Defining In Vivo Complexes of the Murine CD21 and CD23

This information is current as Irina Debnath, Kirstin M. Roundy, Janis J. Weis and John H. of September 25, 2021. Weis J Immunol 2007; 178:7139-7150; ; doi: 10.4049/jimmunol.178.11.7139 http://www.jimmunol.org/content/178/11/7139 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Defining In Vivo Transcription Factor Complexes of the Murine CD21 and CD23 Genes1

Irina Debnath, Kirstin M. Roundy, Janis J. Weis, and John H. Weis2

The expression of the CD21 and CD23 genes is coincident with differentiation from transition 1 B cells (T1) to transition 2 B cells (T2). To define constituents controlling CD21 and CD23 expression, we conducted chromatin immunoprecipitation analyses for candidate transcription factors. We found constitutive binding of Oct-1, NFAT species, YY1, NF-␬B-p52, Pax5, E2A, and RBP-J␬ to CD21 sequences and NF-␬B-p52, Pax5, NFAT species, E2A, and RBP-J␬ to CD23 promoter sequences. Splenic T and B cell subsets displayed constitutive binding of YY1, NF-␬B-p52, Pax5, and Oct-1 to CD21 sequences in B cells but no specific binding of NFATc3 or Pax5 in T cells. Similarly, CD23 sequences demonstrated constitutive binding of NF-␬B-p52 in splenic T and B cells but only Pax5 in B cells. Of the various NFAT species, only a subset were found forming constitutive DNA/

complexes with the CD21, CD23, and IL-2 sequences. Maturing B cells in the marrow possess stable Pax5 complexes on CD19, Downloaded from CD21, and CD23 gene promoters in the nuclei of such cells, even though only CD19 is expressed. The similarity of genetic controlling elements between the CD21 and CD23 genes does not suggest a mechanism for alternative regulation of these genes; however, separation of splenic B cell subsets into T1, T2, marginal zone (MZ), and mature follicular B cells, followed by quan- titative RT-PCR, demonstrated the lack of appreciable CD23 transcripts in CD21؉ MZ cells. We propose an alternative derivation of MZ cells as maturing directly from T1 cells, leaving CD23 transcriptionally inactive in that lineage of cells. The Journal of

Immunology, 2007, 178: 7139–7150. http://www.jimmunol.org/

he murine CD21 gene encodes two distinct proteins, The human and mouse CD21 genes have been scrutinized for known as murine CR1 and CR2, via alternative splicing transcriptional control domains. Both genes have been found to be T (1). These proteins recognize specific complement break- controlled, via reporter assays, by two distinct regions of the gene: down products of C3 and C4 and are critical in generating an the proximal promoter regions and sequences within the first in- optimal Ab response in vivo (2–6). The gene is transcriptionally tron. The human CD21 gene has been shown to possess a number active in maturing and mature B cells and follicular dendritic cells of functional sites, including Sp1, AP1, AP2, and E box sites prox- (7, 8). Expression of the CD21 gene is induced in differentiating B imal to the TATA box sequence, and NF-␬B and heterogeneous

3 by guest on September 25, 2021 cells during the transition 1 B cell (T1) to transition 2 B cell (T2) ribonucleoprotein sites further upstream (Ϫ531 and Ϫ495 relative transition stage in the spleen, after expression of the B cell marker to the transcription induction site, respectively) (13–19). The CD19 is observed in the marrow pre-B cell stage (9–11), but co- mouse CD21 promoter possesses a functional Oct-1 site (equal incident with expression of CD23, the low-affinity IgE binding via EMSA with T and B cell nuclear extracts), a consensus found on B cells and some activated macrophages. B cells in pe- Pax5 binding site (showing a B cell-specific EMSA pattern), and ripheral lymphatic tissues such as the spleen display various levels a conserved NF-␬B site (also showing equal binding via EMSA of CD21 expression with marginal zone (MZ) B cells of the spleen with T and B cell nuclear extracts) all within the first 280 bp of the expressing the highest levels, T2 cells expressing less, and follic- promoter region (7, 20–22). Reporter transfection assays into ular mature (FM) B cells the least. T1, T2, MZ, and FM cells all CD21-expressing, permissive cells with human and mouse CD21 express CD19. CD23 is found on the surface of T2 and FM cells sequences have shown the majority of the transcriptional induction but is absent, or at very low levels, on the surface of MZ cells (12). activity to lie within these first few hundred base pairs of the pro- moter. However, the human gene also contains an inhibitory E box sequence that binds the E2A proteins E12/E47 that is proposed to Division of Cell Biology and Immunology, Department of Pathology, University of limit CD21 expression (18). Utah School of Medicine, Salt Lake City, UT 84112 Transfection of human and mouse CD21 promoter reporter con- Received for publication May 25, 2006. Accepted for publication March 16, 2007. structs into T (CD21 nonexpressing) and B cell lines (expressing The costs of publication of this article were defrayed in part by the payment of page cells) showed virtually identical reporter expression levels (7, 13, charges. This article must therefore be hereby marked advertisement in accordance 14, 22). However, when the first intron sequences were added to with 18 U.S.C. Section 1734 solely to indicate this fact. such constructs, the expression of the CD21 promoter constructs 1 This research was funded, in part, by National Institutes of Health Grant R01 AI2458 (to J.H.W.) and National Institutes of Health Grants R01 AI-32223 and AR- was dramatically reduced in T cells but unaltered in B cells. Anal- 43521 (to J.J.W.). yses with the human intronic sequence localized this tissue-spe- 2 Address correspondence and reprint requests to Dr. John H. Weis, Department of cific repressor element, dubbed CRS (CR2 silencer), to a single Pathology, University of Utah School of Medicine, 15 North Medical Drive East, Salt RBP-J␬ (CBF1) binding site within the intron (14). Transfection of Lake City, UT 84112. E-mail address: [email protected] human K562 cells (which normally do not express CD21) with a 3 Abbreviations used in this paper: T1, transition 1 B cell; BAFF, B cell-activating factor; ChIP, chromatin immunoprecipitation; CsA, cyclosporine; FM, follicular ma- reporter construct demonstrated silencing of such constructs only ture; F-OH, formaldehyde; MZ, marginal zone; NIDO, nidogen; RIPA, radioimmu- with an intact CBF1 site. The mouse first intron of the CD21 gene noprecipitation assay; T2, transition 2 B cell. also possesses three similar RBP-J␬ sites interspersed within a Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 series of repetitive Oct1/YY1/NFAT binding sites. Elimination of www.jimmunol.org 7140 CHROMATIN COMPLEXES OF THE CD21 AND CD23 GENES single repetitive binding sites had no affect upon silencing of re- Antibodies porter function; however, removal of the entire set restored CD21 Mouse monoclonal anti-YY1 (H-10), NFATc1 (7A6), NFATc2 (4G6-G5), promoter activity in mouse T cells (22). Intriguingly, this region of NFATc3 (F-1), NF-␬B-p52 (C-5), Pax5 (A-11), CD3 (PC3/188A), rabbit the first intron is highly conserved in sequence and position be- polyclonal anti-Oct1 (C-21), PU.1 (T-21), and goat polyclonal anti-CD21 tween the mouse, human, and dog CD21 genes. (M-19) Abs were purchased from Santa Cruz Biotechnology. Rabbit poly- The expression of the CD21 gene has also been shown to be clonal anti-NFAT5 (catalog no. ab3446) was purchased from Abcam. Rab- bit anti-␤-actin Ab (catalog no. A2066; Sigma-Aldrich) was used as the influenced by factors and conditions that alter the state of the loading control in Western blot analysis. chromatin. Demethylation of promoter sequences was shown in a variety of human B cells to be correlated with expression; ChIP inhibition of methylation induced expression in normally inac- This protocol was adapted from published procedures (33–35). BALB/c tive cells (23). Additionally, increasing the level of histone splenocytes were isolated, and erythrocytes were lysed using RBC lysis acetylation in human pro-B cells and in murine T cells (via buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA (pH 7.2)), 6 inhibiting histone deacetylase activity) induced expression of washed, and resuspended in PBS with 0.1% BSA. A total of 3 ϫ 10 cells was pelleted for each ChIP reaction, and PBS was removed. DNA/Protein the CD21 genes (21, 23). cross-linking was done by resuspending cell pellets in 1% formaldehyde CD21 and the B cell-specific low-affinity IgE receptor CD23 are and incubated for 10 min at room temperature with rotation. Fifty micro- expressed within the same window of B cell development. CD23 liters of 2.5 M glycine was added to each reaction and incubated for 5 min has been described as possessing an IL-4-dependent enhancer el- with rotation and then centrifuged to pellet cells. Pellet was washed once ϫ ement (24) with possible binding by both STAT 1 and STAT 6 with 1 PBS with 0.1% BSA, and cells were resuspended in radioimmu- noprecipitation assay (RIPA) (50 mM NaCl, 25 mM Tris (pH 7.5), 1 mM (25–27). The CD23 promoter(s) have also been shown to be di- EDTA, 0.1% SDS, 1% w/v deoxycholate, 1% Nonidet P-40, 1% BSA, and Downloaded from rectly regulated by the RBP-J␬ (Notch family), NF-␬B, NFAT, protease inhibitor “Complete Minipill” (Roche)) and incubated for 20 min and Pax5 transcription factors (27–29). Similarities between tran- on ice with intermittent hard vortexing. Lysed samples were sonicated scriptional control of CD21 and CD23 exist beyond sharing the seven times at power setting 4 for 30 s each time. Sonicate was centrifuged for 10 min at 14,000 rpm at 4°C. Supernatant was taken and centrifuged same set of transcriptional control elements. A recent report sug- again to pellet debris. For the input sample, 100 ␮l of the supernatant gested that engagement of the B cell-activating factor (BAFF) re- (named Crude Input) was mixed with 300 ␮l of ChIP elution buffer (10 ceptor with its ligand was required for CD21 and CD23 expres- mM EDTA, 1% (w/v) SDS, and 50 mM Tris-HCl (pH 8.0)), and cross- http://www.jimmunol.org/ sion; animals lacking BAFF showed an absence of CD21 and linking was resolved by heating at 65°C overnight and then purified with CD23 expression (30). The BAFF receptor is known to transduce Qiagen PCR purification columns. Four-hundred microliters of the remain- ing supernatant with 100 ␮l of RIPA, supplemented with 1 protease in- its signal within B cells using an “alternative” activation pathway hibitor mixture (“Complete Minipill”; Roche) per 10 ml of volume, was allowing for translocation of NF-␬B subunits into the nucleus of used for each immunoprecipitation reaction. Samples were precleared for the cell. BAFF receptor engagement has also been linked to NFAT 2 h at 4°C (with rotation) with sheep anti-mouse or sheep anti-rabbit IgG protein activation (31, 32). conjugated Dynabeads (Dynal Biotech) that were previously blocked and washed with 1ϫ PBS with 1 mg/ml BSA. The sequences and tissue-specific expression of the murine For each immunoprecipitation reaction, 1 ␮g of mouse monoclonal or and human CD21 genes are highly conserved, yet clear discrep- rabbit polyclonal Ab was used, except where indicated otherwise. Immu- ancies exist in the analyses of the two genetic elements. Much noprecipitations were done for 2 h rotating at 4°C. Immunoprecipitated by guest on September 25, 2021 of the data generated in both systems has been derived from reactions were added to sheep anti-mouse or sheep anti-rabbit IgG-conju- ϫ reporter analyses (either stable or transient transfections) and in gated Dynabeads blocked and washed with 1 PBS with 1 mg/ml BSA and incubated overnight. Dynabeads were washed, three times with RIPA, twice vitro protein/DNA binding assays. To gain further insight into with RIPA with 0.1 mg/ml herring sperm DNA, twice with RIPA plus 0.1 CD21 gene control, as well as to compare CD21 transcription mg/ml herring sperm DNA plus 500 mM NaCl, twice with RIPA plus 250 mM with a similarly expressed gene, CD23, we initiated a series of LiCl, then one alternate wash with RIPA plus 500 mM NaCl, one wash with in vivo analyses, via the chromatin immunoprecipitation (ChIP) RIPA plus 250 mM LiCl, and, finally, three washes with TE (10 mM Tris (pH 8)-1 mM EDTA). DNA was eluted from the bound beads by adding 200 ␮lof assay, mapping DNA/protein binding complexes that form in ChIP elution buffer to each reaction and incubating overnight at 65°C incu- the nucleus of native, nontransformed cells. We describe in this bator with gentle shaking. Supernatant was then separated from Dynabeads, article constitutive, stable complexes of CD21 and CD23 se- and soluble DNA was isolated. PCR amplification in the presence of quences with NFAT family members, NF-␬B-p52, plus Pax5 [32P]dCTP was done for each sample (input, nonimmune control antisera, and and constituents of the Notch signaling pathway. These data immunoprecipitated samples) and water control using primers specific for CD21, CD23, and CD19 promoter (and CD21 intronic) elements, ␤-actin in- lead us to propose a new model for the development of MZ B Ϫ ϩ tragenic region and nidogen (NIDO) for 30 cycles with 6-s elongation, and cells directly from a CD23 , T1-like cell instead of the CD23 55°C annealing temperature (36). Primer sequences are listed in Table I. PCR T2 intermediate. products were subjected to electrophoresis within a sequencing gel, and the gel was dried and exposed to x-ray film overnight. For PhosphorImager quantification of ChIP assay, the gel was dried and was exposed to a phosphor screen overnight and scanned with a Materials and Methods PhosphorImager (STORM820; Molecular Dynamics). Intensity values for Cell lines, culture conditions, and mouse strains each PCR product were analyzed with Image Quant software (Molecular Dynamics). For relative quantification, a 4-fold dilution series of input was The murine CD21-expressing B cell lymphoma cell 2PK3 was obtained done. The concentration of output samples was taken as “x” (as 400 ␮lof from American Type Culture Collection. It was maintained at 37°C/5% crude input used per immunoprecipitation), and accordingly, the concen- CO2 in RPMI 1640 (Invitrogen Life Technologies) with 10% bovine tration of the input was “0.25ϫ” (as 100 ␮l of crude-input used per input). growth serum (HyClone) and 1% penicillin-streptomycin (Invitrogen Life The data were normalized to the input and to the reference gene NIDO to Technologies). Splenocytes were isolated from adult BALB/c mice (Na- determine the fold enrichment. The fold enrichment value was obtained tional Cancer Institute) (see Figs. 2–7) and maintained in single-cell using the following formula: (ChIP target/ChIP reference)/(input target/ suspensions with RPMI 1640 with 10% bovine growth serum and 1% input reference) ϭ fold enrichment. penicillin-streptomycin. Cells were treated with indicated doses of cy- closporine (CsA) (Sigma-Aldrich) or an equal volume of ethanol Separation and analysis of splenic lymphocyte populations (EtOH) vehicle in medium for indicated times. A/J and A/WySnJ mice (see Fig. 8) were used for bone marrow and splenocyte isolation and For ChIP analysis, splenic B and T cell subsets were purified using Milte- were obtained from The Jackson Laboratory. Splenic B cells were ob- nyi Biotec magnetic beads, according to the manufacturer’s protocol. B220 tained from 6- to 8-wk-old female C57BL/6 mice (see Fig. 9) purchased and CD90 magnetic beads were used for labeling B and T cells, respec- from the National Cancer Institute. tively. The purity of fractionated cells was confirmed by FACS analysis. The Journal of Immunology 7141

Table I. Oligonucleotides used in the ChIP and RT-PCR assays

Forward Reverse

ChIP oligos Intron i8 (CD21) CATCCATATCCATCAGATAGCC CTAGGATCTGGAGTGAAGATCG Intron i10 (CD21) CCCTCATGCACAAAGTCTGATTC TGAAAAATGAAGGCTGAAAAACAGTG Intron i12 (CD21) TAGGGTTTCACAAAGTGGTAC TATAGGGTACTTTGGGGATAGC Promoter P2 (CD21) GGTTTGAAACTGAATTAACTGG GGAAGTCTGCTTTTGGTTCAGG ␤-Actin CTCCATCGTGGGCCGCTCTAG GTAACAATGCCATGTTCAAT NIDO CCAGCCACAGAATACCATCC GGACATACTCTGCTGCCATC IL-2 CCAATATCCTTCCTGTCTAATC TACAATGAACGCCTTCTGTATG CD23 promoter CCAGGTTGTGTCATTTCCTTCCT CTTCTGTGAGCAGTTCATTAATGGC CD19 promoter GCCAAACCATATAGAAGGTCAC CTTCTGCGTGGCTGCGCAGAGGA RT-PCR oligos CD21 (Fig. 6) ATGGGATCCTTGGGTTCGCTC GCTAGGTGAACAAGTGTACCT ␤-Actin CTCCATCGTGGGCCGCTCTAG GTAACAATGCCATGTTCAAT NFATc1 GCCCTCCGGCCGCAGTCTGCG GTTGGGTCTGGTGTTGCCAGAGGAC NFATc2 GCAGTCCCCAAGACGAGCTG CTATACTATCCGGCTCTCCGAACCG NFATc3 TGGATCTCAGTATCCTTTAA CACACGAAATACAAGTCGGA NFATc4 AGCGGGTGTCCCGACCAGTCCAGGTC AGTCTCATAAGCAGGGTCTTCATG NFAT5 CAGAGGGCAGCCGAGGCTCTG CATCGGCATTCCTCAGCTTC NIP45 CGGAGAGGCGCCGGTGGTC TAGCTTCTGGAGCGCCTCCG Downloaded from CD19 AGGAAAAGGAAGCGAATGACT GCCTGGCCAGAGGTAGATGTA CD23 CTCTCCCAGAACCTGAACAGACTC AGCCCTTGCCAAAATAGTAGCAC ADAM10 CAAATGACTGGAGTAGAGGAAGG AAGAACAGGTGATCTGTCTGGAT http://www.jimmunol.org/ For B cell subset separation, total splenocytes were isolated from 6- to tor binding sites but do not always reflect relevant in vivo binding. 8-wk-old C57BL/6 female mice. After RBC lysis, a B cell-enriched pop- Therefore, we sought to increase the stringency of our analyses by ulation was obtained via B220ϩ magnetic bead depletion. B220ϩ cells were stained with Abs against CD21, CD23, and CD24 and resuspended at using ChIP analysis with Abs against the various candidate tran- 5 ϫ 106 cells/ml. Cells were sorted into various B cells subsets with the scription factors. FACSVantage SE. The mouse CD21 promoter element possesses a variety of po- tential sites, including those for Oct-1 (defined by EMSA analysis) RNA preparation, cDNA synthesis, and RT-PCR (38), Pax5, E-box proteins, and NF-␬B (by sequence scanning) Total RNA from cells was isolated using the RNeasy mini kit (Qiagen) and (Fig. 1). Studies on the human CD21 promoter identified biding quantified. cDNA was synthesized by mixing 2 ␮g of RNA, 10 ␮lof5ϫ by guest on September 25, 2021 first-strand buffer, 5 ␮l of 5 mM dNTP, 5 ␮l of 0.1 M DTT, 1 ␮l of 1.25 sites specific for E-box binding proteins (E2A), USF1, AP-2, and mM random primers, 2 ␮l of Maloney murine leukemia virus-reverse tran- Sp1 (17, 18). Within the human/mouse-conserved sequences of scriptase (all were from Invitrogen Life Technologies), and water to a final the first intron of CD21, we have mapped a number of repetitive ␮ volume of 50 l. The reaction mixture was incubated at 37°C for 2 h. Two Oct-1, YY1, and NFAT binding sites by EMSA (22). Addition- microliters of DNase-free RNase (1 mg/ml from Invitrogen Life Technol- ␬ ogies) was then added, and the reaction mixture was incubated for addi- ally, there are three conserved RBJ-J sites identified in this tional 5 min at room temperature, followed by cDNA isolation, using the region by sequence scanning. EMSA analysis identified a func- Qiagen PCR purification kit. Radioactive RT-PCR using 32P was per- tional RBP-J␬ site in the first intron of the human CD21 gene formed as described previously (36). Quantitative real-time RT-PCR was (14). The CD23a promoter contains defined binding sites (from performed as described previously (21, 37). Primer sequences for RT-PCR analysis are listed in Table I. the human and mouse genes) for RBP-J␬, the Stat1/Stat6 IL-4 response element, Pax5, NF-␬B, E proteins, and NFAT family Western blot analysis members (27–29). Splenic or bone marrow B and T cell subsets were purified as described ChIP analysis was performed on total BALB/c splenocytes (Fig. 6 above. Live cells were counted by trypan blue dye exclusion, and 5 ϫ 10 2). Cells used in these analyses were freshly harvested from the cells were resuspended in 100 ␮l of RIPA (50 mM NaCl, 25 mM Tris (pH 7.5), 1 mM EDTA, 0.1% SDS, 1% w/v deoxycholate, and 1% Nonidet spleen and immediately cross-linked with formaldehyde without P-40) with one protease inhibitor “Complete Minipill” (Roche) and 100 ␮l any cell stimulation or activation. Samples were amplified using of PMSF per 10 ml of lysis buffer. The reaction mixture was mixed and primers specific for sequences from the CD21 and ␤-actin genes incubated 15 min on ice with intermittent vigorous agitation. Lysed cells (Table I). As shown in Fig. 2A, titrating the amount of anti-Oct-1 were centrifuged at 14,000 rpm at 4°C for 10 min. The supernatants were pooled, and BCA Protein Assay (Pierce) was performed, according to the Ab increased the immunoprecipitation of sequences from the manufacturer’s protocol, to assay the total protein concentration. Fifteen CD21 gene but not that of the ␤-actin control, which does not micrograms of total protein was used for each Western sample. Rabbit possess Oct-1 binding sites. This data validated our previous anti-actin Ab (Sigma-Aldrich) was used as loading control. Tris-glycine EMSA observations of Oct-1 binding sites within the CD21 gene SDS-PAGE gels (Invitrogen Life Technologies) were used for protein sep- aration and transferred to membrane for Western blot analysis using stan- by showing that these are bound by Oct-1 in vivo. We had dard protocols. expected that moving away from the core Oct binding sites within the CD21 gene would diminish the immunoprecipitation Results of distal regions of the gene (our ChIP protocol generates son- Defining in vivo factor binding sites in the CD21 and CD23 icated DNA fragments of ϳ500–1000 bp in size). This was not genes via ChIP the case for Oct-1 (or precipitations of the CD21 gene using The murine CD21 gene has previously been scrutinized for tran- other transcription factors), suggesting that the gene is highly scription factor binding sites via sequence scanning and EMSA condensed, perhaps due to its telomeric location, giving rise to analysis. These two procedures are useful in defining possible fac- large cross-linked complexes. 7142 CHROMATIN COMPLEXES OF THE CD21 AND CD23 GENES

FIGURE 1. Schematic diagram of mu- rine CD21 promoter and intronic regulatory regions and the murine CD23a promoter. The CD21 promoter is shown divided arbi- trarily into 6 overlapping fragments of ϳ200 bp each (P6-P1). The first half of intron 1 is arbitrarily divided into 12 overlapping fragments of ϳ200 bp each (i1-i12) (22). The arrows denote the genomic fragments of the CD21 and CD23 genes used in this article. Binding sites for specific factors on the CD21 and CD23 genetic elements are shown (see text for explanation). The scale of each gene sequence is noted. Downloaded from

Similar data to that of the Oct-1 ChIP analysis were derived for Abs specific for YY1, NF-␬B-p52, Pax5, NFATc3, E2A, RBP-J␬, NFATc3, YY1, and NF-␬B-p52. These data thus validate the in and Oct-1. The CD23 promoter sequence was isolated with Abs vivo binding of Oct-1, NFATc3, YY1, and NF-␬B-p52 to the specific for all of these transcription factors with the exception of CD21 gene and suggest such analyses can be used to differentially YY1 and Oct-1. The NIDO control sequence was not precipitated http://www.jimmunol.org/ analyze various subsets of splenocytes (see below). by any of these Abs. We expanded these ChIP assays with naive total splenocytes for additional transcription factors and analyzed the products with a Splenic T and B cells demonstrate differential transcription different control sequence (NIDO) and sequences specific for the factor binding to the CD21 gene CD23 promoter (see Fig. 1). As shown in Fig. 3, the CD21 pro- CD21 is expressed in B cells, not in splenic T cells; therefore, we moter and intronic elements were specifically precipitated with sought to determine which of the transcription factors identified in by guest on September 25, 2021

FIGURE 2. ChIP of Oct-1 (A), NFATc3 (B), YY1 (C), and NF-␬B-p52 (D), binding to CD21 intronic and promoter fragments. Splenocytes were treated with 1% formal-

dehyde, lysed, and sonicated. H2O sample is a negative water control (no DNA added). Input sample is the cross-linked, sonicated cell lysate. A total of 0.1, 1, and 5 ␮g of normal mouse IgG or normal rabbit IgG was used as the nonspecific Ab con- trols (Control Ig). A total of 0.1, 1, and 5 ␮g of rabbit polyclonal anti-Oct-1 or mouse monoclonal anti-NFATc3, -YY1, and -NF-␬B-p52 Abs was used. PCR was done using specific oligonucleotides (Ta- ble I) to amplify CD21 intronic fragments (i8, i10, and i12) (see Fig. 1), promoter (P2), and ␤-actin. The Journal of Immunology 7143

the B cell samples. This result illustrated differential binding pat- terns between T and B cells because Pax5 is not expressed by T cells (39). Fig. 4B shows that Oct-1 bound to the CD21 sequences in both T and B cells, whereas PU.1, as expected (40), demonstrated no binding in either cell type. Fig. 4C shows that YY1 also constitu- tively bound to the CD21 sequences in both T and B cells but not the control ␤-actin and IL-2 gene sequences. NFATc3 showed specific binding to the CD21 gene only within the B cell samples, not the T cell subset. As a control, the IL-2 promoter sequence was analyzed for NFATc3 binding. ChIP analysis demonstrated NFATc3 binding to the IL-2 gene (whose transcription is NFAT dependent) was evident in the B cell subset but lacking in the T cell sample. These data indicate that NFATc3 is present in the nucleus of resting B cells (but FIGURE 3. ChIP of transcription factors on the CD21 and CD23 genes. not resting T cells) bound to defined NFAT binding sites (see below). ChIP analysis was performed on total splenocytes. The H2O sample is a water control, and INP is a sample of the sonicated cell lysate (input). M. Similar data for the CD23 gene were obtained from T and B cell control and R. control are mouse- and rabbit-nonspecific Ab controls, re- subsets (Fig. 4D) in that NF-␬B-p52 and Pax5 showed constitutive spectively. The mouse mAbs (M.YY1, M.NF-␬B-p52, M.Pax5, and binding to CD23 sequences in the nuclei of B cells but only NF-␬B- M.E2a) and the rabbit polyclonal Abs (R.RBP-J␬ and R.Oct-1) are as p52 was evident in T cell samples. Downloaded from noted. PCR products are from the CD21 first intron (i8) and promoter (P2), The previous figure suggested that NFATc3 is constitutively the CD23 promoter (CD23), and the mouse NIDO gene. bound to the CD21 gene in B cells but is excluded from the gene in T cells. To further analyze this finding, the effect of CsA on the Figs. 2 and 3 were specific for B cells. Splenic B and T cells were ChIP analyses of the CD21 and CD23 genes was performed. CsA positively selected by magnetic bead sorting using anti-B220 for B blocks the ability of NFAT proteins to move from the cytoplasm to the nucleus; therefore, we anticipated that CsA treatment would cells and anti-CD90 for T cells. Greater than 90% of the selected http://www.jimmunol.org/ cells were of the desired lineage as determined by FACS analysis block binding of the NFATc3 to the CD21 and CD23 genes. As (data not shown). Selected cells were immediately fixed, cross- shown in Fig. 5A, treatment of total splenocytes with CsA, but not linked, and used for ChIP analysis. As shown in Fig. 4A, NF-␬B- the ethanol control, resulted in a loss of NFAT binding to the p52 binding to the CD21 genetic elements was evident in both T CD21 sequences. This analysis was expanded in Fig. 5B by com- and B cell populations, whereas Pax5 binding was only evident in paring the effect of CsA treatment on total splenocytes with that of by guest on September 25, 2021

FIGURE 4. ChIP analysis of purified B and T cell subsets. Splenic B and T cell subsets were purified using magnetic beads (B220 and CD90 magnetic beads were used for isolating B and T cells, respectively). A subset of cells were analyzed by FACS as a control of their purity (data not shown). H2O sample is water control. Input sample is the sonicated cell lysate. One microgram of normal rabbit IgG or normal mouse IgG was used as the nonspecific Ab controls (Contrl), and 1 ␮g of mouse monoclonal anti-NF-␬B-p52, Pax5, YY1, or NFATc3 Abs or rabbit polyclonal anti-Oct-1 or PU.1 Abs was used for immunoprecipitation. PCR was done using specific oligos to CD21 intronic fragments (i8, i10, and i12), promoter (P2), CD23 promoter, or the IL-2 and ␤-actin genes. A, Analysis of NF-␬B-p52 and Pax5 complexes bound to CD21 gene sequences. B, Analysis of Oct-1 and PU.1 complexes bound to CD21 gene sequences. C, Analysis of YY1 and NFATc3 complexes bound to CD21 and IL-2 gene sequences. D, Analysis of NF-␬b-p52 and Pax5 complexes bound to the CD23 promoter sequence. 7144 CHROMATIN COMPLEXES OF THE CD21 AND CD23 GENES

FIGURE 6. NFAT expression by splenic B and T cells. Splenic B and T cells were purified as described in Materials and Methods. A, Total RNA was

isolated from these cell types as well as total spleen (SP), cDNA was prepared, Downloaded from and semiquantitative RT-PCR was performed (55°C annealing temperature) using oligonucleotides specific for NFATc1 (25 cycles), NFATc2 (25 cycles), NFATc3 (27 cycles), NFATc4 (30 cycles), NFAT5 (25 cycles), NIP45 (25 cycles), CD21 (25 cycles), and ␤-actin (16 cycles). C is a water control (no cDNA added). B, Western blot analysis on purified splenic B and T cell sub- sets and total spleen (SP). Fifteen micrograms of total protein was loaded into

FIGURE 5. Cyclosporine treatment of splenocytes alters NFAT complex each well. CD21, ␤-actin, and CD3 Abs were used as controls. http://www.jimmunol.org/ formation on the CD21 and CD23 genes. Single-cell suspension of splenocytes (A–C) or the 2PK3 B cell lines (B) was treated with 10 ␮M CsA or an equal volume of ethanol (Et) vehicle in medium for 16 h before doing ChIP. Addi- tween the two lymphocyte types. In Fig. 6B, the presence of tional fresh, isolated splenocytes (Fr) were analyzed in A. Input is the cross- NFATc1, NFATc2, and NFATc3 protein was also identified by linked sonicated cell lysate from each sample; 1 ␮g of nonimmune mouse IgG Western blot analysis. The Ab regents specific for the NFATc4 and or nonimmune rabbit IgG was used as the isotype controls. One microgram of NFAT5 proteins could not resolve those species in either of these mouse monoclonal anti-YY1 (YY), NFATc3 as marked (A and C) (NF in B), cell samples (data not shown). However, based on the previous and rabbit polyclonal anti-Oct-1 (OC) Ab was used. PCR was done using transcript analysis, it is likely that these proteins are also present oligonucleotides specific for the CD21 intronic fragment (i8), promoter frag- by guest on September 25, 2021 ment (P2), the ␤-actin gene, and CD23 promoter. within T and B cells and that the Ab reagents are deficient for Western blot analysis. ChIP analysis was performed on purified, unstimulated splenic 2PK3 cells, a murine CD21-expressing B cell line. CsA treatment B and T cells with a variety of anti-NFAT Abs (Fig. 7). A diverse of splenocytes diminished binding of NFATc3 and blocked the pattern of NFAT family member binding was evident when com- binding of YY1 and Oct-1 to the gene sequences in the naive paring binding to the CD21, CD23, and IL-2 genetic elements. B splenocytes but only blocked NFATc3 binding to the CD21 gene cells demonstrated complexes with all five NFAT family members, within the 2PK3 B cell line. Previously, we demonstrated that YY1 although preferential complex formations were clearly evident. and Oct-1 binding to this fragment, via EMSA analysis, was di- Thus, the IL-2 promoter sequence was clearly isolated with minished using CsA-treated nuclear extracts (22). These data sug- NFATc1, NFATc2, and NFATc3 reagents, whereas CD23 showed gest that the chromatin architecture of the CD21 gene in normal, little complex formation with NFATc2. Similarly, the CD21 nontransformed cells requires NFAT binding to allow occupancy genomic elements were identified with a variety of the NFAT fam- of YY1 and Oct-1 to their binding sites within the CD21 gene. ily members, including NFATc3. No NFAT complexes were Binding of NFATc3 to the CD23 promoter sequence (Fig. 5C) was found associated with the control NIDO gene. also blocked in the presence of CsA. The analysis of NFAT complexes formed in T cells was more restricted than that seen in the B cell populations. Although Redundant expression and chromatin binding of NF-AT family NFATc3 binding was robust in B cells, it was absent for all of the members genes examined in the T cell populations. Instead, NFATc1, c4, We focused on the NFATc3 protein in the previous assays because and NFAT5 binding was detected with the CD21 gene, while no it is expressed in murine B cells, and the Ab reagents specific for NFAT binding to CD23 sequences was observed. The NFATc1 the protein generated robust data in EMSA supershift and ChIP and NFATc2 proteins were found in association with the IL-2 assays. However, others have shown that animals lacking NFATc3 gene. As seen for the B cell samples, NFAT complexes were not demonstrate normal levels of B cell CD21 expression, indicating found associated with the NIDO gene. that the NFATc3 gene product is dispensable for CD21 transcrip- tion (41). To determine whether other members of the NFAT fam- Pax5 binds the CD21 and CD23 genes independent of active ily are also expressed in murine B cells allowing for redundant transcription control of the CD21 gene, NFAT transcript and protein quantifi- Pax5 has been described as a master transcriptional activator re- cation was performed on splenic T and B cells. In Fig. 6A, RT- quired for B cell lineage commitment and induction of B cell- PCR analysis clearly showed the expression of NFATc1, NFATc2, specific genes (42, 43). One of the primary targets of Pax5 is NFATc3, NFATc4, and NFAT5 in both T and B cell subsets. The CD19, whose promoter possesses multiple Pax5 binding sites (44). accessory signaling protein NIP45 is also equally expressed be- Expression of CD19 is coincident with the expression of Pax5 at The Journal of Immunology 7145

the pro-B stage (43). Animals deficient in Pax5 show B cell arrest at the early pro-B cell stage (45). As shown in Fig. 1, there are also Pax5 binding sites in the CD21 and CD23 promoters, yet the ex- pression of these genes is much later in B cell maturation. There- fore, we were curious whether we could detect Pax5 binding to CD21 and CD23 sequences in marrow B cells (which do not tran- scribe these genes) or whether Pax5 was blocked from binding to these inactive genes. One problem in analyzing mature mouse marrow is that it not only possesses maturing B cell precursors but also mature B cells that recirculate from the spleen and other sites of activation. Thus, FACS analysis of normal, mature bone marrow (Fig. 8A, top two quadrants) shows a small percentage of CD21- and CD23-express- ing cells (ϳ3% of total cells). Animals lacking a functional BAFF receptor (the A/WySnJ strain) (46) produce normal B cells to the splenic T2 stage where they arrest development and differentiation. Thus, mature marrow cells from A/WySnJ mice possess normal immature B cell populations but lack recirculating CD21/CD23 Downloaded from FIGURE 7. positive mature B cells (Fig. 8A, bottom two quadrants). ChIP with NFAT family members. ChIP analysis was ϩ done as described using freshly isolated splenic B and T cells. One B220 B cells, representing immature and mature B cell pop- microgram of nonimmune mouse IgG (M.Control) or nonimmune rabbit ulations, were obtained from the marrow and spleen of the A/J IgG (R.Control) was used as Ab controls. The two different controls mouse and A/WySnJ mice and evaluated for the possession of the were required because the Abs specific for NFATc1, NFATc2, and Pax5 protein by Western blot analysis. As shown (Fig. 8B), compa- NFATc3 were mouse mAbs, while those specific for NFATc4 and rable levels of Pax5 protein is evident in marrow and spleen samples

NFAT5 were rabbit polyclonal Abs. One microgram of Abs was used from the A/J and A/WySnJ mouse. As controls, YY1 protein is also http://www.jimmunol.org/ for each immunoprecipitation. PCR was done using oligonucleotides evident in cell subsets from both animals, but CD21 is primarily found specific for CD21 intronic (i8), promoter (P2) sequences, CD23 pro- moter, and the IL-2 and NIDO genes. Input is the cross-linked sonicated in the A/J splenic sample, which is consistent with the defective BAFF receptor phenotype of A/WySnJ splenic B cells. cell lysate from each sample. H2O represents a negative PCR control with no DNA added. ChIP analysis was performed on the bone marrow cells and splenocytes from the A/J and A/WySnJ mouse for Pax5 binding to the CD19, CD21, and CD23 genes. Nonspecific binding to NIDO by guest on September 25, 2021

FIGURE 8. Pax5 is associated with CD21 and CD23 gene sequences in developing bone marrow B cells. A, FACS analysis of total bone marrow from 10-wk-old A/J (upper panels) and A/WySnJ (A/W) mice (lower panels) double stained with CD19/CD21 or CD23/CD21. B, Western blot analysis on purified B220ϩ splenic and bone marrow B cell populations from 10-wk-old A/J and A/WySnJ (A/W) mice. Total protein (15 ␮g) was loaded into each well. Actin was used as loading control. C, ChIP of Pax5 binding to CD21, CD23, and CD19 promoter fragments from bone marrow and spleen. ChIP assay for Pax5 was performed on total bone morrow cell population from 10-wk-old A/J (A/J BM) and A/WySnJ (A/W BM) mice. PCR was done for CD21, CD23, and CD19 promoter fragments. Total splenocytes from A/J mice (A/J SP) was used as a positive control for Pax5 binding to CD21, CD23, and CD19 promoters. PhosphorImager analysis and quantification of the PCR products were performed as described in Materials and Methods; fold enrichment ϭ (ChIP target/ChIP reference)/(input target/input reference). u, Precipitation with anti-Pax5 Ab; Ⅺ, precipitation with nonspecific Ab control. 7146 CHROMATIN COMPLEXES OF THE CD21 AND CD23 GENES Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 9. Quantification of CD19, CD21, CD23, and ADAM10 transcripts in defined splenic B cell populations. A, Splenic B cells were stained and sorted into discrete populations. Staining protocol was based on using CD21, CD24, and CD23 to elucidate T1 (CD21Ϫ/lowCD24highCD23Ϫ), T2 (CD21highCD24highCD23ϩ), MZ (CD21highCD24highCD23Ϫ), and FM (CD21lowCD24lowCD23ϩ) B cell subsets. Cells were gated with CD23Ϫ or CD23ϩ populations and then sorted by a combination of CD21 and CD24 surface expression. B, Cells from each representative B cell subset were analyzed for transcriptional expression of CD21, CD23, CD19, and ADAM10. Levels of expression for each gene are shown relative to ␤-actin. The data shown are averages of three independent cell sorts. was subtracted from each sample (see Materials and Methods). itation of the spleen samples is higher than the marrow for the The fold enrichment of Pax5 binding vs nonspecific binding was three genes because a higher percentage of cells in the spleen very similar for the different samples examined, regardless of the express Pax5 than in the marrow. gene. Thus, CD19, which is expressed in marrow and spleen, showed, as expected, specific binding of Pax5 to the CD19 pro- The CD21 and CD23 transcription profiles in spleen B cell moter in all three samples. If the Pax5 protein was excluded subsets from binding to the inactive CD21 and CD23 genes in the im- The data in this article and that described by others suggests mature marrow B cells, then no specific binding should have that CD21 and CD23 share many common transcriptional con- been evident in marrow samples compared with the splenic trol regulators. However, the CD21 and CD23 proteins vary samples, which was not the case. Therefore, the CD21 and in the level of surface expression during peripheral B cell devel- CD23 promoters are accessible to Pax5 binding, even though opment. During B cell maturation, the CD21 and CD23 proteins the genes are transcriptionally silent. The relative fold precip- are first found on cells making the T1 to T2 transition in the The Journal of Immunology 7147 spleen (9, 47). T2 cells express both CD21 and CD23. The populations or total splenocytes, it is not likely that our purification CD21 proteins are expressed on the FM B cells and, to an el- schemes resulted in cellular activation. If we were identifying DNA/ evated level, on the surface of MZ cells. The CD23 product is protein complexes from a small subset of activated cells then, for expressed on the surface of T2 cells and FM B cells but not by example, we should have observed NFATc3 binding to gene se- the MZ population (9). quences in the T cell populations because activated T cells clearly The CD23 protein, found on the surface as a homotrimer, is translocate NFATc3 into the nucleus, and CD21, CD23, and IL-2 known to be cleaved from the cell by the ADAM10 protease (48); gene sequences are recognized by that protein. Because this was not therefore, the MZ cells could conceivably lose CD23 from the cell observed in this report, the simplest interpretation of our data is that surface by elevated expression of ADAM10. there are low levels of different NF-␬B and NFAT species that are Previously, it had been shown that CD23 transcripts are de- constitutively present within the nucleus of resting splenic B and T pressed in MZ cells compared with FM and T2 CD23 levels (49) cells, and these proteins can establish binding complexes on target using a semiquantitative RT-PCR analysis. We chose to reanalyze genes. this question using real-time quantitative RT-PCR for CD19, Investigations into the role of the NFAT proteins in the CD21, CD23, and ADAM10 transcripts in sorted T1, T2, MZ, and regulation of CD5 expression may provide clues pertaining to FM cell populations. Splenic B cells were enriched with B220ϩ CD21 and CD23 regulation. The expression of CD5 is clearly up- magnetic beads and stained with Abs specific for CD21, CD23, regulated by NFAT activation following calcium flux (via cross- and CD24 (Fig. 9). CD23-positive (the R5 population) and CD23- linking the BCR-BCR) due, in part, to the presence of multiple negative cells (the R2 population) were then analyzed for relative NFAT binding sites in the promoter of the gene (50–53). Normal Ϫ expression of CD21 and CD24 and sorted (9, 47). The CD23 B-1a cells, however, constitutively express CD5 without requiring Downloaded from CD21ϩ, CD24high cells were identified as MZ cells (R3), whereas exogenous activation. We have previously shown that the expres- the CD23ϪCD21ϪCD24ϩ cells were sorted as T1 cells (R4). The sion of the CD21 gene is sensitive to CsA treatment (thus blocking CD23ϩ cells were similarly sorted into the CD21ϩ, CD24low pop- NFAT protein migration into the nucleus) (22). However, the ex- ulation (FM cells, R6) and the CD21ϩ, CD24high population (T2 pression of CD21 on B cell surfaces is not increased following cells, R7). Total RNA was obtained from these cell populations BCR-mediated activation (our unpublished data). One difference

and analyzed by real-time quantitative RT-PCR (based on ␤-actin between CD5 and CD21 is that NFAT binding sites of the former http://www.jimmunol.org/ equivalence). gene are within the promoter while those in CD21 are within the Transcript analysis of the four B cell subsets indicated that intronic enhancer/silencer. CD21 transcription was highest in the MZ and T2 cells, less for Whether the NFAT proteins are directly facilitating transcrip- the FM cells, and the least in the T1 immature B cells. These tional induction (or repression) at the CD21 intronic site or are data match that of the cell surface expression levels. The CD23 altering the organization state of the chromatin by recruiting transcripts also matched the cell surface staining data in that the histone modification enzymes is not known (7). Our previous T1 and MZ cells had very low levels of transcripts whereas T2 observation that CD21 transcriptional control is influenced by and FM cells had roughly equivalent levels. CD19 was abun- the state of histone acetylation (21) plus our data in this report dantly expressed in all four splenic B cell types as was the showing the exclusion of YY1 and Oct-1 proteins to the CD21 by guest on September 25, 2021 ADAM10 gene. These data confirm that CD23 transcript levels gene following CsA treatment in splenocytes (Fig. 5) may sug- are depressed in MZ cells and that the absence of CD23 on the gest a role for the NFAT binding proteins to alter chromatin surface of MZ cells is due to the absence of CD23 mRNA, not structure in the CD21 gene. A low level of constitutive NFAT heightened ADAM10 production. Thus, even though the CD21 movement into the nucleus of T and B cells may be enough to and CD23 genes share a number of key regulatory mechanisms, satisfy chromosomal organization duties, but large-scale tran- the stage-specific transcriptional control of these two genes is scriptional induction of NFAT target genes would require the distinct. translocation of the cytoplasmic stores of the proteins. Animals deficient in NFATc2 and NFATc3 express CD21 Discussion and CD23 on the surface of splenic B cells (41). MZ B cells are This article has focused on defining in vivo binding of candidate reduced in such animals based on the selective loss of transcription control factors to the CD21 and CD23 genes in ex- CD21highIgMhigh cells. In addition, animals lacking NFATc1 pressing and nonexpressing cells using ChIP. In previous reports, express normal levels of CD21 and CD23 (54). In contrast, we have identified a number of potential protein/DNA complexes animals deficient in NFATc1 show an absence of CD5ϩ B cell based on conserved sequence motifs and EMSA shift data (20, 22). populations (but those deficient in NFATc2 possess normal Such assays are capable of cataloguing the potential of such com- CD5 expression), suggesting that the NFATc1 protein is either plexes forming yet do not address whether such complexes actu- directly responsible for constitutive CD5 expression or the out- ally do develop in the nucleus of the cell. growth of the B-1a cells capable of expressing that surface pro- The ChIP assay defined constitutive binding of NFAT and tein (54). We demonstrate in this article that multiple NFAT NF-␬B species to the CD21 and CD23 (and IL-2) genetic control family members do bind the intronic NFAT binding sites of the regions. These families of transcription factors are notable for their CD21 and CD23 genes and thus may functionally complement cytoplasmic localization until cellular activation. However, we ob- different family members. Indeed, our data showing expression serve nuclear binding complexes of NF-␬B-p52 and NFAT pro- of all five NFAT family members in splenic B cells suggest that, teins in the absence of specific activation. One argument to explain to accurately define the role of NFAT family members via our findings could be that the spleen consists of mixed populations mouse knockout models, strains may need to be developed lack- of activated and unactivated cells, and the sensitivity of the ChIP ing B cell expression of all of the family members (thus the procedure allows for the detection of a small subset of activated genetic equivalent of CsA treatment). cells. We were very careful in these ChIP analyses to prevent The binding of the B cell-specific transcription factor Pax5 to cellular activation. Fresh cells were isolated and immediately the CD21 and CD23 promoters could help define the B cell-spe- cross-linked with formaldehyde or quickly purified and formaldehyde cific expression of these genes. CD19 has been defined as relying treated. Because the same results were obtained from purified cell on Pax5 for B cell stage-specific expression, yet CD19 is expressed 7148 CHROMATIN COMPLEXES OF THE CD21 AND CD23 GENES

FIGURE 10. Alternative pathway for the generation of MZ B cells. A, Schematic for generation of MZ and FM cells from a T2 splenic precursor. B, Schematic for generation of MZ cells from a lineage of T1 cells dis- tinct from the population of T1 cells giving rise to T2 and FM cells. Downloaded from

much earlier in the B cell lineage than CD21 and CD23 (42, 44). imals lacking B cells that can respond to Notch ligands fail to

As shown in Fig. 8, Pax5 binding to the CD21 and CD23 genes establish a MZ cell population (49, 57–59). The CD23 gene http://www.jimmunol.org/ was as evident in immature marrow cells not transcribing those clearly possesses binding sites to positively respond to Notch genes as it was to the CD19 gene, which is being transcribed. ligand activation (via heterodimer binding of RBP-J␬ and the This finding suggests that either additional positive regulatory cytoplasmic Notch binding partner, Notch intracellular frag- components are missing for the CD21 and CD23 genes to be ment (NIC)) (60); thus, it is difficult to envision a transcrip- activated or that Pax5-induced transcription is repressed. Be- tional repression pathway that specifically represses CD23 ex- cause CD23 is differentially expressed in splenic B cell matu- pression in MZ cells while that of CD21 is elevated. ration compared with CD21 (55, 56), two distinct regulatory Interestingly, FM cells of the spleen and peripheral blood B components must exist, one for CD21 and the other for CD23. cells obtained from Notch2-deficient animals (compared with The presence of an inhibitory protein binding to the CD21 pro- wild type) show a reduced mean fluorescent intensity of stain- by guest on September 25, 2021 moter, such as the E2 complex described for the human CD21 ing for CD21 while that for CD23 is unaltered (58). gene (17), could mitigate the action of an active Pax5 protein in An alternative interpretation of these data would suggest that a maturing marrow B cells. MZ-specific lineage of splenic B cells exists that never expresses It was recently reported that BAFF-dependent activation of B CD23 (Fig. 10B). Thus, the T1a subset may produce two lineages, cells was required for the expression of CD21 and CD23 (30). one seeding the MZ compartment and the other the T1b lineage The signaling pathway ascribed to BAFF is that of the NF-␬B that then gives rise to the T2 and FM populations. In this scenario, pathway, requiring receptor interacting protein and NF-␬B-in- CD23 expression does not need to be elevated and then extin- ducing kinase activation and generating the p52 subunit (31, guished but instead is repressed in the entire lineage. The T1b 32). The constitutive binding of the NF-␬B-p52 subunit to the lineage, in contrast, allows for CD23 and CD21 expression in CD21 and CD23 genes in T and B cells (Figs. 3 and 4) (56) T2 and FM cells. The model in Fig. 10B predicts that, although requires a model of low-level translocation of NF-␬B species the CD21 and CD23 genes share a number of transcriptional into the nucleus of both cell types. We have previously shown control elements and binding proteins (61), they still require that NF-␬B-p52 binds to the CD21 and CD23 genes in splenic unique regulatory steps presumably controlled by distinct pro- B cells from BAFF receptor-defective animals (A/WySnJ) as teins. The coordinated expression of CD21 and CD23, con- the wild-type controls (A/J) (56), indicating lack of this tran- trasted with that of CD19, all of which appear to use Pax5, scription factor is not blocking CD21 and CD23 expression in presents a defined testable pathway for transcriptional control in A/WySnJ mouse. transitional B cell subsets, similar to those described for plasma The current model of B cell development in the mouse involves cell maturation and differentiation (62, 63) and B cell marrow a linear maturation pathway of pre-T1 B cell release from the maturation (43). marrow and seeding of such cells into peripheral lymphatic sites such as the spleen (Fig. 10A) (47). T1 cells in the spleen possess low levels of Bcl2 and are susceptible to BCR-induced apoptosis. Our Acknowledgments (and other’s) analysis of T1 cells suggests this group possesses We thank the University of Utah FACS, Oligonucleotide, and Peptide two subsets, one which expresses CD23 but not CD21, a subset Sequencing cores for their assistance. We also like to thank all the we have dubbed T1b (compared with T1a that lack both CD21 members of our laboratories for their assistance and critiques of and CD23 expression) (55, 56). These cells then mature into the this work. T2 subset (CD21ϩ/CD23ϩ). Most models suggest that the T2 cells seed both the FM B cell compartment (CD21ϩ/CD23ϩ) and MZ population (CD21ϩ, CD23Ϫ). The MZ site is unique in Disclosures the splenic niche in that Notch ligand signaling is critical: an- The authors have no financial conflict of interest. The Journal of Immunology 7149

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