For Early B Cell Development the SWI/SNF-Like BAF Complex Is

The SWI/SNF-like BAF Complex Is Essential for Early B Cell Development Jinwook Choi, Myunggon Ko, Shin Jeon, Yoon Jeon, Kyungsoo Park, Changjin Lee, Ho Lee and Rho H. Seong This information is current as of September 26, 2021. J Immunol 2012; 188:3791-3803; Prepublished online 16 March 2012; doi: 10.4049/jimmunol.1103390 http://www.jimmunol.org/content/188/8/3791 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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The SWI/SNF-like BAF Complex Is Essential for Early B Cell Development

Jinwook Choi,*,1 Myunggon Ko,*,1,2 Shin Jeon,* Yoon Jeon,† Kyungsoo Park,* Changjin Lee,* Ho Lee,† and Rho H. Seong*

During the process of B cell development, transcription factors, such as E2A and Ebf1, have been known to play key roles. Although transcription factors and chromatin regulators work in concert to direct the expression of B lineage-specific genes, little is known about the involvement of regulators for chromatin structure during B lymphopoiesis. In this article, we show that deletion of Srg3/mBaf155, a scaffold subunit of the SWI/SNF-like BAF complex, in the hematopoietic lineage caused defects at both the common lymphoid progenitor stage and the transition from pre–pro-B to early pro-B cells due to failures in the expression of B lineage-specific genes, such as Ebf1 and Il7ra, and their downstream target genes. Moreover, mice that were deficient in the expression of Brg1, a subunit of the complex with ATPase activity, also showed defects in early B cell development. Downloaded from We also found that the expression of Ebf1 and Il7ra is directly regulated by the SWI/SNF-like BAF complex. Thus, our results suggest that the SWI/SNF-like BAF complex facilitates early B cell development by regulating the expression of B lineage-specific genes. The Journal of Immunology, 2012, 188: 3791–3803.

lood cells that constitute the three major hematopoietic rearrangement at the Ig H chain locus. The cells with the rear-

lineages—erythroid, myeloid, and lymphoid—originate ranged IgH locus are considered committed B lineage cells. Late http://www.jimmunol.org/ B from pluripotent hematopoietic stem cells (HSCs) in the pro-B (Fr. C) cells expressing pre-BCRs on the surface, a critical fetal liver and the bone marrow after birth (1, 2). HSCs differ- checkpoint for the progression from pro-B cells to pre-B cells, entiate into multipotent progenitors that have lost the capacity for follow early pro-B cells (8). Proper signaling from the pre-BCR is self-renewal but retain the potential for multilineage differentia- required for the allelic exclusion of the IgH chain and the initia- tion; these cells, in turn, give rise to lymphoid-primed multipotent tion of Ig L chain rearrangement in pre-B cells, which eventually progenitors (LMPPs) (3). LMPP populations express high levels results in expression of the BCR (9). of the tyrosine kinase receptor Flt3 (Flk2) and have little poten- B cell specification is controlled by cell-intrinsic transcription tial for erythro-megakaryocyte differentiation (4). LMPPs are factors, such as E2A, paired box protein 5 (Pax5), and early B cell followed by common lymphoid progenitors (CLPs), which are factor, as well as various cytokines produced from the local mi- by guest on September 26, 2021 the precursors of B lymphocytes, NK cells, dendritic cells, and croenvironment, including IL-7. B cell generation is compromised T lymphocytes (5). After the CLP stage, B lineage differentiation in Il7ra-deficient mice, which show a decrease in CLP numbers progresses to pre–pro-B (Fr. A) cells by the upregulation of and impairment in differentiation into the early pro-B cell stage, CD45R/B220 expression on the surface, but not yet of CD19, even though it is not clear whether IL-7 signaling is absolutely CD24, and BP-1 (6, 7). Pre–pro-B cells give rise to early pro-B required for the generation of these cells (10, 11). E2A and Ebf1 (Fr. B) cells, which express CD19 and undergo a complete DH-JH act together as critical transcription factors to generate early pro- B cells from CLPs during B cell differentiation. E2A2/2 mice display significantly fewer LMPPs and CLPs and show defects in *Department of Biological Sciences, Institute of Molecular Biology and Genetics, the transition from pre–pro-B (Fr. A) to early pro-B cells (Fr. B) Research Center for Functional Cellulomics, Seoul National University, Seoul 151- † (12, 13). B cell development in Ebf1-deficient mice is largely 742, Korea; and Cancer Experimental Resources Branch, Research Institute, Na- 2/2 tional Cancer Center, Goyang-si, Gyeonggi-do 410-769, Korea arrested at the pre–pro-B stage, similar to E2A mice. These 1J.C. and M.K. contributed equally to this work. mice fail to express B lineage-specific genes, including Rag1, 2 Rag2, Ig-a (Cd79a, mb-1), Ig-b (Cd79b, B29), l5(Igll1), and Current address: Division of Signaling and Gene Expression, La Jolla Institute for Allergy and Immunology, La Jolla, CA. VpreB1 (Vpreb1), and they do not initiate IgH recombination (14, Received for publication December 2, 2011. Accepted for publication February 7, 15). E2A and Ebf1 also induce Pax5, which is a transcriptional 2012. regulator that is required for B cell commitment and maintenance This work was supported by the National Research Foundation of Korea, in part but not for B lineage specification. In addition, the ectopic ex- through the Research Center for Functional Cellulomics, and in part through National pression of Ebf1 in HSCs from E2A2/2 mice and pre–pro-B cells Research Foundation Grant 2009-0081789 (to R.H.S.). J.C., S.J., and K.P. are sup- 2/2 ported by the BK21 program from the Ministry of Education, Science and Technol- from Il7ra mice rescues B cell development in vitro (10, 11, ogy, Korea. J.C. is supported by a Seoul science fellowship. 14, 16). Address correspondence and reprint requests to Dr. Rho H. Seong, Department of Epigenetic modification and dynamic reorganization of the local Biological Sciences, Institute of Molecular Biology and Genetics (Building 105) and chromatin structure are essential to poise lineage-specific genes for Research Center for Functional Cellulomics, Seoul National University, Kwanak-gu Shinlim-dong San 56-1, Seoul 151-742, Korea. E-mail address: [email protected] expression or repression. The key event in this process is the re- Abbreviations used in this article: ChIP, chromatin immunoprecipitation; CLP, com- cruitment of chromatin-remodeling complexes, which mediate the mon lymphoid progenitor; FO, follicular; HSC, hematopoietic stem cell; LMPP, active orchestration of transcriptional activation or silencing. Thus, lymphoid-primed multipotent progenitor; MZ, marginal zone; Pax5, paired box pro- the spatiotemporal regulation of chromatin reorganization can tein 5; pIpC, polyinosinic-polycytidylic acid; shRNA, short hairpin RNA. have significant impacts on the development and differentiation of Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 the hematopoietic process. The mammalian SWI/SNF-like BAF www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103390 3792 ROLE OF BAF COMPLEX IN EARLY B CELL DEVELOPMENT complex mobilizes nucleosomes by using the energy from ATP (RA36B2)-allophycocyanin; CD43 (S7)-PE; IgM (R6-60.2 or II/41)–PE– hydrolysis and is required for the expression of a number of genes Cy7 and –biotin; CD19 (1D3)–allophycocyanin–Cy7; CD4 (GK1.5)-PE; (17, 18). The SWI/SNF-like BAF complex is composed of mul- CD8 (53-6.7)-FITC, –biotin, and –PE; CD3 (145-2C11)-PE; TCRb (H57- 597)-allophycocyanin; Mac-1 (CD11b, M1/70)-PE; CD71 (C2)-PE; CD24 tiple subunits that are conserved through evolution. Brg1 with (M1/69)-biotin; and BP-1 (6C3)-biotin (all from BD Bioscience). The ATPase catalytic activity, Baf155 (also known as Srg3), Baf170, following Ab conjugates were purchased from eBiosciences: CD16/32 and Baf47 (also known as Snf5) are sufficient to induce remod- (FcgRII/III)-PeCy7; Sca-1 (Ly-6A/E, D7)-allophycocyanin and –PE-Cy7; eling activity in vitro; therefore, these subunits are considered the Mac-1 (CD11b, M1/70)-allophycocyanin Alexa Fluor 750; c-Kit (CD117, 2B8)-allophycocyanin Alexa Fluor 750; CD44 (IM7)-biotin; CD25 core subunits of the complex (17). Srg3 is a murine homolog of (PC61.5)-allophycocyanin; and CD34 (RAM34)-allophycocyanin. B220 Swi3 in yeast and Baf155 in humans (19). Srg3/mBAf155 controls (RA3-6B2)–FITC and IgD (11-26)–PE were purchased from Invitrogen the stability of the SWI/SNF-like BAF complex and, thereby, and Southern Biotech, respectively. serves as a scaffold protein (20, 21). It was also shown that the Semiquantitative and quantitative RT-PCR chromatin-remodeling activity is enhanced in transgenic mice overexpressing Srg3/mBaf155 (22). Total RNAs were purified from cells with TRIzol reagent, following the It is still unknown how the process of hematopoiesis, particu- manufacturer’s instructions (Invitrogen). Equivalent quantities of total RNA were reverse transcribed with SuperScript III (Invitrogen), and di- larly that of early lymphoid lineage differentiation, is epigeneti- luted cDNAs were analyzed by semiquantitative PCR. PCR products were cally controlled, although there are a few studies suggesting its separated on agarose gels and visualized by ethidium bromide. SYBR role during myeloid lineage differentiation (23, 24) and T cell Green PCR mix (Applied Biosystems) or TaqMan Master mix (Applied development (25–27). In this study, we show that the SWI/SNF- Biosystems) was used for quantitative real-time PCR analysis (primer sequences are available upon request), and the results were quantified with like BAF complex plays a critical role in the process of early B StepOnePlus (Applied Biosystems). Downloaded from cell lineage commitment by facilitating the expression of Ebf-1 and Il7ra. Western blot analysis Whole-cell lysates were separated by SDS-PAGE and transferred to Materials and Methods Immobilon-P membrane (Millipore). Blotted proteins were detected using Mice Abs to SRG3, BRG1, b-actin (AC-15; Sigma), or FLAG epitope (M2; Sigma) in TBST (150 mM NaCl, 10 mM Tris-Cl [pH 8], 0.5% Tween-20) http://www.jimmunol.org/ Targeting constructs and strategy for the generation of conditional Srg3/ containing 5% low-fat milk. Antisera against SRG3 and BRG1 were raised mBAF155 knockout mice are described in Fig. 1D. Mice with loxP sites from rabbits in our laboratory. flanking exon 4 of Srg3 (Srg3fl) were backcrossed onto C57BL/6 mice for at least eight generations and were crossed with Mx1-Cre mice for deletion Southern blot analysis of Srg3 at various stages of the hematopoietic lineage. Mx1-Cre mice were purchased from The Jackson Laboratory. All mice were bred and main- Genomic DNA preparation and Southern blotting were done mainly as described (28). Briefly, DNA was purified from B220+CD43+IgM2 pro- tained in specific pathogen-free barrier facilities at Seoul National Uni- fl/fl versity and were used following protocols approved by Institutional B cells obtained from pIpC-treated control and Mx1-Srg3 mice by Animal Care and Use Committees of Seoul National University. phenol-chloroform extraction and resuspended in 10 mM Tris (pH 8) and 1 mM EDTA. PCR analyses of Ig genes were performed with published primers. The PCR product of HSS3 was used as a loading control. DNA

Administration of polyinosinic-polycytidylic acid by guest on September 26, 2021 products amplified by PCR were separated on 1% agarose gels by elec- For the deletion of Srg3, 6–8-wk-old control (Srg3fl/fl or Srg3fl/+)and fl/fl trophoresis and transferred to Hybond-N membranes (Amersham). Srg3 mice crossed with Mx1-Cre mice were injected with 250 mg Southern blotting was probed with 32P-labeled oligonucleotides. polyinosinic-polycytidylic acid (pIpC; GE Healthcare) three to four times every other day i.p. Deletion efficiency was determined by PCR or Western Chromatin immunoprecipitation assay blot analysis. For the chromatin immunoprecipitation (ChIP) assay, a solution of 37% DNA constructs (v/v) formaldehyde was added to the culture medium of PD36 cell line or primary pro-B cells to a final concentration of 1%, and cells were incubated Srg3 Brg1 For sequence-specific knockdown of and , target sequences for 10 min with rocking at 37˚C, followed by washing twice with ice-cold (Srg3,59-CATCCTGGTTTGATTATAA-39 and Brg1,59-CCGTCAA- PBS. Then, cells were lysed for 10 min with SDS lysis buffer. The samples 9 GGTGAAGATCAA-3 ) were cloned into MDH1–PGK–GFP2.0 (kindly were sonicated to an average length of 100–500 bp, and the sonicated cell provided by Dr. Chang-Zhen Cheng, Stanford University) or MSCV– supernatants were diluted 10-fold in dilution buffer (1% Triton X-100, LTRmiR30–PIG (Open Biosystems) retroviral vectors. The FLAG- 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl [pH 8.1], and Protease D tagged BAF57 N mutant and Myc-tagged Srg3 were cloned into Inhibitor mixture [Roche]). Then, lysates were precleared for 1 h with pMIG vectors (kindly provided by Dr. Warren S. Pear, University of salmon sperm DNA/Protein-A agarose (50% slurry), followed by incuba- Pennsylvania). tion overnight at 4˚C with isotype-control anti-rabbit IgG (Upstate), anti- me3 Flow cytometry and cell sorting Brg1, anti-Srg3, or anti-H3K9 (Upstate). The beads were washed se- quentially once with a low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM Single-cell suspensions were prepared from the bone marrow (femur and EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl), a high-salt buffer (0.1% tibia), spleen, lymph node, and thymus by passing through a cell strainer to SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM remove cell debris. To deplete the RBCs, an equal volume of ACK RBC NaCl), and LiCl washing buffer (0.25 M LiCl, 1% Nonidet P-40, 1% lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) was deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]) and twice with 10 added to the cells and incubated on ice for 5 min. Then up to 10 ml PBS mM Tris (pH 8) plus 1 mM EDTA. Cross-linked chromatin–histone plus 0.1% FBS were added to stop the lysis reaction. Cells were counted, complexes were eluted with elution buffer (1% SDS, 0.1 M NaHCO3). and 1–3 3 106 cells were used for Ab staining. Then cells were stained After washing and elution steps, cross-linking was reversed by heating at with mAbs in various combinations in 13 PBS containing 1% heat- 65˚C for 4 h, followed by proteinase K treatment for an additional 1 h. inactivated FBS. Biotinylated Abs were revealed by PerCP- or allophy- DNA was purified with a QIAquick Spin kit (QIAGEN, Seoul, Korea), and cocyanin-conjugated streptavidin (BD Biosciences). Intracellular staining eluted DNA was analyzed by PCR with primers within the Ebf1 or Il7ra was performed following the manufacturer’s protocols (BD Bioscience). promoter regions. The sequences of the primer pairs are as follows: Ebf1 Flow cytometric analyses were performed using FACSCanto II (BD Bio- promoter regions, P1, 59-TCAGACTAGCTCAGAATCTCCCATA-39, P2, science), and FACSAria II (BD Biosciences) was used for cell sorting. 59-ATCAGTAGGTTTTGCCTCTTGC-39, P3, 59-CTACAGCAACCAGC- Data were analyzed using FlowJo software. The following Abs were used: GTCCTC-39, and P4, 59-GGGCTGGAGTTTCCTTTTGT-39; Il7ra pro- biotin-conjugated mouse lineage (Lin) panel that contains anti-B220 moter regions, P1, 59-CCACCTTGCTCAAAAGTAAGC-39 and P2, 59- (RA3-6B2), anti-CD3ε (145-2C11), anti–Gr-1 (RB6-8C5), anti-CD11b GAAGCACGCTTGTATGTGC-39; and Gapdh intron regions, sense, 59- (Mac1, M1/70), and anti–Ter-119 Abs; CD34 (RAM34)-PE; CD117 TTACTTTCGCGCCCTGAG-39 and antisense, 59-GCGGTTCATTCATT- (c-Kit, 2B8)-PE and PE-Cy7; CD127 (IL-7Ra, SB/199)-PE; B220 TCCTTC-39. The Journal of Immunology 3793

In vitro B cell-differentiation assay pro-B cell culture percentage of B220+CD19+ B cells in the bone marrow (14.24 6 6 The murine OP9 stromal cells were maintained in a-MEM supplemented 1.212%) compared with control (26.09 1.456%) (Fig. 2A). In with 5% FBS. For in vitro culture, HSCs or pro-B cells were isolated from accordance with the reduction in B cell frequency, the B cell pIpC-treated control and Mx1-Srg3fl/fl mice by FACSAria II (BD Bio- numbers were also greatly reduced (40.94 6 4.493 versus 17.51 6 sciences) at day 5 or 10 after the final induction and cocultured (BD 3.233 3 105) (Fig. 2B). In the spleen, B cell generation was also Biosciences) with gamma-irradiated (30 Gy) OP9 cells in RPMI 1640 defective in the absence of Srg3 (Fig. 2C). Moreover, Srg3-deficient supplemented with 10% FBS and cytokines. Stem cell factor (25 ng/ml), Flk2/Flt3 ligand (10 ng/ml), and IL-7 (20 ng/ml; all from PeproTech) were mice showed a significant reduction in B cell numbers to half that of used for the culture of hematopoietic stem cells, and IL-7 (20 ng/ml; control mice (Fig. 2D). In addition, interestingly, the percentage of PeproTech) only was used for the culture of pro-B cells. After 3–6 d of follicular (FO) B cells was significantly decreased, whereas the culture, both suspended and adherent cells were harvested, stained with frequency of the marginal zone (MZ) B cells was increased in Mx1- lineage markers, including Gr-1, B220, and CD19, and analyzed using fl/fl a FACSCanto II (BD Biosciences). Srg3 mice (Fig. 2E). The numbers of FO B cells were also reduced by as much as half (Fig. 2F). These results indicate that Srg3 Retroviral infection and bone marrow reconstitution is essential for proper B cell development in both the bone marrow Bone marrow cells were flushed out of femurs and tibias after i.p. injection and the periphery. with 5-fluorouracil at 150 mg/g body weight for 4 d. RBCs were removed Impairment of B-lineage potential in Srg3-deficient lymphoid using ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA). Then, the cells were cultured overnight in complete RPMI 1640 progenitors (10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 50 mg/ml 2-ME, Srg3 is expressed at the early stage of hematopoietic progenitor 1% Glutamax [Life Technologies], 1% nonessential amino acid [Life cells. Thus, we investigated the function of Srg3 in HSC mainte- Downloaded from Technologies], and 1% sodium pyruvate [Life Technologies]) containing nance and the development of lymphoid progenitors. The frequency 5 ng/ml IL-3, 10 ng/ml IL-6, 50 ng/ml stem cell factor, and 50 ng/ml Flt3 2 2 ligand (all from PeproTech) before the initial retroviral infection. The of the Lin IL-7Ra c-Kit+Sca-1+ (LSK) subset was comparable to packaging Phoenix-eco cells were transfected with a control empty vector that of control mice. The subsets of long-term HSCs, short-term and Brg1-short hairpin RNA (shRNA) using a standard calcium phos- HSCs, and the LMPPs were not significantly altered in the absence phate protocol to generate retroviruses. After 48 h, the viral supernatants of Srg3, although a mild decrease in LMPPs was observed (data not were harvested and used to spin-infect HSC-enriched cells for 1.5 h at 2

2500 rpm and 30˚C in the presence of 10 mg/ml polybrene. This proce- shown). The frequency and cell numbers of traditional Lin IL- http://www.jimmunol.org/ dure was repeated three times, followed by the injection of 2–3 3 106 7Ra+c-KitlowSca-1low CLPs, which are the precursors of B and infected cells via the tail vein into each recipient that was lethally 137 T cells, were not significantly altered by Srg3 deletion (Fig. 3A, gamma-irradiated with 1100 rad at 2-h intervals ([ Cs]; GC 3000 Elan; 3B). The CLP population is heterogeneous, and genes involved in MDS Nordion). early B cell development, including Ebf1 and Rag1, are highly Statistical tests transcribed in B cell-restricted CLPs (31–34). Interestingly, CLPs highly expressing AA4.1 on the surface were significantly reduced Two-tailed Student t tests were used to calculate p values where indicated; fl/fl p values , 0.05 were considered statistically significant. in Mx1-Srg3 mice compared with control mice (Fig. 3C). AA4.1hi CLPs are more potent in the generation of B lineage cells, Results but not myeloid lineage cells, than are traditional CLPs (35, 36). To by guest on September 26, 2021 assess whether the Srg3-deficient CLPs have normal lymphoid The SWI/SNF-like BAF complex is required for normal B cell potential, we analyzed the expression of B lineage-specific genes in development these CLPs. Notably, CLPs purified from Srg3-deficient mice We first examined the expression pattern of the core components showed less Ebf1 expression compared with those from control of the SWI/SNF-like BAF complex in different lineage cells. He- mice, whereas the expression of flt3 remained unchanged (Fig. 3D). 2 matopoietic progenitor-enriched populations (Lin ) and B cells It was reported that Ebf1-deficient CLPs express very low levels of expressed Srg3/mBaf155 (hereafter referred to as Srg3) protein B lineage-specific genes (37). We also found that expression of B highly compared with erythroid and myeloid lineage cells (Fig. lineage-specific genes, such as Cd79a (mb-1), Cd79b (B29), Rag1, 1A). We also analyzed Srg3 transcript levels throughout the Rag2, and FoxO1, were significantly reduced in Srg3-deficient lymphoid lineage differentiation. Srg3 was highly expressed in CLPs (Fig. 3E). HSCs (LSKs), CLPs, and pre–pro-B (Fr. A) cells (Fig. 1B). Srg3 expression was gradually increased during B cell maturation from Pro-B cell development was defective in the absence of BAF early pro-B cells to pre-B cells. Brg1, a core subunit of the SWI/ complex SNF-like BAF complex with ATPase activity, also showed a sim- Although Srg3-deficient lymphoid progenitors showed defects in ilar expression pattern (Fig. 1C). the expression of genes that are associated with the development Previous studies showed that the complete deletion of Srg3 of early B-lineage cells, a considerable number of CLPs was still resulted in embryonic lethality during the peri-implantation stage detected in the absence of Srg3. Therefore, we assessed the cell- (29). To overcome this restriction and examine the function of Srg3 specification processes in Mx1-Srg3fl/fl mice. Consistent with the in hematopoietic lineage differentiation, we generated conditional lower numbers of B cells in Srg3-deficient mice, B cell develop- knockout mice (Mx1-Srg3fl/fl) carrying a loxP-flanked exon 4 (Fig. ment was blocked at the early stage of B-lineage cells in the bone 1D–F) and crossed them with Mx1-Cre transgenic mice, in which marrow of Mx1-Srg3fl/fl mice. Analysis of Mx1-Srg3fl/fl mice treatment with pIpC induced efficient gene deletion in hemato- showed that most of the B cells were arrested at the pro-B cell poietic lineage cells (30). After treatment with pIpC, we confirmed stage, resulting in a significant reduction in the frequency of pre- the deletion of Srg3 alleles in genomic DNAs (Fig. 1G). We also B cells and immature B cells compared with those from control confirmed a drastic reduction in the protein levels in B cells puri- mice (Fig. 4A, 4B). The number of pro-B cells was decreased in fied from the bone marrow and spleen of Mx1-Srg3fl/fl mice Mx1-Srg3fl/fl mice, even though the Srg3-deficient mice had a compared with control (Fig. 1H, 1I). We then analyzed the effect of higher percentage of pro-B cells (Fig. 4C). To further define the Srg3 deficiency on the hematopoietic system, especially B cell defective stage in pro-B cell development in Srg3-deficient mice, development, by comparing Mx1-Srg3fl/fl mice with control lit- we subcategorized the pro-B cells using the differential cell surface termates. Srg3-deficient mice showed a drastic decrease in the markers BP-1 and CD24 (HSA) or CD19. The proportion of CD19+ 3794 ROLE OF BAF COMPLEX IN EARLY B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Generation of Srg3 conditional knockout mice. (A) Western blot analysis of SRG3 expression levels from the indicated subsets in the bone marrow. Expression of b-actin was used as a loading control. Representative results of three independent experiments are shown. (B) Relative expression of Srg3 mRNA in sorted lymphoid lineage compartment in the bone marrow using TaqMan real-time PCR analysis. The expression of gapdh served as a loading control. Data are shown as mean 6 SEM (n = 3). (C) Relative expression of Brg1 mRNA in sorted lymphoid lineage compartment in the bone marrow using TaqMan real-time PCR analysis. The expression of gapdh served as a loading control. Data are shown as mean 6 SEM (n =3).(D) Schematic representation of the targeting strategy. (E) Southern blot analysis of genomic DNA from wild-type (+/+) and targeted (flox/+) embryonic stem cells with a probe overlapping exon 3 reveals a different HindIII (7.5 versus 10.2 kb) fragment in the targeted cells. (F) PCR analysis of tail DNA from wild-type (+/+), heterozygous (flox/+), and homozygous (flox/flox) mice using primers in introns 3 and 4 [P1 and P2 or P3 and P4, as indicated by arrows in (D)]. (G) Representative PCR analysis of bone marrow cells after pIpC treatment of control and Mx1-Srg3fl/fl mice. (H) Western blot analysis of SRG3 expression in B220+CD19+ B cells of bone marrow purified from pIpC-treated control, Mx1-Srg3fl/+,andMx1-Srg3fl/fl mice. (I) Western blot analysis of SRG3 expression in splenic B220+CD19+ Bcellspurified from pIpC-treated control and Mx1-Srg3fl/fl mice. B220+, B lineage cells; Bh, BamHI; DT-A, diphtheria toxin A-chain gene; Gr-1+Mac-1+, myeloid lineage cell population H, HindIII; Lin2, hematopoietic progenitor-enriched population; neo, neomycin-resistance gene; Ter-119+, erythroid lineage cells. cells was severely reduced in the pro-B cells of Mx1-Srg3fl/fl mice but higher frequencies of pre–pro-B (Fr. A) cells than did the (Fig. 4D). In addition, Mx1-Srg3fl/fl mice had significantly lower control mice (Fig. 4D, 4E). In fact, the numbers of early and late frequencies of early pro-B cells (Fr. B) and late pro-B cells (Fr. C) pro-B cells were also severely decreased, whereas the number of The Journal of Immunology 3795 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. B cell generation is impaired in the absence of Srg3/mBaf155. (A) Flow cytometric analysis of bone marrow B cells (B220+CD19+) from control and Mx1-Srg3fl/fl mice. Numbers in plots indicate the percentage of populations. (B) Frequencies (left panel) and numbers (right panel) of bone marrow B cells (B220+CD19+) obtained from control and Mx1-Srg3fl/fl mice. Each symbol represents one mouse; the lines show the mean percentage of bone marrow B cells. Error bars represent SEM (n = 12). (C) Flow cytometric analysis of splenic B220+CD9+ cells from control and Mx1-Srg3fl/fl mice. Numbers indicate the percentage of splenic B cells. (D) Total cell numbers of splenic B cells. Each symbol represents one mouse; the lines show the mean value of splenic B cells (n = 7 for control and n = 9 for Mx1-Srg3fl/fl mice). (E) Flow cytometric analysis of splenic B cells from control and Mx1-Srg3fl/fl mice. B220+CD19+ cells were analyzed for the expression of surface molecules CD21 and CD23. Representative dot plot of FO (CD21+CD23+) and MZ (CD212CD23+) B cells. Numbers indicate the percentage of indicated populations. (F) Frequency (left panel) and cell numbers (right panel) of MZ and FO B cells from control and Mx1-Srg3fl/fl mice. Data represent average 6 SEM (n = 6 for each group).

pre–pro-B cells remained largely unchanged (Fig. 4F). In previous Brg1-deficient chimeras using Brg1-shRNA (Fig. 5A, 5B). The studies, Fr. A population was shown to be heterogeneous and to deficiency in Brg1 also significantly decreased the frequency of contain AA4.1+ and AA4.12 cells. AA4.1+ Fr. A cells are sub- B cells in the bone marrow (Fig. 5C). The frequencies of pre-B, divided into A1 and A2, and they are considered the earliest B- immature B, and mature B cells were reduced in Brg1-deficient lineage population (7). Mx1-Srg3fl/fl mice showed significantly chimeras (Fig. 5D). Furthermore, most of the B cells were arrested fewer AA4.1+ Fr. A cells compared with the control mice (Fig. 4G). at the stage of pre–pro-B cells, similarly to Srg3-deficient mice Overall, Srg3 deficiency leads to a defect in the transition from (Fig. 5E). These results indicate that proper activity of the pre–pro-B to early pro-B cells. SWI/SNF-like BAF complex is required for normal B cell development. The deletion of Brg1 in HSCs also leads to defects in early pro-B cell development The SWI/SNF-like BAF complex is required for IL-7Ra Because the impairment of B cell development in Mx1-Srg3fl/fl expression and pro-B cell survival mice appeared to be due to a functional deficiency of the SWI/ Having shown that early pro-B cell development is blocked in the SNF-like BAF complex, it was likely that the inactivation of other absence of Srg3, we speculated that pro-B cells of Mx1-Srg3fl/fl subunits of the complex would also lead to similar defects in mice would be defective in IL-7R signaling, which leads to more B cell development. To confirm this hypothesis, we generated cell death and less proliferation (38, 39). In fact, we found that 3796 ROLE OF BAF COMPLEX IN EARLY B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Srg3-deficient CLPs displayed the reduced expression of B lineage-specific genes. (A) Flow cytometry of CLP (traditional surface marker; Lin2IL-7Ra+c-KitloSca-1lo) fractions from control and Mx1-Srg3fl/fl mice. Numbers indicate percentage calculated from total cells. (B) Total frequency (left panel) and numbers (right panel) of CLP populations. Each symbol represents one mouse; the lines show the mean percentage, and error bars represent SEM (n = 14). (C) Surface expression levels of Flt3 and AA4.1 on CLP cells (left panel) and mean fluorescence intensity (MFI: right panel) of AA4.1 expression. Data are shown as mean 6 SEM (n = 13). Open graphs represent the expression of each surface marker, and gray-filled graphs represent isotype-matched control Abs. (D) Quantitative real-time PCR analysis of Srg3 (left panel), Ebf1 (middle panel), and Flt3 (right panel) in CLPs purified from control and Mx1-Srg3fl/fl mice. Data represent the average 6 SEM of four to six independent experiments. (E) Quantitative real-time PCR analysis of B lineage-specific genes, including Cd79a, Cd79b, Rag1, Rag2, and FoxO1, in CLPs purified from pIpC-treated control or Mx1-Srg3fl/fl mice. Data represent the average 6 SEM (n = 3–5). *p , 0.05, **p , 0.01, ***p , 0.001.

pro-B cells from Mx1-Srg3fl/fl mice failed to upregulate IL-7Ra To analyze the responsiveness of pro-B cells to IL-7 signaling, expression on the surface, especially at the early pro-B cell stage, pro-B cells that were isolated from pIpC-treated control or Mx1- whereas the control pro-B cells showed a high level of expression Srg3fl/fl mice were cultured on OP9 stromal cells with IL-7. Srg3- of IL-7Ra (Fig. 6A, 6B). In addition, we found that the Srg3- deficient pro-B cells were severely reduced on day 3, and fewer deficient pro-B cells showed a much higher frequency of Annexin pro-B cells were observed on day 6 after culturing compared with V+ apoptotic cells compared with the control pro-B cells (Fig. control mice (Fig. 7A). In addition, Srg3-deficient pro-B cells 6C). These results indicate that pro-B cells lacking Srg3, espe- showed a much higher frequency of Annexin V+ cells on day 3 cially early pro-B cells, are more susceptible to apoptosis, which of in vitro culture (Fig. 7B). Srg3-deficient pro-B cells also may explain, at least in part, the severely reduced numbers of early showed a significant reduction in cell proliferation compared with pro-B cells in the bone marrow of Mx1-Srg3fl/fl mice. Notably, the control pro-B cells, as measured by BrdU incorporation after pro-B cells purified from Mx1-Srg3fl/fl mice showed higher levels 3 d of in vitro culture of the pro-B cells (Fig. 7C). These results of proapoptotic Bim expression and lower levels of antiapoptotic indicate that Srg3-deficient pro-B cells were defective in survival Bcl-xL expression (Fig. 6D). as a result of the impairment in IL-7 signaling. The Journal of Immunology 3797

FIGURE 4. Srg3/mBaf155 is essential for the development of pro-B cells. (A) Flow cytometry of B cell subsets of pro-B (IgM2 B220+CD43+), pre-B (IgM2B220+CD432), immature B (IgM+B220+), and mature recir- culating B (IgM2B220high) cells. Numbers indicate the percentage of B cell subsets. (B)

Frequency of subsets, which are described in Downloaded from (A). The horizontal lines show the average percentages. (C) The numbers of cells in the B lineage subsets, as described in (A). Data represent the average 6 SEM of ﬁve to seven independent experiments. (D) Expression of CD19, BP-1, and HSA (CD24) on gated + 2 + B220 IgM CD43 pro-B cells (upper panel). http://www.jimmunol.org/ Pre–pro-B (Fr. B; B220+IgM2CD43+BP-12 HSA+) cells were analyzed for CD19 expression (lower panel). Numbers indicate the percentage. (E) Frequencies of pro-B cell fractions, including Fr. A, Fr. B, and Fr. C, calculated from total bone marrow cells. The horizontal lines show the average percentage. (F) Numbers of subsets, as described in (E). Data represent the average 6 SEM (n = 9–12 by guest on September 26, 2021 mice/group). (G) Flow cytometry of AA4.1 expression (left panel) and percentage of AA4.1+ cells (right panel) on gated pre–pro-B (Fr. A) cells. Individual symbols represent one mouse; the horizontal lines show the mean percentages (n = 5). *p , 0.05, **p , 0.01, ***p , 0.001.

Impaired B lineage-specific gene expression and VDJ analyzed gene expressions in PD36 cells ectopically transduced rearrangement in Srg3/mBaf155-deficient pro-B cells with control or Srg3-shRNAs. We found that the expression of We next examined the expression of genes that are critical for Ebf1 and its target genes was decreased in the absence of Srg3 (Fig. early B cell development in pro-B cells isolated from pIpC- 8C). Brg1-deficient B cells also showed a reduction in the ex- treated control and Mx1-Srg3fl/fl mice. Srg3-deficient pro-B cells pression of B lineage-specific genes (Fig. 8D), which strongly expressed much less E47 (Tcfe2a)andEbf1,aswellastheir indicated that Srg3-deficient pro-B cells fail to express genes that downstream target genes, such as Pax5, Il7ra, Cd79a, Cd79b, are crucial for B cell development. Igll1, Vpreb1, Rag1,andRag2, than did the control pro-B cells In accordance with the reduced expression of Rag1 and Rag2 (Fig. 8A, 8B). However, it is possible that the diminished ex- in Srg3-deficient CLPs and pro-B cells, the expression of the pression of B lineage-specific genes in Srg3-deficient pro-B cells intracellular m-H chain in Srg3-deficient pro-B cells was much is due to fewer early pro-B cells. To rule out this possibility, we lower than in the control pro-B cells (Fig. 8E). To determine 3798 ROLE OF BAF COMPLEX IN EARLY B CELL DEVELOPMENT

FIGURE 5. Impairment in pro-B cell development in the absence of Brg1.(A) Schematic representations of retroviral constructs for shRNA targeting Brg1. Empty control (MSCV–LTRmiR30–PIG vector) and Brg1-shRNA virus express GFP only or both GFP and shRNA targeted to Brg1 transcripts. (B) Reduction in BRG1 expression by shRNA-Brg1 retrovirus. NIH3T3 cells were transduced with control or shRNA- Brg1 viruses, and GFP+ cells were sorted by ﬂow cytometry. Whole-cell extracts were isolated from the transduced cells, and

SRG3 and BRG1 proteins were quantiﬁed Downloaded from by immunoblotting. b-Actin served as a loading control. One of two independent experiments is shown. (C) Flow cytometric analysis of bone marrow B cells (B220+ CD19+) from control and Brg1-deﬁcient chimeras. Numbers indicate the percentage of populations (left panel); summary of the http://www.jimmunol.org/ percentage of B cells (right panel). Indi- vidual symbols represent one mouse; the horizontal lines show the mean percentage (n = 3). (D) Flow cytometry of B cell subsets of IgM2B220+, IgM2B220+, and IgM2B220hi cells. Numbers indicate the percentage of B cell subsets (left panel). Summary of the percentage of subsets de-

scribed above (right panel). The horizontal by guest on September 26, 2021 lines show the mean percentage (n = 3). (E) Expression of CD43 on gated B220+IgM2 B cells and of CD19 on gated B220+IgM2 CD43+ pro-B cells was analyzed to dis- criminate the pre–pro-B fractions. Numbers represent the percentage of each subset. Data are representative of three independent experiments (left panel). Summary of the percentage of subsets described above (right panel). The lines show the mean percentage (n = 3). *p , 0.05, **p , 0.001.

whether the Srg3-deficient pro-B cells failed to undergo V(D)J and the distal VHJ558 gene were also decreased in Srg3-deficient and DJ rearrangement of the IgH (Igh)genes,weanalyzedthe pro-B cells compared with the control pro-B cells (Fig. 8G). genomic DNAs isolated from pro-B cells obtained from control These results indicate that the Srg3-deficient pro-B cells had and Mx1-Srg3fl/fl mice by semiquantitative PCR, followed by defects in the V(D)J rearrangement of the Igh genes. This is Southern blotting. The recombination frequencies of the proxi- consistent with impairment of IL-7R signaling, which is required mal VH locus, including VH7183 and VHQ52, as well as the more for the regulation of VH gene rearrangement, in pro-B cells from fl/fl distant VH locus, including VHGam3.8, VH3609 and VHJ558, Mx1-Srg3 mice. were reduced in Srg3-deficient pro-B cells compared with control pro-B cells. Furthermore, the incidence of rearrangement of The core components of the SWI/SNF-like BAF complex directly bind to the promoter of Il7ra and Ebf1 DH-JH wasalsomuchlowerinSrg3-deficient pro-B cells than control pro-B cells (Fig. 8F). Consistent with the genomic DNA Our results suggest that defects in the expression of B lineage- analysis, the rearranged transcripts of the proximal VH7183 gene specific genes, including Ebf1 and IL-7Ra,onthesurfaceof The Journal of Immunology 3799 Downloaded from http://www.jimmunol.org/

FIGURE 6. Srg3/mBaf155 regulates pro-B cell survival via regulation of IL-7Ra expression on the surface. (A) IL-7Ra expression on pro-B (left panel), pre–pro-B (Fr. A) (middle panel), and early pro-B (Fr. B) (right panel) cells from control and Mx1-Srg3fl/fl mice. (B) Mean fluorescence intensity (MFI) of IL-7Ra expression on pro-B cells from control and Mx1-Srg3fl/fl mice. Data represent the average 6 SEM (n = 4). (C) Expression of Annexin V on pro-B (B220+CD43+IgM2) cells from control and Mx1-Srg3fl/fl mice. Numbers represent the percentage of Annexin V+ cells. (D) Quantitative real-time PCR analysis of gene expressions of Bim-EL, Bcl-xL, and Mcl-1 in pro-B cells purified from control and Mx1-Srg3fl/fl mice by flow cytometry. Data represent the average 6 SEM (n = 3–6 mice/group). **p , 0.01. by guest on September 26, 2021 pro-B cells led to the impairment of pro-B cell development in Srg3 and suggest that it requires the SWI/SNF-like BAF complex Mx1-Srg3fl/fl mice. We confirmed reduction of the Ebf1 transcript for its full functional activity. We also found that SRG3 and in PD36 cells transfected with a small interfering RNA duplex BRG1 bind to the proximal promoter regions of Il7ra in PD36 targeting Srg3 or Brg1 transcripts (Fig. 9A). It is known that the cells and primary pro-B cells (Fig. 9G, 9H). These results col- expression of Ebf1 is controlled through two other promoters lectively indicate that the SWI/SNF-like BAF complex is re- (Fig. 9B): a distal promoter that is regulated by E2A and a quired for the transcriptional activation of Ebf1 and Il7ra in pro- proximal promoter that is regulated by PU.1 and Pax5 (40). B cells. Previous studies showed that PU.1 or Pax5 can cooperate with the BAF complex to activate target genes in various conditions (41– Discussion 44). These results led us to investigate whether SRG3 directly The development of B lineage cells in the bone marrow occurs in regulates the expression of Ebf1 at the proximal region. To test a stepwise manner with critical checkpoints. The rearrangement the potential binding of SRG3, we used ChIP analysis with cross- of Ig genes and the proper expression of B lineage-specific genes linked chromatin extracted from PD36 cells. We found that im- are required for B cell commitment, survival, and proliferation. munoprecipitation with SRG3-specific Abs, but not isotype- Epigenetic changes in the chromatin structure are essential for control Abs, was enriched in the proximal promoter regions controlling the transcriptional regulation of these genes. In (Fig. 9C). BRG1 was also recruited to the same region (Fig. 9C). addition, the chromatin-remodeling complexes cooperate with In addition, ChIP analysis using primary pro-B cells showed the sequence-specific transcription factors to promote the expres- binding of SRG3 and BRG1 to the proximal promoter of the Ebf1 sion of several genes in many cell-specification events (17, 18). gene (Fig. 9D). The forced expression of Ebf1 in LSK cells pu- In this study, we demonstrated that Srg3/mBaf155, a core rified from pIpC-treated Mx1-Srg3fl/fl mice partially rescued the subunit of the SWI/SNF-like BAF complex, is essential for generation of B cells in vitro, whereas the generation of B cells normal B cell development. Our data indicate that the BAF was absent in Srg3-deficient LSKs that were ectopically trans- complex plays a pivotal role at both the CLP stage and the duced with control vectors (0.3937 6 0.2889% versus 3.117 6 transition from pre–pro-B to early pro-B cells by regulating the 0.5429% for B220+CD19+ cells) (Fig. 9E). However, ∼30% of expression of the B lineage-specific genes, such as Ebf1 and the LSKs obtained from control mice differentiated into B cells Il7ra. (Fig. 9E). Although ectopic expression of Ebf1 produced B cell Ebf1-deficient CLPs showed defects in the expression of B progenitors, the recovery of the cells was very inefficient in the lineage-specific genes, although the numbers of CLPs are com- absence of Srg3 (Fig. 9F). These results indicate that Ebf1 is very parable between WT and Ebf12/2 mice (37). E47-deficient mice inefficient in activating B lineage-specific genes in the absence of showed severe reduction in AA4.1hi CLPs (36). Notably, the 3800 ROLE OF BAF COMPLEX IN EARLY B CELL DEVELOPMENT

mice. The first defect is the failure in cell survival and proliferation via IL-7 signaling. We have clearly shown that the Srg3- deficient pro-B cells, especially early pro-B stage cells, failed to express IL-7Ra on the surface, which resulted in more apoptosis and less proliferation of pro-B cells, even in the presence of IL-7. In addition, Srg3-deficient pro-B cells showed a higher expression of Bim and a significantly lower expression of Bcl-xL relative to the control mice. These results are consistent with the decreased expression of IL-7Ra on the surface, which induces Bcl-xL expression after the activation of Stat5 by Jak-mediated phosphorylation (45). Notably, the development of FO B cells was severely impaired by Srg3 deletion, whereas MZ B cells constituted a much higher proportion in the splenic B cell population in the absence of Srg3. These results are consistent with the phenotype in Il7ra2/2 mice, in which the peripheral B cell defects are most apparent in the FO B cell compartment (46). Moreover, it was shown that the FO B cells increase in number, whereas the proportion of MZ B cells decreases in IL-7 trans- genicmice(47). Downloaded from The second defect in Srg3-deficient pro-B cells is the impaired expression of B lineage-specific genes, such as E47 (Tcfe2a), Ebf1,andPax5. Our data also demonstrated that Srg3-deficient pro-B cells expressed less Rag1 and Rag2, which led to the failure of V(D)J rearrangement of the Igh genes and the expression of the FIGURE 7. Srg3-deficient pro-B cells are defective in survival in the cytoplasmic m-chain. Moreover, the reduced expression of the http://www.jimmunol.org/ presence of IL-7. (A) Flow cytometry of residual B220+ pro-B cells pu- downstream target genes of E2A and Ebf1, such as Il7ra, Cd79a, fl/fl rified from pIpC-treated control or Mx1-Srg3 mice 6 d after in vitro Cd79b, Igll1,andPax5, was observed in the absence of Srg3. B culture with OP9 stromal cells. ( ) Expression of Annexin Von pro-B cells Proper functioning of E2A or Ebf1 is required during B lineage from control and Mx1-Srg3fl/fl mice 3 d after in vitro culture (left panel). differentiation. The loss of either E2A or Ebf1 results in a devel- Numbers represent the percentage of Annexin V+ cells. Summary of the percentage of Annexin V+ cells from control and Mx1-Srg3fl/fl mice (right opmental block at the early stage of B cell development (12–14). panel). Each symbol represents one mouse; the horizontal lines show the The recombination of DH-JH segments, as well as the expression mean percentage of Annexin V+ cells (n = 4 for control and n = 6 for Mx1- of B lineage-specific genes, is defective in the absence of either fl/fl C Srg3 mice). ( ) Flow cytometry of the proliferation of pro-B cells, using E2A or Ebf1. Unlike E2A-deficient mice, which show significantly by guest on September 26, 2021 BrdU staining (left panel), purified from pIpC-treated control and Mx1- lower numbers of CLPs and LMPPs (13), Ebf1-deficient mice Srg3fl/fl mice by flow cytometry and then cultured for 3 d on OP9 stromal have normal numbers of CLPs and pre–pro-B cell populations cells with IL-7. Numbers indicate the percentage of BrdU+ cells. Right + (14, 32). The overt characteristics of pro-B cell development panel, Average frequency of BrdU cells. Error bars indicate SEM of three blockade and impaired expression of B lineage-specific genes in independent experiments. 2 2 CLPs in Srg3-deficient mice closely resemble those in Ebf1 / mice. Therefore, a decreased expression of Ebf1 itself and/or surface expression of AA4.1 on CLPs was significantly reduced in downstream target genes is a plausible explanation for the com- the absence of Srg3, whereas Flt3 expression remained largely promised B lineage differentiation potential in lymphoid progen- unchanged. It was noted that AA4.1hi CLPs have the ability to itors and the blockade of early B cell development in the absence give rise to B and T cells, but not myeloid lineage cells, both of Srg3. in vivo and in vitro (31). Consistent with the defects in the ex- In agreement with the finding that the expression of Ebf1 and its pression of AA4.1 on CLPs, Srg3-deficient CLPs displayed de- downstream target genes was reduced in CLPs and pro-B cells creased expression of Ebf1 and its downstream target genes, such obtained from Srg3-deficient mice, our results also provided ev- as Cd79a, Cd79b, Rag1, Rag2, and FoxO1, which are associated idence that the expression of Ebf1 was directly mediated by the with early B cell development (31–34, 37). Although we cannot SWI/SNF-like BAF complex. SRG3 and BRG1 were found to officially exclude the possibility that Srg3 directly regulates the bind to the proximal promoter regions of the Ebf1 gene in pro- expression of AA4.1, it is unlikely because there are still AA4.1+/ B cells. Although the relative activity of the distal promoter of med subsets in CLPs in Srg3-deficient mice. AA4.1+ pre–pro- Ebf1 (Ebf1-a) is higher than the activity of the proximal promoter B cells, but not AA4.12 cells, could develop into B220+CD43+ (Ebf1-b) in the PD36 cell line, Ebf1-b transcript is predominantly pro-B cells (7). Our data showed that Srg3-deficient pre–pro- expressed in all B lineage cells, especially in CLP and pro-B cells B cells completely lack AA4.1+ population, although the number (the relative abundance of Ebf1-b mRNA is .100-fold higher of pre–pro-B cells was normal in Srg3-deficient mice. Therefore, than that that of Ebf1-a) (40). Moreover, it was previously re- the pre–pro-B cells found in Srg3-deficient mice could not gen- ported that recruitment of SWI/SNF-like BAF complex is re- erate early pro-B cells, and these defects might be due to the quired for the functional activity of Pax5, and the expression 2 2 impairment of the expression of B lineage-specific genes at the of Ebf1-b was reduced in Pax5 / pro-B cells, whereas the ex- stage of CLPs in the absence of Srg3. Taken together, this appears pression of Ebf1-a was comparable (40–43). Therefore, it appears to result in the severe reduction in the number of early pro-B cells that the SWI/SNF-like BAF complex may act cooperatively with in these mice. Pax5 to activate the expression of Ebf1 through the proximal Two major defects were observed in pro-B cells, consisting of promoter. Thus, in the absence of Srg3, Ebf1-b expression, which pre–pro-B, early pro-B, and late pro-B cells, from Srg3-deficient is reported to be transcribed by the proximal promoter, would be The Journal of Immunology 3801 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 8. The expression of B lineage-specific genes and rearrangement of IgH gene were defective in Srg3/mBaf155-deficient pro-B cells. (A) Quantitative real-time PCR analysis of expression of Srg3 and B lineage-specific genes in purified pro-B cells from control and Mx1-Srg3fl/fl mice. Data represent the average 6 SEM (n = 6). (B) Quantitative real-time PCR analysis of gene expression involved in pre-BCR signaling and Igh recombination. Data represent the average 6 SEM (n = 6). (C) Semiquantitative PCR analysis of expression of B lineage-specific genes in Srg3-deficient PD36 B cells. Expression of b-actin was used as a loading control. (D) Semiquantitative PCR analysis, described above, of Brg1-deficient PD36 B cells. (E) Flow cytometric analysis of the expression of intracellular m-chain in pro-B cells from the bone marrow of control and Mx1-Srg3fl/fl mice. Numbers indicate the + percentage of m-chain cells. (F) Southern blot analysis of genomic DNA from pro-B cells for VH7183-, VHJ558-, VHQ52-, VHGam3.8-, or VH3609-DJH3 and DH-JH3 rearrangement. Three-fold serial dilutions were analyzed, and input DNAs were normalized by the PCR product of IL-4 locus hypersensitivity site 3 (HSS3). Data are representative of three independent experiments. (G) Semiquantitative PCR analysis of transcripts of rearranged VH7183 or VHJ558 to DJH3. Expression of b-actin was used as a loading control. Wedges in (F), (G) indicate 3-fold serial dilution of input DNAs. Data are representative of two independent experiments. ***p , 0.001.

significantly decreased, and this will affect the early B cell de- of SWI/SNF activity in Srg3-deficient mice. Therefore, we be- velopment. lieve that our results reflect the requirement of SWI/SNF for the Enforced expression of Ebf1 in Srg3-deficient LSK cells res- functional activity of Ebf1, which is consistent with previous cued, although very inefficiently, the generation of B cells in vitro reports (41–43). (GFP2 control: 0.3937 6 0.2889% versus GFP+ Ebf1:3.1176 In summary, our results collectively establish the critical func- 0.5429% for B220+CD19+ cells and control LSK: 28.57 6 tions of the SWI/SNF-like BAF complex in the process of early 1.489%). Therefore, enforced expression of Ebf1 drives, with priming of B lineage specification. The functional requirement for very low efficiency (∼10% of control), the recovery of B cell the BAF complex at both the CLP stage and the transition from pre– progenitors that lack SWI/SNF-like BAF complex. This very pro-B to early pro-B cells is correlated with defects in the ex- low efficiency of the recovery appeared to be due to the absence pression of B lineage-specific genes, such as Ebf1, Il7ra, Tcfe2a, 3802 ROLE OF BAF COMPLEX IN EARLY B CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 9. The SWI/SNF-like BAF complex binds to the promoter regions of Ebf1 and Il7ra.(A) Semiquantitative PCR analysis of Ebf1 transcript level in PD36 cells transfected with small interfering RNA duplexes targeting GFP (si-GFP), Brg1 (si-Brg1), or Srg3 (si-Srg3). Data are representative of two independent experiments. Expression of gapdh served as a loading control. (B) Schematic representation of Ebf1 promoter regions and PCR primer sets for ChIP were depicted by arrows (P1–P4). (C) ChIP analysis of the binding of SRG3 and BRG1 to the Ebf1 proximal promoter locus on cross-linked chromatin from PD36 B cells, immunoprecipitated with anti-SRG3, anti-BRG1, or isotype control Ab (IgG1), followed by PCR amplification of input DNA or immunoprecipitated DNA with specific primers for the proximal promoter region. PCR product of gapdh intron region was used as a negative control. (D) ChIP analysis of cross-linked chromatin from B220+CD43+IgM2 pro-B cells, assessed as described in (C). PCR product of gapdh intron region served as a negative control, and immunoprecipitated chromatin with anti-H3K4me3 was used as a transcriptional activation marker. Shown is a representative of two independent experiments. (E) Flow cytometric analysis of differentiation of B220+ CD19+ B cells from control LSKs or Srg3-deficient LSK cells transduced with vectors expressing empty or Ebf1. Numbers indicate the percentage of B220+ CD19+ B cells. (F) Summary of B220+ CD19+ B cells differentiated from control LSKs or Srg3-deficient LSK cells transduced with vectors expressing empty or Ebf1. Data represent the average 6 SEM (n = 3). (G) ChIP analysis of binding of SRG3 and BRG1 to the Il7ra promoter locus on cross-linked chromatin from PD36 B cells, immunoprecipitated with anti- SRG3, anti-BRG1, or isotype control Ab (IgG1), followed by PCR amplification of input DNA or immunoprecipitated DNA with specific primers for proximal promoter region. PCR product of gapdh intron region was used as a negative control, and enriched fragment immunoprecipitated with anti-PU.1 was used as a positive control. (H) ChIP analysis of cross-linked chromatin from B220+CD43+IgM2 pro-B cells, assessed as described in (F). Data are representative of two independent experiments. *p , 0.05, ***p , 0.001. and Pax5, and in the V(D)J rearrangement of the Igh locus in the epigenetic remodeling of chromatin is essential for the sequential absence of Srg3. Thus, our study suggests that proper control of steps of early B cell development. The Journal of Immunology 3803

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