J.R. Ramezani-Rad P. Leung C.R. Chen C. Richardson A.D. Conner E.M. Benschop R.J. Woodgett J.R. Rickert R.C. Gsk3 is a metabolic checkpoint regulator in B cells.Nat. Immunol. 2017; 18: 303-312Crossref PubMed Scopus (168) Google Scholar). Our results show that GC B cell development relies on mitochondrial oxidative metabolism, possibly fueled by glycolysis. To elucidate how mitochondrial metabolism contributes to the induction of Bcl6 expression, we profiled the metabolome of iGB cells treated with IL-4 or anti-IgG for 6 h, or untreated, without feeder cells (Figure 3A). At this time point, expression levels of Bcl6 and Blimp1 mRNA were unchanged (Figure S3A). The metabolite profiling revealed different alterations between the cells stimulated with IL-4 or anti-IgG (Figure S3B), among which the TCA cycle was the most significantly altered pathway (Figure 3B). Among the TCA cycle metabolites, the amount of αKG was strikingly higher in the cells stimulated with IL-4 than anti-IgG or the unstimulated cells (Figure 3C). Kinetics analysis demonstrated that the amount of αKG was elevated by 6 h and then became almost stable at least until 24 h after stimulation with IL-4 (Figure S3C). However, the lactate:pyruvate ratio was highest in the anti-IgG-stimulated cells, suggesting increased aerobic glycolysis, which consumes pyruvate for producing lactate and limits the TCA cycle (Figure 3D). Similarly, ex vivo GC B cells contained a higher amount of αKG than NB cells (Figure 3E). We next assessed the impact of mitochondrial oxidative metabolism on the intracellular αKG levels. In accord with a previous report that ETC inhibitors lower the αKG level (Fendt et al., 2013Fendt S.-M. Bell E.L. Keibler M.A. Olenchock B.A. Mayers J.R. Wasylenko T.M. Vokes N.I. Guarente L. Vander Heiden M.G. Stephanopoulos G. Reductive glutamine metabolism is a function of the α-ketoglutarate to citrate ratio in cells.Nat. Commun. 2013; 4: 2236Crossref PubMed Scopus (240) Google Scholar), treatment with oligomycin during the IL-4 stimulation significantly decreased the amount of αKG in iGB cells (Figure 3F). Given the parallel effects of oligomycin in suppressing both IL-4-induced αKG accumulation (above) and Bcl6 expression (Figure 1G), as well as in vivo GC development and Bcl6 expression (Figures 2G and 2H), we next sought to reveal the molecular link between αKG and Bcl6 expression. For this purpose, iGB cells were plain cultured with IL-4 under low glucose conditions to limit αKG production via the TCA cycle and supplemented or not with dimethyl-αKG (DMαKG), a cell-permeable αKG derivative (Figure 3G). Under the low glucose conditions, the expression of Bcl6 was mostly abolished, while supplementation with DMαKG restored the expression of Bcl6 but not of Irf4 (Figure 3H). Supplementation with diethyl-succinate (DE-Suc), a cell-permeable derivative of succinate that is a downstream metabolite of αKG in the TCA cycle, did not restore Bcl6 expression but viability of cells cultured under low glucose conditions (Figures 3I and 3J). These data suggest that Bcl6 expression is driven by αKG produced in the glucose-fueled TCA cycle in IL-4-stimulated cells and that this αKG function is distinct from a general role of TCA cycle metabolites on energy production. To test the effect of αKG on GC B cell differentiation, we cultured B1-8hi B cells under the same conditions as above and transferred them into NP-CGG-immunized B6 mice (Figure 3G). B cells cultured with low glucose barely developed into GC B cells in vivo, but supplementation with DMαKG rescued the GC B cell development (Figure 3K). Our findings indicate that IL-4 signaling reprograms mitochondrial metabolism in activated B cells, which in turn accumulates αKG and drives the expression of Bcl6. αKG is a cofactor for αKG-dependent dioxygenases, many of which function as epigenetic modifiers, and the accumulation of αKG is known to activate αKG-dependent histone demethylases (Carey et al., 2015Carey B.W. Finley L.W.S. Cross J.R. Allis C.D. Thompson C.B. Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells.Nature. 2015; 518: 413-416Crossref PubMed Scopus (597) Google Scholar; Chisolm et al., 2017Chisolm D.A. Savic D. Moore A.J. Ballesteros-Tato A. León B. Crossman D.K. Murre C. Myers R.M. Weinmann A.S. CCCTC-Binding Factor Translates Interleukin 2- and α-Ketoglutarate-Sensitive Metabolic Changes in T Cells into Context-Dependent Gene Programs.Immunity. 2017; 47: 251-267.e7Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar; Tischler et al., 2019Tischler J. Gruhn W.H. Reid J. Allgeyer E. Buettner F. Marr C. Theis F. Simons B.D. Wernisch L. Surani M.A. Metabolic regulation of pluripotency and germ cell fate through α-ketoglutarate.EMBO J. 2019; 38: e99518Crossref PubMed Scopus (57) Google Scholar). Thus, we focused on the epigenetic regulation of Bcl6 expression. We found that in GC B cells, the global trimethylation of histone H3 lysine 27 (H3K27me3), a repressive histone mark contributing to chromatin compaction, was decreased and the acetylation of H3K27 (H3K27Ac) was increased, whereas the trimethylation of H3K4 and H3K9 remained mostly unchanged as compared with NB cells (Figure 4A). We next analyzed published chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) data of NB cells and GC B cells, and found that H3K27me3 was reduced in GC B cells in a region spanning from ~140 to ~360 kb upstream of the Bcl6 transcriptional start site (Figure S4A). This region largely overlapped with two enhancer elements (enhancer 1 and 2) that are activated specifically in GC B cells and interact with the Bcl6 promoter region (Bunting et al., 2016Bunting K.L. Soong T.D. Singh R. Jiang Y. Béguelin W. Poloway D.W. Swed B.L. Hatzi K. Reisacher W. Teater M. et al.Multi-tiered Reorganization of the Genome during B Cell Affinity Maturation Anchored by a Germinal Center-Specific Locus Control Region.Immunity. 2016; 45: 497-512Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar; Ryan et al., 2015Ryan R.J.H. Drier Y. Whitton H. Cotton M.J. Kaur J. Issner R. Gillespie S. Epstein C.B. Nardi V. Sohani A.R. et al.Detection of enhancer-associated rearrangements reveals mechanisms of oncogene dysregulation in B-cell lymphoma.Cancer Discov. 2015; 5: 1058-1071Crossref PubMed Scopus (83) Google Scholar). These enhancer regions were enriched in GC B cells with an enhancer mark H3K4me1 together with H3K27Ac (Figure S4A), a modification associated with enhancer activity (Calo and Wysocka, 2013Calo E. Wysocka J. Modification of enhancer chromatin: what, how, and why?.Mol. Cell. 2013; 49: 825-837Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). Knock out of these regions has been reported to result in a defective GC formation, indicating that these enhancers are crucial for Bcl6 expression during GC B cell development (Bunting et al., 2016Bunting K.L. Soong T.D. Singh R. Jiang Y. Béguelin W. Poloway D.W. Swed B.L. Hatzi K. Reisacher W. Teater M. et al.Multi-tiered Reorganization of the Genome during B Cell Affinity Maturation Anchored by a Germinal Center-Specific Locus Control Region.Immunity. 2016; 45: 497-512Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Our ChIP coupled with quantitative PCR (qPCR) analysis confirmed that H3K27me3 was significantly lower in GC B cells than in NB cells, in the same enhancer regions of the Bcl6 locus, except for position −163 kb, where H3K27me3 was equally low in NB cells (Figure 4B). Therefore, we hypothesized that the H3K27me3 marks at the Bcl6 enhancers are actively demethylated to drive the transcription of the Bcl6 locus to promote GC B cell development. To test this hypothesis, we assessed the histone modifications in iGB cells treated with IL-4 or anti-IgG for 6 h in comparison to untreated cells. Our ChIP-qPCR assay revealed that treatment with IL-4 decreased H3K27me3 at enhancer 1 and 2 of the Bcl6 locus, again except for position −163 kb, concomitant with an increase in H3K27Ac on the enhancer and promoter regions (Figures 4C, 4D, and S4B). In contrast, treatment with IL-4 or anti-IgG had no effect on H3K27me3 levels in both enhancer and promoter regions of the Irf4 locus (Chapuy et al., 2013Chapuy B. McKeown M.R. Lin C.Y. Monti S. Roemer M.G.M. Qi J. Rahl P.B. Sun H.H. Yeda K.T. Doench J.G. et al.Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma.Cancer Cell. 2013; 24: 777-790Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar; Raisner et al., 2018Raisner R. Kharbanda S. Jin L. Jeng E. Chan E. Merchant M. Haverty P.M. Bainer R. Cheung T. Arnott D. et al.Enhancer Activity Requires CBP/P300 Bromodomain-Dependent Histone H3K27 Acetylation.Cell Rep. 2018; 24: 1722-1729Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), although anti-IgG increased H3K27Ac in these regions (Figures 4E, 4F, S4C, and S4D). Given the dependence of Bcl6 expression on mitochondrial oxidative metabolism that regulates αKG accumulation, we next assessed whether IL-4-mediated reduction of H3K27me3 at the Bcl6 enhancers depends on this metabolism. First, we demonstrated that treatment with oligomycin resulted in an increase in H3K27me3 at the Bcl6 enhancers (Figure S4E) and a marked decrease in Bcl6 mRNA expression that was partially restored by the addition of DMαKG (Figure S4F). Second, to determine whether αKG is required for the IL-4-mediated loss of H3K27me3 in the Bcl6 enhancers, we treated glucose-restricted iGB cells with DMαKG in the presence of IL-4. Whereas H3K27me3 on enhancer 1 and 2 of the Bcl6 locus was increased by the restriction of glucose, the addition of DMαKG restored it to a lower level (Figure 4G). These data suggested that IL-4 signaling induces epigenetic remodeling of the Bcl6 locus and activates its enhancers through mechanisms depending on mitochondrial oxidative metabolism and αKG. Given the finding that αKG lowers H3K27me3 at the Bcl6 enhancers, we addressed the possible involvement of an αKG-dependent H3K27me3-specific demethylase. Treatment of iGB cells with GSK-J4 (Kruidenier et al., 2012Kruidenier L. Chung C.W. Cheng Z. Liddle J. Che K. Joberty G. Bantscheff M. Bountra C. Bridges A. Diallo H. et al.A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response.Nature. 2012; 488: 404-408Crossref PubMed Scopus (673) Google Scholar), a potent inhibitor of UTX (encoded by Kdm6a), resulted in an increase in H3K27me3 at the Bcl6 enhancers and a concomitant reduction in Bcl6 expression after IL-4-treatment (Figures 5A and 5B ). In addition, the treatment of immunized mice with GSK-J4 during the early stage of the GC formation significantly decreased the number of antigen-specific GC B cells (Figure 5C). Moreover, small hairpin RNA (shRNA)-mediated knockdown of UTX in iGB cells (Figures S5A and S5B) increased H3K27me3 at the Bcl6 enhancers and decreased the expression of Bcl6 after treatment with IL-4 (Figures 5D and 5E). To assess the role of UTX in the αKG-mediated GC formation in vivo, B cells transduced with shControl or shUTX and cultured under low glucose conditions with or without DMαKG were transferred into NP-CGG-immunized B6 mice (Figure S5C). Supplementation with DMαKG restored the GC formation of B cells transduced with shControl but not of those with shUTX (Figure S5D). These data highlight that active H3K27me3-demethylation mediated by UTX with αKG is required for Bcl6 upregulation and GC B cell development. Since the addition of DMαKG alone to iGB cells cultured without IL-4 did not result in an increase in Bcl6 expression (Figure S6A), general activation of UTX by αKG appears insufficient for the Bcl6 induction. Thus, we next sought to clarify the mechanisms by which the genome-wide epigenetic modifier UTX targets the Bcl6 locus after the IL-4 stimulation. The IL-4-IL-4R axis is known to activate transcription factors signal transducer and activator of transcription 3 (STAT3), STAT5, and STAT6, and this was the case for iGB cells (Figure 6A). shRNA-mediated knockdown revealed that STAT6 but not STAT3 or STAT5 was responsible for the upregulation of Bcl6 expression after IL-4 treatment (Figures 6B and S6B). Then, we assessed the effect of STAT6 knockdown in vivo. We transduced in vivo-primed B1-8hi B cells with the STAT