EZH2 Represses the B Cell Transcriptional Program and Regulates Antibody-Secreting Cell Metabolism and Antibody Production This information is current as Muyao Guo, Madeline J. Price, Dillon G. Patterson, of September 28, 2021. Benjamin G. Barwick, Robert R. Haines, Anna K. Kania, John E. Bradley, Troy D. Randall, Jeremy M. Boss and Christopher D. Scharer J Immunol published online 29 December 2017 http://www.jimmunol.org/content/early/2017/12/29/jimmun Downloaded from ol.1701470 Supplementary http://www.jimmunol.org/content/suppl/2017/12/29/jimmunol.170147 Material 0.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published December 29, 2017, doi:10.4049/jimmunol.1701470 The Journal of Immunology EZH2 Represses the B Cell Transcriptional Program and Regulates Antibody-Secreting Cell Metabolism and Antibody Production Muyao Guo,*,† Madeline J. Price,* Dillon G. Patterson,* Benjamin G. Barwick,‡ Robert R. Haines,* Anna K. Kania,* John E. Bradley,x Troy D. Randall,x Jeremy M. Boss,* and Christopher D. Scharer* Epigenetic remodeling is required during B cell differentiation. However, little is known about the direct functions of epigenetic enzymes in Ab-secreting cells (ASC) in vivo. In this study, we examined ASC differentiation independent of T cell help and germinal center reactions using mice with inducible or B cell–specific deletions of Ezh2. Following stimulation with influenza virus or LPS, Ezh2-deficient ASC poorly proliferated and inappropriately maintained expression of inflammatory pathways, B cell–lineage Downloaded from transcription factors, and Blimp-1–repressed genes, leading to fewer and less functional ASC. In the absence of EZH2, genes that normally gained histone H3 lysine 27 trimethylation were dysregulated and exhibited increased chromatin accessibility. Furthermore, EZH2 was also required for maximal Ab secretion by ASC, in part due to reduced mitochondrial respiration, impaired glucose metabolism, and poor expression of the unfolded-protein response pathway. Together, these data demonstrate that EZH2 is essential in facilitating epigenetic changes that regulate ASC fate, function, and metabolism. The Journal of Immunology, 2018, 200: 000–000. http://www.jimmunol.org/ he humoral immune response is initiated when B cells are enhanced metabolism and Ab secretion (1–3). The ASC tran- stimulated to differentiate into Ab-secreting cells (ASC), scriptional program is enabled by the expression of transcription T also known as plasma cells. Irrespective of how they are factors—such as Blimp-1, XBP1, and IRF4, which reinforce and activated or the availability of T cell help, a distinct set of support ASC transcriptional changes (2, 4)—that are coupled to a reprogramming events are required to terminate the B cell–fate reorganization of the epigenome (5–7). Although the transcription program and initiate a new gene expression program that supports factors and genes they regulate have been studied biochemically and genetically, little is known about the role of epigenetic by guest on September 28, 2021 modifiers in ASC function and programming. *Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322; †Xiangya School of Medicine, Central South University, Changsha, 410008, China; Epigenetic modifications are dynamic during distinct stages of ‡Department of Radiation Oncology, Emory University, Atlanta, GA 30322; and xDivision B cell differentiation. In both mice and humans, DNA methylation of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama is primarily lost as B cells differentiate to ASC in response to both at Birmingham, Birmingham, AL 35294 T cell–dependent and –independent stimuli (8–10). Deletion of the ORCIDs: 0000-0003-1347-0848 (M.G.); 0000-0001-6053-5216 (D.G.P.); 0000-0002- 5819-5694 (A.K.K.); 0000-0002-2432-1840 (J.M.B.); 0000-0001-7716-8504 (C.D.S.). maintenance methyltransferase Dnmt1 leads to a reduction in germinal center (GC) B cells (11), but whether de novo DNA Received for publication October 23, 2017. Accepted for publication November 29, 2017. methylation is required for B cell differentiation is not known. This work was supported by National Institutes of Health Grants 1R01AI123733 Histone modifications, characterized by chromatin immunopre- to J.M.B.; P01 AI 125180-02 to J.M.B. and T.D.R.; T32 GM0008490 to R.R.H., cipitation (ChIP) sequencing (ChIP-seq), have cell type–specific A.K.K., and B.G.B.; F31 AI112261 to B.G.B.; and F31 1F31 AI131532 to R.R.H. patterns in naive B cells (nB), ex vivo–differentiated ASC, and in The sequencing data presented in this article have been submitted to the National GC B cells (12–16). However, few studies have examined the role Center for Biotechnology Information’s Gene Expression Omnibus (https://www. ncbi.nlm.nih.gov/geo/) under accession number GSE103195. of histone-modifying enzymes using genetic approaches (5). Address correspondence and reprint requests to Dr. Christopher D. Scharer and Deletion of the histone acetyltransferase, MOZ, reduces GC Prof. Jeremy M. Boss, Emory University, 1510 Clifton Road, Atlanta, GA 30322. B cells and skews responding B cells toward low-affinity IgM+ E-mail addresses: [email protected] (C.D.S.) and [email protected] (J.M.B.) memory B cells (17). Additionally, treatment of mice with histone The online version of this article contains supplemental material. deacetylase inhibitors reduces B cell responses (18), indicating Abbreviations used in this article: actB, activated B cell; AID, activation-induced that both erasing and writing de novo epigenetic modifications is cytidine deaminase; ASC, Ab-secreting cell; ATAC-seq, assay for transposase- accessible chromatin sequencing; CD19-Ezh2KO, Ezh2fl/flCD19Cre/+; ChIP, chroma- an essential process in B cell differentiation. Importantly, epi- tin immunoprecipitation; ChIP-seq, ChIP sequencing; Ctl, control; CTV, CellTrace genetic modifiers are frequent targets of both activating and Violet; DAR, differentially accessible region; DEG, differentially expressed gene; inactivating mutations in lymphomas (19, 20). Therefore, a full ERT2-Cre Ctl, Ezh2fl/+Rosa26CreERT2/+;ERT2-Ezh2KO, Ezh2fl/flRosa26CreERT2/+; EZH2, enhancer of zest 2; FC, fold change; FDR, false discovery rate; FSC, forward understanding of epigenetic mechanisms and targets for distinct light scatter; GC, germinal center; GSEA, gene set enrichment analysis; HA, hem- enzymes is important to manipulate B cell differentiation and to agglutinin; H3K27me3, histone H3 lysine 27 trimethylation; KO, knockout; MEDS, mosaic end double stranded; nB, naive B cell; PC, principal component; PR8, A/PR8/ understand the effects of therapeutics targeting these enzymes. 34 strain of influenza; PRC2, polycomb repressive complex 2; qPCR, quantitative One of the best characterized repressive epigenetic histone PCR; rppm, reads per peak per million; RT-qPCR, quantitative RT-PCR; SSC, side modifications is the trimethylation of histone H3 at lysine scatter; UPR, unfolded-protein response. 27 (H3K27me3), which is mediated by the polycomb repressive Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 complex 2 (PRC2) (21, 22). Enhancer of zest 2 (EZH2) is the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701470 2 EZH2 IN ASC FUNCTION catalytic subunit of the PRC2 complex and functions as an essential were enriched by positive selection of CD138+ cells from the spleens of transcriptional silencer (23–25). EZH2 is upregulated in pre–B cells, mice 3 d post–LPS inoculation. Splenocytes were first stained with CD138- in which it is necessary for VDJ recombination during B cell de- allophycocyanin (14205, clone 281-2; BioLegend) and immunomagnetic enrich- k ment was performed using anti-allophycocyanin microbeads (130-090-855; velopment (26) and to repress germline Ig transcription (27). EZH2 Miltenyi Biotec). Enriched populations were analyzed for purity by flow is expressed at low levels in quiescent, nB, but is highly upregulated cytometry (Supplemental Fig. 1A, 1B). For FACS sorting of cellular division in GC B cells where it facilitates cellular proliferation, protects from samples in Fig. 1: following 3 d LPS inoculation, adoptively transferred activation-induced cytidine deaminase (AID) off-target activity, and CD45.1 cells were stained with CD45.1-PE (12-0453-82, clone A20; Bio- science) and immunomagnetic enrichment was performed using anti-PE represses the differentiation of GC B cells into ASC (15, 16, 28, 29). microbeads (130-048-801; Miltenyi Biotec) prior to FACS sorting as previ- EZH2 interacts with distinct sets of transcription factors, such as ously described (8). BCL6inGCBcells(29)andBlimp-1inASC(30),todirectcell