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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 , 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- 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 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 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 Flow cytometry and cell sorting type–specific gene repression programs. B cell–specific deletion of Ezh2 leads to a loss of GC formation, thereby leading to defects in For staining, cells were resuspended at 1 3 106 cells/100 ml in FACS buffer the formation of ASC (15, 16). However, no studies have directly (13 PBS, 1% BSA, 2 mM EDTA) and blocked with anti-Fc (2.4G2; Tonbo m 3 6 assessed whether or how EZH2 functions in ASC. Biosciences) at a concentration of 0.25 g/1 10 cells for 15 min on ice. The following Abs were used for FACS analysis: B220-PE-Cy7 (60-0452- In this study, we tested the role of EZH2 in two T-independent U100, clone RA3-6132; Tonbo Biosciences), CD43-FITC (553270, clone models of ASC differentiation, one initiated by the T-independent S7; BD Pharmingen), CD19-PerCP-Cy5.5 (65-0193-U100, clone 1D3; Ag, LPS, and the other initiated by influenza infection in the Tonbo Biosciences), CD138-BV711 (563193, clone 281-2; BD Horizon), absence of CD4 T cells. We found that EZH2 was progressively GL7-eFlour 660 (50-5902-82, clone GL-7; Invitrogen), CD45.1-FITC (35-0453- U500, clone A20; Tonbo Biosciences), CD45.2-PerCP-Cy5.5 (65-0454-U100, Downloaded from upregulated in stimulated B cells, with expression peaking in ASC. clone 104; Tonbo Biosciences), CD23-eFlour 450 (48-0232-80, clone B3B4; In addition, B cell–specific genes gained H3K27me3 in their eBioscience), CD21-allophycocyanin-Cy7 (47-0211-80, clone eBio8D9; eBio- promoters as ASC differentiation progressed, indicating that science), IgM-FITC (11-5890-85, clone eB121-15F9; eBioscience), IgD-BV605 EZH2 may repress these genes. Following immunization with the (583003, 11-26c.2a; BD Horizon), CD11b-allophycocyanin-Cy7 (25-0112- T-independent Ag, LPS, or infection with influenza virus in the U100, clone M1/70; Tonbo Biosciences), F4/80-allophycocyanin-Cy7 (123118, clone BM8; BioLegend), Thy1.2-allophycocyanin-Cy7 (105328, clone 30-H12; absence of T cells, mice with a tamoxifen-inducible Ezh2 deletion BioLegend), Ezh2-PE (562478, clone 11/Ezh2; BD Pharmingen), Annexin V- generated fewer ASC. The EZH2-dependent defect was cell FITC (BMS500FI/100; eBioscience), Annexin V-allophycocyanin (17-8007-74; http://www.jimmunol.org/ intrinsic to B cells and resulted in the enhanced expression and Invitrogen), Viability Ghost Dye-Red 780 (13-0865-T500; Tonbo Biosciences), increased chromatin accessibility of B cell genes that gain and Zombie yellow dye (77168; BioLegend). Influenza-specific, PE-conjugated hemagglutinin (HA) tetramers were previously described (35). Cells were H3K27me3 and are normally repressed in ASC, including Blimp- stained for 30 min on ice, protected from light, and fixed with 1% parafor- 1 target genes and inflammatory genes. Ezh2-deficient ASC failed maldehyde. Intracellular staining was performed with the Fixation/ to upregulate oxidative phosphorylation and glycolysis pathways, Permeabilization Kit (555028; BD Biosciences) following the manufac- as well as the unfolded-protein response (UPR), resulting in turer’s protocol. Flow cytometry was performed on a BD Biosciences LSR II decreased secreted Ig. Together, these data demonstrate a critical Flow Cytometer using FACSDiva (version 6.2). Flow cytometry data were processedbyFlowJo(version9.9.6).SortingofnBandASCbyFACSwas role for EZH2 in the programming of T-independent B cell performed on a BD FACSAriaII at the Emory Flow Cytometry Core. The differentiation and define specific roles for EZH2 in regulating following gating strategy preceded all flow cytometry analyses presented. by guest on September 28, 2021 ASC function. Cells were gated on 1) lymphocytes (forward light scatter [FSC]–area 3 side scatter [SSC]–area), 2) singlets (FSC-width 3 FSC-height and SSC-width 3 SSC-height), and 3) live cells (Viability Dye2). Finally, non–B cell lineage Materials and Methods cells were removed from the analyses based on the presence of Thy1.1, F4/80, Mice and CD11c. fl/fl CreERT2 Cre Ezh2 (022626; JAX) (31), Rosa26 (08463; JAX) (32), CD19 Deletion genotyping (06785; JAX) (33), CD45.1 (002014; JAX), C57BL/6J (00664; JAX), and mMT mice (02288; JAX) (34) were purchased from The Jackson Labo- DNA was extracted from purified splenic B cells following tamoxifen in- ratory and bred on site. CD45.2 mMT mice were bred onto the CD45.1 jection using the DNeasy Blood and Tissue kit (Qiagen). A total of 45 ng was background. All animal protocols were approved by the Emory Institu- used in a 35 cycle PCR with a three-primer design that amplified either the tional Animal Care and Use Committee. Mice used for experiments wild-type or deleted alleles. PCR products were resolved on a 1.5% agarose were between 6.5 and 10 wk old and were age and gender matched. gel. The following primers were used: Ezh2.del-fwd 59-GCTAGGCCTG- Cre-mediated deletion was induced by the treatment of 100 ml of 40 mg/ml CTGGTAAATA-39, Ezh2-del-rev 59-AGGAAATGGCAGGGTCTTTAG-39, tamoxifen (J63509; Alfa Aesar) by daily i.p. injection for five consecutive and Ezh2-del 59-CAGTACAATCTCCTGTGTC-39. days. For LPS experiments, 50 mg (ALX-581-008; Enzo Life Sciences) was administered i.v. and mice were analyzed 3 d after inoculation. For ELISA mixed bone marrow chimera experiments, 10 3 106 bone marrow cells fl/+ fl/fl CreERT2/+ ELISA plates (52-333801301F; Evergreen Scientific) were coated with from Ezh2 CD45.1/2 and Ezh2 Rosa26 CD45.2 were mixed at 5 mg/ml capture Ab (5300-01B; Southern Biotech) or 1 mg/ml influenza 1:1 ratios, transferred to lethally irradiated CD45.1 hosts, and the immune HA at 4˚C overnight and blocked with 1% nonfat dry milk at room tem- system allowed to reconstitute for 6 wk. For wild-type cell division ex- 3 6 perature for 2 h. Standard Abs and serum were bound to the plates at 4˚C periments, 20 10 splenic CD45.1 B cells were labeled with CFSE and overnight, plates were washed, and secondary HRP-conjugated goat anti- adoptively transferred into CD45.2 mMT hosts. For competitive cell 3 6 fl/+ mouse IgM or IgG Abs were added for 2 h at room temperature. Plates division experiments, 10 10 splenic B cells from Ezh2 CD45.1/2 and were developed with TMB ELISA peroxidase substrate (800-666-7625; Ezh2fl/flRosa26CreERT2/+CD45.2 mice were mixed at a 1:1 ratio, labeled with m Rockland) and the reaction was stopped with 0.2 M sulfuric acid. Plates CellTrace Violet (CTV), and adoptively transferred to CD45.1 MT hosts. were read at 450 nm with the Gen5 software. + CD4 T cell depletion and influenza infection Quantitative RT-PCR analysis + m For depletion of CD4 T cells, mice were treated with 200 g anti-mouse Total RNA was extracted from B cells and ASC using the RNeasy Mini Kit CD4 (GK1.5, BE0003-1; Bio X Cell) by i.p. injection 3 and 1 d before (Qiagen) and cDNA was synthesized with SuperScriptII Reverse Transcriptase infection. Mice were infected intranasally with 15,000 viral focal units of (Invitrogen) as described (36). Real-time PCR was performed using SybrGreen A/PR8/34 influenza virus (PR8) and analyzed 7 d later. incorporation on a BioRad CFX96 Thermocycler measuring the deleted exon of 9 9 Magnetic enrichment procedures Ezh2 (Ezh2-del-fwd 5 -CAGGATGAAGCAGACAGAAGAG-3 and Ezh2-del- rev 59-TTGTTGCCCTTTCGGGTT-39) and normalized to 18S rRNA (18s-fwd B cells were enriched from splenocytes from naive mice by negative selection 59-GTAACCCGTTGAACCCCATT-39 and 18s-rev 59-CCATCCAATCGGTA- using CD43 microbeads (130-090-862; Miltenyi Biotec). LPS-induced ASC GTAGCCG-39). The Journal of Immunology 3

RNA sequencing Biosystems), and sequenced using 50-bp, paired-end chemistry on a HiSeq2500. Tamoxifen-treated Ezh2fl/fl (control [Ctl]) and Ezh2fl/flRosa26CreERT2/+ (ERT2-Ezh2KO) CD138+ ASC were magnetically enriched 3 d following Assay for transposase-accessible chromatin using sequencing LPS inoculation. Three independent replicates were generated for Ctl and data analysis KO ASC. RNA was isolated using the RNeasy Mini Kit (Qiagen) and sequencing libraries were generated using the mRNA HyperPrep Kit with Raw reads from the assay for transposase-accessible chromatin sequencing poly(A) selection beads (KAPA Biosystems) using 500 ng total RNA as (ATAC-seq) were mapped to the mm9 version of the mouse genome using input according to the manufacturer’s instructions. Final libraries were Bowtie (45) (version 1.1.1) with the default parameters. PCR duplicates quality checked on a bioanalyzer, quantitated by quantitative PCR (qPCR), were removed from all downstream analyses with Picard (http:// pooled at equimolar ratio, and sequenced on a HiSeq2500 using paired- broadinstitute.github.io/picard/). Enriched regions were identified using end, 50-bp sequencing chemistry. Raw fastq files were mapped to the MACS2 (46) (version 2.1.0.20140616). A composite list of all identified mm9 version of the mouse genome using TopHat2 (37) (version 2.0.13) peaks in any sample was generated using the HOMER (47) (version 4.8.2) with the University of California, Santa Cruz mm9 knownGene table (38) “mergePeaks” function. The read depth for each sample was then anno- as the reference transcriptome. PCR duplicates were removed from tated for all peaks and differential accessibility was calculated using the all downstream analyses with Picard (http://broadinstitute.github.io/ GLM function of edgeR (40) (version 3.12.1), controlling for the mouse picard/). Reads that overlapped exons were summarized into unique each sample originated from. Principal component (PC) analysis plots EntrezID genes using the GenomicRanges (39) (version 1.22.4) package in were generated using all differentially accessible regions (DAR) and the R/Bioconductor. Genes that were not expressed at one read per million in vegan (version 2.4-3) package in R/Bioconductor. Genomic annotation of at least three samples were discarded for low expression. Differential ex- peaks was performed using HOMER (47) (version 4.8.2). Peaks that pression was tested using a pairwise test in edgeR (40) (version 3.12.1). overlapped a promoter (+500 and 22000 bp surrounding the transcription For gene set enrichment analysis (GSEA), all detected genes were ranked start site) were k-means clustered using the Biganalytics package (https:// by multiplying the sign of the fold change (FC) (+/2) by the 2log10 of the CRAN.R-project.org/package=biganalytics). Enriched Downloaded from p value. This ranked gene list was used as input for the GSEA preranked motifs were identified using HOMER (47) (version 4.8.2) and the find- analysis. To determine the genes involved in protein secretion and trans- MotifsGenome.pl script. Histograms of read depth surrounding each motif port, the UPR and XBP1 up gene lists were annotated using the Gene were generated using custom R/Bioconductor scripts. Ontology Consortium web portal (41). Genes categorized into protein transport (GO:0015031), protein localization (GO:0008104), or Golgi ChIP-seq vesicle transport (GO:0048193) biological processes were annotated. Se- ChIP was performed as described previously (48). For each immunopre- quencing depth for each gene was normalized to fragments per kilobase cipitation, 10 3 106 CD432 splenic B cells or 1 3 106 CD138+ splenic http://www.jimmunol.org/ per million using custom scripts implemented in R/Bioconductor. ASC were fixed in 1% formaldehyde for 10 min, chromatin was isolated, Preparation of Tn5 for the assay for transposase-accessible and then sonicated to an average size of 400 bp. One microgram of anti- H3K27me3 (07-449; EMD Millipore) was prebound to Dynal Protein chromatin sequencing G magnetic beads (Thermo Fisher Scientific) and DNA–chromatin com- In-house purification of adapter-loaded Tn5 transposase was performed as plexes immunoprecipitated overnight at 4˚C. DNA was reverse cross- previously described (42). Briefly, the pTXB1-Tn5 plasmid (60240; Addgene) linked and purified using a PCR Cleanup Kit (Qiagen). ChIP–DNA was was transformed into C3013 cells (C3013l; NEB) and a single colony grown in diluted 1:20 and enrichment of the Hoxa9 (positive) and Actb (negative) 250 ml of Luria Broth, Miller with 100 mg/ml ampicillin at 37˚C until the loci was tested by qPCR. The remaining ChIP–DNA was used as input for the KAPA HyperPrep Kit (KAPA Biosystems). ChIP-seq libraries were culture reached an A600 of 0.75–0.9. To induce Tn5 expression, isopropyl b-D- 1-thiogalactopyranoside was added to a final concentration of 0.25 mM for 4 h sequenced on a HiSeq2500 using 50-bp, paired-end chemistry. Raw se- at23˚C.Cellswerethenpelletedat2800rpmfor15min,resuspendedin15ml quencing reads were mapped to the mm9 version of the mouse genome by guest on September 28, 2021 HEGX (20 mM HEPES-KOH at pH 7.2, 0.8 M NaCl, 1 mM EDTA, 0.2% using Bowtie (45). Uniquely mappable and nonredundant reads were used Triton X-100, 10% glycerol, complete with Roche protease inhibitors), and for subsequent analyses. HOMER (47) software was used for peak calling lysed with a French pressure cell (9000 lb/in2). The resulting lysate was pelleted and annotation. Data were normalized to reads per peak per million (rppm) at 15,000 rpm for 30 min, and 550 ml of 10% polyethyleneimine was added to as previously described (44) using Eq. 1:   the supernatant on a magnetic stirrer. The precipitate was removed via centri- 3 6 fugation at 12,000 rpm for 10 min. The supernatant was then loaded onto a 1-ml rppm ¼ Reads 3 1 10 : chitin column (S6651S; NEB) and washed with 20 ml HEGX. On-column Unique Reads 3 FRiP loading of Tn5 with preannealed mosaic end double-stranded (MEDS) oligo- nucleotides was achieved by adding 60 nmol of mixed and annealed Tn5 MED- For the generation of scatterplots between nB and ASC samples the rppm p7/-p5 (ME-p7, 59-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-39; read depth was quantile normalized and the log2FC and the average log2 ME-p5, 59-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-39;and rppm between nB and ASC samples were calculated. 9 9 ME-rev, 5 -[phos]-CTGTCTCTTATACACATCT-3 )tothecolumnin1.2ml Data availability HEGX buffer. After 48 h, the column was washed with 20 ml of HEGX to remove unbound MEDS and cleavage of Tn5-MEDS was initiated by adding All sequencing data are available at the National Center for Biotechnology 1.2 ml HEGX with 100 mM DTT to the column. After 48–72 h, small elution Information Gene Expression Omnibus database (https://www.ncbi.nlm. fractions were collected and those with the highest protein concentration were nih.gov/geo/) under the accession number GSE103195. All code and pooled and dialyzed overnight versus 23 Tn5 dialysis buffer (100 mM HEPES data processing scripts are available upon request. at pH 7.2, 0.2 M NaCl, 0.2 mM EDTA, 2 mM DTT, 0.2% Triton X-100, 20% glycerol). Following dialysis, 0.6 vol of glycerol was added to make the final Extracellular flux assays storage buffer (50 mM HEPES at pH 7.2, 0.1 M NaCl, 0.1 mM EDTA, 1 mM A Seahorse Bioanalyzer XFe96 instrument was used for all extracellular DTT, 0.1% Triton X-100, 50% glycerol). Tn5 was dispensed into 50-ml aliquots flux assays. A FluxPak cartridge was hydrated at least 12 h prior to running and placed at 280˚C for long-term storage. each assay with 200 mldH2O in a 37˚C non-CO2 incubator. One hour prior m Assay for transposase-accessible chromatin sequencing to each assay, dH2O was removed and 200 l prewarmed Seahorse Cali- brant solution (103059-000; Agilent) was added to all experimental wells. Tagmentation was performed as described in detail previously (43, 44). For the mitochondrial stress test and measurement of oxygen consumption, Briefly, 2000–4000 cells from Ezh2fl/fl (Ctl) and ERT2-Ezh2KO nB and purified cell populations were washed in Seahorse XF Assay Media, pH ASC from tamoxifen-treated bone marrow chimeras 3 d following LPS 7.4 6 0.1, supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine inoculation were FACS sorted and tagmentation was performed using (G7513; Sigma), and 5.5 mM glucose at 37˚C. For the glycolysis stress test 2.5 ml of Tn5 in 13 TD Buffer (Illumina) in 25 ml total volume for 1 h at and extracellular acidification measurements, purified cell populations 37˚C. Tagmented nuclei were lysed, DNA was purified using a double solid were washed in Seahorse XF Assay Media, pH 7.4 6 0.1, supplemented phase reversible immobilization-bead size selection (0.73 negative fol- with 2 mM L-glutamine. Cells were washed once in appropriate media and lowed by 13 positive selection), and PCR amplified using Nextera counted by flow cytometry using AccuCheck counting beads (PCB100; Indexing Primers (Illumina) and the HiFi HotStart Polymerase (KAPA Invitrogen). Prior to each experiment, CellTak (354420; Corning) was Biosystems) for 14 cycles of PCR. Final libraries were purified using a diluted in sterile 13 PBS to a final concentration of 22.4 mg/ml, and 25 ml second double SPRI-bead size selection (0.23 negative followed by 13 was added to each well of a Seahorse XFe96 cell culture plate, incubated positive selection), quantitated using the Illumina qPCR Quant Kit (KAPA for 20 min at room temperature. The plates were washed with 200 mldH2O 4 EZH2 IN ASC FUNCTION and then 400,000 cells per well were plated and incubated in a 37˚C non- EZH2-mediated repression. functional analysis, CO2 incubator for 45 min prior to beginning the assay. The indicated drugs summarized and condensed by REVIGO (50) (Fig. 1D), indicated were diluted in assay-specific media for injection into each port. For the that these genes were enriched in pathways associated with B cell mitochondrial stress test the ports were as follows: port A, oligomycin (75351; Sigma) was used at a final concentration of 1 mM; port B, carbonyl functions that are repressed in ASC, such as cell cycle (e.g., cyanide-4-(triflurome-thoxy) phenylhydrazone (C2920; Sigma) was used Cdkn1a), response to LPS (e.g., Nfkb1 and Tnf), and Ag presen- at a final concentration of 2.5 mM; and port C was injected a combination tation by MHC (e.g., H2-Ab1 and Cd74). Thus, these data indicate of Rotenone (R8875; Sigma) and Antimycin A (A8674; Sigma), each at a that the progressive upregulation of Ezh2 during B cell differen- final concentration of 1 mM. For the glycolysis stress test the ports were as follows: port A, glucose (G8270; Sigma) was used at a final concentration tiation coincides with repression of the B cell transcriptional of 10 mM; port B, oligomycin was used at a final concentration of 1 mM; program in ASC. and Port C, 2-deoxyglucose (D8375; Sigma) was used at a final concen- tration of 50 mM. ASC differentiation in response to type I, t-independent stimuli requires EZH2 To determine the role of Ezh2 in vivo, we crossed Ezh2fl/fl mice Results (31) with tamoxifen-inducible Rosa26CreERT2 mice (32). Follow- EZH2 is progressively upregulated during type-I, T- ing tamoxifen treatment, we observed genomic rearrangement of independent B cell differentiation the Ezh2 locus in splenic B cells and a significant reduction Ezh2 is progressively upregulated during ex vivo B cell differ- in Ezh2 transcripts, and protein was observed (Supplemental entiation in response to LPS (49). To assess its expression pattern Fig. 1C–E). Ezh2 is essential for B cell development (26, 51); in vivo, we transferred CFSE-labeled CD45.1 B cells into CD45.2 however, it is dispensable for peripheral B cell homeostasis (26). Downloaded from mMT mice and subsequently inoculated the recipients with LPS to Consistent with previous findings, following tamoxifen-induced induce differentiation (8). Distinct divisions (Div0, -1, -3, -5, -8+) deletion of Ezh2, a block in B cell development at the pre-B were FACS isolated and gene expression was quantitated by stage was observed (Supplemental Fig. 1F). However, in the quantitative RT-PCR (RT-qPCR). For consistency across the dif- short time frame following deletion, no defect was observed in the ferentiation models, we define plasma cells that have acquired viability of peripheral splenic B cells or in the frequency of

CD138 expression as ASC. Ezh2 mRNA was upregulated during marginal zone or follicular B cells (Supplemental Fig. 1G–I). http://www.jimmunol.org/ the divisions, peaking in CD138+ ASC at Div8+ (Fig. 1A). EZH2 To test the function of EZH2, ERT2-Ezh2KO and Ezh2fl/fl protein levels were quantified in splenic nB and 3 d after LPS (Ctl) mice were treated with tamoxifen and subsequently stimulation in B220+GL7+CD1382 activated B cells (actB) and inoculated with LPS to induce B cell differentiation. In ERT2 CD138+ ASC. Similar to previous reports (15), EZH2 protein was -Ezh2KO mice, we observed a significant reduction in the fre- relatively low in nB, elevated in actB, and peaked in ASC quency and absolute numbers of splenic and lymph node ASC and (Fig. 1B). actB (Fig. 2A–E). These differences were not due to changes in To determine what genes might be affected by EZH2-mediated the viability of ERT2-Ezh2KO splenocytes (Fig. 2G). Following histone methylation, we performed ChIP-seq for H3K27me3 in nB LPS inoculation, a sharp increase in IgM titers occurred in control T2 and ASC, which were enriched to .94% purity (Supplemental mice; however, ER -Ezh2KO mice failed to reach the same titers by guest on September 28, 2021 Fig. 1A, 1B). We found that 1623 gene promoters gained of serum IgM (Fig. 2H), a reduction that correlated with the de- H3K27me3 in ASC (Fig. 1C), suggesting that these were targets of creased ASC response. IgG levels were also measured by ELISA

FIGURE 1. EZH2 is progressively upregulated during B cell differentiation in response to type-I, T-independent stimuli. (A) The indicated divisions were isolated by FACS (left) and Ezh2 mRNA levels (right) quantitated by RT-qPCR and expressed relative to 18s rRNA as mean 6 SD. These data are representative of three independent experiments. *p , 0.05 by Student two-tailed t test. (B) Protein levels of EZH2 in nB and LPS-induced actB and CD138+ ASC. Data are representative of two experiments. FMO, fluorescence minus one. (C) Scatterplot of the average promoter H3K27me3 in nB and ASC versus the log2FC of H3K27me3 as determined by ChIP-seq. ChIP-seq data are summarized from two biological replicates of nB and ASC. (D) REVIGO (50) plot summarizing Gene Ontology terms for the 1623 genes that gain promoter H3K27me3 in ASC from (C). The Journal of Immunology 5 and showed a similar reduction in ERT2-Ezh2KO mice; however, (CD19-Cre Ctl), and Ezh2fl/fl (Ctl) mice were inoculated with LPS the overall levels of IgG were very low in response to LPS (data and differentiation tested as above. Similar to tamoxifen-induced not shown). Ezh2 deletion, CD19-Ezh2KO mice formed 50% fewer ASC and The expression of Cre recombinase can negatively impact actB compared with controls (Fig. 2I–L, Supplemental Fig. 2C, 2D). rapidly proliferating cells (52). To ensure that the observed phe- Together, these data demonstrated that Ezh2 was required for efficient notype was specific to deletion of Ezh2, we compared hemizygous ASC differentiation in response to type-I, T-independent stimuli. Ezh2fl/+Rosa26CreERT2/+ (ERT2-Cre Ctl) mice with ERT2-Ezh2KO and the Cre2 control mice described above. Three days after LPS EZH2 controls the early T-independent burst of influenza- inoculation, ERT2-Cre Ctl mice had similar frequencies of splenic specific ASC actB and ASC as control mice and significantly more of each During the humoral response to protein Ags and pathogens, a burst of population than ERT2-Ezh2KO mice (Supplemental Fig. 2A, 2B). ASC provides an initial surge of serum Abs before T cell–dependent To further assess this system, the Cre recombinase expressed from processes facilitate affinity maturation and class-switched B cell re- the Cd19 locus was used (33) because it bypasses the develop- sponses (53, 54). In response to the hapten NP, EZH2-deficient mental defects observed in the ERT2-Ezh2KO strain, allowing B cells displayed reduced NP-specific serum Ab titers as early as normal B cell development to occur (26) and facilitating B cell–specific day 7 (15), suggesting defects in early ASC responses. To assess the deletion. In this study, Ezh2fl/flCD19Cre/+ (CD19-Ezh2KO), CD19Cre/+ early T cell–independent differentiation events, CD4 T cells were first Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Maximal ASC differentiation in response to T-independent stimuli requires EZH2. Ezh2fl/fl (Ctl) and ERT2-Ezh2KO (KO) mice were treated with tamoxifen followed by inoculation with LPS and analyzed as follows. (A) Frequency and (B) absolute number of splenic CD138+ ASC 3 d after LPS inoculation. A representative example is plotted on the left and the mean 6 SD of four biological replicates is shown on the right. (C) Frequency of CD138+ ASC from the lymph nodes of mice treated as above. (D) Frequency and (E) absolute number of splenic B220+GL7+ actB. (F) Frequency of B220+GL7+ actB from the lymph nodes of mice treated above. (G) Frequency of viable cells from the spleen. (H) Serum IgM titers measured before (D0) and 3 d after LPS inoculation (D3) for the indicated genotype. Ezh2fl/fl (Ctl) and CD19-Ezh2KO mice were analyzed by flow cytometry as follows. (I) Frequency and (J) absolute numbers of splenic CD138+ ASC. (K) Frequency and (L) absolute numbers of splenic B220+GL7+ actB cells. See also Supplemental Fig. 2. Each point represents independent biological samples and data are summarized as mean 6 SD. The data presented represent between two and five groups of mice containing three to four mice per cohort. Significance was determined by Student two-tailed t test. 6 EZH2 IN ASC FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. EZH2 is required for the early burst of influenza-specific ASC in the absence of CD4 T cells. Ezh2fl/fl (Ctl) and CD19-Ezh2KO (KO) mice were CD4 T cell depleted and infected with PR8 and analyzed by flow cytometry 7 d postinfection. (A) Frequency and (B) absolute number of CD138+ ASC in the bronchial lymph node. (C) Frequency and (D) absolute number of HA-specific ASC cells. (E) Frequency of B220+GL7+ actB in the bronchial lymph node. (F) Frequency of HA-specific actB. (G) Frequency of HA-specific lineage negative (Thy1.12F4/802CD11c2) B cells. (H) ELISA for HA-specific IgM in naive (D0) and 7 d (D7) postinfection of PR8. See also Supplemental Fig. 3. Each point represents independent biological samples and data are summarized as mean 6 SD. The data presented represent between two and five groups of mice containing three to four mice per cohort. Significance was determined by Student two-tailed t test. depleted from CD19-Ezh2KO and control cohorts of mice using an mice were mixed 1:1 and transferred into lethally irradiated anti-CD4 Ab and subsequently infected with PR8. CD4 T cells were CD45.1 hosts. At 4 wk, the percentages of CD45.2 and CD45.1/2 fully depleted at both the time of PR8 infection and the assay end point donor cells in the periphery of recipient mice were similar at day 7, at which no GC B cells were detected (Supplemental Fig. 3A, (Fig. 4A). After 6 wk, mice were treated with tamoxifen (as 3B). At day 7, control mice exhibited expanded lymph node CD138+ above) to induce EZH2 deletion and subsequently inoculated with ASC, whereas the frequency and absolute number of ASC were sig- LPS. Three days after LPS, both ERT2-Ezh2KO (CD45.2) and nificantly reduced in CD19-Ezh2KO mice (Fig. 3A, 3B). Ag-specific, control cells were identified (Fig. 4B) and a significant reduction PR8-responding cells were identified using B cell tetramers against HA in CD138+ ASC in the spleen (Fig. 4C) and lymph nodes (Fig. 4D) (35), and the frequency of HA+ ASC, HA+ actB, and total HA+ cells was observed in cells transferred from ERT2-Ezh2KO mice. was significantly reduced in CD19-Ezh2KO compared with control Additionally, the frequency of actB was significantly reduced mice (Fig. 3C–G, Supplemental Fig. 3C, 3D). Additionally, assessment (Fig. 4E, 4F). These data indicate that EZH2 performs an essential of Ab levels revealed an increase in HA-specific IgM titers from day cell-intrinsic function during LPS-induced B cell differentiation. 0 to 7 in control mice but significantly reduced levels in CD19- EZH2 represses B cell transcription factor networks Ezh2KO at mice at day 7 (Fig. 3H). Together, these data define an T2 essential role for EZH2 in the formation of T-independent ASC in Transcriptome profiling was performed on tamoxifen-treated ER response to both nonprotein and protein Ags. -Ezh2KO or control ASC following LPS stimulation to determine the molecular consequences of Ezh2 deletion. In ERT2-Ezh2KO Cell-intrinsic requirement for EZH2 in ASC differentiation ASC, 1498 genes were significantly upregulated (false discovery To determine a cell-intrinsic role for Ezh2 in ASC differentiation, rate [FDR] , 0.05, log2FC . 1) whereas only 79 were down- bone marrow from CD45.2 ERT2-Ezh2KO and CD45.1/2 control regulated (Fig. 5A). Although not entirely, these data are largely The Journal of Immunology 7 Downloaded from

FIGURE 4. Cell-intrinsic requirement for EZH2 in ASC differentiation. The bone marrow from Ezh2fl/+CD45.1/2 (Ctl) and ERT2-Ezh2KO CD45.2 (KO) mice was transferred to lethally irradiated CD45.1 wild-type hosts and analyzed at 4 wk for chimerism. (A) At 6 wk, the mice were treated with tamoxifen and inoculated with LPS and analyzed 3 d later. (B) Frequencies of chimeric populations following LPS treatment. Quantitation of CD138+ ASC fre- quencies from the (C) spleen and (D) lymph nodes of LPS inoculated mice. Quantitation of B220+GL7+ actB frequencies from the (E) spleen and (F) lymph nodes of LPS-inoculated mice. Each point represents independent biological samples and data are summarized as mean 6 SD. Data are representative of two independent experiments with four to five mice per group. Significance was determined by Student two-tailed t test. http://www.jimmunol.org/ consistent with a repressive function for EZH2. Confirming the and ASC, suggesting they are normally repressed in both cell types. tamoxifen-induced deletion in ERT2-Ezh2KO ASC, an 18-fold To further understand the consequences of Ezh2 deletion, we reduction in the transcripts across Ezh2’s deleted exons was ob- performed ATAC-seq (43, 44) on nB and ASC from ERT2 served compared with wild type (Supplemental Fig. 4A). More- -Ezh2KO and control mice. PC analysis of all DAR revealed a over, in ERT2-Ezh2KO ASC, Ezh2 was the most significantly large difference between nB and ASC regardless of EZH2 status downregulated gene by an FDR differential of 10113. GSEA defined by PC1 (Supplemental Fig. 4C), indicating that in the (55, 56) indicated that, in the absence of Ezh2, genes involved in absence of EZH2, most differentiation-associated chromatin ac- the regulation of inflammation and NF-kB–mediated TNF-a sig- cessibility changes occurred normally. PC2 separated control and by guest on September 28, 2021 naling and the pathway were upregulated (Fig. 5B). During ERT2-Ezh2KO ASC but not nB, which contained minimal DAR B cell development, EZH2 repressed the cell cycle inhibitor (Supplemental Fig. 4D), indicating chromatin accessibility dif- Cdkn2a and prevented p53 activation (51); thus, EZH2 may be ferences were primarily specific to Ezh2-deficient ASC. performing a similar role in ASC. Promoter accessibility of genes upregulated in ERT2-Ezh2KO To determine if the differentially expressed genes (DEG) were ASC was annotated in nB and ASC and the patterns were cate- direct targets of EZH2, we compared the change in promoter gorized by k-means clustering. Three distinct patterns of promoter H3K27me3 enrichment between nB and ASC with the change in chromatin accessibility were observed: k1, promoters that lost expression between ERT2-Ezh2KO versus control ASC. We found accessibility between nB and ASC; k2, promoters that were that 923 genes were upregulated in ERT2-Ezh2KO ASC and gained minimally accessible in nB, remained such in control ASC yet promoter H3K27me3 compared with nB (Fig. 5C, 5D), indicating gained accessibility in ERT2-Ezh2KO ASC; and k3, promoters that that these DEG are direct repression targets of EZH2. Bcl6 was were inaccessible in nB and control ASC and only gained ac- among the top genes that gained promoter H3K27me3 in wild-type cessibility in ERT2-Ezh2KO ASC (Fig. 5E). We found that pro- ASC and was increased in expression in ERT2-Ezh2KO ASC, sug- moters of DEG in ERT2-Ezh2KO ASC were more accessible than gesting that other B cell transcription factors could be dysregulated. those in control ASC. Direct comparison with control ASC GSEA analysis was performed using a set of transcription factors revealed 1317 loci that gained accessibility in ERT2-Ezh2KO ASC specifically expressed in follicular B cells compared with bone compared with only 347 that decreased (Fig. 5F). Examples of marrow ASC (57) and showed that this set of transcription factors chromatin accessibility changes include specific regions located in were significantly upregulated in ERT2-Ezh2KO ASC (Fig. 5B). Bcl6 and (k1), Hoxa1 (k2), and Eml6 (k3) (Fig. 5G). Other examples of genes that were significantly upregulated in ERT2 The failure to repress key B cell transcription factors suggested -Ezh2KO ASC and demonstrated extensive gains in H3K27me3 in that such factors may contribute to a unique accessibility footprint ASC included the developmental transcription factor Klf4,inflam- in ERT2-Ezh2KO ASC. Analysis of DNA sequence motifs within matory genes Ifit3 and Lta, and cell cycle inhibitors Cdkn1a and Slfn1 regions of increased accessibility revealed an enrichment for (Fig. 5G, Supplemental Fig. 4B). These results indicate that EZH2 CTCF, ETS, OCT, and IRF family transcription factor binding functions to epigenetically silence key B cell genes involved in in- sites (Fig. 6A). Although CTCF was expressed at similar levels in flammatory responses. nB and ASC, the presence of CTCF sites within DAR suggests a potential defect in rearranging the three-dimensional genome EZH2 controls chromatin accessibility in ASC during ASC differentiation (58). Transcription factor footprinting The derepression of 923 genes could be directly attributed to a for the occurrence of each motif revealed unique patterns at each failure to gain H3K27me3 in ASC. However, an additional 575 binding site and clear increases in accessibility surrounding each DEG gained expression and were marked by H3K27me3 in both nB motif in ERT2-Ezh2KO compared with control ASC (Fig. 6A). 8 EZH2 IN ASC FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. EZH2 is a transcriptional repressor in ASC. RNA sequencing (RNA-seq) was performed on enriched splenic ASC from tamoxifen-treated fl/fl T2 Ezh2 (Ctl) and ER -Ezh2KO (KO) mice 3 d after LPS inoculation. (A) Volcano plot summarizing DEG (FDR , 0.05, log2FC . 1) between Ctl and KO ASC. RNA-seq data represent three biological replicates of Ctl and KO ASC. (B) Top GSEA gene sets. (C) The log2FC for DEG was plotted versus the log2FC in promoter H3K27me3 enrichment between nB and ASC. (D) Heatmap of H3K27me3 enrichment for 2 kb surrounding promoters of 1498 up- regulated DEG. Data were ranked by the change in H3K27me3 in ASC versus nB. Color bars map distinct clusters of genes from (C). ATAC-seq was performed on Ctl and KO nB and ASC from tamoxifen-treated bone marrow chimeras 3 d following LPS inoculation (see Fig. 4). (E) Promoter accessibility (ATAC-seq) heatmap for genes in (D) was categorized using k-means clustering (k = 3). Three independent replicates of Ctl nB, Ctl ASC, and KO ASC, and two replicates of KO nB are shown. (F) ATAC-seq–derived volcano plot showing DAR (FDR , 0.05, log2FC . 1) comparing KO and Ctl ASC. (G) For the indicated genes, the change in expression by RNA-seq is plotted with a genome plot depicting chromatin accessibility and H3K27me3-enrichment data for each locus. ATAC-seq data are summarized as mean of three biological replicates for Ctl nB, Ctl ASC, and KO ASC, and two replicates for KO nB. H3K27me3 ChIP-seq data represent the mean of two biological replicates for nB and ASC. Promoter regions are highlighted. FPKM, fragments per kilobase per million; rpm, reads per million. See also Supplemental Fig. 4.

Increased accessibility could be due to an increase in expression was performed using a high-confidence set of Blimp-1–repressed of a transcription factor family member that binds to a specific target genes derived from transcriptional profiling of genetic motif, such as the ETS factor SPIB that is upregulated, or due to a deletions and ChIP-seq data (30). Of the 109 genes that over- failure to recruit EZH2 to a region. Thus, these data depict the lapped in the data sets, 96% demonstrated higher expression in the epigenetic derepression of loci controlled by EZH2 through in- absence of EZH2 (Fig. 6B). Examples included the ETS family creases in gene expression and chromatin accessibility at sites transcription factor gene Spib, which was upregulated and normally repressed in ASC. contained one of the strongest Blimp-1 binding sites (Fig. 6C, 6D). Additionally, genes for Klf2, a transcription factor involved in Blimp-1–repressed genes are upregulated in the absence of B cell homing and migration (62); the TLR Tlr9; and Btg1, which EZH2 functions as a negative regulator of cellular proliferation and ap- Blimp-1 can elicit gene repression through physical interactions optosis (63, 64); all contained Blimp-1 binding sites at regions that with epigenetic modifiers (59–61), including EZH2 (30). GSEA gained H3K27me3 in ASC and were upregulated in the absence of The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Blimp-1 target genes are upregulated in EZH2-deficient ASC. (A) ATAC-seq–based transcription factor footprinting histograms for motifs enriched in KO versus Ctl ASC from Fig. 5F. The significance of enrichment for each motif is indicated and the enriched motif is displayed. Histograms summarize three independent replicates of ATAC-seq data from Ctl and KO ASC. rpm, reads per million. (B) GSEA assessing the enrichment of Blimp-1– repressed genes (30) in Ctl and KO RNA sequencing (RNA-seq) data. (C) Representative RNA-seq data summaries for the indicated genes. Data are representative of three biological replicates and are summarized as mean 6 SD. (D) Genome plots depicting the loci in (C). ASC–Blimp-1 data were previously reported and normalized to reads per million (30). H3K27me3 ChIP-seq data represent the mean of two biological replicates for nB and ASC.

EZH2. These data provide an in vivo link between EZH2 and Blimp- Consistent with previous results (8), eight cellular divisions were 1 in ASC and demonstrate that epigenetic modifications via EZH2 typically observed with control B cells (Fig. 7B, 7C). In contrast, ERT2 may be required for the Blimp-1–mediated gene repression program. -Ezh2KO B cells initially proliferated normally through three cellular divisions but were reduced in number at all subsequent divisions. The Deletion of Ezh2 impairs the proliferation of actB B cell activation marker GL7 was progressively upregulated in control Recent studies in B cells and B cell–derived lymphomas demonstrated cells through eight divisions. However, ERT2-Ezh2KO B cells failed to that inhibition or depletion of EZH2 resulted in cell cycle arrest, im- gain additional GL7 expression after three divisions (Fig. 7D, 7E). paired proliferation, and apoptosis (16, 28, 65–67). Indeed, a cell cycle Analysis of differentiation kinetics through expression of CD138 T2 gene set was one of the most downregulated gene sets in ER revealed that B cells from control and ERT2-Ezh2KO mice both -Ezh2KO ASC (Fig. 5B). Additionally, the EZH2 target gene Cdkn1a achieved seven to eight divisions before an accumulation of CD138+ (16), which encodes the P21 cell cycle inhibitory kinase, is upregulated ASC were observed, however the ERT2-Ezh2KO CD138+ cells were T2 in ER -Ezh2KO ASC (Supplemental Fig. 4B). These data suggest reduced in frequency (Fig. 7F, 7G). These data indicated that in the that the observed decrease in actB and ASC was due to an EZH2- absence of EZH2, a defect in the ability to proliferate, possibly through mediated defect in the proliferative phase of B cell differentiation. To the failure to repress negative regulators of the cell cycle, contributes to T2 test this hypothesis, tamoxifen-treated CD45.2 ER -Ezh2KO and the reduced numbers of differentiated ASC. CD45.1/2 control B cells were labeled with CTV, transferred into congenically marked CD45.1 mMThosts,andstimulatedtodifferen- EZH2 is required for the metabolic programming of ASC tiate by inoculation with LPS. After 3 d, ERT2-Ezh2KO and control Although a subset of ERT2-Ezh2KO B cells could undergo eight B cells were analyzed for their ability to divide and differentiate (Fig. 7A). rounds of division and differentiate into ASC, the molecular 10 EZH2 IN ASC FUNCTION phenotype of these cells indicated that they were likely dysfunc- mTOR pathway in follicular lymphoma (71), suggesting the re- tional (Fig. 5). To directly measure the Ab-secreting capacity of duced IgM secretion in ASC may be due to a metabolic defect. these cells, control and ERT2-Ezh2KO ASC—derived following To test the hypothesis that ERT2-Ezh2KO ASC were metabol- LPS-induced differentiation in vivo—were purified, equal num- ically distinct, extracellular flux assays were performed to probe bers cultured for 3.5 h, and Ab titers from the resulting superna- both mitochondrial respiration and glucose metabolism. The ox- tant measured by ELISA. On a per-cell basis, ERT2-Ezh2KO ASC ygen consumption rate was measured as a readout for mitochon- secreted 50% fewer molecules of IgM (Fig. 8A). However, no drial respiration. At basal levels, ERT2-Ezh2KO ASC consumed difference in the levels of IgM mRNA transcripts (Fig. 8B) or significantly less oxygen than control ASC (Fig. 8F). Following intracellular IgM protein levels (Fig. 8C) were observed between inhibition of ATP synthase with oligomycin, similar declines were control and ERT2-Ezh2KO ASC, indicating that the IgM defi- observed in oxygen consumption. Maximal respiration rates were ciency may lie in protein secretion. also greater in control ASC compared with ERT2-Ezh2KO, as Consistent with this idea, GSEA indicated that the UPR and shown by the addition of carbonyl cyanide-4-(triflurome-thoxy) genes upregulated by the transcription factor XBP1 (68) failed to phenylhydrazone to the system. This suggests that control ASC be induced in ERT2-Ezh2KO ASC (Fig. 8D). Annotation of genes are more capable of using the oxidative phosphorylation metabolic involved in protein secretion and transport revealed that these pathway than ERT2-Ezh2KO. Because metabolism is a balance genes were more highly expressed in control than in ERT2 between multiple metabolic pathways and the expression of the -Ezh2KO ASC, consistent with decreased IgM levels in the cul- glucose transporter Glut1 was upregulated after LPS stimulation ex tures of ERT2-Ezh2KO ASC. XBP1-mediated induction of the vivo (72), the ability of ASC to metabolize glucose was assessed. The

UPR pathway is also important for increased mitochondrial extracellular acidification rate, which is measured by pH changes Downloaded from function (69), and ASC require a high metabolic capacity to associated with excretion of lactic acid generated from pyruvate (73), support synthesis and secretion of Ab molecules (70). Indeed, can be used to measure rates of glucose metabolism. Addition of GSEA revealed the expression of oxidative phosphorylation and glucose to glucose-deprived ASC resulted in enhanced lactate se- glycolysis metabolic pathways were enriched in control but not cretion in control compared with ERT2-Ezh2KO ASC, resulting in a ERT2-Ezh2KO ASC (Fig. 8E). Moreover, EZH2 regulates the significantly higher ability to perform glycolysis (Fig. 8G). These http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Deletion of Ezh2 impairs B cell activation and proliferation in vivo. B cells isolated from tamoxifen-treated Ezh2fl/+CD45.1/2 (Ctl) and Ezh2fl/flRosa26CreERT2/+CD45.2 (KO) mice were labeled with CTV, transferred into CD45.1 mMT mice, and inoculated with LPS as above. (A) Frequencies of transferred populations in the spleens of mice 3 d following LPS treatment. (B) Histograms and (C) absolute numbers of CTV labeled in Ctl and KO cells. (D) Representative flow cytometry plot and summary of the frequency of GL7+ cells. (E) The absolute number of B220+GL7+ cells in each division from (D). (F) Representative and summary of the frequency of CD138+ cells from the above cells. (G) The absolute number of CD138+ cells in each division from (F). For summary graphs, the mean 6 SD is shown. Data are representative from two independent experiments containing cohorts of five animals each. Significance was determined by paired Student t test. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 8. EZH2 is required for maximal Ab secretion and ASC metabolism. Ezh2fl/fl (Ctl) and ERT2-Ezh2KO (KO) mice were treated with tamoxifen and inoculated with LPS as above and analyzed as follows. (A) ELISA measuring IgM titers at 3.5 h of culture from 1 3 106 LPS-induced splenic CD138+ ASC from Ctl and KO mice. (B) Expression of IgM transcripts measured by RNA sequencing (RNA-seq) from splenic Ctl and KO CD138+ cells following LPS inoculation. See Fig. 5. (C) Measurement of intracellular IgM in Ctl and KO CD138+ ASC. Representative flow cytometry data are plotted and the mean fluorescence intensity (MFI) summarized as mean 6 SD. (D) GSEA assessment of RNA-seq data from Fig. 5A for the indicated gene sets. “Xbp1 up” by guest on September 28, 2021 genes were previously described (68). False discovery corrected q value is shown. Genes involved in protein secretion and transport are indicated in red below. (E) GSEA assessment of RNA-seq data from Fig. 5A for the indicated gene sets. (F) Extracellular flux analysis of Ctl and KO ASC described above. Oxygen consumption rate (OCR) before and after treatment with the indicated pharmacological inhibitors. Data represent the combination of two inde- pendent experiments containing cohorts of four animals. Each point represents the mean 6 SD. (G) Extracellular acidification rate (ECAR) of ASC as in (F), measured at steady state without glucose, after addition of glucose, oligomycin, and 2-deoxy-D-glucose (2DG). Data represent the combination of two independent experiments containing cohorts of four animals. Each point represents the mean 6 SD. Significance determined by Student two-tailed t test.

data therefore define EZH2 as an essential upstream regulator of ASC was Cdkn1a, which encodes the G1/S cell cycle checkpoint inhibi- metabolic potential. tor P21 (74). ChIP-seq data showed that Cdkn1a has increased H3K27me3 across its promoter and proximal upstream sequences, Discussion confirming a previously described direct role for EZH2 in its regulation In this article, we tested whether EZH2 regulated ASC differen- (15, 16, 28). In vivo, B cells undergo approximately eight cell divisions tiation using both type-I and type-II T-independent B cell responses before differentiating into ASC (8). Ezh2-deficient cells went through in vivo. Consistent with other data (26), we found that EZH2 is not fewer divisions, resulting in fewer cells that acquired the actB phe- required for homeostasis of naive follicular and marginal zone notype (GL7+) and fewer CD138+ ASC, consistent with the continued B cells. However, immunization with T-independent Ags or pro- activity of cell cycle inhibitors. This result is consistent with findings in tein Ags in the absence of T cell help generated poor B cell re- multiple myeloma in which EZH2 is overexpressed and pharmaco- sponses in Ezh2-deficient mice. The role of EZH2 was B cell logical inhibition limits cell growth (75, 76). Thus, the failure to repress intrinsic as Ezh2-deficient B cells also poorly differentiated into cell cycle inhibitors, such as Cdkn1a, is a likely mechanism leading to ASC in mixed bone marrow chimeric mice. As EZH2 is the decreased numbers of observed ASC. continued target of therapeutic intervention, these data have im- Ezh2-deficient ASC also failed to repress the transcriptional plications for the effect of inhibitors on normal humoral immune program associated with mature B cells, including the accumu- responses. lation of H3K27me3 at genes, such as Ciita which functions in The poor differentiation of Ezh2-deficient ASC could be due, in MHC class II Ag processing, B cell transcription factors like Bcl6, part, to a defect in the ability of Ezh2-deficient ASC to upregulate as well as NF-kB–mediated inflammatory genes. Transcriptome genes involved in the cell cycle. In fact, the top upregulated gene profiling of discrete divisions in vivo following B cell activation sets in Ezh2-deficient ASC were inflammatory and p53 pathways, with LPS revealed that the third cell division following activation with each consisting of genes that function to negatively regulate was the first stage in which gene repression occurred. This divi- the cell cycle. Among the most induced genes within these sets sion also coincided with the downregulation of NF-kB–regulated 12 EZH2 IN ASC FUNCTION genes (8). Ezh2-defient cells displayed a proliferation defect at ASC require the continued activity of Blimp-1 to sustain the UPR and division three as well, suggesting that this stage may be an initial secrete Abs (68). These results pose that EZH2 may also be con- differentiation checkpoint requiring the repression of a set of tinually required for these ASC functions. If ASC require the constant genes to continue the process. activity of transcription factors and epigenetic enzymes to maintain The failure to repress B cell–associated transcriptional programs metabolic potential, this suggests that throughout their lifetime ASC was also associated with the enrichment of transcription factor may be able to adapt to nutrient availability and respond to envi- binding motifs in regulatory regions of those genes. For example, ronmental cues. These data therefore identify an Ezh2-dependent in Ezh2-deficient ASC, CTCF and the ETS family transcription epigenetic process that facilitates ASC metabolism. factor binding motifs were significantly more accessible. The chromatin landscape associated with CTCF within the MHC class Acknowledgments II region is reorganized during B cell differentiation in response to We acknowledge the members of the Boss laboratory for scientific contri- LPS (58), and CTCF is known to function in the maintenance of butions and editorial input to the project, the Emory Flow Cytometry Core GC B cells (77). It is possible that this architecture may not be for FACS expertise, the Emory Integrated Genomics Core for sequencing properly organized in the absence of EZH2. The most differen- library quality control, and the New York University Genome Technology tially regulated ETS family member was Spib, which is normally Center for Illumina sequencing. silenced in ASC through direct repression by Blimp-1 (30, 78). Furthermore, genes normally repressed in both B cells and ASC Disclosures and marked by H3K27me3, including Hoxa1 and Eml6, failed to The authors have no financial conflicts of interest. remain repressed. 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