LSD1 Cooperates with Noncanonical NF-κB Signaling to Regulate Marginal Zone B Cell Development

This information is current as Robert R. Haines, Christopher D. Scharer, Jenna L. Lobby of October 2, 2021. and Jeremy M. Boss J Immunol published online 6 September 2019 http://www.jimmunol.org/content/early/2019/08/30/jimmun ol.1900654 Downloaded from

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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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 6, 2019, doi:10.4049/jimmunol.1900654 The Journal of Immunology

LSD1 Cooperates with Noncanonical NF-kB Signaling to Regulate Marginal Zone B Cell Development

Robert R. Haines, Christopher D. Scharer, Jenna L. Lobby, and Jeremy M. Boss

Marginal zone B cells (MZB) are a mature B cell subset that rapidly respond to blood-borne pathogens. Although the transcriptional changes that occur throughout MZB development are known, the corresponding epigenetic changes and epigenetic modifying pro- teins that facilitate these changes are poorly understood. The histone demethylase LSD1 is an epigenetic modifier that promotes plasmablast formation, but its role in B cell development has not been explored. In this study, a role for LSD1 in the development of B cell subsets was examined. B cell–conditional deletion of LSD1 in mice resulted in a decrease in MZB whereas follicular B cells and bone marrow B cell populations were minimally affected. LSD1 repressed in MZB that were normally upregulated in the myeloid and follicular B cell lineages. Correspondingly, LSD1 regulated chromatin accessibility at the motifs of transcription factors known to regulate splenic B cell development, including NF-kB motifs. The importance of NF-kB signaling was examined

through an ex vivo MZB development assay, which showed that both LSD1-deficient and NF-kB–inhibited transitional B cells Downloaded from failed to undergo full MZB development. expression and chromatin accessibility analyses of in vivo– and ex vivo–generated LSD1-deficient MZB indicated that LSD1 regulated the downstream target genes of noncanonical NF-kB signaling. Addition- ally LSD1 was found to interact with the noncanonical NF-kB p52. Together, these data reveal that the epigenetic modulation of the noncanonical NF-kB signaling pathway by LSD1 is an essential process during the development of MZB. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/ cell progenitors develop through multiple stages to be- Specific signaling mechanisms drive the MZB or FoB cell fate come mature naive B cells capable of generating a hu- decision. When immature B cells enter the periphery, they undergo B moral immune response. In the bone marrow, common positive selection through tonic BCR signaling to promote survival lymphoid progenitors progress through the pro-B and pre-B cell (3). The strength of tonic BCR signaling influences immature stages, during which the BCR is rearranged to generate a func- B cell fate with stronger signals promoting FoB commitment tional yet diverse repertoire of B cells (1). BCR-expressing immature and weaker signals promoting MZB commitment (3). Immature B cells migrate to the spleen, where they undergo transitional B cell B cells must experience two additional signaling pathways to (TrB) development, resulting in the formation of follicular B cells further commit to the MZB fate. The first is NOTCH2 signaling by guest on October 2, 2021 (FoB) and marginal zone B cells (MZB). FoB circulate throughout through interaction with the NOTCH ligand DLL1, which is the periphery and facilitate humoral immune responses to Ag and expressed by splenic venules in the red pulp and marginal zone give rise to memory B cells and long-lived plasma cells (1). MZB (1). The second is BAFFR-dependent activation of noncanonical localize to the splenic marginal sinus and rapidly respond to blood- NF-kB signaling (1). Both pathways are necessary for MZB cell borne pathogens, primarily forming short-lived plasmablasts (1, 2). development and function in a synergistic manner (1). Throughout cell fate commitment, MZB acquire a transcrip- tional identity distinct from FoB that confers specific functional Department of Microbiology and Immunology, Emory University School of Medi- capabilities (1, 2). For example, MZB express high levels of S1pr1 cine, Atlanta, GA 30322 to facilitate homing to the marginal zone (4) and downregulate ORCIDs: 0000-0001-9574-4341 (R.R.H.); 0000-0001-7716-8504 (C.D.S.); 0000- the FoB genes Itgb7, Cxcr4, and Ccr7 that facilitate homing to 0002-1395-2583 (J.L.L.); 0000-0002-2432-1840 (J.M.B.). secondary lymphoid organs (5). is highly expressed in MZB, Received for publication June 13, 2019. Accepted for publication August 5, 2019. providing an enhanced capacity to proliferate in response to Ags This work was supported by the following National Institutes of Health grants: R01 such as bacterial LPS (6). MZB can rapidly respond to other TLR AI123733 and P01 AI125180 to J.M.B., T32 GM0008490 to R.R.H., and F31 AI131532 to R.R.H. agonists (7) and display a concomitant increase in innate immune The sequences presented in this article have been submitted to the National Center sensor molecules relative to FoB, including TLR3, TLR7, TLR9, for Biotechnology Information Omnibus (https://www.ncbi.nlm. NOD1/2/3, and NLRC4 (8). Although the MZB transcriptome is nih.gov/geo/query/acc.cgi?acc=GSE132227) under accession number GSE132227. characterized, the epigenetic modifications acquired during B cell Address correspondence and reprint requests to Dr. Jeremy M. Boss, Emory University, development that establish it are not well studied. Additionally, 1510 Clifton Road, Room 3001, Atlanta, GA 30322. E-mail address: [email protected] the enzymes that facilitate splenic B cell epigenetic remodeling The online version of this article contains supplemental material. are not known. Abbreviations used in this article: ATAC-seq, assay for transposase-accessible fl/fl Cre/+ Lysine-specific demethylase 1 (LSD1) is a histone demethylase chromatin sequencing; CKO, Kdm1a Cd19 conditional knockout; CreWT, Kdm1a+/+ Cd19Cre/+ wild-type; DAR, differentially accessible region; DEG, dif- that targets H3K4me1, H3K4me2, H3K9me1, and H3K9me2 ferentially expressed gene; eMZB, ex vivo–derived MZB; FDR, false discovery through FAD-dependent amine oxidation (9). LSD1-based modi- rate; FoB, follicular B cell; FSC, forward scatter; GSEA, gene set enrichment analysis; IP, immunoprecipitation; LSD1, lysine-specific demethylase 1; MZB, fication of chromatin results in the fine-tuning of target gene ex- marginal zone B cell; NP-40, Nonidet P-40; PCA, principal component analysis; pression, which is critical for driving cellular development (9). qPCR, quantitative PCR; RHD, ; RNA-seq, RNA sequenc- Regarding B cell differentiation, LSD1 promotes plasmablast for- ing; rppm, reads per peak per million; SSC, side scatter; TrB, transitional B cell. mation and decommissions active enhancers at Blimp-1, PU.1, and Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 IRF4 binding sites through H3K4me1 demethylation and repression

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900654 2 LSD1 REGULATES MARGINAL ZONE B CELL DEVELOPMENT of chromatin accessibility (10). LSD1 also promotes germinal 13 penicillin/streptomycin, 10 mM HEPES, and 1 mM sodium pyruvate) center formation by repressing plasma cell genes, such as Prdm1 at 150,000 cells/ml, then 1 ml was plated onto OP9 cells after gently aspirating and Irf4, through enhancer decommissioning facilitated by inter- off OP9 media. Ten nanograms BAFF (8876-BF-010; R&D Systems) was added to the wells. Cells were placed in a 37˚C incubator for 3 d, then un- action with BCL6 (11). Despite evidence highlighting a critical derwent analyses. Where indicated, 800 nM of the IkB kinase inhibitor IKK- role for LSD1 in the epigenetic regulation of B cell differentiation, 16 (SML1138; Sigma-Aldrich) (17) or DMSO was added to the wells. its in vivo role during B cell development has not been explored. Flow cytometry In this study, mice with B cell–conditional deletion of LSD1 6 were used to examine its function throughout B cell development. Cells were resuspended at a concentration of 10 cells/100 mlFACS 3 Phenotyping revealed that LSD1 was dispensable for the devel- buffer (1 PBS, 2 mM EDTA, 1% BSA). Cells were stained with Fc block (2.4G2; BD Biosciences) for 15 min and then followed with opment of bone marrow B cell subsets and FoB but was required specific Ab fluorophores for 1 h at 4˚C, then washed with 10 volumes for MZB formation. RNA sequencing (RNA-seq) analysis of LSD1- of FACS buffer. The following Abs or stains were used as indicated: deficient MZB and FoB showed that LSD1 functions as a transcrip- B220-PE-Cy7 (RA3-6B2; Tonbo Biosciences), CD1d-BV605 (1B1; BD tional repressor in MZB. Assay for transposase-accessible chromatin Biosciences), CD11b-allophycocyanin-Cy7 (M1/70; Tonbo Biosciences), CD21-allophycocyanin (B-ly4; BD Biosciences), CD23-eFluor450 (B3B4; sequencing (ATAC-seq) analysis revealed a chromatin modulatory eBioscience), CD43-PE (S7; BD Biosciences), CD45.1-FITC (A20; role for LSD1 at motifs of transcription factors critical for MZB de- Tonbo Biosciences), CD45.2-PerCP-Cy5.5 (104; Tonbo Biosciences), velopment, including NF-kB. Experiments using an ex vivo MZB CD90.2-allopycocyanin-Cy7 (30-H12; BioLegend), CD93-PE (AA4.1), development system indicated pathway overlap between LSD1 F4/80-allophycocyanin-Cy7 (BM8; BioLegend), GL7-eFluor660 (GL-7; and noncanonical NF-kB signaling. LSD1 and NF-kB p52 also eBioscience), IgD-BV650 (IA6-2; BD Biosciences), IgM-FITC (II/41; k eBioscience), Ly6A/E-PerCP-Cy5.5 (E13-161.7; BioLegend), Ly6C-PerCP- interact following noncanonical NF- B stimulation. Overall, these Cy5.5 (HK1.4; BioLegend), Zombie Yellow Fixable Viability Dye (kit no. Downloaded from data identify LSD1 as a key transcriptional and epigenetic mod- 423104; BioLegend), and Live/Dead Fixable Blue Dead Cell Stain Kit for ifier during MZB development. UV Excitation (L23105; Thermo Fisher Scientific). An LSRFortessa X-20 was used for analysis and a FACSAria II was used for sorting (BD Biosciences). All flow cytometry data were analyzed with FlowJo v9.9.5. Preceding all flow Materials and Methods cytometry analyses presented is the following gating strategy: 1) lymphocytes Data availability (forward scatter [FSC] area by side scatter [SSC] area), 2) singlets (FSC width by FSC-height), 3) singlets (SSC width by SSC height), 4) live cells (viability – – – – Sequencing data are available through accession GSE132227 at the Na- dye ), 5) exclusion of non-B cell lineage cells (Thy1.1 F4/80 CD11b ). http://www.jimmunol.org/ tional Center for Biotechnology Information Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE132227). RNA-seq and data analysis Mice and animal procedures B220+CD93–GL7–CD23–CD21hiCD1d+ MZB and B220+CD93– GL7–CD23+CD21midCD1d– FoB were targeted to ensure pure, naive fl/fl LSD1 floxed mice (Kdm1a , 023969; The Jackson Laboratory) (12) were populations. Fifty thousand cells were isolated via FACS into 600 ml fl/fl Cre/+ a gift from Dr. David Katz (Emory University). Kdm1a Cd19 con- RLT buffer (79216; Qiagen) containing 1% 2-ME. Similarly, for TrBs fl/fl ditional knockout (CKO) mice were created from crossing Kdm1a mice (B220+CD93+) and ex vivo–derived MZB (eMZB) (B220+CD21+CD1d+), Cre/+ +/+ Cre/+ to Cd19 mice (006785; The Jackson Laboratory) (13). Kdm1a Cd19 2000 cells were isolated per sample via FACS into 300 ml RLT buffer as wild-type (CreWT) mice were used as wild-type controls because of the above. Cells were lysed by vortexing for 1 min on high, and External RNA fl/fl CreERT2/+ hemizygous nature of the Cd19 allele (14). Kdm1a Rosa26 mice Controls Consortium spike-in RNA transcripts (18) were added (5 mlof by guest on October 2, 2021 CreERT2/+ were created from crossing Rosa26 mice (008463; The Jackson 1:2000 dilution per sample) to enable downstream mRNA/cell calculations. fl/fl Laboratory) (15) with Kdm1a mice. C57BL/6 wild-type mice were Total RNA was isolated using a the Quick-RNA MicroPrep Kit (R1050; obtained from The Jackson Laboratory (000664) and were bred on site. Zymo Research). For 50,000 cell preps, all RNA was used to create libraries Experiments were performed on mice 8–12 wk old. with an mRNA HyperPrep Kit (KK8581; KAPA Biosystems). For 2000 cell 7 For mixed bone marrow chimera experiments, 1–2 3 10 bone marrow preps, all RNA was used to synthesize cDNA with the SMART-Seq v4 Ultra fl/fl Cre/+ +/+ Cre/+ cells from Kdm1a Cd19 CD45.1 mice and Kdm1a Cd19 Low Input RNA Kit (634891; Takara), and libraries were generated with the CD45.1/0.2 mice were mixed in a 1:1 ratio and injected into host CD45.2 Nextera XT DNA Library Prep Kit (FC-131-1096; Illumina). An Agilent B6 wild-type mice (000664; The Jackson Laboratory) that were lethally Bioanalyzer was used to quality control check each library. Libraries were irradiated with 900 rad (two doses of 450 rad 6 h apart). Host mice were pooled at an equimolar ratio and sequenced on a HiSeq 2500 system (Illu- given water containing 2% sucrose, 10 mg/ml neomycin (N5285; Sigma- mina) using 50-bp paired-end chemistry. Aldrich), and 125 ng/ml polymyxin B (P0972; Sigma-Aldrich) for 2 wk Raw sequencing reads were mapped to the mm10 genome using STAR and were analyzed 6 wk postirradiation. v2.2.1 (19). Gene counts were determined with the GenomicAlignments For the adoptive transfer experiment, CD43 (Ly-48) MicroBeads 2 package v1.18.1 (20) using the mm10 transcriptome database. A gene was (130-097-148; Miltenyi Biotec) were used to magnetically purify CD43 considered detected if all samples from a sample group had at least splenic B cells from naive mice according to the manufacturer’s instructions. 0.1 mRNA/cell (the minimum External RNA Controls Consortium Purified splenic B cells were stained with CD23-PE (B3B4; BioLegend), transcript/cell concentration to detect at least 90% of that particular + then CD23 cells were magnetically depleted, leaving a population of MZB. transcript across all samples) or one RPM. The edgeR package v3.24.3 MZB were transferred into mMT host mice via i.v. injection into the tail. (21) was used to determine differentially expressed genes (DEG) based fl/fl CreERT2/+ For CreERT2-based deletion of LSD1, Kdm1a Rosa26 mice on both relative (false discovery rate [FDR] #0.05, no fold change were treated with 40 mg total of tamoxifen. Tamoxifen powder (T5648; cutoff) and absolute changes (FDR #0.05, no fold change cutoff) Sigma-Aldrich) was dissolved in corn oil previously described (10). Mice in expression as previously described (22). All DEG determined are listed received daily injections i.p. for 5 d (8 mg per day). Following five ad- in Supplemental Table I. KEGG pathway analysis was performed with ditional days of rest, mice underwent experimental analysis. DAVID (23). Significance for DEG overlap comparisons was determined with Fisher exact test. Observed/expected ratios were calculated through Ex vivo development of MZB permutation testing against gene sets randomly chosen from all detected OP9-DL1 and OP9-GFP mouse stromal cell lines (16) were obtained from genes (1000 permutations per test). Significance for boxplot expression Dr. Juan Carlos Zu´n˜iga-Pflu¨cker (University of Toronto). In a 24-well plate, data were determined with Student two-tailed t test. PreRanked Gene Set 100,000 OP9-DL1 or OP9-GFP cells were plated in 1 ml of OP9 media Enrichment Analysis (GSEA) (24) was performed on all detected tran- (10 g/l a-MEM powder [12561; Thermo Fisher Scientific], 2.2 g/l sodium scripts ranked based on the following equation: bicarbonate [pH 7], 5% heat-inactivated FBS, 13 penicillin/streptomycin). ð2 ð P ÞÞ CD93+ cells were enriched from splenocytes from naive mice by first sign of fold change log10 edgeR value staining cells with CD93-PE (AA4.1; BioLegend), then performing immu- nomagnetic enrichment of CD93-PE–stained cells with anti-PE microbeads ATAC-seq and data analysis (130-105-639; Miltenyi Biotec) according to the manufacturer’s instruc- tions. Enriched cells were resuspended in B cell media (RPMI 1640, 10% Ten thousand FACS-isolated cells were used for ATAC-seq (25). Libraries heat-inactivated FBS, 0.05 mM 2-ME, 13 nonessential amino acids, were generated as described previously (26). Raw sequencing reads were The Journal of Immunology 3 mapped to the mm10 genome using Bowtie v1.1.1 (27). Peaks were Cruz Biotechnology), Lamin B (B-10, sc-374015; Santa Cruz Bio- called using MACS2 v2.1.1 (28) and annotated using HOMER v4.10 technology), or control IgG (sc-2025; Santa Cruz Biotechnology) were (29). Data were normalized to reads per peak per million (rppm) as bound to the beads overnight by rotating at 4˚C. The beads were wash previously described (30). A Benjamini–Hochberg FDR #0.05 and a twice in 1 ml wash/binding buffer, then mixed with 300 mg of fold change $1.5 were required for significance. All differentially extract in a final volume of 300 ml brought up with IP lysis buffer. accessible regions (DAR) determined are listed in Supplemental Coimmunoprecipitation was performed by rotating for 3 h at 4˚C. Table II. Motif analysis was performed with the HOMER program Beads were washed four times with 1 ml IP lysis buffer. Protein were findMotifsGenome.pl using randomly generated genomic sequences eluted from beads by boiling for 10 min in Laemmli sample buffer with as background. PageRank analysis (31) was performed using raw 5% 2-ME and 100 mM DTT. ATAC-seq data and mRNA/cell gene expression data. Only tran- scription factors with a PageRank score .0.003inatleastonesample Western blotting group of the indicated comparison were included to filter out less- important factors (corresponds to the top 10% of all transcription Protein were resolved with SDS-PAGE, transferred to a PVDF mem- factors). Significance for boxplot enrichment data were determined brane overnight, and probed with primary mouse anti-human Abs di- with Student two-tailed t test. lutedinTBST(150mMNaCl,20mMTris,2mMKCl,0.1%Tween20) containing 5% nonfat dry milk. Abs to the following were used: actin Quantitative RT-PCR (AC-15, sc-69879, diluted 1:1000; Santa Cruz Biotechnology), LSD1 (diluted 1:400), p52/p100 (diluted 1:1000), Lamin B (diluted 1:100), Cells were pooled from six replicate cultures, and B220+ cells were b-tubulin (D-10, sc-5274, diluted 1:100; Santa Cruz Biotechnology). enriched by first staining with B220-allophycocyanin (RA3-6B2; Tonbo Membranes were treated with a solution of HRP-conjugated sheep anti- Biosciences), then performing immunomagnetic enrichment of B220- mouse secondary Ab (A6782; Sigma-Aldrich) diluted 1:3300 in TBST allophycocyanin–stained cells with anti-allophycocyanin microbeads containing 5% nonfat dry milk and were visualized with the Chemi Hi (130-090-855; Miltenyi Biotec). Fifty thousand cells were added to Resolution application using a ChemiDoc MP imaging system (Bio-Rad

600 ml RLT buffer (Qiagen) containing 1% 2-ME. Total RNA was Laboratories). Downloaded from isolated using a Quick-RNA MicroPrep Kit (R1050; Zymo Research) and reverse transcribed with SuperScript II Reverse Transcriptase Immunofluorescence (18064014; Invitrogen). cDNA was diluted 5-fold, and quantitative Spleens were prepared for staining as previously described (34). Slides were PCR (qPCR) was performed with a CFX96 instrument (Bio-Rad stained with a primary Ab mixture of CD169-FITC (MOMA-1, MCA947F, Laboratories) using SYBR Green incorporation. Gene expression diluted 1:50; Bio-Rad Laboratories), IgM-Biotin (II/41, 553436, diluted was calculated relative to 18S rRNA. Sequences for all primers are listed 1:35; BD Biosciences), and CD1d (1B1, 123551, diluted 1:200; Bio- in Supplemental Table III. Significance was determined with Student two- Legend) in serum blocking buffer. Slides were secondary stained with anti- http://www.jimmunol.org/ tailed t test. FITC-A488 (polyclonal; A11090, diluted 1:100; Life Technologies) and Protein purification streptavidin-Hilyte555 (60666, diluted 1:20; AnaSpec) in serum blocking buffer. Slides were imaged on a Zeiss Axio Observer Z1 at room tem- To quantify LSD1 protein levels in CKO and CreWT naive B cells, CD43– perature with acquisition software Zen v2.0.0.0 (Blue Edition) using tiling splenic naive B cells from both strains of mice were magnetically and z-stacking. Images were taken with a 203 Plan-Apochromat 203/0.8 purified (130-097-148; Miltenyi Biotec) according to the manufac- objective (420650-9901; Zeiss) and an Axiocam 506 mono digital camera turer’s instructions. Cells were washed in PBS, pelleted at 3000 rpm (Zeiss). Image files were analyzed with ImageJ v1.52k (National Institutes for5minat4˚C,andresuspendedin13 volume of cold RIPA buffer of Health). (20% glycerol, 50 mM Tris [pH 8], 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40 [NP-40], 1 mM PMSF). Results by guest on October 2, 2021 Resuspended cells were put on ice for 20 min and centrifuged at 15,000 LSD1 regulates MZB development rpm for 10 min at 4˚C. Resulting supernatant containing the protein was stored at 280˚C. LSD1 regulates B cell differentiation to plasma cells (10, 11), Protein from the Raji human B cell line (32) either untreated or treated but its role in B cell development has not been explored. To with 2 mg/ml anti-CD40 Ab (5C3, 334304; BioLegend) was isolated from nuclear and cytosolic fractions using a modified protocol (33). Cells were examine its role from the pro-B to mature B cell stage, CD19- washed with cold PBS, washed with 53 volumes of hypotonic buffer based B cell–conditional LSD1 deletion mice (CKO) (10) and Cre/+ (10 mM HEPES [pH 7.9], 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl2, Cd19 control mice (CreWT) were phenotyped by flow 10 mM KCl, 1 mM DTT, 0.05% NP-40, 0.5 mM PMSF), resuspended in cytometry. Compared with CreWT mice, the bone marrow of 33 volumes of hypotonic buffer, put on ice for 10 min, and homogenized by passing through an insulin syringe 5–10 times. A 0.13 volume of su- CKO mice exhibited similar numbers of total B cells, pro-B cells, crose restore buffer (67.5% sucrose, 10 mM DTT, 13 sucrose restore pre-B cells, immature B cells, and mature B cells (Fig. 1A–C). buffer salts [103 sucrose restore buffer salts: 500 mM HEPES, pH 7.9, The spleen of CKO mice exhibited similar numbers of total 10 mM KCl, 2 mM EDTA]) was added and mixed by inverting two to three B cells, TrBs, and FoB, but there was a 1.5-fold reduction in MZB times. Nuclei were pelleted at 3000 rpm for 5 min at 4˚C. Supernatants were centrifuged at 15,000 rpm for 10 min at 4˚C, and the resulting su- (Fig. 1D–F). A significant reduction in MZB was also observed in pernatant containing the cytoplasmic fraction was stored at 280˚C until CKO mice using two alternative MZB gating strategies (Fig. 1G, application. 1H). LSD1 protein was not detected in CKO splenic naive For Western blotting, nuclei were resuspended in 13 volume of cold B cells (Supplemental Fig. 1A), confirming knockout in this RIPA buffer. Resuspended nuclei were put on ice for 20 min, sonicated for 30 s, and centrifuged at 15,000 rpm for 10 min at 4˚C, and the resulting population. CKO and CreWT spleens were examined by im- supernatant containing the nuclear fraction was stored at 280˚C. If the munofluorescence for markers IgM (total B cells), Cd1d (MZB), downstream application was coimmunoprecipitation, nuclei were resus- and CD169 (marginal zone macrophages) to determine if pended in 53 volumes of cold immunoprecipitation (IP) lysis buffer CKO splenic marginal zones were morphologically normal. (20 mM Tris [pH 8], 135 mM NaCl, 20% glycerol, 1% NP-40, 1 mM Compared with CreWT, CKO spleens displayed characteristic PMSF, complete protease inhibitor mixture [05892970001; Sigma- + Aldrich]). Resuspended nuclei were rotated for 20 min at 4˚C and marginal zone architecture outlined by CD169 marginal zone centrifuged at 15,000 rpm for 10 min at 4˚C, and the resulting supernatant macrophages (Fig. 2, green). White pulp regions also displayed containing the nuclear fraction was stored at 280˚C. normal patterns of IgM+ B cells and CD1d+ MZB (Fig. 2, blue, red), suggesting that the decrease in CKO MZB is due to a Coimmunoprecipitation developmental defect instead of a splenic architectural defect. Thirty microliters of Dynabeads M280 sheep anti-mouse IgG magnetic B-1 B cell frequencies were assessed in the spleen and perito- beads (11201D; Thermo Fisher Scientific) were washed four times with neal cavity of CKO mice (Supplemental Fig. 1B–D). Both 1mlwash/bindingbuffer(PBSwith1%BSA)byrotatingfor10minat 4˚C. Three micrograms of primary Ab specific to LSD1 (B-9, sc- compartments exhibited a significant increase in B-1b cells, 271720; Santa Cruz Biotechnology), p52/p100 (C-5, sc-7386; Santa whereas the peritoneal cavity exhibited alterations in B-1 and B-2 4 LSD1 REGULATES MARGINAL ZONE B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. B cell conditional LSD1 deletion results in fewer MZB. (A–C) Flow cytometry analysis of the expression of developing B cell markers in the bone marrow of unstimulated naive CreWT and CKO mice. Analysis of CreWT and CKO total cell numbers of the following B cell populations are shown: (A) B220+ Bcells;(B)IgM–B220+CD43+ pro-B cells and IgM–B220+CD43– pre-B cells; (C)IgM+B220mid immature B cells and IgM+B220hi mature B cells. (D–H) Flow cytometry analysis of the expression of developing B cell markers in the spleen of unstimulated CreWT and CKO mice. Analysis of CreWT and CKO total cell numbers of the following B cell populations are shown: (D)B220+ Bcells;(E) B220+CD93+CD23– T1 and B220+CD93+CD23+ T2 B cells; (F)B220+CD93–CD21hiCD23– MZB and B220+CD93–CD21midCD23+ FoB; (G)B220+CD93–CD21hiCD1d+ MZB; and (H)B220+CD93–IgM+IgD– MZB. All data are representative of at least two independent experiments using three to five mice per group. Error bars represent mean 6 SD. Significance was determined by Student two-tailed t test. *p , 0.05, **p , 0.01, ***p , 0.001. population frequencies, suggesting that LSD1 regulates B-1 cytometry. MZB exhibited significantly lower frequencies of CKO cell development. The ability of LSD1-deficient MZB to re- cells compared with CreWT (Fig. 3A, 3B), which is reflected in a spond to the T-independent Ag LPS was examined by adop- 3–4-fold reconstitution ratio favoring CreWT cells over CKO tively transferring CKO and CreWT MZB in a 1:1 ratio into cells. Splenic FoB and T1 B cells exhibited similar reconsti- B cell–deficient mMT host mice. CKO MZB exhibited a 2-fold tution ratios between CreWT and CKO cells, but T2 B cells reduction in plasmablast formation (Supplemental Fig. 1E), were skewed in favor of CreWT (Fig. 3A, 3C), supporting a verifying the known role that LSD1 has in regulating plasma- defect in LSD1-deficient splenic B cell development. Lymph blast differentiation (10). node B cells, pro-B cells, and bone marrow immature B cells To examine the intrinsic nature of CKO B-2 cell development, also had similar frequencies; however, pre-B cells and bone mixed bone marrow chimeras were established using an equal ratio marrow mature B cells displayed partially skewed reconstitu- of CD45.1 CKO and CD45.1/2 CreWT bone marrow in lethally tion ratios (Fig. 3D–F). Overall, these data demonstrate that irradiated CD45.2 wild-type hosts. The frequencies and ratios of LSD1 regulates MZB development in a cell-intrinsic manner reconstituted host B cell compartments were analyzed with flow (Fig. 3G). The Journal of Immunology 5

FIGURE 2. B cell conditional LSD1 deletion mouse

spleens display normal marginal zone architecture. Downloaded from Immunofluorescence staining for CD169 (green), IgM (blue), and CD1d (red) in the spleens of CreWT and CKO mice. Images are at original magnifica- tion 310. Scale bar, 400 mm. http://www.jimmunol.org/ by guest on October 2, 2021

LSD1 functions as a transcriptional repressor in MZB (8, 35). Known MZB genes were upregulated in MZB CreWT, The gene regulatory role that LSD1 plays in splenic B cell de- including the homing S1pr1, the transcription factor Myc, velopment was examined by performing RNA-seq on CKO and and the NOTCH2 target Dtx1 (4, 6, 36). Similarly, known FoB CreWT MZB and FoB. Principal component analysis (PCA) was genes were upregulated in FoB CreWT, including the homing performed on all 9690 detected genes (Fig. 4A). Principal com- receptors Ccr7, Cxcr4, and Itgb7, as well as the transcription ponent 1 stratified CreWT samples by cell type and MZB CreWT factors Bach2 and Klf2 (5, 37, 38). Using GSEA (24), the data from CKO. PC2 further stratified MZB CKO from all samples. above were compared with two previous MZB studies (8, 35) Intriguingly, FoB CreWT and CKO samples did not stratify by (Supplemental Fig. 2A). Genes upregulated in MZB CreWT were either principal component, indicating little transcriptional varia- significantly enriched for previously identified MZB genes whereas tion due to LSD deletion in those cell types. These data suggest genes upregulated in FoB CreWT were significantly enriched for that LSD1 regulates the MZB transcriptional program. Further- previously identified FoB genes, validating the datasets. Compari- more, the alignment of MZB CKO and FoB cell types suggests sons between CKO and CreWT samples for each cell type identified that MZB CKO transcriptomes possess FoB-like qualities. 323 DEG between MZB CKO and MZB CreWT but only 48 DEG To identify transcriptional differences between cell types and the between FoB CKO and FoB CreWT, supporting the conclusion from effects of LSD deletion, differential gene expression analyses were the PCA that LSD1 primarily regulates the MZB transcriptome. performed on three sample group comparisons: MZB CreWT MZB CKO had 297 upregulated DEG and only 26 downregulated versus FoB CreWT, FoB CKO versus FoB CreWT, and MZB CKO DEG, suggesting that LSD1, similar to plasmablasts and germinal versus MZB CreWT (Fig. 4B). The comparison of MZB and FoB center B cells (10, 11), mainly plays a repressive role in regulating CreWT cells revealed that 1887 genes were significantly upregulated MZB transcription. in MZB, whereas only 101 genes were significantly upregulated in DEG across all samples were assessed and organized based on FoB, confirming that MZB and FoB possess distinct transcriptomes function (Fig. 4C). Signaling genes upregulated in MZB CKO that 6 LSD1 REGULATES MARGINAL ZONE B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Reduction of LSD1-deficient MZB is cell intrinsic. (A–F) Flow cytometry analysis of the expression of developing B cell markers in the spleen and bone marrow of unstimulated mixed bone marrow chimera mice reconstituted in a 50:50 ratio of CKO and CreWT bone marrow. Analysis of CreWT and CKO frequencies of the following B cell populations are shown: (A) splenic B220+CD93–CD21midCD23+ FoB and B220+CD93–CD21+CD23– MZB; (B) splenic B220+CD93–CD21hiCD1d+ MZB; (C) splenic B220+CD93+CD23– T1 B cells and B220+CD93+CD23+ T2 B cells; (D) lymph node B220+ B cells; (E) bone marrow IgM–B220+CD43+ pro-B cells and IgM–B220+CD43– pre-B cells; and (F) bone marrow IgM+B220mid immature B cells and IgM+B220hi mature B cells. (G) Ratios of CreWT/CKO frequencies of B cell populations per mouse. All data are representative of two independent experiments using five mice per group. Error bars represent mean 6 SD. Significance was determined by Student paired two-tailed t test. *p , 0.05, **p , 0.01, ***p , 0.001. are known to play a role in B cell development included Cdkn2c promotes MZB formation by inhibiting basic helix-loop-helix and Flt3. CDKN2C is a cyclin-dependent kinase inhibitor that transcription factors such as E2A (41); and IRF1 and IRF7, suppresses cell cycle G1 progression and is known to regulate which regulate the expression of IFN response genes (42). Genes splenic B cell homeostasis (39). Loss of FLT3-ligand in mice encoding surface involved in adhesion and migration results in a cell extrinsic increase in MZB and decrease in FoB, were upregulated as well and included Cxcr4 and Itgb7,which suggesting an important role for FLT3 signaling in splenic B cell facilitate homing to the bone marrow and gut, respectively (5). development (40). Genes encoding transcriptional regulators im- These two genes exhibited a significant increase in surface ex- portant for B cell development were overexpressed in MZB CKO. pression on MZB (Supplemental Fig. 2B, 2C), validating the gene These included the transcription factors BACH2 and BCL6, which expression data. BACH2 and BCL6 are also known to promote are critical for bone marrow B cell development (37); ID3, which germinal center B cell formation (43), but there was no significant The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 4. LSD1 functions as a transcriptional repressor in MZB. (A) Top two principal components from PCA of z-score normalized mRNA/cell expression of all 9690 detected genes in all samples. (B) Scatterplots of log2 fold change versus log2 fragments per kilobase per million (log2FPKM) data from differential expression analysis comparing MZB CreWT (orange) and FoB CreWT (green), FoB CKO (blue) versus FoB CreWT (green), and MZB CKO (red) versus MZB CreWT (orange). (C) Heatmaps of z-score normalized mRNA/cell expression of genes in KEGG pathways or functional categories. (D) GSEA plot displaying the enrichment of the top 200 most significant genes upregulated in MZB CreWT relative to FoB CreWT within the by guest on October 2, 2021 MZB CKO versus MZB CreWT ranked gene list. (E) Overlapping DEG between the indicated comparison. Significance was determined by Fisher exact test. Observed/expected (obs/exp) refers to the ratio of observed DEG overlap over expected overlap according to a permutation test. increase in germinal center B cells in CKO spleens (Supplemental of LSD1, there is lineage dysregulation into a myeloid-type cell. Fig. 2D). Thus, LSD1 is important for establishing the transcriptional Genes critical for FoB function such as Klf2, Bach2, Itgb7, and identity of MZB during splenic B cell development, partly through Cxcr4 were upregulated in MZB CKO, implying defective MZB repressing FoB and myloid lineage transcriptional programs. development through aberrant expression of FoB genes. To further k explore this effect, the top 200 significant genes upregulated in LSD1 represses chromatin accessibility at NF- B motifs FoB CreWT compared with MZB CreWT were analyzed for en- The effect that LSD1 deficiency has on chromatin accessibility in richment in MZB CKO genes using GSEA (Fig. 4D). The results FoB and MZB was addressed by performing ATAC-seq on the same displayed a significant enrichment of FoB CreWT genes in the sample groups as RNA-seq. PCA on all 94,161 peaks showed that MZB CKO cells. Furthermore, the 297 upregulated DEG in MZB samples separated by both cell type and LSD1 deletion status CKO were tested for significant overlap with the 101 upregulated (Fig. 5A). MZB CKO samples separated more from MZB CreWT DEG in FoB CreWT (Fig. 4E). A total of 56 genes overlapped samples by PC2 compared with the separation between FoB CKO between the two groups, which was 18.3-fold more than expected and CreWT samples, suggesting that LSD1 has a larger impact on by chance. Additional example FoB genes include those that en- chromatin accessibility in MZB than FoB. code JUND, a transcription factor that promotes Bcl6 expression Differential accessibility analysis was performed on three in germinal center B cells (44), CD200, a receptor that is over- sample group comparisons: MZB CreWT versus FoB CreWT, FoB expressed on B cell neoplasms and regulates antitumor immunity CKO versus FoB CreWT, and MZB CKO versus MZB CreWT (45), and IL-21R, which binds IL-21 to regulate B cell prolifer- (Fig. 5B). Comparison of FoB CreWT and MZB CreWT revealed ation, differentiation, and survival (46). Also, because some MZB thousands of DAR, indicating that these cell types are very distinct CKO upregulated DEG are highly expressed in myeloid cells, at the level of chromatin accessibility. Compared with their such as Csf2ra, Irf1, Irf7, and Itgam, and pre-B cells can be CreWT counterparts, MZB CKO had 1014 total DAR whereas transdifferentiated into macrophages by altering the pre-B cell FoB CKO had 678 total DAR, with DAR increasing in accessibility transcriptional network (47), MZB CKO genes were tested for being more numerous than DAR decreasing in accessibility for both enrichment of myeloid progenitor (48) and macrophage lineage comparisons. Thus, LSD1 regulates chromatin accessibility in both (49) genes (Supplemental Fig. 2E). MZB CKO genes were signif- MZB and FoB—but to a greater extent in MZB—and plays more of icantly enriched for these gene sets, suggesting that in the absence a repressive role in both cell types. 8 LSD1 REGULATES MARGINAL ZONE B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. LSD1 regulates chromatin accessibility at NF-kB motifs. (A) Top two principal components from PCA of z-score normalized rppm ac- cessibility data of all 94,161 detected peaks in all samples. (B) Volcano plots of 2log10FDR versus log2 fold change (FC) from differential accessibility analysis on three different sample group comparisons. (C and D) Heatmap displaying 2log2 (p values) of top significantly enriched transcription factor family motifs for the indicated DAR groups from (B). Boxplots depict rppm enrichment of chromatin accessibility for the indicated sample group at specific transcription factor motifs. Significance was determined by Student two-tailed t test. (E) Scatterplots of log2FC data from PageRank analysis versus log2FC data from RNA-seq analysis on three different sample group comparisons. Points correspond to transcription factors with a PageRank score .0.003 (top 10%) in at least one of the compared sample groups. A red point indicates a DEG in the given comparison. (F) Heatmap of z-score normalized mRNA/cell expression of p52 target genes predicted by PageRank analysis. (G) Gene track examples of rppm chromatin accessibility data for DAR that increase in accessibility in MZB CKO that map to a p52 motif and a p52 target DEG predicted by PageRank analysis. The Journal of Immunology 9

To understand what transcription factor binding motifs were factors for 1) highest percent DEG of all target regulated genes, enriched within DAR, motif enrichment analysis was performed and 2) chromatin accessibility changes at motifs mapping to these and enrichment p values for top motifs were plotted as a heatmap DEG. EBF1, p52, and STAT6 had the highest percentage of MZB for the CreWT, FoB CKO, and MZB CKO sample group com- CKO DEG of their target regulated genes (Supplemental Fig. 3C). parisons (Fig. 5C, 5D, Supplemental Fig. 3A). For the MZB and Of these factors, only p52 exhibited a significant increase in ac- FoB CreWT comparison (Fig. 5C), ETS factor motifs were highly cessibility in MZB CKO compared with MZB CreWT (Supplemental enriched in both DAR groups. ETS factors such as SPIB, SPI1, Fig. 3D), suggesting that without LSD1, p52 fails to properly repress ETS1, and FLI1 are known to regulate splenic B cell development target gene expression and chromatin accessibility, possibly through a (50–52) and may be influencing chromatin accessibility in these direct interaction. cell types. Transcription factor binding motifs for bHLH, POU, The biological roles of transcription factors were explored by Rel homology domain (RHD), and RUNT factors were more examining their PageRank-determined target genes. PBX2, which enriched in MZB CreWT DAR, suggesting a role for these factors was identified as important in MZB CreWT compared with FoB in regulating MZB chromatin accessibility. Certain bHLH factors CreWT, is not known to have a role in B cell development. Here, such as TCF3 (E2A), TCF4 (E2-2), and MYC regulate the for- PBX2 was predicted to upregulate 59 DEG in MZB CreWT that mation and function of splenic B cells (6, 41, 53). Both POU were determined to be involved in a number of processes via KEGG factors and RHD factors are required for normal splenic B cell pathway analysis, the most significant being carbon metabolism development (54–56). IRF and KLF factor motifs were more and mismatch repair (Supplemental Fig. 3E). Example genes in- enriched in FoB CreWT DAR. Because KLF2 and IRF4 activate clude the metabolic enzymes Got1, H6pd, and Prps2 and the DNA genes important for FoB function (38, 42), they may influence repair enzymes Mlh1 and Rpa3. These data suggest a novel role chromatin accessibility to exert their transcriptional regulation. for PBX2 in MZB function. In MZB CKO, EBF1 was predicted to Downloaded from For the CreWT versus CKO comparisons (Fig. 5D, Supplemental have a dysregulated transcriptional network upon LSD1 deletion Fig. 3A), ETS factor motifs were highly enriched in all DAR despite not having dysregulated chromatin accessibility at target groups. LSD1 has been shown to modulate chromatin accessibility motifs, suggesting an indirect LSD1-mediated regulatory effect. at ETS motifs and binding sites in plasmablasts or a B cell line EBF1 is known to regulate genes involved in the BCR signaling (10, 57), providing further evidence for B cell–specific LSD1- pathway and does so in an LSD1-dependent manner with the based regulation at these sites. MZB CKO upregulated DAR genes Pik3cg and Rasgrp3 (Supplemental Fig. 3F), suggesting a http://www.jimmunol.org/ were enriched for CTCF, KLF, RHD, and RUNT binding motifs, possible role for LSD1 in regulating this process (61). p52 was whereas MZB CreWT DAR were enriched for bHLH, GATA, predicted to upregulate 23 DEG in MZB CKO, including the HSF, and POU binding motifs. LSD1 has been shown to interact transcriptional regulators BACH2 and ID3, the receptors S100a10 directly with the bHLH factors MYOD and TAL1 (58, 59) and the and TLR2, and the signaling molecules PIK3CG, RASGRP3, and RHD factor p65 (60), supporting the possibility that LSD1 may SPATA13 (Fig. 5F). p52 binding motifs were assessed individually cooperate with transcription factors of the same families during for overlap with DAR. Four of these mapped to the p52 target B cell development to exert its effects on chromatin accessibility DEGs Crisp3, Id3, S100a10, and Sapcd1 (Fig. 5G, Supplemental and gene expression. Fig. 3G). Nineteen other p52 motif-containing DAR were located by guest on October 2, 2021 PageRank analysis was used to integrate RNA-seq data and throughout the genome (Supplemental Fig. 3H). In addition to the ATAC-seq data to rank transcription factors in each sample group above, 10 DAR that did not contain a p52 motif mapped to p52 by predicted importance based on the expression and identity of its target DEG (Setbp1 and Tlr2) (Supplemental Fig. 3I). Together, target genes (31). The PageRank score of the top 20 transcription these data suggest a role for LSD1 in directly repressing the ex- factors (out of 639 analyzed) per sample group were plotted as a pression of p52 target genes by limiting chromatin accessibility at heatmap (Supplemental Fig. 3B). Reflecting their shared precursor p52 binding sites. origins, the analysis indicated that FoB and MZB share many top factors, including SPIB, ETS1, ELF1, p50, and p52. Some tran- LSD1 regulates ex vivo MZB development induced by NOTCH2 k scription factors are unique to certain sample groups, such as PBX2 and noncanonical NF- B signaling to MZB, indicating a more prominent role in target gene regulation The above analysis suggested a dependence of LSD1 on non- for these factors in these sample groups. canonical NF-kB signaling through p52, a critical factor for MZB PageRank analysis between MZB CreWT and FoB CreWT formation (54). To assess the relevance of LSD1 in noncanonical (Fig. 5E) identified factors known to be important for the forma- NF-kB signaling during MZB development, CKO and CreWT tion and function of MZB and/or FoB were identified and include B220+CD93+ TrBs were cultured on OP9-DL1 cells, which BACH2, BCL6, EBF1, FLI1, MYC, p52, and TCF4 (6, 37, 52, 53, stimulate NOTCH2 signaling through Delta-like–1 ligand ex- 55, 61). Other factors identified such as MEIS3 and PBX2 have pression, and in the presence of BAFF to stimulate noncanonical not been shown to play a role in B cell development but are known NF-kB signaling (1). Precultured TrB displayed similar population to regulate the development of other cell types (62, 63). Between levels between CKO and CreWT mice and exhibited low surface FoB CKO and FoB CreWT, transcription factors such as NF-kB expression of the MZB surface markers CD21 and CD1d (Fig. 6A, p52 were determined to be more important in FoB CKO despite 6B). After 3 d in culture, 14–20% of all CreWT TrB developed into the relatively unchanged FoB CKO transcriptome and lack of a B220+CD21+CD1d+ eMZB, whereas only 6–10% of CKO TrB de- splenic FoB phenotype in CKO mice. Between MZB CKO and veloped into eMZB (Fig. 6C). Cells were developed under additional MZB CreWT, the transcription factors BACH2, CIC, EBF1, p52, conditions, including controls for BAFF and the Delta-like–1 ligand STAT6, and TCF3 were determined to be more important to the DL1 (Supplemental Fig. 4A). LSD1-deficient cells developed into MZB CKO transcriptional program, implying LSD1-dependent significantly fewer MZB under all conditions, suggesting a defect regulation of their downstream target genes. in both NOTCH2 and noncanonical NF-kB signaling. To ensure The transcription factors most likely to cooperate with LSD1 to that the defect was due to the absence of LSD1 in splenic B cell directly regulate target genes through modulation of chromatin development and not earlier stages, Kdm1afl/flRosa26CreERT2/+ (IKO) accessibility were determined by filtering MZB CKO by PageRank and Kdm1afl/flRosa26+/+ (wild type) CD93+ TrB were cultured score (.0.5 or ,20.5) and then analyzing filtered transcription ex vivo as above at day 5 after tamoxifen treatment (Fig. 6D, 10 LSD1 REGULATES MARGINAL ZONE B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. LSD1 regulates ex vivo MZB development. (A) Flow cytometry analysis of B220 and CD93 expression on PE+ enrichments of CD93-PE– stained spleens from CreWT and CKO mice. (B) CD21 and CD1d expression on gated populations from part A. (C) CD21 and CD1d expression on B220+ CKO or CreWT cells after 3 d of being cultured with OP9-DL1 cells in the presence of BAFF. (D) CD21 and CD1d expression on B220+ IKO or wild-type (WT) cells after 3 d of being cultured with OP9-DL1 cells in the presence of BAFF. Cells were cultured 5 d after a 5 d tamoxifen treatment regimen. (E)Top two principal components from PCA of z-score normalized mRNA/cell expression of all 9843 detected genes in all samples. (F) Scatterplots of log2 fold change versus log2 fragments per kilobase per million (log2FPKM) data from differential expression analysis for the indicated sample group comparisons. (G) GSEA plot displaying the enrichment of the top 200 most significant genes upregulated in MZB CreWT relative to FoB CreWT within the eMZB CreWT versus TrB CreWT ranked gene list. (H) GSEA plots displaying the enrichment of the top 200 most significant genes upregulated in MZB CKO relative to MZB CreWT within two ranked gene lists: TrB CKO versus TrB CreWT and eMZB CKO versus eMZB CreWT. (I) Overlapping DEG between the indicated comparison. Significance was determined by Student two-tailed t test (A, C, and D) and Fisher exact test (I). Observed/expected (obs/exp) refers to the ratio of observed DEG overlap over expected overlap according to a permutation test. ***p ,0.001.

Supplemental Fig. 4B). The same defect was observed, indicating performed on LSD1-deficient and -sufficient eMZB and TrB. PCA that the reduction in eMZB CKO cells is likely due to a defect in indicated that TrB CreWT stratified from eMZB CreWT but not splenic B cell development. eMZB CKO (Fig. 6E), suggesting that changes induced by To determine whether LSD1 regulated NOTCH2 target and/or NOTCH2 and/or NF-kB signaling normally observed in eMZB NF-kB target genes during eMZB development, RNA-seq was CreWT are not occurring in eMZB CKO. Minimal stratification The Journal of Immunology 11 was observed between TrB CKO and TrB CreWT, indicating that hematopoietic stem cell development and survival (70), CDH17, a LSD1 does not have a strong role in regulating the TrB tran- cadherin that regulates early B cell development (71), and ID3, a scriptional program. Differential expression analysis was per- transcription factor that promotes MZB formation (41). Total formed on the indicated sample groups (Fig. 6F). The 3220 total expression of these 37 genes in both MZB CKO and eMZB CKO DEG observed between TrB CreWT and eMZB CreWT showed were significantly higher than their CreWT counterparts (Fig. 7C). that the two cell types were transcriptionally distinct, with eMZB Additionally, p52 motifs mapping to these genes exhibit a sig- CreWT upregulating MZB genes such as Myc, Dtx1, and S1pr1 nificant increase in chromatin accessibility in MZB CKO, sup- and TrB CreWT upregulating TrBs genes such as Myb and Sox4. porting that they are repressed by LSD1 (Fig. 7D). Using GSEA, MZB genes from this study and two others (8, 35) Noncanonical NF-kB signaling through the transcription fac- were shown to be significantly enriched in eMZB CreWT genes tors p52 and RelB is critical for splenic B cell development, as (Fig. 6G, Supplemental Fig. 4C), validating the ex vivo MZB indicated by B cell–conditional knockout of Nfkb2 and Relb (55). development assay as a viable method for testing MZB develop- To confirm the role of noncanonical NF-kB signaling during ex ment. CKO comparisons showed 46 total changes in gene ex- vivo MZB development, the NF-kB inhibitor IKK-16 (17) was pression between TrB CKO and TrB CreWT and 108 total changes applied to ex vivo MZB cultures of C57BL/6 wild-type cells in gene expression between eMZB CKO and eMZB CreWT, with (Supplemental Fig. 4I). Cultures treated with 800 nM of IKK-16 most changes being increases (Fig. 6F). eMZB CKO genes, but exhibited a significant decrease in eMZB compared with control not TrB CKO genes, were significantly enriched for MZB genes cells treated with DMSO, showing that noncanonical NF-kB (Fig. 6H). Additionally, 30 out of the 72 eMZB CKO upregulated signaling is critical for ex vivo MZB development and suggesting DEG overlapped the 297 MZB CKO upregulated DEG (Fig. 6I, that both inhibition of NF-kB signaling and LSD1 deficiency af- 11.1-fold more than expected by chance), including homing re- fect a similar pathway. To assess pathway overlap, LSD1-deficient Downloaded from ceptors Cxcr4 and Itgb7. These data further support a repressive cultures were treated with IKK-16 (Fig. 7E). CKO inhibitor cul- role for LSD1 during MZB development and suggest a similar role tures exhibited a significant decrease in eMZB compared with for LSD1 during both in vivo and ex vivo MZB development. both CKO DMSO cultures and CreWT inhibitor cultures, but this MZB CKO and eMZB CKO genes were tested for enrichment in decrease was not completely additive. These data imply a degree NOTCH2 and NF-kB target genes, which were acquired from the of overlap between pathways affected by both LSD1 deletion and Molecular Signatures Database (64) and, in the case of NF-kB NF-kB inhibition. http://www.jimmunol.org/ target genes, the PageRank analysis from this study and publica- To confirm that genes from Fig. 7B are regulated by both LSD1 tions involving genetic deletion of NF-kB signaling transcription and NF-kB signaling, RNA was collected from B cells from factors (54, 55, 65–67). NOTCH2 target genes were not significantly the four culture conditions displayed in Fig. 7E (Supplemental enriched in MZB CKO or eMZB CKO (Supplemental Fig. 4D). Of Fig. 4J) and quantitative RT-PCR was performed to assess the all 21 NF-kB target gene sets tested, seven gene sets were sig- expression of the nine genes that are DEG in both MZB and nificantly enriched in MZB CKO, whereas four gene sets were eMZB (Csrp1, Ehd2, Eno1b, Il2ra, Ly6c2, Padi2, Pqlc1, Ptprv, significantly enriched in eMZB CKO (Supplemental Fig. 4E). Of Spata13). The genes Ccr7, JunB, and Tap1, which are known these sets, two were enriched in both MZB CKO and eMZB CKO targets of canonical NF-kB signaling (72–74), were used as neg- by guest on October 2, 2021 and represent genes aberrantly upregulated when either Nfkb2 or ative controls. RT-qPCR revealed that the genes Csrp1, Il2ra, Relb are deleted in splenic naive B cells treated with BAFF. Im- Ly6c2, Padi2, Ptprv, and Spata13 were significantly upregulated in portantly, genes upstream of both NOTCH2 signaling and NF-kB CreWT inhibitor cultures compared with CreWT DMSO cultures, signaling, such as Notch2 and the BAFF receptor (Tnfrsf13c), indicating their repression by NF-kB signaling (Fig. 7F). The six were not dysregulated in MZB CKO, TrB CKO, or eMZB CKO significant genes from above plus the gene Ehd2 were significantly (Supplemental Fig. 4F). Thus, in the absence of LSD1, these data upregulated in CKO inhibitor cultures relative to CreWT DMSO suggest a defect in the expression of noncanonical NF-kB sig- cultures, indicating possible pathway overlap. The three conical naling target genes but not NOTCH2 signaling target genes. NF-kB signaling genes were not differentially expressed in any condition, supporting that the gene dysregulation observed is due k LSD1 and NF- B cooperate to regulate MZB development to a defect only in noncanonical NF-kB signaling. Flow cytometry Genes regulated by both LSD1 and NF-kB signaling were further was used to validate the surface expression of Ly6A, which was analyzed to understand how LSD1 and NF-kB intersect. Of the found to be significantly increased upon LSD1 deletion and NF-kB genes encoding transcription factors that form a functional NF-kB inhibition (Fig. 7G). complex, Nfkb2, Rela, Relb, and Rel are induced in eMZB com- Endogenous interaction of LSD1 with p52 was examined by pared with TrB, whereas Nfkb1 is not (Fig. 7A), implying that p50 coimmunoprecipitation experiments performed in the Raji hu- complexes play a lesser role in ex vivo MZB development than man B cell line (32), from which sufficient quantities of protein p52 complexes. This is further supported by the finding that of all could be obtained. To induce p52 nuclear translocation, Raji p50 and p52 target genes from PageRank analysis and GSEA gene cells were treated with anti-CD40 Ab, and nuclear and cyto- sets used above (Supplemental Fig. 4G), eMZB express 70% of all plasmic fractions were assessed (Fig. 7H). At 4 h posttreatment, p52 genes, whereas they only express 42% of all p50 genes nuclear p52 levels increased whereas its cytoplasmic precursor (Supplemental Fig. 4H). Genes regulated by p52 complexes in p100 levels decreased, indicating that anti-CD40 Ab treatment splenic naive B cells treated with BAFF (54, 55) or predicted to be successfully stimulated p52 nuclear translocation. Coimmuno- regulated by p52 by PageRank analysis were examined for gene precipitation of p52 and LSD1 was performed on Raji nuclear expression changes in MZB CKO and eMZB CKO. A total of lysate at 4 h after anti-CD40 Ab treatment (Fig. 7I). LSD1 37 genes that were DEG in both sample groups or were a DEG immunoprecipitated with p52 and p52 immunoprecipitated with in one group and trending up in the other group are displayed LSD1, indicating that the two proteins are found within the same (Fig. 7B). Genes include those that PTPRV, a protein ty- complex. Together, these data confirm a critical regulatory role rosine phosphatase that mediates -induced cell cycle exit (68), for noncanonical NF-kB signaling and demonstrate a coopera- CSRP1, a LIM-domain protein that suppresses cell proliferation tive relationship between this signaling pathway and LSD1 in and development (69), Ly6A, a surface protein that promotes MZB development. 12 LSD1 REGULATES MARGINAL ZONE B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 7. LSD1 and NF-kB cooperate to regulate MZB development. (A) FPKM expression of NF-kB transcription factors. (B) Heatmaps of z-score normalized mRNA/cell and FPKM expression of 37 genes that are DEG in both sample groups or are a DEG in one group and trending up in the other. DEG are denoted by an asterisk (*). (C) Box plots of mRNA/cell or FPKM expression of the 37 genes displayed in (B). (Figure legend continues) The Journal of Immunology 13

Discussion vivo, suggesting that LSD1 may regulate MZB developmental This study establishes a clear role for LSD1 in MZB development pathways aside from BAFF-induced noncanonical NF-kB signaling. and begins to define the epigenetic mechanisms that govern splenic PageRank data suggested that LSD1 regulates EBF1 target genes B cell development. LSD1 promoted MZB formation and func- associated with BCR signaling (Fig. 5E, Supplemental Fig. 2F). tioned to repress gene expression in both in vivo– and ex vivo– This process is critical for the bifurcation of TrB into MZB and FoB generated MZB. LSD1 also regulated chromatin accessibility in and may represent either a direct or indirect LSD1 target pathway MZB at motifs of transcription factors that are critical for splenic that influences splenic B cell development. B cell development, including NF-kB. Bioinformatic analyses of NF-kB transcription factors have been shown to influence the RNA-seq and ATAC-seq data pointed toward LSD1 having a epigenome (76, 77), but not in the context of B cell development. major regulatory effect on p52 target genes and p52 motifs that The data presented in this article strongly implies regulation of mapped to these genes. Experiments involving the application p52 target genes by LSD1 in MZB. In macrophages, LSD1 di- of an NF-kB inhibitor to ex vivo MZB development indicated rectly interacts with and demethylates p65 to stabilize it and pathway overlap between LSD1 and noncanonical NF-kB signal- amplify the inflammatory response to LPS (60). It is possible that ing. The presence of LSD1 and p52 in the same complex further LSD1 demethylates a component of the NF-kB heterodimers that demonstrated cooperation between a DNA binding protein and an form downstream of noncanonical NF-kB signaling, resulting in epigenetic modifier to drive the bifurcation of the development of the altered epigenome and transcriptome observed in MZB CKO. the MZB and FoB cell fates. However, given that LSD1 demethylates H3K4me1 in plasma- MZB and FoB display hundreds of DEG, many of which have blasts and germinal center B cells (10, 11), it is also possible that been shown to guide their development and give rise to their unique LSD1 is functioning with noncanonical NF-kB transcription fac- Downloaded from cellular attributes (1, 2). This study begins to reveal the epigenetic tors to demethylate surrounding H3K4 residues to repress target mechanisms behind these differences in gene expression and de- genes. This function is supported by the predominantly repressive fines LSD1 as a critical epigenetic regulator for a subset of MZB- role that LSD1 has in MZB in regulating transcription and chro- specific genes. LSD1 represses multiple genes that are typically matin accessibility. A similar effect has been observed with the upregulated in FoB, including those that encode the key FoB LSD1 homolog LSD2, as it can interact with c-Rel to demethylate transcription factor KLF2 and its target migratory surface recep- promoter-based chromatin (78). tors CXCR4 and ITGB7 (5, 38). Regardless of regulating these The finding that B cell conditional deletion of LSD1 impairs http://www.jimmunol.org/ genes, CKO MZB localize normally in the splenic marginal zone MZB development but not FoB development raises questions as to (Fig. 2). LSD1 also represses the genes that encode the Blimp-1 how LSD1 selectively affects one cell fate over another. LSD1 is repressors BACH2 and BCL6 (37), suggesting that CKO MZB only modestly upregulated in MZB compared with FoB (Fig. 3A), may respond poorly to Ag. Although MZB generate plasmablasts suggesting that strict abundance of mRNA and potentially protein in response to TLR agonists in a Blimp-1–dependent manner (75), levels are not likely the major mechanism. All five NF-kB family the response of LSD1-deficient MZB to the TLR4 agonist LPS has member genes are expressed similarly in MZB and FoB. Thus, no been observed to be significantly defective in part because of NF-kB transcription factor is present only in MZB to facilitate

LSD1 regulating Blimp-1 target genes and other key plasmablast LSD1-dependent gene regulation. Genes expressed exclusively by guest on October 2, 2021 genes (10). Based on this observation, it will be difficult to discern by MZB may be influencing NF-kB–based LSD1 activity. For the extent to which LSD1 target genes that are dysregulated during example, the noncanonical IKK kinase IKKε promotes gene reg- MZB development, such as Bach2 and Bcl6, actually contribute ulatory capabilities of a p52–p65 NF-kB complex (79) and is to MZB differentiation. Despite lack of evidence for an LSD1- significantly upregulated in MZB compared with FoB (8), sug- specific role in MZB function, it is clear that LSD1 promotes the gesting increased p52 activity in MZB. The high expression of BAFF-initiated p52-dependent development of TrB into MZB. both Nfkb2 and Rela in eMZB (Fig. 6A) in addition to previous p52 target genes significantly upregulated in both CKO MZB and work showing that both p52- and p65-based NF-kB complexes are eMZB represent the highest-confidence genes that contribute to capable of repressing genes through epigenetic mechanisms (80, the observed decrease in MZB development and include the reg- 81) supports the possibility of a p52–p65 complex functioning ulators of lymphocyte proliferation and development Ptprv, Csrp1, with LSD1 as a transcriptional repressor during MZB develop- Ly6a, Cdh17, and Id3 (41, 68–71). Of these genes, Id3 is partic- ment. Also, because cell signaling is known to drive cell fate bi- ularly critical for MZB formation, and although deletion of Id3 furcation in other hematopoietic developmental pathways (82, 83), results in fewer MZB (41), the effect of Id3 overexpression has it is possible that gene programs induced by NOTCH2 signaling not been studied in the context of splenic B cell development. may be influencing LSD1-based selectivity. Even though it was previously shown that LSD1-deficient naive The epigenetic reprogramming of lymphocytes during devel- B cells globally downregulate cell cycle genes and exhibit de- opment is crucial for proper immune system formation and function creased proliferation in response to LPS (10), no cell cycle genes (84). Developing B cells in the bone marrow exhibit distinct were downregulated in CKO MZB or eMZB, implying a different patterns of chromatin accessibility and histone modifications (85), mechanism for LSD1-dependent MZB development. Furthermore, and this study demonstrates that the epigenome remains dynamic dozens of additional genes associated with multiple pathways are throughout splenic B cell development as well. Therapeutic tar- dysregulated in LSD1-deficient MZB in vivo compared with ex geting of histone modifying enzymes is used to treat numerous

(D) Box plot of rppm enrichment of chromatin accessibility at p52 motifs that map to the 37 genes displayed in (B). (E) Flow cytometry analysis of CD21 and CD1d expression on B220+ CKO or CreWT cells after 3 d of being cultured with OP9-DL1 cells in the presence of BAFF. Cells were treated with either the NF-kB inhibitor IKK-16 (inhib.) or DMSO. (F) Quantitative RT-PCR expression data relative to 18S expression of select genes. Canonical NF-kB target genes are denoted by an asterisk (*). (G) Flow cytometry analysis of Ly6A for the four populations of cells gated in part E. (H) Western blot of Raji cell nuclear and cytoplasmic lysates collected following 0, 2, and 4 h of anti-CD40 Ab treatment. (I) Coimmunoprecipitations from Raji cell nuclear extracts at 4 h following anti-CD40 Ab treatment. All flow cytometry data are representative of two independent experiments using four to five mice per group. Error bars represent mean 6 SD. 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Supplemental Figure 1 – LSD1 regulates B-1 B cell populations and MZB differentiation (A) Western blot of LSD1 protein quantified from splenic naïve B cells purified from three CKO mice and three CreWT mice. (B) Flow cytometry analysis of the expression of B-1 cell markers on (B) IgM+ splenic cells, (C) IgM+ peritoneal cavity cells, and (D) IgM+ CD23– peritoneal cavity cells from C. (E) Flow cytometry analysis of CD138+ plasmablasts resulting from the LPS-induced differentiation of congenically labeled MZB that were adoptively transferred into MT host mice. All flow cytometry data are representative of at least two independent experiments using three to five mice per group. Error bars represent mean ± SD. Significance was determined by Student’s two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001.

Supplemental Figure 2 – Validation of marginal zone B cell and follicular B cell gene expression (A) GSEA plots displaying the enrichment of the top 200 most significant genes upregulated in MZB or FoB from two different studies (8, 35) within the MZB CreWT vs. FoB CreWT ranked gene list. (B, C) Flow cytometry analysis of ITGB7 (B) or CXCR4 (C) expression on MZB and FoB that are CKO or CreWT. (D) Flow cytometry analysis of germinal center B cell markers GL7 and Fas on B220+ splenic cells that are CKO or CreWT. (E) GSEA plots displaying the enrichment of myeloid progenitor genes (48) and macrophage genes (49) within the MZB CreWT vs. FoB CreWT ranked gene list. All flow cytometry data are representative of at least two independent experiments using three to five mice per group. Error bars represent mean ± SD. Significance was determined by Student’s two-tailed t-test. *P<0.05, ***P<0.001.

Supplemental Figure 3 – LSD1 represses gene expression and chromatin accessibility at p52 motifs (A) Heatmap displaying –log2(P values) of top significantly enriched transcription factor family motifs for the indicated DAR group from B. Boxplots depict rppm enrichment of chromatin accessibility for the indicated sample group at specific transcription factor motifs. Significance was determined by Student’s two-tailed t-test. (B) Heatmap of the –log2(PageRank) score of the top 20 transcription factors per sample group. (C) Heatmaps displaying the percentage of MZB CKO DEG in total predicted target genes of the transcription factors listed. (D) Boxplot rppm enrichment of chromatin accessibility at the indicated motifs mapping to DEG that are predicted target genes of the transcription factor that binds to the motif. (E) Top five KEGG pathways of PBX2 target genes, expression of Pbx2, and example predicted target genes. (F) Same as E but with EBF1. (G-I) Gene track examples of rppm chromatin accessibility data for MZB CKO DAR that increase in accessibility and map to (G) a p52 motif and p52-target MZB CKO upregulated DEG, (H) a p52 motif, and (I) a p52-target MZB CKO upregulated DEG.

Supplemental Figure 4 – Inhibition of NF-κB disrupts ex vivo marginal zone B cell development (A,B) Flow cytometry analysis on (A) B220+ CKO or CreWT cells after three days of being cultured in the indicated conditions, and (B) B220+ IKO or WT cells after three days of being cultured in the indicated conditions following five days after a five-day tamoxifen treatment regimen. (C) GSEA plots displaying the enrichment of the top 200 most significant genes upregulated in MZB from two different studies (Shi 2015(35) and Kleiman 2015(8)) within the eMZB CKO and eMZB CreWT ranked gene list. (D) GSEA plots displaying the enrichment of Notch2 pathway genes within two ranked gene lists: MZB CKO vs. MZB CreWT and eMZB CKO vs. eMZB CreWT. (E) GSEA results for the 21 indicated NF-κB gene sets within the two ranked gene lists from A. Red = significantly enriched. (F) Heatmaps displaying the expression of components of canonical NF-kB, non- canonical NF-kB, and NOTCH2 signaling pathways. (G) Venn diagram displaying genes that are regulated by p50 and p52. (H) p50 and p52 regulated genes that are expressed in eMZB CreWT samples. (I,J) Flow cytometry analysis on either NF-κB inhibitor IKK-16 (inhib.) or DMSO treated cultures of (I) C57BL/6 wild-type cells after three days of being cultured with OP9-DL1 cells in the presence of BAFF and (J) APC+ enrichments of B220- APC-stained CKO and CreWT cultures. All flow cytometry data are representative of at least two independent experiments using three to five mice per group. Error bars represent mean ± SD. Significance was determined by Student’s two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001.