The Histone Demethylase LSD1 Regulates B Cell Proliferation and Plasmablast Differentiation

This information is current as Robert R. Haines, Benjamin G. Barwick, Christopher D. of September 23, 2021. Scharer, Parimal Majumder, Troy D. Randall and Jeremy M. Boss J Immunol 2018; 201:2799-2811; Prepublished online 19 September 2018;

doi: 10.4049/jimmunol.1800952 Downloaded from http://www.jimmunol.org/content/201/9/2799

Supplementary http://www.jimmunol.org/content/suppl/2018/09/18/jimmunol.180095 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 86 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/201/9/2799.full#ref-list-1

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The Histone Demethylase LSD1 Regulates B Cell Proliferation and Plasmablast Differentiation

Robert R. Haines, Benjamin G. Barwick,1 Christopher D. Scharer, Parimal Majumder, Troy D. Randall,2 and Jeremy M. Boss

B cells undergo epigenetic remodeling as they differentiate into Ab-secreting cells (ASC). LSD1 is a histone demethylase known to decommission active enhancers and cooperate with the ASC master regulatory Blimp-1. The contribution of LSD1 to ASC formation is poorly understood. In this study, we show that LSD1 is necessary for proliferation and differentiation of mouse naive B cells (nB) into plasmablasts (PB). Following LPS inoculation, LSD1-deficient hosts exhibited a 2-fold reduction of splenic PB and serum IgM. LSD1-deficient PB exhibited derepression and superinduction of involved in immune system processes; a subset of these being direct Blimp-1 target–repressed genes. Cell cycle genes were globally downregulated without

LSD1, which corresponded to a decrease in the proliferative capacity of LSD1-deficient activated B cells. PB lacking LSD1 Downloaded from displayed increased histone H3 lysine 4 monomethylation and chromatin accessibility at nB active enhancers and the binding sites of transcription factors Blimp-1, PU.1, and IRF4 that mapped to LSD1-repressed genes. Together, these data show that LSD1 is required for normal in vivo PB formation, distinguish LSD1 as a transcriptional rheostat and epigenetic modifier of B cell differentiation, and identify LSD1 as a factor responsible for decommissioning nB active enhancers. The Journal of Immunology, 2018, 201: 2799–2811. http://www.jimmunol.org/ umoral immunity against pathogens is achieved through short-lived PB that secrete mostly IgM (3). The second phase the function of Ab-secreting cells (ASC). In response to requires the formation of germinal centers that produce PC and H Ag, the ASC compartment is generated from the dif- memory B cells. nB interactions with T cell–independent Ags, ferentiation of naive B cells (nB) and is populated by short-lived, such as bacterial LPS, primarily results in the rapid generation of mitotically active plasmablasts (PB) and long-lived, noncycling PB through an extrafollicular response (4). plasma cells (PC) (1). Depending on the Ag, nB can give rise to As nB differentiate to ASC, they undergo widespread changes in a variety of responses, each evolved to efficiently neutralize the expression mediated by transcription factors, such as Blimp-1, target pathogen in an Ag-specific manner (2). nB interactions with XBP-1, IRF4, and PU.1 (1, 5). To support the demand of constant T cell–dependent Ags results in a two-phase response. The first phase, substantial Ab production, ASC upregulate the expression of by guest on September 23, 2021 known as the extrafollicular response, results in the generation of genes that function in metabolic processes (6) as well as production, modification, and trafficking (7). Transcriptional changes in ASC are accompanied by changes in the epigenome. Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322 For example, in response to LPS, global and specific increases in 1 Current address: Department of Hematology and Medical Oncology, Emory Uni- occur in the newly formed PB that is accompa- versity School of Medicine, Atlanta, GA. nied by alterations in chromatin accessibility at enhancers (8) and 2Current address: Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL. a reciprocal decrease in DNA methylation (9). PB also exhibit ORCIDs: 0000-0001-9574-4341 (R.R.H.); 0000-0001-9810-3566 (B.G.B.); 0000- alterations in H3K4me2, H3K4me3, H3K9ac, and H3K27me3 0001-7716-8504 (C.D.S.); 0000-0002-2432-1840 (J.M.B.). and chromatin accessibility at Blimp-1 binding sites (10). Blimp-1 Received for publication July 9, 2018. Accepted for publication August 22, 2018. recruits the chromatin-remodeling and histone-modifying com- This work was supported by the following National Institutes of Health grants: R01 plexes BAF, NuRD, and PRC2 to regulate its target genes in PB AI123733 and P01 AI125180 to J.M.B., T32 GM0008490 to R.R.H. and B.G.B., F31 (10). Within the PRC2 complex, the histone methyltransferase AI131532 to R.R.H., and F31 AI112261 to B.G.B. EZH2 is critical for PB formation through H3K27me3-linked re- The sequencing data presented in this article have been submitted to the National Center pression of transcription factor networks (11). However, the de- for Biotechnology Information’s Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE114777) under accession number GSE114777. gree to which other epigenetic-modifying enzymes regulate ASC Address correspondence and reprint requests to Dr. Jeremy M. Boss, Emory differentiation and how they influence promoter and enhancer University, 1510 Clifton Road, Room 3001, Atlanta, GA 30322. E-mail address: chromatin throughout this process remains poorly understood. [email protected] Lysine-specific demethylase 1 (LSD1) is a monoamine oxidase The online version of this article contains supplemental material. that demethylates H3K4me1, H3K4me2, H3K9me1, and H3K9me2 Abbreviations used in this article: ASC, Ab-secreting cell; ATAC-seq, assay for through a flavin adenine dinucleotide–dependent amine oxidation transposase-accessible chromatin sequencing; ChIP-seq, chromatin immunoprecipi- mechanism (12, 13). The protein structure of LSD1 consists of an tation sequencing; CKO, conditional knockout; CreWT, Lsd1+/+Cd19Cre/+ wild-type; CTV, CellTrace Violet; DAR, differentially accessible region; DEG, differentially enzymatically active amine oxidase–likedomainaswellasSWIRM expressed gene; ERCC, External RNA Controls Consortium; FC, fold change; and Tower domains that facilitate protein/protein interactions FDR, false discovery rate; FSC, forward scatter; GEO, Gene Expression Omnibus; GO, ; GSEA, gene set enrichment analysis; IKO, inducible knockout; (14). By interacting with lineage-specific chromatin–modifying LSD1, lysine-specific demethylase 1; nB, naive B cell; PB, plasmablast; PC, plasma complexes, LSD1 regulates multiple cellular differentiation pathways cell; qPCR, quantitative PCR; qRT-PCR, quantitative RT-PCR; RNA-seq, RNA se- including embryonic stem cell differentiation (15), neurogenesis quencing; SSC, side scatter; WT, wild-type. (16), and hematopoiesis (17). Known complexes in which LSD1 Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 functions as a coactivator or corepressor include those containing www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800952 2800 LSD1 REGULATES B CELL DIFFERENTIATION

CoREST (18), HDAC1/2 (18), the androgen (12), and the 20 ng/ml IL-2 (14-8021; eBioscience), and 5 ng/ml IL-5 (14-8051; (19). Importantly, LSD1 is the only histone eBioscience) as previously described (24). demethylase proven to decommission enhancers during cellular Flow cytometry differentiation by demethylating the active enhancer modification 6 H3K4me1 (15). In the context of PC differentiation, LSD1 has Cells were resuspended at a concentration of 10 cells/100 mlFACS buffer (13 PBS, 2 mM EDTA, and 1% BSA). Cells were stained with been shown to interact with Blimp-1 (20). The extent to which Fc block (BD Biosciences) for 15 min, stained with Ab fluorophores LSD1 regulates transcriptional and epigenetic changes that occur for1honice,thenwashedwith10volofFACSbuffer.Thefollowing during B cell differentiation has not been determined. Abs were used: B220-PE-Cy7 (RA3-6B2; Tonbo Biosciences), CD11b- In this study, LSD1 expression was found to specifically increase allophycocyanin-Cy7 (M1/70; Tonbo Biosciences), CD45.1-FITC (A20; Tonbo Biosciences), CD45.2-PerCP-Cy5.5 (104; Tonbo Biosciences), as PB form during B cell differentiation, indicating a potential role for CD90.2-allophycocyanin-Cy7 (30-H12; BioLegend), F4/80-allophycocyanin- this protein during the process. Conditional genetic deletion of Lsd1 Cy7 (BM8; BioLegend), CD138-BV711 (281-2; BD Biosciences), in mice was used to examine its function in PB formation. LSD1 CD21-allophycocyanin (B-ly4; BD Biosciences), CD43-PE (S7; BD Bio- was necessary for normal LPS-induced differentiation of nB into sciences), Fas-BV421 (Jo2; BD Biosciences), GL7-eFluor660 (GL-7; CD138+ PB. LSD1-repressed genes were involved in immune eBioscience), CD23-eFluor450 (B3B4; eBioscience), IgM-FITC (II/41; eBioscience), Annexin V–allophycocyanin (kit no. 88-8007-72; eBio- system processes, including Blimp-1 target genes. Cellular pro- science), and Zombie Yellow Fixable Viability Dye (kit no. 423104; liferation was also impaired in LSD1-deficient B cells, and this BioLegend). An LSR II was used for analysis and a FACSAria II was was coupled to reduced expression of cell cycle genes. Mechanis- used for sorting (BD Biosciences). All flow cytometry data were ana- tically, LSD1 reduced local chromatin accessibility at nB-active lyzed with FlowJo version 9.9.5. Preceding all flow cytometry analyses presented is the following gating strategy: 1) lymphocytes (forward scatter Downloaded from enhancers and PU.1, IRF4, and Blimp-1 target binding sites. Without [FSC] area by side scatter [SSC] area), 2) singlets (FSC width by FSC height), LSD1, H3K4me1 accumulated at enhancers of LSD1-regulated 3) singlets (SSC width by SSC height), 4) live cells (viability dye2), and genes, supporting a role for this epigenetic factor in modulating 5) exclusion of non–B cell lineage cells (Thy1.12F4/802CD11b2). gene expression. Cumulatively, this study shows that LSD1 is ELISA required for normal B cell proliferation and differentiation and functions as a transcriptional rheostat and epigenetic modifier Flat-bottom ELISA plates (M9410-1CS; Sigma-Aldrich) were coated with goat anti-mouse Ig (5300-05B; Southern Biotechnology). Plates were incubated throughout this process. http://www.jimmunol.org/ overnight at 4˚C, washed (13 PBS with 0.05% Tween 20), blocked with 1% nonfat dry milk for 2 h at room temperature, washed, incubated with diluted Materials and Methods serum samples or mouse IgM (5300-01B; Southern Biotechnology) for 2 h at Data availability room temperature, washed, incubated with 1:1000 diluted HRP-conjugated goat anti-mouse IgM (1021-05; Southern Biotechnology) for 2 h at room temperature, Sequencing data are available through accession number GSE114777 (https:// and washed. Plates were incubated with tetramethylbenzidine ELISA peroxidase www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE114777) at the National substrate (TMBE-1000; Rockland) for 20 min at room temperature (50 ml/well) Center for Biotechnology Information Gene Expression Omnibus (GEO). to develop a color reaction. Fifty microliters of 0.2 M sulfuric acid was applied to each well to stop the HRP color reaction. After 20 min, OD at 450 nm (OD450) Animal protocols was collected by a Synergy HT Multi-Mode Microplate Reader (BioTek). by guest on September 23, 2021 Lsd1fl/flCd19Cre/+ mice were generated from breeding Cd19Cre/+ mice (21) RNA sequencing and data analysis (006785; The Jackson Laboratory) with Lsd1fl/fl mice received as a gift from Dr. D. Katz [previously described (22)]. Lsd1fl/flRosa26CreERT2/+ mice For each sample, 50,000 cells were isolated via FACS into 600 mlofRLT were generated from breeding Rosa26CreERT2/+ mice (23) (008463; The buffer (Qiagen) containing 1% 2-ME. Samples were vortexed for 1 min Jackson Laboratory) with Lsd1fl/fl mice. Experiments were performed on on high to lyse cells, and External RNA Controls Consortium (ERCC) mice 8–12 wk old. All experiments were approved by the Emory Uni- spike-in transcripts (26) were added (5 ml of 1:2000 dilution per sample) versity Institutional Animal Care and Use Committee. Sequences used for to enable downstream mRNA/cell calculations. Total RNA was isolated PCR genotyping of mice are listed in Supplemental Table III. For LPS using a Zymo Research Quick-RNA MicroPrep Kit (11-328M). One experiments, mice were i.v. injected into the tail vein with 50 mg LPS from microgram of RNA per sample was used to create libraries with a KAPA Salmonella minnesota R595 (Enzo Life Sciences) diluted 1:1 in PBS as mRNA HyperPrep Kit (KK8581). An Agilent Bioanalyzer was used to quality previously described (24). Spleens were isolated on day 3 for the analyses check each library. Libraries were pooled at an equimolar ratio and sequenced presented. For influenza experiments, mice were infected intranasally with on a HiSeq 2500 System (Illumina) using 50 bp paired-end chemistry. 15,000 viral focal units of A/PR8/34 (PR8) influenza virus (25). Medias- Raw sequencing reads were mapped to the mm9 genome using TopHat2 tinal lymph nodes were isolated on day 7 for the analysis presented. (27). Gene counts were determined with the Bioconductor package In some experiments, B cells were adoptively transferred to naive mice. GenomicAlignments (28) using the mm9 transcriptome database. A gene CD43 (Ly-48) MicroBeads (130-097-148; Miltenyi Biotec) were used to was considered detected if all three samples from a sample group had at magnetically purify CD432 splenic B cells from naive mice according to the least 0.2 mRNA per cell, which was the minimum ERCC transcript/cell manufacturer’s instructions. Purified cells were either transferred into mMT concentration to detect at least 90% of that particular transcript across all host mice (i.v. injected into the tail) or stained with 5 mM of CellTrace Violet samples. The Bioconductor package edgeR (29) was used to determine (CTV; C34557; Invitrogen) for 20 min at room temperature protected from differentially expressed genes (DEG) based on both relative (false discovery light. CTV-stained cells were washed with 5 vol of B cell media (RPMI rate [FDR] # 0.05, no fold change [FC] cutoff) and absolute changes (FDR # 1640, 10% heat-inactivated FBS, 0.05 mM 2-ME, 13 nonessential amino 0.05, no FC cutoff) in expression as previously described (9). All DEG de- acids, 13 penicillin/streptomycin, 10 mM HEPES, and 1 mM sodium py- termined are listed in Supplemental Table I. Gene ontology (GO) analysis ruvate) and were adoptively transferred into mMT host mice. was performed with the Database for Annotation, Visualization, and Integrated Tamoxifen was used to activate Cre-mediated recombination in Discovery functional annotation software (30). Preranked gene set enrichment Rosa26CreERT2/+ mice. Tamoxifen powder (T5648; Sigma-Aldrich) was analysis (GSEA) (31) was performed on all detected transcripts ranked dissolvedin100%EtOHataconcentrationof40mg/300ml. The ta- based on the following equation: moxifen was solubilized with 10 min of sonication, then 1 ml of corn oil ð2 ð p ÞÞ prewarmed to 37˚C was added per 300 ml of EtOH. The solution was sign of fold change log10 edgeR value sonicated for 15 additional minutes and then ethanol was evaporated to yield a 40 mg/ml tamoxifen/corn oil solution. Mice received daily 100-ml Observed/expected ratios were calculated through permutation testing against injections of 40 mg/ml tamoxifen i.p. for 5 d. Following five additional gene sets randomly chosen from all detected genes (1000 permutations per test). days of rest, mice underwent experimental analyses. Assay for transposase-accessible chromatin sequencing and Ex vivo B cell differentiation data analysis CD432 splenic B cells purified as above were cultured at a concentration of Assay for transposase-accessible chromatin sequencing (ATAC-seq) (32) was 0.5 3 106 cells/ml in B cell media with 20 mg/ml LPS (L2630; Sigma-Aldrich), performed using 10,000 FACS-isolated cells that were resuspended in nuclei The Journal of Immunology 2801

lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2,and0.1% cross-linked in 1% formaldehyde for 10 min, and chromatin lysate was IGEPAL CA-630) and centrifuged at 500 3 g for 30 min. Samples were prepared and sonicated to generate fragments averaging 500 bp in length. transposed with 25 ml of tagmentation reaction mixture (23 Tagmentation For immunoprecipitations, anti-H3K4me1 (07-436; MilliporeSigma) and Buffer and 1 ml Tagmentation Enzyme from the Illumina Nextera DNA anti-IgG (12-370; MilliporeSigma) Ab was used. Protein G beads (10004D; Library Prep Kit) at 37˚C for 1 h, diluted 2-fold in tagmentation clean-up Invitrogen) were used to isolate the chromatin/Ab complexes. After washing, buffer (300 mM NaCl, 100 mM EDTA, 0.6% SDS, and 1.6 mg proteinase K), the immunoprecipitated chromatin was eluted in 1% SDS and incubated and incubated for 30 min at 40˚C. Size selection with solid phase reversible overnight at 65˚C to reverse the formaldehyde-induced cross-links. The immobilization beads was used to isolate low m.w.–transposed DNA that DNA was purified and used as a template in real-time PCR. DNA was was amplified with 23 KAPA HiFi HotStart ReadyMix and Nextera quantitated by qPCR using a five-point genomic DNA standard curve and indexing primers (Illumina). To enrich for low m.w. DNA, a second size an iCycler (Bio-Rad Laboratories). qPCR mixtures contained 5% DMSO, selection with solid phase reversible immobilization beads was performed one SYBR Green (BioWhittaker Molecular Applications), 0.04% gelatin, post-PCR. Libraries were quality checked and sequenced as above. 0.3% Tween 20, 50 mM KCl, 20 mM Tris (pH 8.3), 3 mM MgCl2, 0.2 mM Raw sequencing reads were mapped to the mm9 genome using Bowtie deoxynucleoside triphosphate, and 100 nM of each primer. Sequences for (33). Peaks were called using MACS2 (34) and annotated using HOMER all primers are listed in Supplemental Table III. All experiments were (35). Data were normalized to reads per peak per million as previously performed for a total of six times from independent preparations of described (36). The Bioconductor package edgeR (29) was used to de- chromatin. The data were averaged and plotted with respect to the input termine differentially accessible regions (DAR). A Benjamini–Hochberg chromatin. A Student two-tailed t test was used to determine statistical FDR #0.05 and a log2 FC $1 were required for significance. All DAR significance. determined are listed in Supplemental Table II. Observed/expected ratios were calculated through permutation testing against peaks randomly chosen from all peaks detected with ATAC-seq (1000 permutations per Results test). Tissue-specific enhancer data were previously determined (9). For H3K4 methylation is remodeled during B cell differentiation odds ratio calculations, the reference group was all ATAC-seq peaks de- Downloaded from tected (71,925), and significance was determined using Fisher exact test. Motif H3K4me2 is broadly associated with enhancers, promoters, and analysis was performed with the HOMER program findMotifsGenome.pl gene bodies and is correlated with transcription in multiple cell using randomly generated genomic sequences as background. types and organisms, including mammalian immune system cells Chromatin immunoprecipitation sequencing data analysis (15). The dynamics of H3K4 methylation in B cell differentiation was examined by comparing the changes in H3K4me2 enrichment Public datasets were downloaded from GEO. Peaks were called using between nB (40) and PB derived from an ex vivo LPS differen-

MACS2 and annotated using HOMER. For H3K4me2 scatter plots, the http://www.jimmunol.org/ reads per million data from nB and PB were quantile normalized. tiation model (10) (Fig. 1A). The comparison revealed that in PB, 6209 genomic regions gained (red, Ι), whereas 6592 regions lost Quantitative RT-PCR H3K4me2 (blue, ΙΙ), indicating that H3K4me2 is remodeled Per sample, 50,000 cells were sorted via FACS into 600 ml of RLT–2-ME throughout B cell differentiation. To examine the relationship be- and processed as with RNA sequencing (RNA-seq). Total RNA was tween the changes in H3K4me2 and gene expression, regions that reverse transcribed with SuperScript II Reverse Transcriptase (18064014; gained and lost H3K4me2 were mapped within 20 kb of all DEG Invitrogen). cDNA was diluted 5-fold, and quantitative PCR (qPCR) was between nB versus LPS-induced PB defined previously (9). Com- performed with a CFX96 instrument (Bio-Rad Laboratories) using SYBR Green incorporation. Gene expression was calculated relative to ERCC tran- parison of the log2 FC of the DEG mapping to regions that gained or lost H3K4me2 modifications in PB indicated that changes script no. 130. Sequences for all primers are listed in Supplemental Table III. by guest on September 23, 2021 A Student two-tailed t test was used to determine statistical significance. in gene expression were positively associated with changes in Chromatin immunoprecipitation assays H3K4me2 (Fig. 1B) and suggests that this mark is remodeled and correlated with transcription during PB formation. For chromatin immunoprecipitation sequencing (ChIP-seq) analyses, public To understand how H3K4me2 was remodeled, the expression of datasets were downloaded from GEO. Peaks were called using MACS2 and annotated using HOMER. For H3K4me2 scatter plots, the reads per million known H3K4 demethylases was analyzed from data derived from data from nB and PB were quantile normalized. discrete B cell divisions during differentiation to PB in vivo in For ChIP-qPCR assays, PB and nB for ChIP were collected from response to LPS (9). Kdm1a and Kdm5c, encoding the H3K4 LPS-inoculated and naive mice, respectively. PB were collected by staining histone demethylases LSD1 and JARID1C, respectively, were splenocytes with CD138-allophycocyanin (281-2; BioLegend) and then were purified with Anti-Allophycocyanin MicroBeads (130-090-855; Miltenyi Biotec). progressively upregulated throughout the differentiation pro- CD432 splenic B cells were purified as above and represent nB. ChIP-qPCR cess (Fig. 1C). Other members of the family were not induced assays were performed as described previously (37–39). Briefly, cells were or expressed at appreciable levels in dividing B cells or PB.

FIGURE 1. H3K4me2 is remodeled throughout B cell differentiation. (A) Scatter plot of genomic regions significantly enriched for H3K4me2 in nB and

LPS-induced PB (37,887 total peaks). Regions that gain or lose H3K4me2 in PB by a log2 FC of at least one are red and blue, respectively. (B) Box plots of the log2 FC of the expression of genes differentially expressed from nB versus LPS-induced PB that map within 20 kb of at least one H3K4me2 peak within PB upregulated regions (red box, Ι) and PB downregulated regions (blue box, ΙΙ). Significance was determined by Wilcoxon rank sum test. (C) Expression in mRNA/cell per division of known H3K4me2 demethylases (9). Error bars represent mean 6 SD. 2802 LSD1 REGULATES B CELL DIFFERENTIATION

LSD1 was chosen for further investigation because of its known ELISA from the same cultures showed a significant reduction in interaction with key ASC regulatory transcription factor Blimp-1 secreted IgM in CKO as compared with CreWT (Fig. 2B). The re- (20) and for its ability to decommission enhancers by catalyzing duced number of PB was not due to cell death, as CKO and CreWT H3K4me2/me1 to an unmethylated H3K4 ground state (15). B cell cultures showed no difference in the frequency of apoptotic or necrotic cells (Supplemental Fig. 2A). The consequence of in vivo LSD1 is required for normal PB formation Lsd1 deletion on B cell differentiation was assessed by inoculating To examine the role of LSD1 during B cell differentiation, a floxed CKO and CreWT mice with LPS and analyzing spleens at the peak Lsd1 mutant allele (22) was bred onto the Cd19Cre/+ background B cell response time point of 3 d (41). Compared with CreWT mice, (21) to generate a B cell–specific conditional knockout (CKO). CKO mice exhibited a significant reductioninthefrequencyandtotal Efficient Lsd1 deletion in FACS-isolated CKO splenic nB and number of CD138+ PB as well as a significant reduction in serum CD138+ PB was observed (Supplemental Fig. 1A), and Lsd1 IgM titers (Fig. 2C, 2D). Consistent with the ex vivo data, this defect mRNA levels were decreased significantly in CKO nB and PB was not due to an increase in cell death as CKO and CreWT populations (Supplemental Fig. 1B). spleens showed no difference in apoptotic or necrotic nB and Splenic B cells from naive CKO mice and Cd19Cre/+ control mice PB (Supplemental Fig. 2B). The defect was not due to a de- (Lsd1+/+Cd19Cre/+ wild-type [CreWT]) were purified and cultured crease in B cell activation as there was no difference in splenic ex vivo in the presence of LPS, IL-2, and IL-5 (24) to induce dif- GL7+-activated B cells (Supplemental Fig. 2C). To assess whether ferentiation. Flow cytometry performed on day 3 showed that CKO LSD1 only functions in LPS-stimulated B cell differentiation, B cell cultures exhibited a significant reduction in the frequency and CKO and CreWT mice were inoculated with the A/PR8/34 (PR8) total number of CD138+ PB (Fig. 2A). Secreted Ab measured by strain of influenza. At 7 d postinfection, mice were sacrificed and Downloaded from http://www.jimmunol.org/

FIGURE 2. LSD1 is required for PB formation. (A)Flowcytometry analysis of B220 and CD138 expres- sion in ex vivo–differentiated CreWT and CKO splenic B cells cultures (left) and quantification of CD138+ PB (right). (B) IgM media titer of B cell cultures from (A). (C) B220 and CD138 expression in CreWT and by guest on September 23, 2021 CKO splenocytes on day 3 after LPS inoculation (left) and quantification of CD138+ PB (right). (D) IgM se- rum titer of mice from (C) directly before (D0) and on day 3 after LPS inoculation (D3). (E) B220 and CD138 expression in adoptively transferred CreWT and CKO splenocytes on day 3 after LPS inoculation (left) and quantification of CD138+ PB (right). (F) B220 and CD138 expression in ex vivo–differentiated WT and IKO splenic B cells cultures (left) and quantification of CD138+ PB (right). (G) IgM media titer of B cell cul- tures from (F). (H) B220 and CD138 expression in adoptively transferred WT and IKO splenocytes on day 3 after LPS inoculation (left) and quan- tification of CD138+ PB (right). (I) IgM serum titer of mice from (H)on day 3 after LPS inoculation. 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 , 0.01, ***p , 0.001. The Journal of Immunology 2803 the mediastinal lymph nodes were harvested and analyzed. Flow Because the IKO system deletes Lsd1 from all cells within the mouse, cytometry showed a significant reduction in PB formed by both the effect of Lsd1 deletion on B cells was tested by purifying splenic frequency and total number in CKO mice (Supplemental Fig. 2D). B cells from tamoxifen-treated IKO and WT mice and transferring ELISA showed a significant reduction in IgM titers in the serum them separately into mMT host mice. Following LPS inoculation and (Supplemental Fig. 2E). These data indicate that LSD1 is required analysis as above, host mice that received IKO B cells again exhibited for normal B cell differentiation. a significant reduction in the frequency and total number of PB, The intrinsic nature of the defect was examined by purifying and respectively (Fig. 2H). ELISA was performed on host serum and adoptively transferring congenically marked CKO (CD45.1) and revealed that IKO cell recipient hosts had a significant reduction in CreWT (CD45.1/2) splenic B cells in a 1:1 ratio into mMT (CD45.2) serum IgM titers (Fig. 2I). Thus, ablation of LSD1 using multiple host mice, which lack B cells (42). Hosts were inoculated with LPS mechanisms of genetic deletion demonstrated that B cell differen- 1 d after transfer, and spleens were harvested and analyzed at day 3. tiation and humoral immune responses are impaired in the absence Compared with the CreWT B cell compartment, the CKO B cell of LSD1, and this defect is cell intrinsic. compartment exhibited a significant reduction in the frequency and total number of CD138+ PB, respectively (Fig. 2E). Together, these LSD1 regulates the PB transcriptional program data show that the requirement of LSD1 for normal B cell differ- To elucidate the molecular program altered by LSD1 deficiency entiation is intrinsic to the adoptively transferred B cells. during PB formation, RNA-seq was performed on FACS-isolated The above results were furthersupportedbybreedingLsd1 B220+GL72CD1382 nB and CD138+ PB from both CKO and floxed alleles to the tamoxifen-inducible Rosa26CreERT2/+ allele Lsd1fl/fl (WT) splenocytes 3 d after LPS inoculation. Hierarchical

(23) (inducible knockout [IKO]). Efficient Lsd1 deletioninIKO clustering and principal component analysis of all expressed genes Downloaded from splenic nB was achieved compared with Lsd1fl/fl WT cells after showed that samples were stratified by both cell type and Lsd1 tamoxifen treatment (Supplemental Fig. 1C). Purified splenic deletion status, indicating an LSD1-dependent effect in both nB B cells from naive IKO mice and WT mice following tamoxifen and PB (Fig. 3A, 3B). Calculations of total mRNA per cell showed treatment were cultured ex vivo as above. Similar to the CKO B cells, the expected increase in mRNA levels from nB to PB (9), but no IKO B cells exhibited a significant reduction in PB formation and difference between CKO and WT samples, indicating that Lsd1

secreted IgM as compared with their respective controls (Fig. 2F, 2G). deletion did not affect global cellular mRNA levels (Fig. 3C). In http://www.jimmunol.org/

FIGURE 3. LSD1 regulates the PB transcriptional program. (A) Heatmap by guest on September 23, 2021 of hierarchical-clustered, z-score– normalized expression data (mRNA/ cell) of all 11,909 detected genes be- tween the indicated groups. (B)Top two principal components (PC1, PC2) from principal component analysis (PCA) of z-score–normalized mRNA/ cell expression of genes from (A). Circles represent 99% confidence in- tervals. (C) Average mRNA per cell per sample group. (D) Volcano plots of DEG between the indicated com- parison. Plotted are 2log10(FDR) and log FC based off of differential ex- pression analysis of absolute changes in gene expression (9). LSD1 is indi- cated in each plot (red dot). (E)GSEA analysis of PB WT and PB CKO using a gene set of the top 200 most sig- nificant 1R DEG. (F) DEG exhibiting derepression in PB. (G)GSEAanaly- sis of PB WT and PB CKO using a gene set of the top 200 most signifi- cant 2R DEG. (H) DEG exhibiting superinduction. Error bars represent mean 6 SD. Significance was deter- minedbyedgeR.*FDR, 0.05. 2804 LSD1 REGULATES B CELL DIFFERENTIATION the WT setting, 1428 genes were downregulated and 6050 genes Blimp-1 target–repressed genes are regulated by LSD1 were upregulated in PB as compared with nB (Fig. 3D; referred to One possible explanation for the transcriptional dysregulation hereafter as DEG groups 1R and 2R, respectively). Comparison observed in CKO PB is LSD1-dependent dysregulation of essential between WT and CKO nB found 38 downregulated and 382 up- transcription factors. However, this did not appear to be the case, regulated genes in CKO nB (DEG groups 3R and 4R, respectively). as genes encoding nB transcription factors (BACH2, BCL6, ETS1, Likewise, comparison between CKO and WT PB identified 41 IRF8, PAX5, and SPIB) and PB transcription factors (Blimp-1, downregulated and 471 upregulated genes in CKO PB (DEG groups IRF4, and XBP-1) were appropriately expressed and regulated 5R and 6R, respectively). These data show that LSD1 predomi- (Supplemental Fig. 3G). However, Blimp-1 has been shown to nantly functions as a transcriptional repressor in this system and recruit histone-modifying complexes to facilitate gene repression identify genes that are dysregulated in its absence. (10) and can physically interact with LSD1 (20). To determine The global functions of LSD1-dysregulated genes were inves- if direct Blimp-1 target genes were dysregulated when LSD1 was tigated by performing GO analysis on DEG groups (Supplemental deleted, Blimp-1–activated and –repressed gene sets (10) were Fig. 3A). The top enriched GO term for 6R DEG was immune tested for enrichment in ranked gene lists derived from the com- system process. This was further supported by GSEA of the WT parisons nB WT versus PC WT, nB WT versus nB CKO, and PB and CKO PB RNA-seq data comparisons to all HALLMARK and WT versus PB CKO (Fig. 4A). As expected in the WT setting, the KEGG gene sets (Supplemental Fig. 3B), which revealed that GSEA of WT nB and PB identified genes up- and downregulated genes involved in processes such as the inflammatory response, cell by Blimp-1 (Fig. 4A, top). No enrichment involving Blimp-1 signaling, complement cascade, coagulation, cellular adhesion, and target genes was observed when comparing WT and CKO nB cytokine/cytokine receptor interactions were upregulated in the (Fig. 4A, middle). In contrast, when comparing WT and CKO Downloaded from absence of LSD1. For example, LSD1-deficient PB upregulated PB, Blimp-1–repressed target genes failed to be fully down- the proinflammatory genes C1qa, C1qb, C1qc, C3, C4bp, Cd14, regulated (Fig. 4A, bottom), suggesting that LSD1 deficiency and Tlr7 as well as the IFN-g response genes Ifit2, Ifitm3, Il10ra, leads to derepression of Blimp-1 target genes. Examples included Usp18, Vamp5,andVcam1. These data, therefore, show that the genes Mpeg1 and Sell as described above (Fig. 3F), a gene LSD1 normally represses proinflammatory signals during B cell encoding a glycoprotein found in neutrophil azurophilic granules differentiation. with putative amidase activity [Plbd1 (59)], and those involved in http://www.jimmunol.org/ Interestingly, the top enriched GO term (immune system pro- response to bacteria [Tlr1 (60)] and signaling [Evl (61), Hck (62), cess) for genes upregulated in CKO PB compared with WT controls Hvcn1 (63), Il10ra (64); Fig. 4B]. Gene expression levels were (1R) matched the top GO term for genes downregulated in WT PB validated with qRT-PCR as above and confirmed significant compared with WT nB (6R). This implies that genes downregulated derepression in CKO PB (Supplemental Fig. 3H). These data in- when cells normally differentiate from nB to PB were derepressed dicate that LSD1 is, in part, responsible for repressing genes in the absence of LSD1. This was also supported by the finding inhibited by Blimp-1. that between DEG groups 1R (normally repressed in PB) and 6R (upregulated in CKO PB), there were 143 genes that overlapped, LSD1 promotes B cell proliferation which was 2.5-fold more than expected, by chance (Supplemental Annotation of DEG to HALLMARK and KEGG gene sets by GSEA by guest on September 23, 2021 Fig. 3C). No significant overlap was observed between 1R and 5R identified proliferation and cell cycle genes downregulated in CKO (Supplemental Fig. 3C). To further examine the derepressed genes, PB as compared with WT (Supplemental Fig. 4A). This is consistent genes were ranked by expression differences between WT and CKO with fewer PB observed following LPS-mediated in vivo B cell PB and compared with the 200 most significant DEG from 1R (genes differentiation, suggesting that LSD1 may regulate B cell prolifer- repressed in PB) using GSEA (Fig. 3E). This analysis showed that ation. To test if there was a proliferation defect, the proliferative genes aberrantly upregulated in CKO PB were significantly enriched capacity of CreWT and CKO B cells in response to LPS was for genes normally repressed in WT PB, further underscoring the quantified by CTV staining of purified splenic B cells that were importance of LSD1 in gene repression during PB formation. cultured ex vivo as above. Cultures were analyzed at 24, 36, 48, Additional analyses using gene sets defining follicular splenic 60, and 72 h (Fig. 5A, Supplemental Fig. 4B). CKO B cell cultures B cells and PC (7) corroborated the above GSEA results accumulated significantly fewer cells at 60 and 72 h compared (Supplemental Fig. 3D). Example genes that exhibited this ex- with CreWT B cell cultures. Moreover, when assessed by division, pression pattern included those that function in complement ac- CKO cultures produced fewer total cells after 36 h and fewer tivation [Cfp (43)], homing [Itgb7 (44) and Sell (45)], response to CD138+ PB at all time points after 24 h of culture. bacteria [Mpeg1 (46)], signaling [Lsp1 (47), Plaur (48), and The in vivo proliferation defect was characterized by purifying Siglecg (49)], and survival [Gimap4 (50); Fig. 3F]. These results CKO and CreWT B cells, staining them with CTV, and adoptively were validated by quantitative RT-PCR (qRT-PCR) with CreWT transferring them into a mMT host and inoculating them with LPS control cells, confirming a significant derepression in CKO PB as above. CKO cells had significantly reduced cells in divisions (Supplemental Fig. 3E). two through nine, and the total number of CD138+ PB in later di- GSEA performed with the top 200 upregulated DEG from group visions was decreased substantially (Fig. 5B). Together, these data 2R (normally upregulated in PB) revealed that some genes in WT indicate that LSD1 is critical for normal B cell proliferation in PB were superinduced in the absence of LSD1 (Fig. 3G), which response to LPS. was validated as above (Supplemental Fig. 3D). Example genes that exhibited this expression pattern included Ly6c, which en- LSD1 regulates chromatin accessibility at ETS and IRF codes a PC surface marker (51), and those that function in ad- transcription factor motifs hesion [Amigo2 (52)], homing [Cd68 (53)], proliferation [Cd300a ATAC-seq was performed on the same samples as RNA-seq to assess (54) and Uchl1 (55)], response to virus [Ifitm3 (56)], and signaling the global effects of LSD1 deficiency on active regulatory elements [Cd28 (57) and Dusp14 (58);Fig.3H].Theseresultswere during B cell differentiation. Principal component analysis of validated with qRT-PCR as above, confirming significant su- ATAC-seq data showed that samples stratified by both cell type and perinduction (Supplemental Fig. 3F), and highlight LSD1 as a Lsd1 deletion status (Fig. 6A), supporting a chromatin regulatory transcriptional rheostat throughout B cell differentiation. role for LSD1 in nB and PB. Differential accessibility analysis The Journal of Immunology 2805

FIGURE 4. Blimp-1 target–repressed genesareregulatedbyLSD1.(A) GSEA analysis for enrichment of Blimp-1–activated genes (left) and Blimp-1–repressed genes (right) (10) in all detected genes ranked by ex- pression difference between the in- dicated sample group comparisons. (B) Example Blimp-1 target–repressed DEG. Error bars represent mean 6 SD. Significance was determined by edgeR. *FDR , 0.05. Downloaded from http://www.jimmunol.org/ indicated that in the WT setting, PB lost 19,461 and gained 12,646 which contained nB-accessible regions that normally would be accessibility regions compared with nB (Fig. 6B, referred to inaccessible in PB, were enriched for motifs of transcription hereafter to as DAR groups 1A and 2A, respectively). Examining factors known to be important in nB development and matura- WT and CKO sample comparisons of the same cell type found that tion, including ETS1 (65), RUNX1 (66), and PAX5 (67). Group both CKO nB and CKO PB underwent mostly targeted increases 2A DAR, which contained newly accessible regions in PB, were in chromatin accessibility (groups 4A and 6A, respectively). enriched for motifs of transcription factors known to be impor- To gain insight into the transcription factors associated with tant for PC formation and function, including E2A (68), OCT2 LSD1-regulated chromatin in PB, motif analysis (35) was per- (69), and IRF4 (70). Group 6A DAR, which were more accessible formed on DAR groups 1A, 2A, and 6A (Fig. 6C). Group 1A DAR, because of loss of LSD1, were primarily enriched for motifs of the by guest on September 23, 2021

FIGURE 5. LSD1 promotes B cell proliferation. (A) Flow cytometry analysis of CTV in adoptively trans- ferred CreWT and CKO splenic B cells on day 3 after LPS inoculation (left) and quantification of total cells and total CD138+ PB per division (right). (B) Total cells and total CD138+ PB per division of ex vivo– differentiated CreWTand CKO splenic B cells at five time points. Data are representative of at least two inde- pendent experiments using three to five mice per group. Error bars repre- sent mean 6 SD. Significance was determined by Student two-tailed t test. *p , 0.05, **p , 0.01, ***p , 0.001. 2806 LSD1 REGULATES B CELL DIFFERENTIATION

FIGURE 6. LSD1 regulates chro- matin accessibility at ETS and IRF transcription factor motifs. (A)Toptwo principal components (PC1, PC2) from PCA of z-score–normalized rppm val- ues of all ATAC-seq peaks (72,519 to- tal) and sample group 99% confidence intervals (black ovals). (B) Heatmap depicting DAR between the indicated comparisons. rppm 6 5kbaroundthe peak is shown. rppm, reads per peak

per million. (C) Heatmap displaying Downloaded from

2log2(p values) for transcription factor binding motifs enriched in the indi- cated DAR identified through HOMER known motif analysis. (D) Gene tracks of example DAR mapping to a 6R gene. Transcription factor family motifs are indicated by colored dashes under http://www.jimmunol.org/ each track. by guest on September 23, 2021

transcription factor families ETS and IRF and were modestly indicated that 1A, 2A, and 6A DAR significantly overlapped ac- enriched for motifs of the transcription factor families MADS and tive enhancers from various cell types, which is expected given POU, suggesting that LSD1 functions to restrict chromatin accessi- that enhancers can be shared between cell types (73), but the highest bility at the binding sites of these factors. The occurrence of several degree of overlap for 1A and 6A DAR was observed with nB active motifs, including , , and Sox3, do not occur in any of the enhancers. Conversely, DAR rarely occurred at active promoters DAR groups (Fig. 6C), suggesting specificity for the identified (containing H3K27ac and H3K4me3, but not H3K4me1). nB ac- motifs. Example 6A DAR that contained motifs of transcription tive enhancers overlapping with 1A, 2A, and 6A DAR were tested factors belonging to these families and that also mapped to a 6R for enrichment of H3K4me2 in WT nB and PB cells (Fig. 7D). DEG are displayed (Fig. 6D; Arpp21, Atp10a, Med12l, Per3, Both 1A and 6A nB enhancers exhibited a significant decrease in Serpina3g,andSlc16a7). Overall, these data link PB-based chromatin H3K4me2, whereas 2A nB enhancers exhibited a significant in- closure with LSD1 at sites enriched with ETS, IRF, MADS, and POU crease, demonstrating that 1A and 6A nB enhancer regions normally family motifs and suggests a functional relationship between LSD1 lose LSD1-target H3K4 methylation in PB. Overall, these data imply and transcription factors of these families. that LSD1 functions to decommission nB active enhancers. Motif analysis was performed on 1A and 6A nB enhancers to LSD1 restricts chromatin accessibility at nB enhancers in PB gain insight into the transcription factors possibly bound at Analysis of DAR revealed 2.05-fold more overlap than expected LSD1-regulated nB enhancers (Fig. 7E). In both enhancer sets, the between DAR groups 6A and 1A compared with no significant most significantly enriched motifs included ETS and IRF family overlap between groups 5A and 1A, indicating that LSD1 restricts factors. These data suggest that LSD1 restricts chromatin acces- chromatin accessibility at regions normally accessible in nB (Fig. 7A). sibility at enhancers containing ETS and IRF motifs during PB To determine if LSD1-specific DAR occurred at nB regulatory differentiation. regions, 1A, 2A, and 6A DAR were analyzed for enrichment of the The relationship between LSD1 and transcription factors was active chromatin histone modifications H3K4me1 and H3K27ac from explored by analyzing published ChIP-seq data for the factors published nB datasets (71) (Fig. 7B). Compared with 2A, both 1A PU.1, IRF4, and Blimp-1 from ex vivo LPS-induced PB (10). PU.1 and 6A DAR were significantly enriched for both marks, sug- was chosen because it is an ETS family transcription factor known gesting that in nB, 1A and 6A DAR were located at cis-regulatory to regulate the development and differentiation of B cells and also elements. The overlap of DAR and active enhancers [containing interact with IRF factors (5, 74). IRF4 was chosen because of H3K4me1 and H3K27ac, but not H3K4me3 (72)] in several cell its clear and critical role during B cell differentiation (70). All types was assessed by an odds ratio (Fig. 7C). The analysis binding sites per transcription factor were analyzed for H3K4me2 The Journal of Immunology 2807

FIGURE 7. LSD1 restricts chro- matin accessibility at nB enhancers (enh) in PB. (A) Overlap between DAR group comparisons 1A versus 6A and 2A versus 6A. The ratio of observed DAR overlap over ex- pected overlap according to a per- mutation test is referred to as obs/ exp. (B) ChIP-seq reads per million enrichment of nB H3K4me1 (left) and H3K27ac (right) for DAR groups 1A, 2A, and 6A. (C) Log2 odds ratios of DAR group enrich- ment with active enh and active promoters from six different cell types. (D) Boxplot of ChIP-seq en-

richment of nB and PB H3K4me2 Downloaded from for nB enh mapping to 1A DAR and 6A DAR. (E) Top significantly enriched transcription factor motifs identified through HOMER de novo motif analysis for 1A nB enh and 6A nB enh. (F) Boxplot of ChIP-seq enrichment of nB and PB H3K4me2 http://www.jimmunol.org/ for PU.1 binding sites, IRF4 bind- ing sites, and Blimp-1 binding sites mapping to 6R DEG. (G) Boxplot of chromatin accessibility of the indi- cated sample groups at 6R PU.1, 6R IRF4, and 6R Blimp-1 regions. (H) Gene tracks of example transcrip- tion factor binding sites mapping to a 6R gene that exhibit significant increases in chromatin accessibility by guest on September 23, 2021 in PB CKO. Significance was de- termined by Wilcoxon rank sum test (B), Fisher exact test (C), or Student two-tailed t test (D, F, and G). rppm, reads per peak per million.

enrichment in WT nB and PB (Supplemental Fig. 5A). All three transcription factor binding site groups exhibited a significant in- sets of binding sites exhibited a significant decrease in H3K4me2, crease in chromatin accessibility in LSD1-deficient PB. For a suggesting a role for LSD1 at these sites. Binding of each of the negative control, regions that were bound by SOX2, a transcrip- above factors was found to occur within the 594 group 6A DAR tion factor not involved in regulating B cell differentiation (75), (170, 17, and 34, respectively), suggesting that these transcription were analyzed as above (6R SOX2, 294 binding sites; Supplemental factors bind at LSD1-regulated chromatin and potentially con- Fig. 5C, 5D). No significant differences were found. Example PU. tribute to the recruitment of LSD1 (Supplemental Fig. 5B). These 1, IRF4, and Blimp-1 transcription factor binding sites identified results do not preclude the action of additional factors from in the above analyses are displayed (Fig. 7H; Hmgcll1, L3mbtl3, influencing LSD1-regulated chromatin. and Timd2). These data support a chromatin-remodeling role for The role of LSD1 at PU.1, IRF4, and Blimp-1 binding sites LSD1 at PU.1, IRF4, and Blimp-1 target binding sites that map to that map to LSD1-regulated genes were examined. To start, tran- LSD1-regulated genes. scription factor binding sites were mapped within 100 kb of genes upregulated in LSD1-deficient PB (6R DEG), resulting in three Aberrant accumulation of H3K4me1 at LSD1-regulated loci distinct groups of transcription factor binding sites (6R PU.1, 512 The effect that LSD1 had on chromatin accessibility during B cell binding sites; 6R IRF4, 290 binding sites; 6R Blimp-1, 144 binding differentiation was mainly restrictive and occurred at enhancer sites). Each group was assessed for enrichment of the LSD1 target regions, implying that LSD1 demethylates the active enhancer histone modification H3K4me2 in WT nB and PB (10, 40) (Fig. 7F). histone modification H3K4me1 in this system. To determine if this The analysis found that H3K4me2 levels decreased in PB compared was the case, H3K4me1 levels at LSD1-regulated DAR (Fig. 6, with nB for all three groups, suggesting a role for LSD1 at these group 6A) were assayed by ChIP. Chromatin was prepared from sites. To explore this further, chromatin accessibility data from CreWT nB, CKO nB, CreWT PB, and CKO PB and H3K4me1 this study were examined similarly (Fig. 7G). Each of the three ChIP-qPCR was performed on a set of nine regions previously 2808 LSD1 REGULATES B CELL DIFFERENTIATION

FIGURE 8. Aberrant accumula- tion of H3K4me1 at LSD1-regulated loci. (A and B) ChIP-qPCR for H3K4me1 enrichment displayed as percentage of input at the indicated genomic regions. Data are combined from two independent experiments using three mice per group. Error bars represent mean 6 SD. Signi- ficance was determined by Student two-tailed t test. *p , 0.05, **p , 0.01, ***p , 0.001.

defined (Figs. 6D, 7H). Regions mapping to the derepressed genes Mechanistically, LSD1-regulated chromatin accessibility and Downloaded from Med12l, Slc16a7,andL3mbtl3 and the superinduced genes Arpp21, H3K4me1 levels at many, but not all, nB active enhancers and Atp10a, Per3,andHmgcll1 exhibited significant increases in PU.1, IRF4, and Blimp-1 binding sites mapping to LSD1- H3K4me1 in CKO PB compared with CreWT PB (Fig. 8A, repressed genes as the cells differentiated to PB. Together, these Supplemental Fig. 6A, 6B). Of these DAR, those that mapped to data show that LSD1 modulates PB transcriptional networks through the genes Arpp21, Atp10a, Hmgcll1, and L3mbtl3 mapped to a nB epigenetic reprogramming.

active enhancer (Fig. 7C). LSD1 deletion resulted in the derepression and superinduction http://www.jimmunol.org/ To further explore the role of LSD1 in regulating H3K4me1, 12 of hundreds of genes in PB. Previous studies have shown that additional potential enhancer regions mapping to LSD1-regulated LSD1 deletion results in gene derepression through failure to genes (Figs. 3F, 3H, 4B) were chosen based on 1) transcription factor remove H3K4 methylation (15, 17). This same observation was binding of PU.1, Blimp-1, or IRF4; and 2) presence of H3K4me1 in noted for just more than half of the genes/regions assessed in this nB reported previously (10, 71). Regions mapping to the derepressed study. Some of the regions that showed increased H3K4me1 in genes Hck, Sell,andSiglecg and the superinduced genes Amigo2, the absence of LSD1 mapped to DAR. Together, these obser- Cd28,andIfitm3 exhibited significant increases in H3K4me1 in PB in vations suggest that there may be multiple mechanisms that al- the absence of LSD1 (Fig. 8B, Supplemental Fig. 6C). Five out of six low the decommissioning of active enhancer regions with the regions, including all three derepressed genes, mapped to a nB active first mechanism relying on LSD1 to demethylate active histone by guest on September 23, 2021 enhancer (Supplemental Fig. 6D). Other regions did not reach H3K4me1/2 modification and functioning as a regulator of chromatin statistical significance for increases in H3K4me1 in the absence accessibility. Additional mechanisms may rely on other demeth- of LSD1, suggesting that the role of LSD1 is specific to certain ylases that belong to the JARID/Jumanji family of (79). regions. LSD1 deletion in B cells caused a compound effect of decreased Some of the regions significant for H3K4me1 increases were proliferation and differentiation. This was not due to increased cell further examined in the context of other model systems. Using death or a defect in B cell activation. Decreased proliferation and in vitro–derived effector CD8+ T cell ChIP-seq data of the enhancer differentiation were also observed in an ex vivo B cell–differentiation modifications H3K4me1 and H3K27ac, as well as IRF4 binding (76), culture system, arguing against defective in vivo homing. The data four regions were identified as IRF4-bound enhancers (Supplemental cannot rule out that reduced proliferation contributed to reduced Fig. 7A). Ifitm3 and L3mbtl3 were identified as responsive to changes differentiation but given that LSD1 modulates the expression of in IRF4 expression in IRF42/+-exhausted CD8+ T cells (77). Atp10a hundreds of non–cell cycle genes, it is likely that other factors and Siglecg were identified as being bound by BATF and NFAT in are contributing to the phenotype. For example, CD28-deficient addition to IRF4 and represent chronic infection signature genes B cells produce more short- and long-lived PC (80); thus, CD28 (77). Using ATAC-seq data and PU.1 and LSD1 ChIP-seq data from overexpression observed in this system may result in fewer PB. mouse-engrafted MLL-AF9 primary acute myeloid leukemia cells Also, it may be possible that certain gene regulatory effects of either treated or not treated with an LSD1 inhibitor (78), regions LSD1 that contributed to the phenotype were undetected because mapping to Serpina3g, Ifitm3, L3mbtl3, and Siglecg were identi- the RNA-seq experiment was performed only on the nB and PB fied as being bound by LSD1 and PU.1 and exhibiting increased stages and not any activated B cells from the divisions in be- accessibility upon pharmacological inhibition of LSD1 (Supplemental tween. Future comparisons between nB and activated B cells, as Fig. 7B). These analyses support our conclusions that the regions well as activated B cells to PB, may shed additional light on the examined represent regulatory regions that LSD1 decommissions. specific role of LSD1 during key stages of B cell differentiation. The data display a proliferation defect, but both WTand CKO PB Discussion underwent a sufficient number of divisions within the same time This study demonstrates a critical in vivo role for LSD1 during period to differentiate. This indicates that the WT and LSD1-deficient B cell differentiation in response to the T cell–independent Ag cells proliferate to the same extent and argues that the gene expression LPS. In LSD1-deficient PB, derepression and superinduction of and chromatin dysregulation observed is due to LSD1 deletion and immune system process genes was observed with some dere- not a reduced capacity to proliferate. The decreased expression of pressed genes being targets of Blimp-1 repression. LSD1 also cell cycle genes corresponds with the phenotype, but the most sub- positively regulated the expression of cell cycle genes, which was stantial changes in gene expression were increases. Also, ATAC-seq concordant with a decrease in proliferation upon LSD1 deletion. and ChIP-qPCR data indicate that LSD1 primarily represses active The Journal of Immunology 2809 chromatin. Together, these data argue against a role for LSD1 Acknowledgments directly activating gene expression and indicate that the effect on We acknowledge members of the Boss Laboratory for scientific contribu- cell cycle gene expression may be indirect. Cell cycle regulator tions and editorial input. We thank the Emory Flow Cytometry Core for genes such as Rb1, family genes, or , which have been FACS, the Emory Integrated Genomics Core for sequencing library quality observedinothersystemstoaffectB cell differentiation (81, 82), control, and the New York University Genome Technology Center for were not aberrantly expressed, implying an alternative mecha- Illumina sequencing. nism for cell cycle gene downregulation and reduced prolifera- tion. A number of genes aberrantly upregulated in the absence of Disclosures LSD1 may be contributing. For example, CD300a was super- The authors have no financial conflicts of interest. induced in LSD1-deficient PB. It has been shown that CD300a negatively regulates BCR stimulus–induced B cell proliferation References (54), suggesting that overexpression in the LPS-stimulation model 1. Nutt, S. L., P. D. Hodgkin, D. M. Tarlinton, and L. M. Corcoran. 2015. The may dampen B cell proliferation. generation of antibody-secreting plasma cells. Nat. Rev. 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