Identification of Pax5 Target in Early Differentiation Clare Pridans, Melissa L. Holmes, Matthew Polli, James M. Wettenhall, Aleksandar Dakic, Lynn M. Corcoran, Gordon This information is current as K. Smyth and Stephen L. Nutt of September 26, 2021. J Immunol 2008; 180:1719-1728; ; doi: 10.4049/jimmunol.180.3.1719 http://www.jimmunol.org/content/180/3/1719 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identification of Pax5 Target Genes in Early B Cell Differentiation1

Clare Pridans,2,3*† Melissa L. Holmes,2* Matthew Polli,* James M. Wettenhall,* Aleksandar Dakic,* Lynn M. Corcoran,* Gordon K. Smyth,* and Stephen L. Nutt4*

The Pax5 is essential for B cell commitment in the mouse, where it represses lineage-inappropriate expression while simultaneously activating the B cell program. In this study we have performed a global gene expression screen of wild-type and Pax5-deficient pro-B cells in an attempt to identify the crucial Pax5 targets in early B lym- phopoiesis. These studies have identified 109 Pax5 targets comprising 61% activated and 39% repressed genes. Interestingly, Pax5 directly regulates the genes encoding a number of transcription factors that are required at the pre-B cell stage of differentiation, including Irf8, Spib, and Ikzf3 (Aiolos), suggesting that a key function of Pax5 is to activate secondary transcription factors that

further reinforce the B cell program. Pax5 is also required for the expression of many genes known to be involved in adhesion and Downloaded from signaling, indicating that Pax5 modulates the homing and or migration properties of B cell progenitors. Finally, Pax5 also represses a cohort of genes that are involved in multiple biological processes, many of which are not typically associated with B cells. These include the repression of the adhesion molecule Embigin, which is expressed in bone marrow progenitors, T cells, and myeloid cells but is specifically repressed by Pax5 in B cells. The Journal of Immunology, 2008, 180: 1719–1728.

he B lymphocytes are produced in a stepwise process that they are not committed to the B cell lineage and are able to http://www.jimmunol.org/ from self-renewing hemopoietic stem cells (HSCs)5 in the differentiate into virtually all hemopoietic cell lineages in vitro and T fetal liver and postnatal bone marrow (BM). In recent in vivo (7–10). years it has become apparent that this process is controlled by a Pax5 promotes B lymphopoiesis by activating B cell-specific complex transcription factor network that both activates lineage- genes such as those involved in pre-BCR signaling, including specific gene expression (lineage specification) and restricts the Cd19 (11), Cd79a (mb-1) (4, 12), and Blnk (13), as well as Igll5 differentiation options of HSCs and their progeny (lineage com- and VpreB1 (14). Although the inability to express the pre-BCR mitment) (1–3). Although there has been extensive analysis of the was potentially the cause of the developmental block in the ab- transcription factors that regulate the initial steps in B lymphopoi- sence of Pax5, the introduction of functionally rearranged Igh and by guest on September 26, 2021 esis, relatively little is known about the molecular targets of these chimeric Igh-Ig␤ transgenes into the Pax5 mutant background was factors that ultimately mediate the commitment process. unable to progress B cell development beyond the early pro-B cell The transcription factor Pax5 is essential for B lymphopoiesis, stage (15). Pax5 also functions to repress genes whose expression as development is arrested at an early pro-B cell stage in the BM is not usually associated with the B cell program. The Pax5-de- Ϫ/Ϫ of Pax5-deficient mice (4, 5). These Pax5 pro-B cells can be pendent repression of the csf1r and Notch1 genes illustrates at the propagated in vitro in the presence of IL-7 and stromal cells and molecular level how developmental options are suppressed in maintain an early B cell phenotype characterized by the expression committed B lymphocytes, as these cells are no longer responsive ␭ of B cell-specific transcripts such as Cd79b (B29), Igll5 ( 5), and to the myeloid cytokine M-CSF or to the -inducing Notch1 VpreB1 and D-J recombination events at the Igh (4, 6). Pax5- ligand Delta-like 1 (7, 16, 17). Pax5 also functions to repress genes deficient pro-B cells, however, display a remarkable phenotype in associated with multipotency such as Flt3, which, although re- quired for early hemopoiesis, must be silenced by Pax5 to allow B *The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia; and lymphopoiesis to proceed (18). A more global approach to iden- † University of Western Sydney, Richmond, Australia tifying target genes using cDNA microarray technology has con- Received for publication September 4, 2007. Accepted for publication November firmed that many Pax5-repressed genes are normally expressed in 28, 2007. non-B cell lineages and interestingly found that a number of those The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance are reactivated during the physiological down-regulation of Pax5 with 18 U.S.C. Section 1734 solely to indicate this fact. during differentiation, whereas many B cell-specific 1 This work was supported by a Pfizer Australia Research Fellowship (to S.L.N.), and genes are positively regulated by Pax5 (19, 20). the National Health and Medical Research Council of Australia. As an alternative approach to identify potential Pax5 target 2 C.P. and M.L.H. contributed equally to this work. genes, we performed a screen of a mature B cell cDNA microarray 3 Current address: Cambridge Institute for Medical Research, Department of to compare gene expression between wild-type and Pax5Ϫ/Ϫ pro-B Haematology, Hills Road, Cambridge CB2 OXY, U.K. cells. This screen has resulted in the identification of Ͼ100 genes 4 Address correspondence and reprint requests to Dr. Stephen L. Nutt, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, representing both Pax5-activated and -repressed targets, many of Australia 3050. E-mail address: [email protected] which were not detected in the previous studies. These genes are 5 Abbreviations used in this paper: HSC, hemopoietic stem cell; BM, bone marrow; known or predicted to perform a diverse range of functions within CLP, common lymphoid progenitor; EMB, Embigin; ER, ; NIA 15k, the cell and highlight the dual function of Pax5 to repress inap- 15,000 clone mouse cDNA library of National Institute of Aging; Sdc4, syndecan-4. propriate gene expression while further activating essential com- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 ponents of the B cell program.

www.jimmunol.org 1720 GENE REGULATION BY Pax5

Table I. Oligonucleotide primer sequences used in semiquantitative RT-PCR analysis of potential Pax5 target genesa

5Ј Primer Sequence 3Ј Primer Sequence Gene (5Ј33Ј) (5Ј33Ј)

Ikzf3 ATGACAACAGCAGACCAACCAG TGTAGTTGGCATCGAAGCAGTG Arpc5l GAACGAGCCCAGGGTGTAGTCC TGGTCCATTGTCAGTCCCTTCTTC Blr1 GACATGGGCTCCATCACATA GTGCCTCTCCAGGATTACCA Cbfb GACCAGAGGAGCAAGTTCGAG GAGTTCTTCTTCGAGCCTCTTC Cd19 GAGAGGCACGTGAAGGTCATTG CATGGCTCTGAGCTCCAGTATC Tcfe2a TGGCACTTACAGTGGGACTTC ATGGAGACCTGCATCGTAGTTG Ebf1 ATGTTTGGGATCCAGGAAAGC CAGGGTTCTTGTCTTGGCCTT Emb TGTACACAGGGACCAACGGAGACG TGTTGCCCATTTTAGTTGTATTGA Ep400 GAGCTGGCTGACTTTATGGAAC GCTCCTTCCTCACATAAACAGG Flt3 GTGACTGGCCCCCTGGATAACGAG TCCAAGGGCGGGTGTAACTGAACT Frmd4b GGGCTCGAGGTGGCAAGTT CCAGTGGGGGTATGAGGTAGTTTA Gpx1 GGTTTCCCGTGCAATCAGTTCG GCCGCCTTAGGAGTTGCCAGAC Hprt GGGGGCTATAAGTTCTTTGC TCCAACACTTCGAGAGGTCC Lax1 GAACTCAGAGCCCAGCACTCGG GGAGGCAGAGTCAACGATGGAG Lcp1 TATCGGAGGTGGACAGAAGG ACCCTTGCTCCGATTTTTCT Lsp1 GAGAGTTCTCACCAAGCCAAAG TTCTGCTCCCACAGACTTTTCT Nfatc1 CCGATAGCACTCTGGACCTG GTAGCTGCACAATGGGGTGT Downloaded from Plcg2 GTGGAGACGAAGGCAGACAG CTGCAGGACGTAGCCTGTTC Pten GCTGAGAGACATTATGACACCG GCGCCTCTGACTGGGAATTGTG Spib GCTGGCTTCAAGCTCATGACAC TTGGCCTGTAGCACTTGAACGG Syvn1 GTGATGGGCAAGGTGTTCTT CACGGAGTGCAGCACATACT Tmsb10 GGCTCTTCCTCCACATCACGA AAGAAAACCGAGACGCAGGAGAAG Irf8 CAGGAGGTGGATGCTTCCATC GCACAGCGTAACCTCGTCTTC

Blnk CTGCCGCACCATCCCCACTAC GTCACAGGCGCCAGCATACCAG http://www.jimmunol.org/ Atp1b1 CGAGGCCTACGTGCTAAACAT GTATCCGCCCATCCCAAAGTA Ccnd3 CAGCGCTGCGAGGAGGATGTCTTC CACGGCAGCCAGGTCCCACTTGAG Sdc4 AGCCTCCCCGACGACGAAGAT ACGCCCGCCACCCACAACC Cd24a TTCCCCAAATCCAAGTAACG AACCTGTGCCCAATTTCAAGTG Snx2 TCGCAGAAGCCACAGAAGAGGT GAGCGGGTGGCACGATGTAAC Rbm3 AAACACATCAGACACAACAGAATA TGAGCTCCCAAGGTAGT Igf2 GCCCCTGAAAGCACCCATCC AGCCGTGAAAGCACCCATCC

Ϫ/Ϫ Materials and Methods Rag1 pro-B cell lines in a saturated loop design. The scanned microar- by guest on September 26, 2021 Mice ray images were quantified using SPOT image analysis software (www. hca-vision.com). Statistical analysis of the microarray data used the Bio- Pax5Ϫ/Ϫ (5) mice were maintained on a C57BL/6 background and geno- conductor software package LIMMA (www.bioconductor.org). Quality typed as described (4). assessment using MA plots resulted in three of the NIA 15k arrays being discarded due to apparent hybridization problems. Expression values were Cell lines and in vitro culture conditions print-tip loess normalized, with spot quality weights used to down-weight spots with unusually small or large scanned sizes (23). The duplicate spots Pro-B cell lines were derived and propagated as described (4). The Pax5- for each probe were combined using a common correlation algorithm that (ER) fusion (Pax5ER) and control ER retroviral uses both within and between array variability to assess statistical signif- Ϫ/Ϫ ␮ vectors were introduced into Pax5 pro-B cells and activated by 1 M icance (24). Clones were ranked for differential expression between ␤ Ϫ Ϫ ϩ ϩ -estradiol as previously reported (6). Some experiments were performed Pax5 / and Pax5 / using empirical Bayes modified t statistics (25). ␮ in the presence of 50 g/ml cycloheximide (Sigma-Aldrich). p values were adjusted for multiple testing using the Benjamini and Hochberg algorithm to control the false discovery rate. Raw data from the cDNA microarray NIA 15k arrays is available from the Gene Expression Omnibus (GEO) A B lymphocyte cDNA library was a generous gift from C. Estes and M. database (www.ncbi.nlm.nih.gov/projects/geo) as series GSE9345. Bento Soares of the University of Iowa (Iowa City, IA) (21). The microar- Identification of cDNA clones ray was prepared at the Australian Genome Research Facility (Melbourne, Australia), where the inserts from 10,000 randomly generated clones were Plasmid DNA from differentially expressed clones was prepared and the PCR amplified, purified, and spotted onto glass slides in duplicate. A sec- insert sequenced. Clones identities were determined using the basic local ond screen with an identical strategy used microarrays printed with the alignment search tool (BLAST). 15,000 clone mouse cDNA library of the National Institute on Aging (NIA 15k) (22). Total RNA was isolated from four pro-B cell lines (108 cells) RT-PCR using TRIzol (Invitrogen Life Technologies) and mRNA isolated using the Oligotex mRNA isolation kit (Qiagen). mRNA (4 ␮g) was reverse tran- Total RNA was isolated from sorted B cell populations using TRIzol (In- scribed using SuperScript II (Invitrogen Life Technologies) according to vitrogen Life Technologies) and reverse-transcribed with Moloney murine instructions from the manufacturer. The purified cDNA samples were la- leukemia virus reverse transcriptase (Promega) according to the manufac- beled with the fluorescent dye esters N-hydroxysuccinimide (NHS)-Cy3 turer’s instructions. cDNA was amplified using the gene-specific primers and NHS-Cy5 (Amersham Biosciences), and the microarrays were hybrid- listed in Table I. cDNA input was normalized to Hprt. ized for 16 h at 42°C with each of the fluorescently labeled probes in 25% Flow cytometry formamide, 5ϫ SSC, 0.1% SDS, 5 ␮g/ml Cot-1 DNA (Invitrogen Life Technologies), 10 mg/ml poly(A) (Sigma-Aldrich), and 10 mg/ml salmon The labeled mAbs CD157, CXCR5 (Blr1), and Syndecan-4 (Sdc4) were sperm DNA (Sigma-Aldrich). Slides were washed at room temperature purchased from BD Pharmingen. The mAbs against B220, CD19, Flt3 with 1ϫ SSC plus 0.2% SDS and then 0.1ϫ SSC plus 0.2% SDS followed c-Kit, CD24a, and Embigin (Emb) were purified and conjugated in our by two stringent washes (0.1ϫ SSC) before scanning on a GenePix 4000B laboratory. HSC, common lymphoid progenitor (CLP), and B cell popu- (Molecular Devices). For the B lymphocyte screen, nine arrays were used lations were sorted as previously described (26, 27). For flow cytometry, to compare one Pax5ϩ/ϩ and three Pax5Ϫ/Ϫ pro-B cell lines. For the single-cell suspensions were prepared from the BM of 12-day old mice and NIA15k screen, 12 arrays were used to compare Pax5ϩ/ϩ, Pax5Ϫ/Ϫ, and stained with the appropriate mAb in PBS containing 2% FCS. Biotinylated The Journal of Immunology 1721

Table II. List of potential Pax5-activated genesa

GenBank or RefSeq Fold Change Adjusted p Validation Accession No. Gene Description (Log2) t Statistic Value (Ref.)

NM_009844 Cd19 CD19 antigen Ϫ4.19 Ϫ46.4 5E-18 Known (11) NM_172656 Als2cr2 Amyotrophic lateral sclerosis 2 (juvenile) Ϫ2.39 Ϫ33.3 8E-16 In vitro region, candidate 2 NM_026386 Snx2 Sorting nexin 2 Ϫ1.87 Ϫ31.9 2E-15 In vitro AK037030 Prkd2 Protein kinase D2 Ϫ1.43 Ϫ31.2 2E-15 AK014486 Mlstd2 Male sterility domain containing 2 Ϫ1.24 Ϫ27.8 1E-14 NM_011417 Smarca4 SWI/SNF-related, matrix-associated, actin-dependent Ϫ1.01 Ϫ27.3 2E-14 Ex vivo regulator of , subfamily a, member 4 (Brg1) XM_283022 Ikzf3 IKAROS family zinc finger 3 (Aiolos) Ϫ2.13 Ϫ25.1 7E-14 Ex vivo NM_008131 Glul Glutamate-ammonia ligase (glutamine synthetase) Ϫ1.27 Ϫ23.9 2E-13 In vitro AK028838 Stk4 Serine/threonine kinase 4 Ϫ1.07 Ϫ23.3 2E-13 NM_207246 Rasgrp3 RAS, guanyl releasing protein 3 Ϫ1.83 Ϫ23.1 3E-13 NM_026444 Cs Citrate synthase Ϫ1.04 Ϫ22.8 3E-13 NM_178087 Pml Promyelocytic leukemia Ϫ1.76 Ϫ22.3 4E-13 BC037730 H3f3b H3 histone, family 3B Ϫ0.87 Ϫ21.3 1E-12 In vitro NM_016791 Nfatc1 Nuclear factor of activated T cells, cytoplasmic, Ϫ1.09 Ϫ21.0 1E-12 Ex vivo calcineurin-dependent 1 MMU87620 Spib Spi-B transcription factor (Spi-1/PU.1 related) Ϫ1.45 Ϫ21.0 1E-12 Ex vivo NM_011925 Cd97 CD97 antigen Ϫ1.20 Ϫ21.0 1E-12 NM_009846 Cd24a CD24a antigen (HSA) Ϫ1.77 Ϫ21.0 1E-12 Ex vivo Downloaded from NM_009763 Bst1 Bone marrow stromal cell antigen 1 (CD157 antigen) Ϫ1.39 Ϫ20.5 1E-12 Ex vivo NM_022309 Cbfb b Ϫ0.83 Ϫ20.4 1E-12 Ex vivo NM_009201 Slc1a5 Solute carrier family 1 (neutral amino acid Ϫ1.57 Ϫ20.3 1E-12 In vitro transporter), member 5 NM_028809 Arpc5l Actin related protein 2/3 complex, subunit 5-like Ϫ0.94 Ϫ20.1 2E-12 Ex vivo XM_204234 Cecr2 Cat eye syndrome chromosome region, candidate 2 Ϫ1.36 Ϫ20.1 2E-12 Ex vivo NM_198703 Wnk1 WNK lysine deficient protein kinase 1 Ϫ1.02 Ϫ19.8 2E-12 In vitro NM_001025572 Ankrd12 Ankyrin repeat domain 12 Ϫ1.02 Ϫ19.8 2E-12 http://www.jimmunol.org/ NM_017464 Nedd9 Neural precursor cell expressed, developmentally Ϫ1.24 Ϫ19.6 3E-12 down-regulated gene 9 NM_011548 Tcfe2a Transcription factor E2a Ϫ0.87 Ϫ19.4 3E-12 In vitro NM_008511 Lrmp Lymphoid-restricted membrane protein (Jaw1) Ϫ1.46 Ϫ19.2 4E-12 Ex vivo NM_001039519 Gtf2a2 General transcription factor II A, 2 Ϫ1.18 Ϫ19.2 4E-12 NM_144833 Zfp410 Zinc finger protein 410 Ϫ7.48 Ϫ17.7 1E-11 NM_133910 Tbc1d14 TBC1 domain family, member 14 Ϫ1.03 Ϫ17.6 2E-11 NM_016895 Ak2 Adenylate kinase 2 Ϫ0.84 Ϫ17.5 2E-11 AK157720 Bcl7a B-cell CLL/lymphoma 7A Ϫ1.23 Ϫ17.5 2E-11 NM_008142 Gnb1 Guanine nucleotide binding protein, b1 Ϫ0.66 Ϫ17.4 2E-11 AL772319 Pax5 Paired box gene 5 Ϫ1.76 Ϫ17.4 2E-11 NM_008031 Fmr1 Fragile X mental retardation syndrome 1 homolog Ϫ0.79 Ϫ17.0 2E-11 by guest on September 26, 2021 BC055044 Epb4.1l2 Erythrocyte protein band 4.1-like 2 Ϫ0.67 Ϫ16.5 4E-11 NM_001033293 Uap1l1 UDP-N-acetylglucosamine pyrophosphorylase 1-like 1 Ϫ1.62 Ϫ16.5 4E-11 NM_170588 Cpne1 Copine 1 Ϫ0.87 Ϫ16.4 4E-11 AK137389 Ebf1 Early B Cell Factor 1 Ϫ1.17 Ϫ16.2 5E-11 Ex vivo NM_031185 Akap12 A kinase (PRKA) anchor protein (gravin) 12 Ϫ1.70 Ϫ16.1 5E-11 NM_172514 Tmem71 Transmembrane protein 71 Ϫ0.99 Ϫ15.8 7E-11 NM_008321 Id3 Inhibitor of DNA binding 3 Ϫ1.07 Ϫ15.5 1E-12 Ex vivo NM_028780 Tm9sf1 Transmembrane 9 superfamily member 1 Ϫ0.90 Ϫ14.8 2E-10 AK150817 Pyhin1 Pyrin and HIN domain family member 1 Ϫ1.67 Ϫ14.7 2E-10 Ex vivo NM_019635 Stk3 Serine/threonine kinase 3 Ϫ1.08 Ϫ14.7 2E-10 NM_009727 Atp8a1 ATPase, aminophospholipid transporter (APLT), Ϫ1.16 Ϫ14.6 3E-10 class I, type 8A, member 1 NM_010731 Zbtb7a Zinc finger and BTB domain containing 7a (LRF) Ϫ1.77 Ϫ14.6 3E-10 NM_016856 Cpsf2 Cleavage and polyadenylation specific factor 2 Ϫ0.72 Ϫ14.5 3E-10 Ex vivo NM_027184 Ipmk Inositol polyphosphate multikinase Ϫ0.68 Ϫ14.5 3E-10 NM_172456 Endogl1 Endonuclease G-like 1 Ϫ1.10 Ϫ14.4 3E-10 NM_028769 Syvn1 Synovial inhibitor 1, synoviolin Ϫ0.68 Ϫ14.4 3E-10 Ex vivo NM_175048 Gimap4 GTPase, IMAP family member 4 Ϫ1.03 Ϫ14.3 4E-10 NM_001002846 Fbxl12 F-box and leucine-rich repeat protein 12 Ϫ1.04 Ϫ14.1 5E-10 NM_009009 Rad21 RAD21 Ϫ0.70 Ϫ13.8 7E-10 NM_010703 Lef1 Lymphoid enhancer binding factor 1 Ϫ1.88 Ϫ13.5 9E-10 Known (6) NM_133739 Tmem123 Transmembrane protein 123 Ϫ.077 Ϫ13.3 1E-09 NM_007655 Cd79a CD79A antigen (Iga, mb-1) Ϫ2.58 Ϫ13.3 1E-09 Known (12) NM_011671 Ucp2 Uncoupling protein 2 (mitochondrial, proton carrier) Ϫ1.23 Ϫ13.2 1E-09 AK153056 D3Ucla1 Unknown Ϫ0.63 Ϫ12.4 4E-09 AK032345 Tcf4 Transcription factor 4 (E2-2) Ϫ0.52 Ϫ7.56 4E-06 AK017215 5033414 Unknown Ϫ0.71 Ϫ7.35 6E-06 D02Rik AK050833 Clcn4Ϫ2 Chloride channel 4Ϫ2 Ϫ0.40 Ϫ1.98 1E-01

NIA 15k NM_009721 H3019E05 Atp1b1 ATPase, Naϩ/Kϩ transporting, b1 polypeptide Ϫ2.29 Ϫ16.0 9E-09 In vitro NM_008528 H3057F11 Blnk B-cell linker (BASH, SLP-65) Ϫ2.27 Ϫ10.2 3E-06 Known (13) NM_001081636 H3041D11 Ccnd3 Cyclin D3 Ϫ1.13 Ϫ10.5 2E-06 In vitro NM_016809 H3010F04 Rbm3 RNA binding motif protein 3 Ϫ0.91 Ϫ9.19 1E-05 In vitro NM_011521 H3138F12 Sdc4 Syndecan 4 (ryudocan) Ϫ1.02 Ϫ9.34 9E-06 Ex vivo NM_009261 H3141C09 Strbp Spermatid perinuclear RNA binding protein Ϫ1.03 Ϫ9.42 8E-06 In vitro

a Potential Pax5 activated genes as identified by cDNA microarray analysis of gene expression between Pax5-deficient and wild-type pro-B cells. Genes are listed in order of statistical significance (moderated t-statistic). Adjusted p values represent false discovery rate bounds. Validation was performed on either pro-B cell lines (in vitro) or on sorted B220ϩc-Kitϩ cell (ex vivo) from wild-type and Pax5Ϫ/Ϫ BM. Known target genes are indicated with the appropriate reference (Ref). The Pax5 transcript is truncated after 2inPax5Ϫ/Ϫ cells. Only those cDNAs identified from the NIA 15k array that were confirmed by RT-PCR are shown. 1722 GENE REGULATION BY Pax5

Table III. List of potential Pax5 repressed genesa

GenBank or RefSeq Log2-Fold Adjusted p Validation Accession # Gene Description Change t Statistic Value (Ref.)

NM_019391 Lsp1 Lymphocyte specific 1 2.71 33.9 7E-16 Ex vivo NM_008505 Lmo2 LIM domain only 2 3.30 23.5 2E-13 In vitro NM_007415 Parp1 Poly (ADP-ribose) polymerase family, member 1 1.94 22.4 4E-13 In vitro AK009211.1 Lcp1 Lymphocyte cytosolic protein 1 1.13 21.9 6E-13 Ex vivo NM_008160 Gpx1 Glutathione peroxidase 1 1.88 21.2 1E-12 Ex vivo NM_008533 Cd180 CD180 antigen (RP105) 1.45 19.6 3E-12 XM_127380.2 H2afy H2A histone family, member Y 1.66 19.4 3E-12 NM_008761 Fxyd5 FXYD domain-containing ion transport regulator 5 1.70 18.7 5E-12 In vitro NM_008960 Pten Phosphatase and tensin homolog 1.19 18.5 6E-12 Ex vivo NM_007551 Blr1 Burkitt lymphoma receptor 1 (CXCR5) 0.91 17.9 1E-11 Ex vivo NM_010581 Cd47 CD47 Ag 1.43 17.4 2E-11 NM_009741 Bcl2 B-cell leukemia/lymphoma 2 1.16 17.3 2E-11 NM_013472 Anxa6 Annexin A6 0.87 17.3 2E-11 NM_194052 Rtn4 Reticulon 4 1.03 17.3 2E-11 Ex vivo AK161168 Ttc3 Tetratricopeptide repeat domain 3 0.95 17.2 2E-11 AK076070.1 Csnk1a1 Casein kinase 1, ␣1 0.83 17.1 2E-11 NM_172842 Lax1 Lymphocyte transmembrane adaptor 1 1.55 16.5 4E-11 Ex vivo BC010489 Sept6 Septin 6 1.24 16.4 4E-11 Downloaded from AK088605 Ggta1 Glycoprotein galactosyltransferase ␣1,3 1.38 16.3 4E-11 XM_203393 Prr6 Proline-rich polypeptide 6 0.86 16.2 5E-11 NM_011972 Pol Polymerase (DNA directed), ␫ 0.78 16.2 5E-11 In vitro NM_172285 Plcg2 Phospholipase C, ␥2 1.28 15.9 7E-11 Ex vivo NM_008714 Notch1 Notch gene homolog 1 1.18 15.6 9E-11 Known (16) NM_016776 Mybbp1a MYB binding protein (P160) 1a 1.11 14.9 2E-10 Ex vivo

NM_134086 Slc38a1 Solute carrier family 38, member 1 0.66 14.4 3E-10 http://www.jimmunol.org/ NM_019993 Aldh9a1 Aldehyde dehydrogenase 9, subfamily A1 0.71 14.4 3E-10 NM_008774 Pabpc1 Poly A binding protein, cytoplasmic 1 0.74 14.2 4E-10 Ex vivo NM_009983 Ctsd Cathepsin D 0.65 14.0 5E-10 AK030569 Dynlt1 Dynein light chain Tctex-type 1 0.95 14.0 5E-10 NM_023326 Bmyc Brain expressed myelocytomatosis oncogene 0.99 13.8 6E-10 NM_026446 Rgs19 Regulator of G-protein signaling 19 0.95 13.7 7E-10 NM_022964 Lat2 Linker for activation of T cells family, member 2 0.88 13.7 7E-10 NM_025282 Mef2c Myocyte enhancer factor 2C 0.99 13.4 1E-09 AK146784 Mark4 MAP/microtubule affinity-regulating kinase 4 0.84 13.4 1E-09 BC104333 Serpinb1a Serine (or cysteine) peptidase inhibitor, 1.18 10.7 3E-08 In vitro clade B, member 1a by guest on September 26, 2021 NM_029337 Ep400 E1A binding protein p400 (mDomino) 0.25 5.27 3E-04 Ex vivo

NIA 15K NM_001039392 H3001H10 Tmsb10 Thymosin ␤ 10 1.65 14.0 4E-08 Ex vivo NM_010514 H3024B07 Igf2 Insulin-like growth factor 2 2.44 8.47 2E-05 In vitro NM_054040 H3132F10 Tulp4 Tubby like protein 4 1.42 9.19 1E-05 In vitro NM_010330 H3024E05 Emb Embigin 0.93 9.12 1E-05 Ex vivo NM_025378 H3107D05 Ifitm3 Interferon induced transmembrane protein 3 1.59 8.51 2E-05 In vitro NM_145148 H3057G09 Frmd4b FERM domain containing 4B 1.10 5.35 3E-03 Ex vivo NM_207652 H3066H08 Tsc22d1 TSC22 domain family, member 1 1.17 5.89 8E-04 Ex vivo

a Potential Pax5 repressed genes as identified by cDNA microarray analysis of gene expression between Pax5-deficient and wild-type pro-B cells. Genes are listed in order of statistical significance (moderated t statistic). Adjusted p values represent false discovery rate bounds. Validation was performed on either pro-B cell lines (in vitro) or sorted B220ϩc-Kitϩ cells (ex vivo) from wild-type and Pax5Ϫ/Ϫ BM. Known target genes are indicated with the appropriate reference (Ref). Additional cDNAs identified from the NIA 15k array are indicated. mAb were revealed by PE-streptavidin (SouthernBiotech). Cells were an- Aldrich). HRP was visualized with the 3-amino-9-ethylcarbazole substrate alyzed on a FACScan flow cytometer (BD Biosciences) and cell sorting kit (Vector Laboratories). was conducted on high-speed flow cytometers (Vantage SE DiVa; BD Biosciences). Dead cells were excluded by propidium iodide staining. Results Generation of anti-mouse Emb Ab Identification of Pax5 target genes using cDNA microarrays G7.43.1, the anti-Emb mAb, was generated by immunizing a rat with hu- To further our understanding of gene regulation by Pax5 in early man 293T cells infected with a retrovirus-expressing mouse Emb. Super- Pax5Ϫ/Ϫ B cell development, we performed cDNA microarray analysis on natants from hybridoma clones were screened against pro-B cells. Ϫ/Ϫ The specificity of G7.43.1 was determined by Western blot analysis. wild-type and Pax5 pro-B cells. As starting material we took advantage of the ability to grow pure populations of pro-B cells in Immunohistochemistry long-term cultures in the presence of IL-7 and a ST-2 stromal Tissues were embedded, stored, sectioned, and stained as described (28). layer. Previous analysis has confirmed that these pro-B cells retain Abs used were unlabeled anti-Emb and biotinylated anti-CD19. Anti- many of the features of the equivalent populations in vivo (4). The GL117 was an isotype control. Emb and GL117 were detected with mouse experimental setup involved a pairwise comparison of three anti-rat Abs conjugated to HRP (BD Pharmingen) and anti-CD19 with Ϫ/Ϫ ϩ/ϩ streptavidin-alkaline phosphatase (SouthernBiotech). Alkaline phosphatase Pax5 pro-B cells lines and a single wild-type control, SB was visualized with the Fast Blue kit (Vector Laboratories), with endog- (7). Hybridizations were performed on microarrays printed with enous phosphatases blocked by the addition of 2 mM levamisole (Sigma- two mouse cDNA libraries, a custom cDNA microarray consisting The Journal of Immunology 1723 Downloaded from http://www.jimmunol.org/

FIGURE 1. Expression of potential Pax5 target genes in pro-B cells in vitro. A, cDNA was prepared from established Pax5-deficient (Ϫ/Ϫ) and wild-type (ϩ/ϩ) pro-B cell lines, and transcripts of the indicated genes were analyzed by semiquantitative RT-PCR using 5-fold serial dilutions of the cDNA. B, Comparison of target gene expression in three independent FIGURE 2. Expression of potential Pax5 target genes in pro-B cells ex ϩ ϩ Pax5Ϫ/Ϫ pro-B cell lines. Genes were amplified using semiquantitative vivo. cDNA was prepared from sorted BM pro-B cells (B220 c-Kit )of RT-PCR. The cDNA input in A and B was normalized according to the the indicated genotype, and transcripts of the indicated genes were ana- expression of the Hprt gene. C, Flow cytometric analysis of CD24a, lyzed by semiquantitative RT-PCR using 5-fold serial dilutions of the CXCR5 (Blr1), and Emb expression in Pax5-deficient (Ϫ/Ϫ) and wild-type cDNA. The cDNA input was normalized according to the expression of the by guest on September 26, 2021 (ϩ/ϩ) pro-B cell lines. Genotypes are as indicated on the panels. Hprt gene. A, Potential Pax5-activated genes whose expression is higher in wild-type pro-B cells. B, Potential Pax5-repressed genes whose expression is higher in Pax5-deficient pro-B cells. C, Comparison of Tcfe2a expres- sion between in vitro propagated and ex vivo sorted pro-B cells. of 10,000 randomly selected cDNAs from an IgMϩ mature B cell library and the NIA 15k library (22) derived from diverse sources including embryonic, neural, and malignant samples. The combi- nation of these two libraries allowed us to maximize the identifi- target gene Cd19 (4, 11) which was the most differentially ex- cation of both B cell and non-B cell-specific Pax5 target genes. All pressed transcript on the B cell array (Table II). As expected Pax5 hybridizations were performed in duplicate with the Cy3 and Cy5 itself was also differentially expressed. Several other known Pax5 fluorochromes swapped between each experiment and with three targets, including Blnk (13), Lef1, Cd79a (6), Notch1 (16), Ikzf3 independent Pax5Ϫ/Ϫ pro-B cell lines. (29), and Ebf1 (29, 30), were also identified, confirming the va- The custom B cell microarray screens yielded a large number of lidity of the screen. Importantly, analysis of the expression of a clones with statistically significant differential expression in selection of the potential Pax5 target genes confirmed the differ- Ϫ Ϫ Pax5Ϫ/Ϫ (4919 probes at a false discovery rate of Ͻ5%). Approx- ential expression in three independent Pax5 / pro-B cell lines imately 200 of the most highly differentially expressed clones were (Fig. 1B and data not shown). sequenced and identified by database searches. As expected from Of the potential Pax5-activated genes, a striking 28% were nu- a library comprised of random clones, the differentially expressed clear involved in various aspects of transcription, 15% genes were represented by multiple cDNA clones (16 genes were were cell surface receptors, 15% were involved in intracellular present in two or three copies in the initial 200 clones). The NIA signaling pathways, and a further 15% were of unknown function. 15k microarray screens yielded another 1491 differentially ex- The remaining genes have been implicated in a variety of cellular pressed clones at a false discovery rate of Ͻ5%. Fifty-two of the functions such as metabolism, transport, and cytoskeletal integrity. most significant clones were sequence confirmed. Overall, ϳ60% The Pax5-repressed genes contained a relatively large proportion of the clones had reduced expression in Pax5Ϫ/Ϫ pro-B cells, rep- of cell surface (26%) and intracellular signal-transducing (17%) resenting potential Pax5-activated genes, and 40% were more proteins, with nuclear proteins (21%) and metabolic enzymes strongly expressed in the absence of Pax5, representing Pax5-re- (12%) making up a significant fraction of the clones. pressed genes. The sequence-validated candidates are shown in Tables II and III. Approximately 50% (54 of 109) has been con- Confirmation of the differential expression of firmed by independent methodologies as outlined in the Tables II Pax5-regulated genes in vivo and III and Figs. 1 and 2. This list includes a number of previously Although the pro-B cell culture system provided an abundant and identified Pax5 regulated genes, most notably the canonical Pax5 pure source of mRNA with which to conduct the array screening, 1724 GENE REGULATION BY Pax5

FIGURE 4. Direct regulation of Irf8, Ikzf3, Syvn1, and Spib by Pax5. RT-PCR analysis of gene expression in Pax5Ϫ/Ϫ pro-B cell lines comple- mented with a retrovirus expressing the Pax5-ER fusion protein or the ER alone. Cell lines were induced by 1 ␮M ␤-estradiol (E2) for8hinthe presence of absence of 50 ␮g/ml cycloheximide (CHX). cDNAs were nor- malized using Hprt. Induction of Cd19 was a positive control. Downloaded from expressing an inducible Pax5-ER fusion protein and subsequently proven using direct promoter analysis. To assess to what extent the differentially expressed genes identified here were direct Pax5 tar- gets, we used the Pax5-ER system to induce Pax5 activity in the presence and absence of the protein translation inhibitor cyclohex-

imide and assessed the expression of the relevant genes by RT- http://www.jimmunol.org/ PCR. In this manner the transcription factors Ikzf3 (encoding for Aiolos) and Spib, which play important roles at the pre-B and mature B cell stages of differentiation, as well as the E3 ubiquitin ligase Syvn1 appear to be direct Pax5 targets (Fig. 4). Interestingly, Irf8 (Icsbp), which was identified using a candidate approach by us FIGURE 3. Cell surface expression of potential Pax5 target genes ex and others (20), also appeared to be directly regulated by Pax5 vivo. A, BM cells from 2-wk-old mice of the indicated genotype were (Fig. 4). Despite its reduced expression in the absence of Pax5, we stained with B220 and c-Kit to identify the pro-B cell population (boxed). were unable to induce Ebf1 using this system.

Number indicates the percentage of total BM cells. B, Cells gated as in A by guest on September 26, 2021 were analyzed for expression of CD157, Emb, Sdc4, and CD24a. CD19 Emb is broadly expressed in hemopoiesis and repressed by and Flt3 were included as known targets, being activated and repressed by Pax5 upon B cell commitment Pax5, respectively. Wild-type cells are indicated by a solid line and Pax5- deficient cells by a dotted line. One gene that was strongly and specifically expressed in Pax5- deficient pro-B cells in vitro and in vivo was Emb (Fig. 1). Emb is a transmembrane glycoprotein belonging to the Ig superfamily that analysis of differentially expressed genes in pro-B cells in vivo is expressed in the early stages of mouse embryogenesis and is provided an important additional validation to exclude any differ- postulated to mediate -cell-substratum adhesion (31). ences between the genotypes that resulted from differences in the Analysis of Emb during normal hemopoiesis revealed expression ability of the cells of the two genotypes to be propagated in culture. in T cell and myeloid cells but not B cells (Fig. 5A and data not Indeed Lmo2, which was identified as a Pax5-repressed gene from shown). Specific analysis of progenitors showed that Emb was the B cell array and confirmed as being differentially expressed in strongly expressed in HSC- and CLP-enriched populations and pro-B cell cultures, was not differentially regulated on ex vivo later repressed in pro- and pre-B cells (Fig. 5A). This was a vir- isolated pro-B cells (data not shown). Interestingly, the expression tually identical expression pattern to that of Flt3, which we have of Tcfe2a, which encodes the essential regulator of early B cell shown is directly repressed by Pax5 upon B cell lineage commit- development, E2A, was also reduced in Pax5-deficient pro-B cell ment (18). cultures, yet the expression of Tcfe2a was actually increased in To further analyze the Emb expression domain, we generated a mutant pro-B cells ex vivo (Table II and Fig. 2C). specific mAb by immunizing rats with the 293T cell line express- Semiquantitative RT-PCR on cDNA isolated from FACS-sorted ing full-length mouse Emb. Supernatants from hybridoma clones B220ϩc-Kitϩ pro-B cells from either genotype confirmed the dif- were initially screened by flow cytometry on wild-type and ferential expression of the many of the candidates (Fig. 2). In a Pax5Ϫ/Ϫ pro-B cells lines. A single clone, G7.43.1, with specific number of cases, the putative target genes were cell surface mol- reactivity against Pax5-deficient pro-B cells was identified and fur- ecules against which mAbs were available and expression could be ther characterized. The specificity of G7.43.1 was confirmed by assessed by flow cytometry. In this manner, CD157, CD24a, and Western blotting of parental and Emb-transfected 293T cells (data Sdc4 were shown to rely to varying degrees on Pax5 (Fig. 3), not shown). whereas CXCR5 (Blr1) was confirmed as being expressed in Pax5- Analysis of Pax5Ϫ/Ϫ BM confirmed the differential expression deficient pro-B cell cultures (Fig. 1C). of Emb protein on pro-B cells in vivo (Fig. 3). A detailed flow We have previously shown that Pax5 directly regulates the ex- cytometric analysis of the hemopoietic compartment using biotinyl- pression of a variety of genes in pro-B cells including Cd19 and ated anti-Emb Ab revealed very strong expression in thymocytes, a Flt3 (6, 18). In those cases, direct regulation was initially inferred finding that was confirmed by immunohistochemistry (Fig. 5B). Emb by the complementation of Pax5Ϫ/Ϫ pro-B cells with a retrovirus was also expressed at a moderate level in mature peripheral CD4ϩ The Journal of Immunology 1725

Discussion Pax5 is essential for many aspects of B cell biology, including the initiation of B cell lineage commitment in the BM, V-DJ recom- bination of the Igh locus, and the maintenance of the B cell fate in more mature cells (33). In an attempt to understand the molecular events mediated by Pax5 in lineage commitment and early B cell differentiation, we used a cDNA microarray approach to identify genes regulated by Pax5. The putative Pax5 target genes identified here, as well as those isolated in the recent studies (19, 20), provide a wealth of material to further define existing roles and identify new functions of Pax5 in B cells.

The role of Pax5-activated genes in B cells Analysis of the microarray data revealed that Pax5 potentially ac- tivated the majority of targets identified thus far, as expression was relatively higher in wild-type pro-B cells. Numerous genes in- volved in signaling have been previously identified as Pax5 targets,

including Cd19, Cd79a, Lef1 (6), and Blnk (13), were confirmed in Downloaded from the current screen. Other signaling molecules include Cd97,a transmembrane receptor involved in intracellular signaling in as- sociation with G proteins in immune cells (34), and Cd24a (heat- stable Ag or HSA), a surface glycoprotein expressed on immature hemopoietic cells involved in cell adhesion and activation that has a minor role in B lymphopoiesis in the BM (35). Pax5 also acti- http://www.jimmunol.org/ vates Bst1 (encoding CD157) and Sdc4, two receptors that play roles in mature B cells. Bst1Ϫ/Ϫ mice have delayed B1 B cell development and impaired T-independent Ab responses (36), whereas Sdc4 is expressed broadly in the B cell lineage and is involved in adhesion to the extracellular matrix (37). Pax5 also activates a variety of intracellular signaling molecules including Rasgrp3, which is required for BCR-induced signaling and prolif- eration (38), along with a number of other molecules with less

characterized roles in lymphocyte signaling including protein ki- by guest on September 26, 2021 nase D2 (Prkd2), Lrmp, Stk3, Stk4, and Wnk1. FIGURE 5. Analysis of Emb expression within the hemopoietic system. The potential outcome of the altered expression of a group of A, cDNA was prepared from sorted HSC, CLP, pro-B (B220ϩc-Kitϩ) and adhesion receptors and intracellular signaling molecules may be pre-B (B220ϩCD25ϩ) cells as well as cultivated Pax5-deficient (Ϫ/Ϫ) and manifested in a defect in homing or adhesion. Indeed, lymphoid wild-type (ϩ/ϩ) pro-B cell lines. Expression of Emb and Flt3 was ana- progenitors undergo a dramatic change in cell migration and lyzed by semiquantitative RT-PCR. The cDNA input was previously nor- adhesion at B cell commitment, where committed early B cells malized according to the expression of the control Hprt gene. B, Flow adhere to a specialized BM niche consisting of IL-7-expressing cytometric analysis of Emb expression within the hemopoietic system of stromal cells. In contrast, uncommitted progenitors adhere to IL- wild-type mice. In the histograms, Emb staining is indicated by a dotted 7-negative BM stroma (39). Interestingly, Nedd9 (also called line and the solid line indicates the isotype control. C, Histological exam- HEF1), which is an important component of the - ination of Emb expression in the spleen and thymus of wild-type mice. linked signaling pathway initiated by ligation of the BCR (40), was Anti-CD19 (␣CD19) was used to identify the B cell areas and anti-GL117 (␣GL117) is an isotype control. dependent on Pax5 for its expression. In agreement with these results, Pax5Ϫ/Ϫ pro-B cells have recently been shown in a Trans- well assay to have increased CXCL12-induced migration and, most significantly, have greatly impaired integrin-mediated sub- ϩ strate adhesion compared with wild-type pro-B cells (20). Pax5 is and CD8 T cells and NK cells, and at a very high level in myeloid also regulates the cellular proliferation that is required for the ex- cells (Fig. 5B and data not shown). In keeping with the RT-PCR data, pansion phase of BM lymphopoiesis, first by IL-7 and then as a the majority of BM, spleen, and lymph node B cells did not express result of signals received from the pre-BCR, as Pax5 regulates Emb (Fig. 5, B and C). Initial attempts to use the anti-Emb Ab to Ccnd3 (cyclin D3), which is crucial for the proliferation of early B assess the function of this poorly characterized molecule in non-B cells (41). cells has not been successful, as the addition of the Ab did not affect thymopoiesis using fetal thymic organ culture, mature T cell prolif- eration, or NK cell cytolytic function (S. L. Nutt and M. Polli, un- Pax5 activates downstream transcription factors published observations). Anti-Emb did however partially deplete involved in B cell differentiation CD8ϩ T cells when injected in vivo (S. L. Nutt and M. Polli, unpub- It is striking that many of the Pax5-activated genes identified here lished observations). Interestingly, we and others have recently shown were nuclear proteins. A number of the genes identified were well- that Emb is re-expressed in activated B cells and plasma cells and that known regulators of B cell differentiation and function, including this expression correlated with the posttranslational inhibition of Pax5 Ikzf3 (Aiolos), Spib, and Irf8 (Icsbp), as well as the previously function (32). Thus Emb expression in B cells represents a convenient identified regulators Lef1 (6) and Ebf1 (30). In most cases this marker for impaired Pax5 function. regulation appeared to be direct, suggesting that Pax5 functions in 1726 GENE REGULATION BY Pax5 part to reinforce the B cell program by further activating the down- ␤10), Cd47, and Lat2. Two genes involved in neutrophil migration stream transcriptional cascade. Interestingly, Ikzf3 and Ebf1 ex- were also identified as potential Pax5-repressed genes: L-plastin pression was also dependent on Pax5 in the chicken DT40 B cell (murine Lcp1) (53) and Lsp1 (54). In addition, glutathione perox- line, although that study did not determine whether the regulation idase-1 (Gpx1) expression was up-regulated in Pax5 deficient was direct (29). Although these factors have many functions within pro-B cells. This enzyme is primarily responsible for the intracel- the B cell lineage, it is interesting to note that they all have roles lular degradation of hydrogen peroxide and is the predominant in early B cell development, particularly at the pre-B cell stage (2, antioxidant enzyme expressed by osteoclasts and BM macro- 3, 42). The genes encoding two E proteins, Tcfe2a (encoding E2A) phages (55). and Tcf4 (encoding E2-2), were also differentially expressed, as The gene encoding the Ig superfamily receptor Emb appears to was the inhibitor of E proteins, Id3. E2-2 is required for optimal be a particularly interesting Pax5-repressed gene, as it was ex- pro-B cell expansion (43), whereas Id3 functions to inhibit growth pressed both in progenitor populations as well as in non-B cells. To and induce apoptosis in pro-B cells as well as late stage B cells (44, further our study of Emb, we produced a mAb that detects Emb in 45). The differential expression of Tcfe2a and Id3 was surprising, flow cytometry, immunohistochemistry and Western blotting. as previous studies have not found these genes to be Pax5 depen- These studies demonstrate that while virtually all B cells are Emb- dent (4, 46). Although the available genetic evidence suggests that low or negative, myeloid cells express an extremely high level of Tcfe2a acts upstream of Pax5 during B cell commitment (47), the Emb whereas T cells are Emb-intermediate. Finally, Emb is in- recent finding that Ebf1 also acts both upstream and downstream duced during the terminal differentiation of conventional B cells of Pax5 raised the possibility that a similar situation occurred with and its expression is maintained in plasma cells, whereas perito-

Tcfe2a (30). Comparison of cultured and ex vivo isolated pro-B neal B1 B cells and splenic marginal zone B cells constitutively Downloaded from cells revealed that the differential expression of Tcfe2a was an express Emb (32). Emb is thought to be involved in mediating artifact of adaptation of the cells to in vitro propagation, as the adhesion to the extracellular matrix and is a member of a small expression of Tcfe2a in pro-B cell analyzed ex vivo did not depend gene family that also contains Basigin (CD147) a protein ex- on Pax5 and was actually slightly increased in Pax5Ϫ/Ϫ pro-B pressed on a variety of cell lineages including erythrocytes and T cells. The factors that regulate Tcfe2a expression are unknown, but cells (56). Future loss-of-function studies are required to test the

IL-7 has been predicted to promote expression in early B cells (1). importance of the Pax5-mediated down-regulation of Emb for B http://www.jimmunol.org/ Because both the pro-B cell cultures and a number of Pax5 target cell differentiation. genes, including Nmyc, are exquisitely sensitive to IL-7 (6), small Several Pax5-repressed genes have important functions in B changes in the culture conditions may result in this apparent dif- cells. This includes Cd180 (also known as RP105), which encodes ferential expression. Similarly, while our previous studies did not a receptor for LPS that triggers Ig class switching and secretion find Id3 to be directly regulated by Pax5 (6), it was subsequently (57). The Pten-encoded phosphatase functions as a negative reg- reported that Id3 is induced by pre-BCR signaling, a process that ulator of the PI3K signaling pathway upon engagement of the TCR is also Pax5-dependent (13). These findings highlight the fact that or BCR (reviewed in Ref. 58). Although BM B lymphopoiesis the regulation of specific targets may be context specific and con- occurred normally after the conditional deletion of Pten in B cells, firm the importance of assessing differential gene expression using the resultant lower signaling threshold through the BCR promoted by guest on September 26, 2021 equivalent primary cell populations directly isolated from the BM. marginal zone and B1 B cell development and rescued germinal A number of other transcription factors whose function in B center formation in signaling deficient, Cd19Ϫ/Ϫ mice (59). As cells is less well known were also identified. Of these, the calcium- well as being essential for many aspects of peripheral B cell biol- inducible factor Nfatc1 is important for T cell function. Nfatc1Ϫ/Ϫ ogy, the inhibition of Pax5 function and the down-regulation of its lack B1a B cells (48), and their follicular B cells are hyporespon- expression is a crucial event in initiating plasma cell differentiation sive to BCR and CD40L-mediated signals (49). It is possible that (29, 32, 60). In line with this, it has been shown that many Pax5- NFATc1 fulfills a function in pre-BCR signaling, although this has repressed genes, including, Flt3, Mef2c, Igj, and Emb are re-ex- not been investigated. pressed during late B cell differentiation (19, 32). Several cofactors and chromatin regulators were also differen- tially expressed, including Cbfb (core-binding factor-␤). Cbf␤ is a transcriptional coactivator that interacts with Runx family mem- Comparison of the target genes identified in this study with bers, such as Runx1, a factor that is essential for many aspects of published reports hemopoiesis including B cell development (50). The gene coding The microarray data set presented in this study complements two for the catalytic subunit of the SWI/SNF-related complex, recent screens for Pax5 target genes performed by the Busslinger Smarca4 (also known as Brg1), which is essential for T cell de- laboratory (19, 20). These studies also used cDNA microarrays to velopment (51), was moderately differentially expressed in both compare the transcriptomes of in vitro cultivated Pax5Ϫ/Ϫ and arrays. Taken together, the number of nuclear proteins that are wild-type pro-B cells. The initial study used subtractive hybrid- activated by Pax5 suggests that a key function of this factor is to ization to enrich for differentially expressed genes and found 110 activate the expression of a panel of genes that further reinforce the putative Pax5-repressed genes (19), whereas that of Schebesta et B cell transcriptional program. al. used the mouse “lymphochip” (61) to identify 170 Pax5-acti- vated transcripts (20). A variety of models of Pax5 deficiency were Many Pax5-repressed genes are associated with then used to extensively validate the target genes. In comparison, non-B cell lineages our study has used two different cDNA microarrays for the screen- The expression of lineage inappropriate genes such as Csf1r, Flt3, ing, a cDNA clone set from the National Institute of Aging that and Notch1 in Pax5Ϫ/Ϫ pro-B cells (reviewed in Ref. 52) has pro- does not contain specifically selected hemopoietic cDNAs and a vided support for the concept of lineage priming and led to a model custom array produced from a mature IgMϩ normalized cDNA whereby Pax5 functions to repress this promiscuous gene expres- library. In addition, other small variables would be expected to sion and thus restrict progenitors to the B cell fate. The Pax5- slightly alter the gene lists; for example the prior studies have used repressed genes identified here include a number of genes associ- a 3-fold cutoff for differential expression whereas our studies have ated with broad hemopoietic expression such as Tmsb10 (thymosin used statistical significance only. The Journal of Immunology 1727

Comparison of the data presented here on Pax5-activated genes 17. Tagoh, H., R. Ingram, N. Wilson, G. Salvagiotto, A. J. Warren, D. Clarke, revealed that 18 of the 66 genes listed in Table II (or 27%) were M. Busslinger, and C. Bonifer. 2006. The mechanism of repression of the my- eloid-specific c-fms gene by Pax5 during B lineage restriction. EMBO J. 25: found in the previous report (20). Similarly, 12 of the 43 repressed 1070–1080. genes (28%) were found in the prior study (19). The overlap be- 18. Holmes, M. L., S. Carotta, L. M. Corcoran, and S. L. Nutt. 2006. Repression of Flt3 by Pax5 is crucial for B-cell lineage commitment. Genes Dev. 20: tween the lists can, however, be further enhanced by only com- 933–938. paring those genes in our study that were validated by an inde- 19. Delogu, A., A. Schebesta, Q. Sun, K. Aschenbrenner, T. Perlot, and pendent methodology (RT-PCR and/or flow cytometry), as only M. Busslinger. 2006. Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells. Immunity 24: 269–281. 50% of the genes in our study were further examined. Using this 20. Schebesta, A., S. McManus, G. Salvagiotto, A. Delogu, G. A. Busslinger, and approach, the overlap between these studies was now 39%. Thus, M. Busslinger. 2007. Transcription factor activates the chromatin of key it appears that the use of distinct screen techniques have, while genes involved in B cell signaling, adhesion, migration, and immune function. Immunity 27: 49–63. identifying many common genes such as Notch1, Igf2, Emb, Spib, 21. Soares, M. B., M. F. Bonaldo, P. Jelene, L. Su, L. Lawton, and A. Efstratiadis. Ikzf3, Sdc4, and Ccnd3, proven complementary by detecting ad- 1994. Construction and characterization of a normalized cDNA library. Proc. ditional unique target genes, for example Syvn1, Frm4, and Natl. Acad. Sci. USA 91: 9228–9232. 22. Kargul, G. J., D. B. Dudekula, Y. Qian, M. K. Lim, S. A. Jaradat, T. S. Tanaka, Tmsb10. M. G. Carter, and M. S. Ko. 2001. Verification and initial annotation of the NIA In summary, we have identified numerous novel Pax5 target mouse 15K cDNA clone set. Nat. Genet. 28: 17–18. 23. Smyth, G. K., and T. Speed. 2003. Normalization of cDNA microarray data. genes whose expression is potentially activated or repressed by Methods 31: 265–273. Pax5. These genes mediate diverse biological functions in B cells 24. Smyth, G. K., J. Michaud, and H. S. Scott. 2005. Use of within-array replicate such as migration, signaling, and the regulation of gene expression spots for assessing differential expression in microarray experiments. Bioinfor- matics 21: 2067–2075. Downloaded from and demonstrate the plethora of processes that are regulated by 25. Smyth, G. K. 2004. Linear models and empirical bayes methods for assessing Pax5 to promote B cell lineage commitment and subsequent differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3: differentiation. Article3. 26. Nutt, S. L., D. Metcalf, A. D’Amico, M. Polli, and L. Wu. 2005. Dynamic reg- ulation of PU.1 expression in multipotent hematopoietic progenitors. J. Exp. Med. Acknowledgments 201: 221–231. We thank Jason Brady, Kate Elder, Jaclyn Gilbert, and Matthew Ritchie for 27. Polli, M., A. Dakic, A. Light, L. Wu, D. M. Tarlinton, and S. L. Nutt. 2005. The development of functional B lymphocytes in conditional PU.1 knock-out mice. technical assistance. Blood 106: 2083–2090. http://www.jimmunol.org/ 28. Pulendran, B., M. Karvelas, and G. J. Nossal. 1994. A form of immunologic Disclosures tolerance through impairment of germinal center development. Proc. Natl. Acad. Sci. USA 91: 2639–2643. The authors have no financial conflict of interest. 29. Nera, K. P., P. Kohonen, E. Narvi, A. Peippo, L. Mustonen, P. Terho, K. Koskela, J. M. Buerstedde, and O. Lassila. 2006. Loss of Pax5 promotes plasma cell References differentiation. Immunity 24: 283–293. 1. Singh, H., K. L. Medina, and J. M. Pongubala. 2005. Contingent gene regulatory 30. Roessler, S., I. Gyory, S. Imhof, M. Spivakov, R. R. Williams, M. Busslinger, networks and B cell fate specification. Proc. Natl. Acad. Sci. USA 102: A. G. Fisher, and R. Grosschedl. 2007. Distinct promoters mediate the regulation 4949–4953. of Ebf1 gene expression by interleukin-7 and Pax5. Mol. Cell. Biol. 27: 579–594. 2. Nutt, S. L., and B. L. Kee. 2007. The transcriptional regulation of B cell lineage 31. Huang, R. P., M. Ozawa, K. Kadomatsu, and T. Muramatsu. 1993. Embigin, a member of the immunoglobulin superfamily expressed in embryonic cells, en-

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