An Intronic Silencer Regulates B Lymphocyte Cell-And Stage-Specific Expression of the Human Complement Receptor Type 2 (CR2, CD21) Gene

Home , CD4, Complement receptor 2

An Intronic Silencer Regulates B Lymphocyte Cell- and Stage-Specific Expression of the Human Complement Receptor Type 2 (CR2, CD21) Gene

This information is current as Karen W. Makar, Christine T. N. Pham, Marlin H. Dehoff, of September 23, 2021. Siobhan M. O'Connor, Susan M. Jacobi and V. Michael Holers J Immunol 1998; 160:1268-1278; ; http://www.jimmunol.org/content/160/3/1268 Downloaded from

References This article cites 79 articles, 52 of which you can access for free at: http://www.jimmunol.org/content/160/3/1268.full#ref-list-1

Why The JI? Submit online. http://www.jimmunol.org/

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on September 23, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. An Intronic Silencer Regulates B Lymphocyte Cell- and Stage-Speciﬁc Expression of the Human Complement Receptor Type 2 (CR2, CD21) Gene1

Karen W. Makar,2* Christine T. N. Pham,2† Marlin H. Dehoff,* Siobhan M. O’Connor,† Susan M. Jacobi,† and V. Michael Holers3*

Human CR2 (CD21) is a B lymphocyte protein whose surface expression is restricted primarily to the mature cell stage during development. To study the transcriptional mechanisms that govern cell- and stage-restricted CR2 expression, we first performed transient transfection analysis using constructs extending from ؊5kbto؉75 bp (؊5 kb/؉75) in the CR2 promoter. The promoter was found to be broadly active, with no evidence of cell- or stage-specific reporter gene expression. However, the addition of a 2.5-kb intronic gene segment (containing a DNase I hypersensitive site) to the (؊5-kb/؉75) construct resulted in appropriate Downloaded from -reporter gene expression, defined as the silencing of the (؊5-kb/؉75) promoter activity only in non-CR2-expressing cells. Inter estingly, appropriate reporter gene expression required stable transfection of the constructs in cell lines, suggesting nuclear matrix or chromatin interactions may be important for appropriate CR2 gene expression. Importantly, transgenic mice also required the intronic silencer to generate lymphoid tissue-specific reporter gene expression. Some transgenic founder lines did not demonstrate reporter gene expression, however, indicating that additional transcriptional regulatory elements are present in other regions of http://www.jimmunol.org/ the CR2 gene. In summary, these data support the hypothesis that human CR2 expression is regulated primarily by an intronic silencer with lineage- and B cell stage-specific activity. The Journal of Immunology, 1998, 160: 1268–1278.

uman CR2 (CD21, C3d,g/EBV receptor) is a 145-kDa that occurs in the IgMlow/B220intermediate/IgDϪ developmental glycoprotein and a member of the regulators of comple- stage (reviewed in Ref. 12). An identical CR2 expression pat- ment activation gene family found on human chromo- tern is found using the class I:anti-class I transgenic system (13) H Ϫ some 1q32 (reviewed in Refs. 1 and 2). CR2 is a high afﬁnity in which CR2 immature B cells undergo editing, and then receptor for the activation fragments of complement component transition to an IgMintermediate/B220high/IgDϩ/CR2ϩ phenotype

C3 termed iC3b and C3d,g (3, 4). In addition, CR2 mediates EBV after becoming tolerant to self Ag (11) (Y. Kozono, D. by guest on September 23, 2021 infection by serving as the receptor for the EBV intrinsic mem- Nemazee, and V. M. Holers, unpublished observations). In ad- brane protein gp350/220 (5, 6). CR2 is also a B cell receptor for dition to B cells, human CR2 is also expressed on follicular CD23 (7) and possibly IFN-␣ (8). dendritic cells (14), early thymocytes (15), a small subset of CR2 expression on human B lymphocytes was first studied by CD4ϩ and CD8ϩ peripheral T lymphocytes (16, 17), and epi- Tedder et al., and found to be limited to the mature and early thelial cells (18). activated stages during development (9). Using flow-cytometric Some studies have reported the immortalization of human pre-B analysis, CR2 was undetectable on normal bone marrow-de- cells with EBV, possibly using CR2 as a receptor, and that CR2 is rived pre-B or immature B cells, and was also lost at the plasma on a small subpopulation of CD19ϩ/IgMϪ pre-B cells and pre-B cell stage. Using a second anti-CR2 mAb, CR2 expression was cell malignancies (19). However, based on the analysis of CR2 again found to be limited to IgMϩ/IgDϩ bone marrow cells and expression on normal human B cells and well-characterized B cell to be absent from IgMϩ/IgDϪ cells (10). Studies using the hen lines (9, 10), in addition to the pattern of expression of mouse CR2 egg lysozyme:anti-hen egg lysozyme dual transgenic system (20), which has marked structural and functional similarities with have shown that mouse CR2 is among the first cell surface human CR2 (21–23), it is apparent that the expression of human markers detected on bone marrow B cells after the escape of CR2 on the great majority of developing B cells appears at a com- these cells from negative selection by self Ag (11), a process parable point as murine CR2. That point is during the transition from IgMϩ/IgDϪ to IgMϩ/IgDϩ cells. *Departments of Medicine and Immunology, Division of Rheumatology, University CR2 plays an important role in the generation of a normal † of Colorado Health Sciences Center, Denver, CO 80262; and Department of Med- immune response. Mice lacking CR2 show greatly impaired icine, Division of Rheumatology, Washington University School of Medicine, St. Louis, MO 63110 humoral immune responses, particularly in the generation of a Received for publication July 7, 1997. Accepted for publication October 22, 1997. switched IgG response (24–26). In addition, coupling the CR2 The costs of publication of this article were defrayed in part by the payment of page ligand C3d directly to an Ag results in a 10,000-fold decrease charges. This article must therefore be hereby marked advertisement in accordance in the amount of Ag necessary to generate an IgG response (27). with 18 U.S.C. Section 1734 solely to indicate this fact. Several other studies have demonstrated a role for human CR2 1 This work was supported by National Institutes of Health RO-1 AI31105 and Smyth in the activation and proliferation of B lymphocytes: 1) cross- Professorship in Rheumatology (V.M.H.). linking of CR2 leads to increased [Ca2ϩ] and proliferation in 2 The first two authors contributed equally to these studies. i the presence of suboptimal amounts of phorbol esters, T cell 3 Address correspondence and reprint requests to Dr. V. Michael Holers, Division of Rheumatology Box B-115, University of Colorado Health Sciences Center, 4200 East factors, or anti-IgM (28, 29); 2) coligation of CR2 with mIgM Ninth Ave., Denver, CO 80262. E-mail address: [email protected] results in substantially enhanced expression of c-fos mRNA

(30); 3) CR2 is noncovalently associated with CD19 (31), a Materials and Methods membrane glycoprotein capable of promoting B cell activation RT-PCR for endogenous CR2 expression (32); 4) the interaction of CR2 with CD23 in the presence of Total cellular RNA was prepared from 1 ϫ 108 cells using the Chirgwin IL-4 and CD40-ligand results in enhanced B cell production of technique (53). The RT reaction using 4 ␮g total RNA proceeded for 60 IgE (33, 34); 5) CR2 ligation induces homotypic adhesion of min at 30°C using RT with both oligo(dT) and random primers (Invitrogen tonsillar B lymphocytes and B cell lines (30, 35); and 6) EBV Corp., San Diego, CA). The reaction mixture was then split into equal binding to CR2 on resting B cells increases CD23 mRNA levels aliquots and used directly in the PCR reaction with the following oligo- Ј Ј independently of viral gene expression (36). nucleotide primers: CR2, 5 -GGCAGGAAAACTTTCTATATGG-3 and 5Ј-ATGAGGGGCAGGTTGGCTCC-3Ј (corresponding to positions 2271 CR2 is one of a number of non-Ig B lymphocyte stage-specific to 2291 and 2787 to 2768, respectively, of the CR2 cDNA sequence (54)), markers expressed during development and activation (reviewed in and MCP, 5Ј-ACATACCTAACTGATGAGACCCACAGA-3Ј (corre- Refs. 19 and 37). Other genes of this type include CD10, CD19, sponding to nucleotides 1 to 28 of the MCP cyto-1 exon (55)) and 5Ј- CD20, CD22, CD23, CD40, and HLA class II. Several transcrip- CAAGCCACATTGCAATATTAGCTAAGCCACA-3Ј (corresponding to nucleotides 1323 to 1293 of the MCP cDNA sequence (56)). MCP is ex- tional factors such as B-specific activation protein, Oct-1, and pressed in all bone marrow-derived cell lines tested to date (55). The PCR Oct-2 have been shown to play important roles in the transcrip- reactions proceeded at 95°C ϫ 1 min, 56°C ϫ 1 min, and 72°C ϫ 2 min tional control of CD19, CD20, and CD22, which are three proteins for 20 cycles (Perkin-Elmer Cetus, Norwalk, CT). PCR products were elec- whose expression is also primarily restricted to B cells (38–40). trophoresed on a 1.8% agarose gel. However, the regulatory elements that control cell- and stage-spe- Nuclear runoff analysis cific expression of these genes have not yet been clearly identified Nuclear runoff analysis was performed as described (57) with the following Downloaded from (37, 41, 42). In addition, there is little understanding of the mo- changes. Transcription proceeded for 20 min at 30°C. RNA was harvested lecular signals that lead to transcription of genes that, like CR2, are and hybridized to DNA slot blots with 2.5 ␮g of target DNA at 42°C for primarily activated after the negative selection processes that occur 72 to 96 h. Blots were washed in 2ϫ SSC, 0.1% SDS twice for 5 min at in the bone marrow. 20°C, followed by washing in 0.2ϫ SSC, 0.1% SDS twice for 10 min at In B cell lines, infection with EBV or transfection with con- 55°C. The previously described CR2 cDNA-containing plasmid 4 pBSCR2–12 (54), lacking the poly(A) tail, was in the pBS (Bluescript; structs directing the synthesis of the EBV transactivator EBNA-2 Stratagene, San Diego, CA) plasmid. A CR2–5Ј genomic DNA-containing http://www.jimmunol.org/ leads to increased expression of CR2 in addition to CD23 (43, 44). plasmid substrate was created as a 418-bp dsDNA PCR product encoding Recently, EBNA-2 has been shown to transactivate CD23 and sev- the sequence from ϩ1toϩ418 of the CR2 gene hnRNA transcript using Ј eral other target genes at least in part by relieving repression me- oligonucleotide primers 5 -ATTCGAATTCAGCTGCTTGCTGCTC CAGCCTTGCC-3Ј and 5Ј-ATTCGAATTCAGAAACTTTCCTGCCG diated by the regulatory protein CBF1 (45). CBF1 binds to the CACGCTCCA-3Ј. The PCR product was subcloned into pBS using the CD23 promoter site previously defined as the EBNA-2 response flanking EcoRI oligonucleotide sites. To assure the quantity and quality of element (46, 47). EBNA-2 also has been shown to act as a tran- RNA transcripts, ␤-actin and Alu (57) probes were also used as targets. scriptional activator by interactions with PU.1 (48) and with the Autoradiographs were developed using Kodak XAR-5 film. Densitometric general transcription factor TFIIE and a novel coactivator p100 analysis was performed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

(49). The mechanisms by which EBV or EBNA-2 increases CR2 by guest on September 23, 2021 expression, though, are not yet known. Other studies have sug- Cloning of CR2 regulatory elements gested that HTLV-1 infection of T cells can also up-regulate CR2 ␭ Phage genomic subclones of a previously described regulators of com- expression (50); however, the specific mechanisms by which this plement activation-2 yeast artificial chromosome clone containing the CR2 occurs are also unknown. gene (58) were created using the Lambda DASH II BamHI cloning vector We have previously cloned and sequenced the human CR2 pro- (Stratagene, La Jolla, CA) (library gift of Dr. Dennis Hourcade, Washing- Ϫ ϩ Ϫ ϩ ton University School of Medicine, St. Louis, MO). The library was moter-containing region from 1253 bp to 75 bp ( 1253/ 75) screened using genomic clones encompassing both the 5Ј regulatory region (51). We demonstrated that this region contains potential binding (pBSCR2gH-3; see Fig. 2A for location of this and other subclones) (51) sites for the widely expressed transcription factors SP1 and AP-1/ and a 4.4-kb BamHI-SalI subclone (pBSCR2gA8B-1) containing the ex- Ј AP-2. In addition, we showed that the (Ϫ1253/ϩ75) region could treme 5 sequences of the previously described cosmid A8.1 (54). This strategy ensured that any clone hybridizing with both probes would contain direct transcription of a CAT reporter gene when transiently trans- genomic elements spanning the first intron of the human CR2 gene. One fected into the CR2-positive Raji B cell line (51). However, neither such ␭ phage clone, designated ␭ CR2g1.12, was identified and further we nor others (52) have been able to demonstrate that this partic- characterized for these studies. This clone contained approximately 18 kb ular region contains regulatory elements that control cell- or stage- of DNA spanning from approximately 9.5 kb 5Ј of the first CR2 exon and Ј specific CR2 transcription. extending approximately 3.5 kb into the 5 end of the A8.1 cosmid clone. Analysis of human genomic DNA paired with this clone using informative To utilize human CR2 as a model of B lymphocyte transcrip- restriction enzyme digests demonstrated identical band sizes, indicating tional regulation, we extended our analysis further 5Ј of the tran- that the phage clone was unaltered during yeast artificial chromosome and scriptional initiation site and extensively characterized the proxi- subsequent ␭ phage cloning and represents authentic human genomic DNA mal CR2 5Ј promoter elements. Because we found that these (data not shown). Likewise, partial DNA sequence analysis described in Results further confirmed this conclusion. The sequence of the 2.5-kb additional upstream gene segments still did not confer cell- and XbaI-SacI intronic fragment described in this work has been entered in stage-specific expression, we searched for other regulatory ele- GenBank under accession number U34741. ments throughout the CR2 gene. In this study, we report the iden- Plasmid constructs tification of an intronic element that regulates both cell- and stage- specific CR2 transcription by utilizing a transcriptional silencing The plasmid constructs shown in Figure 3A were made using a neomycin mechanism. The silencer element requires chromosomal integra- (neo) reporter gene (59) (generous gift of Dr. Tim Ley, Washington Uni- versity School of Medicine). The CR2(Ϫ1253/ϩ75)-neo and CR2(Ϫ149/ tion for its activity, suggesting that nuclear matrix or chromatin ϩ75)-neo constructs were created by subcloning DNA from the previously interactions may be necessary for its effects. described plasmid pBSCR2 (Ϫ1253/ϩ75) (51). The CR2(Ϫ5-kb/ϩ75)- neo reporter construct was created by excision of the 5-kb SacI fragment (pBSCR2gS-2) of ␭ CR2g1.12 (see Fig. 2A also) and subcloning into the Ϫ Ϫ ϩ 4 Abbreviations used in this paper: EBNA, Epstein-Barr virus nuclear Ag; CAT, unique SacI site at 149 bp in CR2( 149/ 75)-neo. Constructs contain- chloramphenicol transferase; CBF, c-promoter binding factor; CRS, complement re- ing the 2.5-kb intronic region described in Results were created by excision ceptor 2 silencer; hnRNA, heterogeneous nuclear RNA; HS, hypersensitive site; of a 2.5-kb XbaI-SacI fragment of pBSCR2gS-1 (using a polylinker XbaI MCP, membrane cofactor protein; neo, neomycin; SCR, short consensus repeat. site at the 3Ј end) and subcloning into a 3Ј XbaI site in both CR2(Ϫ5-kb/ 1270 CELL- AND STAGE-SPECIFIC SILENCING IN THE HUMAN CR2/CD21 GENE

ϩ75)-neo and CR2(Ϫ1253/ϩ75)-neo downstream of the SV40 polyade- To screen for potential founders in the populations of putative trans- nylation sequence. genic pups, tail DNA was purified and tested by dot blot using the BamHI- HindIII fragment encoding the neo reporter cDNA. Founders that were Transfection and reporter analysis identified using this means were confirmed by Southern blot analysis. Con- firmed founders were then subsequently backcrossed onto the B10.D2 Raji cells used for transfection were maintained in suspension culture in strain and followed by tail blot analysis. F1 and F2 mice were used for the Iscove’s medium containing 10% FCS, supplemented with 2 mM glu- analysis of reporter gene expression presented in this study, unless other- tamine, penicillin, and streptomycin at 37°C in 5% CO2. All other cells wise noted. were maintained in suspension culture in RPMI 1640 containing the same To analyze neo reporter gene mRNA expression in individual murine additives and 10% FCS (Reh were grown in 15% FCS). Approximately 12 tissues, RNA was first isolated using the Chirgwin technique (53). In some to 15 h before transfection, cells were fed and resuspended in media at a experiments, S1 analysis was performed using the CR2(Ϫ149/ϩ75)-neo ϫ 5 density of 5 10 cells/ml. plasmid that had been linearized within neo using BglII and end labeled at For transient transfections of cells, plasmid DNA (50 ␮g) was trans- 7 that site using T4 kinase. The control for endogenous murine mRNA quan- fected into 2 ϫ 10 cells by electroporation with 125 ␮g of carrier salmon ␤ tity and quality in these studies was 2-microglobulin (63). This probe was sperm DNA, using conditions as described below and a BTX Transfector prepared by digestion with EcoRI, followed by calf intestinal phosphatase 300 (Biotechnologies and Experimental Research, San Diego, CA). As an ␮ treatment and end labeling with T4 polynucleotide kinase. In other exper- internal control to correct for transfection efficiency, 1 to 5 g of RSV-neo iments, neo reporter mRNA expression was analyzed using RT-PCR. For (60) was cotransfected with neo reporter constructs. this analysis, 50 ␮g of RNA from each tissue was first treated with 10 U For stable transfection studies, neo reporter constructs (15 ␮g) directed ϫ RNase-free DNase I (Boehringer Mannheim Corp., Indianapolis, IN) for 30 by CR2 promoter regions (see also Fig. 3A) were cotransfected into 2 min at 37°C in 0.1 M sodium acetate, 5 mM MgCl , pH 5, followed by 7 ␮ 2 10 cells by electroporation using a BTX transfector with 125 g of carrier ethanol precipitation. DNase I-treated RNA was resuspended in TE at 1 salmon sperm DNA and 1.5 ␮g of either the pMon (gift of Drs. Sandip ␮ ␮ ␮ g/ l, and 1 g was utilized in each RT-PCR reaction. RT reactions were Downloaded from Godambe and David Chaplin, Washington University School of Medicine) primed with a 1:1 mixture of oligo(dT) and random primers, according to (Jiyoye and SKW-1 cell lines) or pRep4 (Stratagene) (Jiyoye, Reh, and manufacturer’s specifications (GeneAmp; Perkin-Elmer Cetus). Parallel K562 cell lines) hygromycin-resistance plasmid. The following transfec- samples with and without Moloney murine leukemia virus RT were used to tion conditions were established by pilot experiments and used for transient assure that templates were mRNA and not DNA derived. To further assure transfection experiments and to develop stably transfected lines: Raji, 1200 ␮ ␮ ␮ that amplified bands were mRNA derived, a strategy was designed to detect F and 225 V; Jiyoye, 800 F and 225 V; and SKW-1 and K562, 600 F spliced neo mRNA using the oligonucleotides 5Ј-CCTTACTTCTGTGGT and 225 V. Reh cells were transfected using 1 ␮g plasmid DNA and 0.1 ␮g Ј Ј Ј ␮ GTG-3 and 5 -CCTCATTAAAGGCATTCC-3 . These oligonucleotides pRep-4 hygromycin-resistance plasmid in addition to 10 g DEAE-dextran generate a 115-bp PCR fragment that results from splicing out 66 bp of the http://www.jimmunol.org/ (Pharmacia, Piscataway, NJ) at 950 ␮F and 300 V. All cells were main- SV40-derived 3Ј untranslated sequence at positions (1568)TAAG/GTA tained in the absence of selective medium for 48 h after transfections and AAT... to ...TTTTAG/ATTC(1641), a common splicing event, as previ- were then placed into 200 ␮g/ml (Reh), 300 ␮g/ml (K562), or 400 ␮g/ml ously described (64). PCR was performed using the GeneAmp RNA PCR (Jiyoye and SKW-1) of hygromycin (Calbiochem, San Diego, CA) in kit (Perkin-Elmer Cetus), according to manufacturer’s directions, using 2 RPMI medium, as above. After the initial outgrowth of a stable resistant mM MgCl and amplified 35 cycles using 94°C ϫ 1 min, 54°C ϫ 1 min, population, RNA was harvested as above for S1 analysis, and DNA was 2 and 72°C ϫ 1 min. In preliminary experiments, the 115-bp mRNA-derived harvested in parallel for Southern blot analysis. PCR fragment was cloned using the TA vector system (Invitrogen Corp.), The levels of neo mRNA and other reporter mRNA in transfection and nucleotide sequence analysis of the fragment confirmed the identity studies were determined by ImageQuant following PhosphorImager (Mo- and sequence of the spliced product. lecular Dynamics) analysis. The ␤-actin probe described above also served

The positive control for endogenous murine RNA quality and relative by guest on September 23, 2021 as an internal control for RNA quantity and quality in transient transfec- quantity for these studies is Crry/p65 (65), a gene expressed in all murine tions of neo reporter constructs. To determine the relative copy number of tissues (65, 66), which was detected using the oligonucleotides 5Ј-CACT stably incorporated neo reporter plasmid equivalents, DNA from stably GCCCAGCCCCATCAC-3Ј, derived from short consensus repeat (SCR) 1 transfected cell lines was digested with HindIII and BamHI to release the in the cDNA sequence, and 5Ј-CGAGATACACATTTGGCCAG-3Ј, de- neo-encoding cassette from the surrounding sequences of the expression rived from SCR 5 in the cDNA sequence (65). PCR conditions were iden- plasmid. Following transfer to nitrocellulose, the blot was probed using a tical, except for a hybridization temperature of 58°C. This oligonucleotide 2-kb BglII-BamHI neo cDNA probe. The internal control used to assure pair results in the creation of a 953-bp spliced PCR product from mRNA equivalent amounts of genomic DNA in each lane was a 1.8-kb EcoRI that extends over ﬁve exon-intron junctions in the Crry gene. fragment of the human CGL-1 cDNA (61).

DNase I hypersensitivity analysis of nuclear chromosomal DNA DNase I hypersensitivity analysis was performed as previously described Results (62). After isolation of nuclei and treatment with increasing amounts of CR2 mRNA expression is cell and stage specific DNase I, followed by overnight proteinase K digestion, DNA was ex- Previous results using flow-cytometric analysis of B cell lines have tracted with phenol:CHCl3, CHCl3 alone, and ethanol precipitated and re- solubilized into TE buffer. DNA was then digested with BamHI, electro- shown that CR2 surface expression is limited to the mature B phoresed, transferred to nitrocellulose, and probed with a 4.4-kb probe lymphocyte stage (9). To determine whether this reflects the pres- from the 5Ј end of cosmid A8.1 (see Fig. 3A), whose 3Ј anchored end is the ence or absence of CR2 mRNA, and to establish the CR2 pheno- BamHI site indicated. A 0.6-kb XbaI-BamHI fragment of the A8.1 cosmid type of both B and non-B cell lines, we have developed a sensitive Ј with the same 3 end was also used as a probe. A PhosphorImager and method to detect CR2 mRNA using RT-PCR (Fig. 1A). For these ImageQuant software were used to analyze the autoradiographs from the CR2-negative cell lines. studies, we utilized B cell lines that have been phenotyped by Ig heavy and light chain rearrangements and other non-CR2 surface Creation and analysis of transgenic mice markers to the pre-B (Nalm-6, Nall-1, Reh), mature (Raji, Jiyoye), To create transgenic mice using the CR2(Ϫ5-kb/ϩ75)-neo constructs with or late Ig-secreting (SKW-1, DHL-4) stages (67, 68). or without the intronic regulator described in Results, plasmid DNA was Using this method, we found that the pre-B cell lines Nalm-6, first linearized using KpnI. FVB female pups (21–28 days old) were used Nall-1, and Reh had no detectable CR2 mRNA, as evidenced by as embryo donors. The mice were superovulated using 5 IU pregnant the lack of the 516-bp CR2 band. These and all other cells dem- mares’ serum gonadotropin. After 44 h, 5 IU human chorionic gonadotropin was injected i.p., and mice were then placed with fertile males for onstrated a 329-bp MCP-specific band as a control for mRNA mating. Following this, oviducts were then dissected and flushed with 50 quality and RT efficiency. This result confirms our previous flow- ␮g/ml bovine testis hyaluronidase, embryos were sorted, and fertile em- cytometric, Northern blot, and S1 nuclease analysis, in which we bryos were injected with a solution of MTE (10 mM Tris, pH 8, 0.25 mM could not detect CR2 in the pre-B cell lines by any technique (data EDTA, and Na salt) containing 2 ng/ml of construct. Following injection, embryos were transferred into pseudopregnant recipient females, which not shown). In contrast, mature B cell lines expressing surface were then housed in a clean specific pathogen-free barrier for gestation and CR2 (Raji, Jiyoye) had clearly detectable CR2 mRNA expression. lactation. Two later stage Ig-secreting cell lines SKW-1 and DHL-4, which The Journal of Immunology 1271

have no detectable CR2 by flow-cytometric, Northern, or S1 nuclease analysis, also had no detectable CR2 signal. In two T cell lines tested (HSB-2, CEM), no CR2 mRNA was detected. Other T cell lines such as Jurkat, which are representative of the small subset of CR2ϩ peripheral T cells, did demonstrate a CR2 mRNA signal (data not shown). Several non-B cell lines, including mac- rophage (THP-1, U937) and erythroid (K562), also had no detectable CR2 mRNA. The IMR-90 fibroblast cell line was also CR2 negative (data not shown). Southern blot analysis of the PCR products using a CR2 cDNA probe also confirmed these results, except that, on prolonged ex- posure, the Ig-secreting cell lines SKW-1 and DHL-4 had a very faint CR2 signal (ϽϽ1% of Raji) that could be detected (data not shown). Overall, these studies have shown that the surface expression of CR2, unlike Ig heavy chain, is closely linked to the presence of mature mRNA. In addition, they have allowed us to use these cell lines as in vitro models of B lymphocyte CR2 expression. Downloaded from Lineage- and stage-specific CR2 expression is controlled primarily at the level of transcriptional initiation To determine whether the presence of mature CR2 mRNA and protein is controlled primarily by the process of transcriptional initiation, we performed nuclear runoff analysis using specific DNA target probes containing either the initial 418 bp of the CR2 http://www.jimmunol.org/ hnRNA (pBSCR2(ϩ1/ϩ418)) or a portion of the CR2 cDNA that excludes the poly(A) tail (pBSCR2–12) (Fig. 1B). Positive controls included ␤-actin and Alu, and the pBS plasmid itself served as a negative control in each line. Densitometric analysis of the experiment in Figure 1B is shown in Figure 1C. Data for each cell line are expressed as the value of the CR2-specific probe, pBSCR2–12 or pBSCR2(ϩ1/ϩ418), over the value of the pBS-

negative control. Quantitation of the CR2-specific signal in the by guest on September 23, 2021 mature B cell line, Raji, shows a twofold increase over the pBS- negative control, and a 4.5- to 15-fold increase relative to the CR2- specific signals seen with the other cell lines. These results demonstrate that cell- and stage-specific CR2 mRNA expression is controlled primarily by the initiation of transcription. They also suggest that CR2 is part of the developmental program that controls B lymphocyte stage-specific gene expression first by transcriptional activation and then by inactivation of the promoter upon transition to the Ig-secreting plasma cell stage. Fi- nally, they further validate the use of these cell lines as models in FIGURE 1. A, Gel analysis of the RT-PCR products from whole cell which to test transcriptional activation and silencing using CR2 RNA. A 516-bp CR2 product is detected only in Raji and Jiyoye, both promoter constructs and probes. mature B cell lines (upper panel). A 329-bp MCP product is present in all Two DNase I hypersensitive sites are present in the human CR2 lanes (lower panel), confirming the quality of RNA and the RT reaction. Molecular weight markers (MWM) at left are ⌽X174 DNA-HaeIII digest gene fragments. B, Nuclear runoff analysis using the CR2 cDNA plasmid Since DNase I hypersensitive sites are often indicative of impor- pBSCR2–12, a plasmid (pBSCR2 (ϩ1/ϩ418)) containing the first 418 bp tant regulatory elements, we scanned the CR2 gene for the pres- ␤ of CR2 hnRNA, negative control pBS, and positive controls 12-actin and ence of DNase I hypersensitive sites as a method to identify po- Alu. Nuclear transcription demonstrated by specific hybridization of the tential regulatory regions within the CR2 gene. Using an approach nascent RNA to pBSCR2–12 and pBSCR2(ϩ1/ϩ418), as compared with able to detect hypersensitive sites from approximately 14 kb up- pBS alone, is seen in Raji only. C, Densitometric analysis of the experiment shown in B. Data are expressed as the ratio of pBSCR2 over pBS for stream to 10 kb downstream of the CR2 gene, we identified two each cell line to normalize for background differences. A specific CR2 hypersensitive sites using Raji cell nuclei (Fig. 2B) and the A8B-1 RNA signal is indicated by a ratio greater than one. The pre-B (Nall-1, probe shown in Figure 2A. The site labeled HS1 was further Reh), Ig-secreting (SKW-1, DHL-4), and T cell (CEM) lines did not show mapped using flanking subclones of pBSCR2gH-3 (see Fig. 2A)to a specific signal with either CR2 probe, while the pBSCR2–12 signal is be within the proximal 300 bp upstream of the ϩ1 site within the increased 7 (Reh)-, 5.5 (Nall-1)-, 5.8 (SKW-1)-, 4.5 (DHL-4)-, and 5.7 CR2 promoter, consistent with our identification of this region as (Cem)-fold, and the pBSCR2(ϩ1/ϩ418) signal is increased 14.9 (Reh)-, containing the transcriptional initiation site and regulatory sites 7.5 (Nall-1)-, 5.2 (SKW-1)-, 4.6 (DHL-4)-, and 11.2 (Cem)-fold in Raji (data not shown). HS2 was mapped to be within a 2.5-kb XbaI- cells relative to the other cell lines. Data were generated using ImageQuant SacI segment of the first intron. Hypersensitive analysis was also software (Molecular Dynamics). performed using cell lines representative of earlier and later stages of B cell development as well as the non-B cell, K562. Using the 1272 CELL- AND STAGE-SPECIFIC SILENCING IN THE HUMAN CR2/CD21 GENE Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. A, Map of the CR2 gene, including genomic cosmid and phage subclones, probes, and informative restriction enzyme sites within the region used to localize the DNase I hypersensitive sites identified in B. Restriction endonuclease sites shown are: S, SacI; H, HindIII; E, EcoRI; X, XbaI; K, KpnI; and B, BamHI. B, DNase I hypersensitivity analysis of Raji and K562 cell nuclei using increasing amounts of DNase I and the probes identified as A8B-1 and the XbaI-BamHI fragment of A8B-1. Hypersensitive sites identified as HS1 and HS2 using Raji nuclei are shown by arrows, and their positions are included in the CR2 map in A.

A8B-1 probe and the XbaI-BamHI fragment shown in Figure 2A, DNA sequences within active areas of the CR2 promoter that bind both hypersensitive sites were determined to be cell and stage spe- nuclear factors, and found that essentially identical gel-shift pat- cific, as they could only be detected in the CR2ϩ cells (Fig. 2B, terns were observed in cell types independent of CR2 expression Table I). status (data not shown). Thus, although clearly an active promoter, Previous analysis of the 5Ј promoter for human CR2 has shown this 5Ј region does not contain the elements that are necessary to that it does not demonstrate cell type specific activity (52). These confer stage- and cell-specific transcription of the CR2 gene. results were confirmed using a series of CAT constructs containing deletions of the 5Ј promoter from Ϫ1253 relative to the transcrip- Identification of a transcriptional silencer near HS2 within the tional initiation site (data not shown). Additionally, we identified first intron of the CR2 gene Since we were unable to recreate appropriate regulation of the CR2 Table I. CR2 DNase I hypersensitive sites are cell and stage specific a gene using 5Ј promoter constructs from ϩ75 bp to Ϫ5 kb (see below), our studies focused on the second cell- and stage-specific hypersen- Cell Line Cell Type CR2 Expression HS1 HS2 sitive site (HS2) located within the first intron of the CR2 gene. To determine the functional importance of the region containing the HS2 Reh Pre-B ϪϪϪ Jiyoye Mature B ϩϩϩsite, we used a 2.5-kb XbaI-SacI fragment (that contained the HS2 Raji Mature B ϩϩϩsite) in a series of constructs using the CR2 promoter driving a neo SKW-1 Ig-secreting lymphoblastoid ϪϪϪreporter gene. As controls, we also created the same constructs with- K562 Erythroid ϪϪϪ out the XbaI-SacI fragment (Fig. 3A). An additional construct con- a Summary of CR2 expression and DNase I hypersensitivity analysis performed tained only the Ϫ149/ϩ75 region, but without the intronic segment. on nuclei from cell lines representing different stages of B cell development in addition to a non-B cell (K562). HS1 and HS2 correspond to the two hypersensitive sites Transient transfection of these constructs and analysis using a probe first identified using Raji nuclei in Figure 2B, and their positions in the human CR2 detecting correctly initiated transcripts showed no evidence of appro- gene are noted in Figure 2A. Experiments using CR2-negative cell lines were analyzed using a PhosphorImager and ImageQuant software (Molecular Dynamics). priate regulation using a panel of cell lines representing B and non-B The Journal of Immunology 1273 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. A, Neo reporter constructs used in the transient and stable transfection analyses shown in B, Table II, and the transgenic experiments shown in Figure 4 and Table IV. CRS, indicates the presence of the XbaI-SacI fragment, as described in the text. *, Indicates the relative position of the BglII restriction site used to label the probe for S1 nuclease analysis. -ˆ-Indicates the relative position of the spliced RT-PCR product used in experiments described below. B, Representative results of S1 nuclease analyses of stably transfected cell lines including pre-B (Reh, CR2Ϫ), mature B (Jiyoye, CR2ϩ), Ig-secreting (SKW-1, CR2Ϫ), and non-B (K562, CR2Ϫ). Genomic equivalents of CR2(Ϫ5-kb/ϩ75)-neo vs CR2(Ϫ5-kb/ϩ75)-neo CRS (as determined by PhosphorImager analysis of Southern blots and normalized to 1) for each cell line in this analysis are: Reh, 0.6:1; Jiyoye, 0.7:1; SKW-1, 2.7:1; and K562, 0.6:1. w.t., Indicates untransfected wild-type cells.

cells, as well as different stages of B cell development (summarized in of at least two to three experiments with each line and, because of Table II). the method used, represent pools of many clones of stably trans- However, because some regulatory regions are known to not fected cells. function in transient transfection analysis (69), we generated stably In these studies, the CRS-containing segment is cloned both down- transfected cell lines. When the (Ϫ5 kb/ϩ75)-neo reporter con- stream of the polyadenylation sequences in the neo reporter gene and struct containing the 2.5-kb intronic segment was stably cotrans- in the antisense orientation. The finding of neo reporter mRNA ex- fected into cell lines with a hygromycin-resistance plasmid, and pression in many cell types transiently transfected with this construct neo reporter mRNA expression was measured immediately after (Table II), in stably transfected Jiyoye cells (Fig. 3B), and in lym- the outgrowth of a polyclonal hygromycin-resistant cell line, ap- phoid tissues in transgenic animals (see below) essentially rules out propriate reporter expression was observed (Fig. 3B). Appropriate trivial explanations for our findings, such as the intronic segment in- expression was due to the apparent transcriptional silencing by an terfering nonspecifically with neo reporter mRNA in Reh, SKW-1, element (designated from here as CRS (CR2 Silencer)) in the and/or K562 cells. These results demonstrate the presence of a tran- 2.5-kb intronic region that acts on the (Ϫ5-kb/ϩ75) promoter re- scriptional silencer that is active in non-CR2-expressing cell lines of gion in non-CR2-expressing cells. Importantly, in this analysis, pre-B and Ig-secreting B cell stages, in addition to non-B lineage cell comparable genomic equivalents of neo reporter DNA were types represented by K562 cells. Interestingly, this silencing effect is present in each matched pair of transfected cells (see figure leg- only seen following stable transfection of the construct that contains end). The experiments shown in Figure 3B are also representative it, implying that nuclear matrix and/or chromatin interactions may be 1274 CELL- AND STAGE-SPECIFIC SILENCING IN THE HUMAN CR2/CD21 GENE

Table II. Effect of the CRS element on neo mRNA expression in transient transfectionsa

Constructs/Ratio of test:control RSV-neo mRNA

Cell Line (lineage) (Ϫ149/ϩ75) (Ϫ1253/ϩ75) (Ϫ5kb/ϩ75) (Ϫ1253/ϩ75)-CRS (Ϫ5kb/ϩ75)-CRS

Reh (pre-B, CR2Ϫ) 30.0 29.0 54.5 35.0 25.0 (n ϭ 1) Jiyoye (mature, CR2ϩ) 44.5 45.7 63.0 61.0 39.7 (n ϭ 4) Raji (mature, CR2ϩ) 30.0 29.0 54.5 35.0 25.0 (n ϭ 2) CEM (T cell, CR2Ϫ) 6.0 4.3 11.0 5.5 3.3 (n ϭ 2) K562 (eryth., CR2Ϫ) 15.5 9.0 13.0 6.0 5.0 (n ϭ 3)

a Mean of relative ratios of neo reporter mRNA derived from the constructs shown in Figure 3A to cotransfected RSV-neo reporter mRNA from transiently transfected cell lines as shown. “CRS” indicates the presence of the 2.5-kb XbaI-SacI fragment as described in the text. Data are derived by PhosphorImager following S1 analysis; the number (n) of independent experiments used to calculate these results is shown below each cell line. Neo mRNA is still detected in both CR2-expressing and non-CR2-expressing cell lines with all constructs. Downloaded from necessary for its regulatory activity. In addition, this silencing can act transgenic mouse, which, as expected, has no neo mRNA-derived ␤ at a distance and in the antisense orientation. S1 signal, but does from the internal control, 2-microglobulin. Because the S1 analysis generated a relatively weak signal in Tissue-specific silencing by the CRS element in transgenic mice bone marrow, we were concerned that low levels of expression in To confirm the silencing effect of the CR2 intronic segment, we other tissues might be missed by this type of analysis. To alleviate http://www.jimmunol.org/ made transgenic mice using the CR2(Ϫ5-kb/ϩ75)-neo reporter construct with and without the 2.5-kb intronic sequence. As dis- cussed above, mouse CR2 is expressed on B lymphocytes in a stage-specific manner similar, if not identical, to human CR2. Mouse CR2 is also expressed on follicular dendritic cells, as is human CR2. One difference between the two species is the lack of mouse CR2 expression on T cells or thymocytes (70), whereas human CR2 is found on a subpopulation of peripheral T cells and early thymocytes. On the other hand, like human CR2, mouse CR2 by guest on September 23, 2021 is not expressed in the kidney, heart, liver parenchyma, or other nonlymphoid tissue types. Thus, with some minor differences, the expression of mouse CR2 largely parallels that of human CR2. We performed parallel pronuclear injections using linearized CR2(Ϫ5-kb/ϩ75)-neo in addition to the CR2(Ϫ5-kb/ϩ75)-neo- CRS constructs. Four founders were identified by tail DNA dot- blot analysis, and further confirmed by Southern blot, from mice injected with the CRS-containing construct, and three founders with the non-CRS-containing construct. Table III summarizes the characteristics of each founder. Figure 4A demonstrates the result of an S1 analysis of RNA derived from progeny of founder 1419, which contains the CRS element. A neo mRNA signal essentially identical to the signal from the transfected positive control Jiyoye cells is found only in RNA from spleen and bone marrow, but not liver, kidney, or heart. Shown for comparison also is spleen and liver RNA from a non-

Table III. Characteristics of transgenic founders and progeny a FIGURE 4. A, S1 nuclease analysis of progeny of founder 1419 dem- Founder Copy Transgene onstrates speciﬁc neo mRNA signal in spleen and bone marrow, but not Construct Line Number Transmission liver, kidney, or heart. Control for mouse RNA quality and quantity shown Ϫ ϩ ␤ CR2( 5kb/ 75)-neo-CRS 1419 63 Yes below is 2-microglobulin. Also shown is nontransgenic spleen and liver as CR2(Ϫ5kb/ϩ75)-neo-CRS 1424 5 Yes negative controls and stably transfected Jiyoye cells as a positive control. Ϫ ϩ CR2( 5kb/ 75)-neo-CRS 1425 10 Yes B, RT-PCR analysis of neo reporter and endogenous Crry/p65 mRNA ex- CR2(Ϫ5kb/ϩ75)-neo-CRS 1418 2 No pression in progeny of founders 1424 and 1515. Results are shown with CR2(Ϫ5kb/ϩ75)-neo 1515 55 Yes and without the addition of RT. While the CRS-containing transgenic line Ϫ ϩ CR2( 5kb/ 75)-neo 1523 15 Yes 1424 expresses neo mRNA only in the lymphoid tissues spleen, thymus, CR2(Ϫ5kb/ϩ75)-neo 1514 20 No and bone marrow, the non-CRS-containing transgenic line 1515 expresses a Copy number is determined from ImageQuant following PhosphorImager anal- neo mRNA in all six tissues. A Crry/p65-speciﬁc 953-bp band as a control ysis of dot blots and comparison to known plasmid copy numbers of control neo for mRNA quality is found in each tissue. plasmid. The Journal of Immunology 1275

Table IV. Summary of neo mRNA expression in transgenic mice a

Founder/Construct Spleen Liver Kidney Thymus B. marrow Heart Muscle 1419/CR2(Ϫ5kb/ϩ75)-neo-CRS ϩϪϪ ϩ ϩ ϪϪ 1424/CR2(Ϫ5kb/ϩ75)-neo-CRS ϩϪϪ ϩ ϩ ϪϪ 1425/CR2(Ϫ5kb/ϩ75)-neo-CRS ϪϪϪ Ϫ ND ND ND 1418/CR2(Ϫ5kb/ϩ75)-neo-CRS ϪϪϪ Ϫ ND ϪϪ 1515/CR2(Ϫ5kb/ϩ75)-neo ϩϩϩ ϩ ϩ ϩϩ 1523/CR2(Ϫ5kb/ϩ75)-neo ϪϪϪ ND ND ϪϪ 1514/CR2(Ϫ5kb/ϩ75)-neo ϪϪϪ ND ND ϪϪ

a DNase-treated tissue RNA was analyzed using the RT-PCR assay shown in Figure 4B. Each result in progeny of transmitting founders was repeated using at least two animals. A Crry mRNA-derived RT-PCR signal used as a control for RNA quality could be ampliﬁed from each tissue sample, including those not expressing neo mRNA (data not shown).

this concern, we performed the majority of analyses using a more In summary, these results support the conclusion that the 2.5-kb sensitive RT-PCR assay. For these experiments, we utilized a set CR2 intronic segment contains a silencing activity. Importantly, in of oligonucleotides that produce a correctly spliced neo mRNA progeny of two founders (1419 and 1424), the silencing activity Downloaded from product of 115 bp (see Materials and Methods). Subcloning of this allows reporter gene expression only in the thymus, spleen, and PCR product, followed by nucleotide sequence analysis, conﬁrmed bone marrow, which are the tissues in which human CR2 is ex- the identity of this spliced product. This approach, with the addi- pressed. In organs such as liver, kidney, heart, and muscle, which tional control of performing the PCR reaction on a parallel tube do not express CR2 in humans or mice, neo mRNA expression is without RT, allowed us to determine the presence of spliced neo silenced. In the absence of the intronic element, neo reporter mRNA in the indicated tissues and eliminated the possible effects mRNA is found widely in the progeny of one founder (1515). This of contamination of RNA by genomic DNA sequences. is identical to the results of stable transfection analyses in human http://www.jimmunol.org/ Figure 4B shows the results of an RT-PCR analysis of RNA cell lines, in which the promoter region in this construct (Ϫ5 kb/ from transgenic mice. As can be seen, a neo mRNA-derived band ϩ75) is active in all cell types independent of endogenous CR2 in the CRS-containing 1424 founder line is seen in the lymphoid expression status. Interestingly, the presence of neo mRNA in the tissues: spleen, thymus, and bone marrow, but not liver, kidney, or thymus of CRS-containing founders suggests that this construct heart. In contrast, the 1515 founder line, generated with the directs expression in a human pattern with regard to T cells, as (Ϫ5 kb/ϩ75) CR2 promoter region without the CRS, demon- mouse thymocytes do not express CR2, but early thymocytes in strated a neo mRNA-derived RT-PCR signal in all tissues tested. humans do.

In this analysis, mouse Crry/p65, which is expressed in all cell The sequence of the 2.5-kb CRS intronic element is shown in by guest on September 23, 2021 types, serves as a positive control, confirming the quality of the Figure 5. Before sequencing of the segment, we were concerned RNA in which a neo mRNA-derived signal is not seen. that it might contain one or more pseudo-exons previously de- Table IV summarizes results of identical studies of neo mRNA scribed in this general region as remnants of a primordial CR2/ expression in all transgenic founder lines. In this analysis of seven CR1 gene shared with mouse (71). However, sequence analysis different tissue types, neo mRNA expression in the 1419 and 1424 demonstrated no evidence of such pseudo-exons within the CRS founder lines is only detected in the lymphoid tissues, while neo (data not shown). mRNA expression was found in every tissue tested in the 1515 founder line. Additionally, the 1515 founder line expressed neo mRNA in muscle tissue, while the CRS-containing founders did Discussion not (data not shown). These results, which agree with the data In these studies, we examined the specific mechanisms that govern generated using S1 analysis, are consistent with our results using human CR2 gene regulation. By using a sensitive RT-PCR stably transfected cell lines, and provide strong evidence for a method, we first confirmed that human CR2 gene expression is tissue-specific silencer within the CRS. stage and cell specific, and also showed that the presence of CR2 Interestingly, in four transgenic founder lines, no signal was mRNA correlates with surface CR2 expression. This is in contrast found in any tissue. One CRS-containing founder, 1418, was a low to Ig gene expression, in which one sees intracellular heavy chain copy animal and did not transmit the transgene; therefore, only the mRNA before the detection of surface protein. Using this method, founder itself could be studied. The other CRS-containing founder, we also defined a panel of B and non-B cell lines, in which we 1425, transmitted the neo reporter gene, but no tissues that were tested the stage- and cell-specific activity of elements in both the studied expressed neo mRNA. With regard to the other two non- proximal 5Ј CR2 promoter and the intronic silencer. Although we CRS-containing founders, one (1514) was infertile and thus did not have not specifically excluded the participation of other mecha- transmit the transgene. The founder itself was studied, and neo nisms playing a role in regulating CR2 expression, our nuclear mRNA was not detected in any tissue studied. The third founder, runoff analysis determined that CR2 mRNA expression is con- 1523, transmitted the transgene, but did not express neo mRNA in trolled primarily by the presence or absence of transcriptional any tissue studied. Although our numbers are small and no defin- initiation. itive conclusion can be drawn yet to account for this result, this Through stable transfections and transgenic studies, our findings observation is most likely due to the insertion of the transgene into strongly support the hypothesis that both the tissue specificity and transcriptionally silent regions in these founders. Alternatively, developmentally restricted expression of the human CR2 gene are this could indicate that other regulatory regions outside of those controlled primarily by an intronic silencer that we have desig- included in our constructs are important for determining neo nated CRS. This CRS element is able to confer cell- and stage- mRNA expression levels, but not tissue specificity, in specific expression of the CR2 proximal promoter when the re- transgenic mice. porter constructs containing these elements are stably integrated 1276 CELL- AND STAGE-SPECIFIC SILENCING IN THE HUMAN CR2/CD21 GENE

be integrated stably into the chromatin to mediate its effects. This strongly suggests that nuclear matrix or chromatin interactions are necessary for functional silencing of human CR2. This result is reminiscent of several observations demonstrating important roles for nuclear matrix interactions in the control of the Ig ␮ heavy chain enhancer activity (76) and ␬ gene expression in transgenic mice (77). In addition, developmentally regulated alterations in chromatin structure have been suggested to play a role in Ig heavy chain allelic exclusion (78). The possibility that CR2 genetic elements may interact with matrix is supported by the observation that both the CRS region and the 5Ј promoter contain AT-rich sequences with structural fea- tures of matrix attachment regions (79). Additionally, the CRS contains a 9- of 11-bp match with the consensus sequence for a Drosophila topoisomerase II binding site (Fig. 5) (80) as well as a DNase I hypersensitive site. Both of these sites are characteristics of matrix attachment regions and may indicate that the silencer activity within the CRS is related to interactions between this in- Downloaded from tronic region and nuclear matrix. Several potential mechanisms, such as the necessity of matrix to localize specific protein com- plexes with silencing activity to the gene, or to control the access of such factors to genetic elements, are consistent with our results to date. Furthermore, specific silencing mechanisms, such as the binding of transcriptional repressors, competition for transactivator factor binding sites, and interference with protein-protein interac- http://www.jimmunol.org/ tions necessary for promoter/enhancer activity, could work in con- cert with developmental changes in chromatin structure or acces- sibility to modulate the expression of CR2. Our results showing that four of seven transgenic founders are nonexpressors of neo reporter mRNA suggest the surrounding chromatin can have profound effects on the expression of our FIGURE 5. Sequence of the 2.5-kb XbaI-SacI intronic segment that transgenes. It is likely that other CR2 genomic elements, such as a contains the CRS-silencing activity. The position of the CBF1 sequence is locus control region or insulator, are required to achieve copy by guest on September 23, 2021 underlined (solid) at position 687–693. The 9- of 11-bp match with the number dependence and site of integration independence in trans- topoisomerase II consensus site, GTNA/TAC/TATTNATNNG, is also un- genic animals. Observations in other transgenic settings have derlined (dotted) (see Discussion) (80). shown that such regulatory elements are necessary to obtain high levels of expression, but do not necessarily control the specificity of expression (Ref. 69 and references therein). into the genome. In the absence of the CRS, or when the construct In the founder lines that did express neo mRNA, the CRS ap- containing it is only transiently transfected into cells, the CR2 pears to restrict expression to lymphoid tissues in transgenic mice. proximal promoter is active, regardless of endogenous CR2 However, the pattern of expression in this transgenic setting could expression. be more human-like, with T cell and thymocyte expression, or The silencer activity we have described in this work shows func- more mouse-like, with no such expression. The finding of neo tional similarities to intronic silencers identified in other eukary- reporter mRNA expression in the thymus indicates the former pos- otic systems. For instance, the CD4 gene is inactive early in thy- sibility is more likely. mocyte development, becomes active at a defined developmental CR2 is a gene that is activated at an important point during B point (the CD4ϩCD8ϩ double-positive stage), and then is inacti- lymphocyte development. It has been proposed that the addition of vated again in CD8ϩ single-positive T cells. This regulated pattern CR2 and other genes at this point arms the B cell with receptors of expression also appears to be controlled primarily by an intronic that are capable of interacting with environmental signals such as silencer in combination with an active promoter (72–74). Both the activated complement C3 (11). Whether the activation of genes at CD4 and CR2 intronic elements function when placed outside of this developmental point is a stochastic process, is regulated by the coding region of the reporter gene and in the reverse cytokines or cell-cell interactions, or is possibly controlled by a orientation. combination of mechanisms, is currently unknown. Further dis- In a recent report, Hu and colleagues identified intronic se- section and identification of specific nucleotide target sites within quences in the murine CR2 gene that partially silence the 5Ј prox- the CRS element may provide an appropriate assay system in imal promoter of CR2 in T cells, but not in B cells (75). Analysis which to test putative signaling and transcriptional regulatory of subfragments of this intronic region indicates that the tissue- mechanisms that are active at this point. specific expression is most likely controlled by both positive and Of interest is the presence at positions 687 to 693 of a perfect negative regulatory elements, although the specific factors have match with the heptamer binding site for CBF1, GTGGGAA (Fig. not yet been identified and it is unknown whether this region can 5) (46). CBF1 is a ubiquitously expressed protein that acts as a mediate developmentally restricted expression of murine CR2 in B repressor within the CD23 gene (45). EBNA-2, which increases cells. expression of both CD23 and CR2, has been shown to interact with One notable difference between the silencer characterized in this CBF1 and relieve CBF1-mediated repression of CD23 in addition report and the murine CR2 silencer is the necessity for the CRS to to the EBNA-2-responsive EBV latency promoter (Cp) and the The Journal of Immunology 1277 promoter for the EBV protein LMP-1. Unlike CD23, the CR2 5Ј 18. Birkenbach, M., X. Tong, L. E. Bradbury, T. F. Tedder, and E. Kieff. 1992. proximal promoter from (Ϫ1253/ϩ75) could not be transactivated Characterization of an Epstein-Barr virus receptor on human epithelial cells. J. Exp. Med. 176:1405. significantly by EBNA-2 in coexpression experiments (C. Pham 19. Uckun, F. M. 1990. Regulation of human B-cell ontogeny. Blood 76:1908. and V. M. Holers, unpublished observations), and does not contain 20. Takahashi, K., Y. Kozono, T. J. Waldschmidt, D. Berthiaume, R. J. Quigg, an identical match to a consensus CBF1 binding site. If the puta- A. Baron, and V. M. Holers. 1997. Mouse complement receptors type 1 (CR1; CD35) and type 2 (CR2; CD21): expression on normal B cell subpopulations and tive CBF1 site within the CR2 intronic segment is found to act as decreased levels during the development of autoimmunity in MRL/lpr mice. a functional silencer and an EBNA-2 response element, this will J. Immunol. 159:1557. 21. Molina, H., T. Kinoshita, K. Inoue, J.-C. Carel, and V. M. Holers. 1990. A link CR2 and CD23 transcription to a common mechanism. In this molecular and immunochemical characterization of mouse CR2: evidence for a regard, it is of some theoretic interest that CR2 is a receptor for single gene model of mouse complement receptors 1 and 2. J. Immunol. 145: CD23. Coordination of expression by a common regulatory mech- 2974. 22. Kurtz, C. B., M. S. Paul, M. Aegerter, J. J. Weis, and J. H. Weis. 1989. Murine anism would be an elegant way to help link activities of these two complement receptor gene family. II. Identification and characterization of the proteins. murine homolog (Cr2) to human CR2 and its molecular linkage to Crry. J. Im- munol. 143:2058. 23. Pramoonjago, P., J. Takeda, Y. U. Kim, K. Inoue, and T. Kinoshita. 1993. Ligand Acknowledgments specificities of mouse complement receptor type 1 (CR1) and 2 (CR2) purified from spleen cells. Int. Immunol. 5:337. The authors thank Tim Ley for ␤-actin, Alu, and CGL-1 plasmids; Dan 24. Molina, H., V. M. Holers, B. Li, Y. Fang, S. Mariathasan, J. Goellner, Link for fgr-neo and actin plasmids; Dennis Loh for the pSFFV-lacZ plas- J. Strauss-Schoenberger, W. R. Karr, and D. D. Chaplin. 1996. Markedly im- mid; Kathy Liszewski and John Atkinson for the MCP primers; Alec paired humoral immune response in mice deficient in complement receptors 1 and 2. Proc. Natl. Acad. Sci. USA 93:3357. Cheng for technical assistance; Joe Anderson of UCHSC Cancer Center 25. Ahearn, J. M., M. B. Fischer, D. A. Croix, S. Georg, M. Ma, J. Xia, X. Zhou, Downloaded from transgenic facility for injection of pronuclei and help with the initial breed- R. G. Howard, T. L. Rothstein, and M. C. Carroll. 1996. Disruption of the Cr2 ing and characterization of transgenic animals; and John Stolpa for his locus results in a reduction in B-1a cells and in an impaired B cell response to initial work on the development of the spliced neo mRNA RT-PCR de- T-dependent antigen. Immunity 4:251. 26. Croix, A. D., J. M. Ahearn, A. M. Rosengard, S. Han, G. Kelsoe, M. Ma, and tection assay. M. C. Carroll. 1996. Antibody response to a T-dependent antigen requires B cell expression of complement receptors. J. Exp. Med. 183:1857. 27. Dempsey, P. W., M. E. Allison, S. Akkaraju, C. C. Goodnow, and D. T. Fearon.

References 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired http://www.jimmunol.org/ 1. Cooper, N. R., M. D. Moore, and G. R. Nemerow. 1988. Immunobiology of CR2, immunity. Science 271:348. the B lymphocyte receptor for Epstein-Barr virus and the C3d complement frag- 28. Carter, R. H., M. O. Spycher, Y. C. Ng, R. Hoffman, and D. T. Fearon. 1988. ment. Annu. Rev. Immunol. 6:85. Synergistic interaction between complement receptor type 2 and membrane IgM 2. Hourcade, D., V. M. Holers, and J. P. Atkinson. 1989. The regulators of com- on B lymphocytes. J. Immunol. 141:457. plement activation (RCA) gene cluster. Adv. Immunol. 45:381. 29. Bohnsack, J. F., and N. R. Cooper. 1988. CR2 ligands modulate human B cell 3. Iida, K., L. Nadler, and V. Nussenzweig. 1983. Identification of the membrane activation. J. Immunol. 141:2569. receptor for the complement fragment C3d by means of a monoclonal antibody. 30. Luxembourg, A. T., and N. R. Cooper. 1994. Modulation of signaling via the B J. Exp. Med. 158:1021. cell antigen receptor by CD21, the receptor for C3dg and EBV. J. Immunol. 153:4448. 4. Weis, J. J., T. F. Tedder, and D. T. Fearon. 1984. Identification of a 145,000 Mr membrane protein as the C3d receptor (CR2) of human B lymphocytes. Proc. 31. Matsumoto, A. K., J. Kopicky-Burd, R. H. Carter, D. A. Tuveson, T. F. Tedder, Natl. Acad. Sci. USA 81:881. and D. T. Fearon. 1991. Intersection of the complement and immune systems: a by guest on September 23, 2021 5. Fingeroth, J. D., J. J. Weis, T. F. Tedder, J. L. Strominger, P. A. Biro, and signal transduction complex of the B lymphocyte-containing complement recep- D. T. Fearon. 1984. Epstein-Barr virus receptor of human B lymphocytes is the tor type 2 and CD19. J. Exp. Med. 173:55. C3d receptor CR2. Proc. Natl. Acad. Sci. USA 81:4510. 32. Carter, R. H., and D. T. Fearon. 1992. CD19: lowering the threshold for antigen 6. Nemerow, G. R., R. Wolfert, M. E. McNaughton, and N. R. Cooper. 1985. Iden- receptor stimulation of B lymphocytes. Science 256:105. tification and characterization of the Epstein-Barr virus receptor on human B 33. Aubry, J. P., S. Pochon, P. Graber, K. U. Jansen, and J. Y. Bonnefoy. 1992. CD21 lymphocytes and its relationship to the C3d complement receptor (CR2). J. Virol. is a ligand for CD23 and regulates IgE production. Nature 358:505. 55:347. 34. Henchoz, S., J. F. Gauchat, J. P. Aubry, P. Graber, S. Pochon, and 7. Aubry, J.-P., S. Pochon, J.-F. Gauchat, A. Nueda-Marin, V. M. Holers, P. Graber, J. Y. Bonnefoy. 1994. Stimulation of human IgE production by a subset of anti- C. Siegfried, and J.-Y. Bonnefoy. 1994. CD23 interacts with a new functional CD21 monoclonal antibodies: requirement of a co-signal to modulate epsilon extracytoplasmic domain involving N-linked oligosaccharides on CD21. J. Im- transcripts. Immunology 81:285. munol. 152:5806. 35. Kansas, G. S., and T. F. Tedder. 1991. Transmembrane signals generated through 8. Delcayre, A. X., F. Salas, S. Mathur, K. Kovats, M. Lotz, and W. Lernhardt. MHC class II, CD19, CD20, CD39, and CD40 antigens induce LFA-1-dependent 1991. Epstein-Barr virus/complement C3d receptor is an interferon-␣ receptor. and independent adhesion in human B cells through a tyrosine kinase-dependent EMBO J. 10:919. pathway. J. Immunol. 147:4094. 9. Tedder, T. F., L. T. Clement, and M. D. Cooper. 1984. Expression of C3d re- 36. Roberts, M. L., A. T. Luxembourg, and N. R. Cooper. 1996. Epstein-Barr virus ceptors during human B cell differentiation: immunofluorescence analysis with binding to CD21, the virus receptor, activates resting B cells via an intracellular the HB-5 monoclonal antibody. J. Immunol. 133:678. pathway that is linked to B cell infection. J. Gen. Virol. 77:3077. 10. Campana, D., G. Janossy, M. Bofill, L. K. Trejdosiewicz, D. Ma, V. Hoffbrand, 37. Kehrl, J. H., A. Riva, G. L. Wilson, and C. Thevenin. 1994. Molecular mecha- D. Y. Mason, A. M. Lebacq, and H. K. Forster. 1985. Human B cell development. nisms regulating CD19, CD20 and CD22 gene expression. Immunol. Today 15: I. Phenotypic differences of B lymphocytes in the bone marrow and peripheral 432. lymphoid tissue. J. Immunol. 134:1524. 38. Kozmik, Z., S. Wang, P. Dorfler, B. Adams, and M. Busslinger. 1992. The pro- 11. Hartley, S. B., M. P. Cooke, D. A. Fulcher, A. W. Harris, S. Cory, A. Basten, and moter of the CD19 gene is a target for the B-cell-specific transcription factor C. C. Goodnow. 1993. Elimination of self-reactive B lymphocytes proceeds in BSAP. Mol. Cell. Biol. 12:2662. two stages: arrested development and cell death. Cell 72:325. 39. Thevenin, C., B. P. Lucas, E. J. Kozlow, and J. H. Kehrl. 1993. Cell type- and 12. Loffert, D., S. Schaal, A. Ehlich, R. R. Hardy, Y.-R. Zou, W. Muller, and stage-specific expression of the CD20/B1 antigen correlates with the activity of K. Rajewsky. 1994. Early B-cell development in the mouse: insights from mu- a diverged octamer DNA motif present in its promoter. J. Biol. Chem. 268:5949. tations introduced by gene targeting. Immunol. Rev. 137:135. 40. Wilson, G. L., V. Najfeld, E. Kozlow, J. Menninger, D. Ward, and J. H. Kehrl. 13. Tiegs, S. L., D. M. Russell, and D. Nemazee. 1993. Receptor editing in self- 1993. Genomic structure and chromosomal mapping of the human CD22 gene. reactive bone marrow B cells. J. Exp. Med. 177:1009. J. Immunol. 150:5013. 14. Reynes, M., J. P. Aubert, J. H. M. Cohen, J. Audouin, V. Tricottet, J. Diebold, 41. Andersson, K. B., K. E. Draves, D. M. Magaletti, S. Fujioka, K. L. Holmes, and M. D. Kazatchkine. 1985. Human follicular dendritic cells express CR1, C. Law, and E. A. Clark. 1996. Characterization of the expression and gene CR2, and CR3 complement receptor antigens. J. Immunol. 135:2687. promoter of CD22 in murine B cells. Eur. J. Immunol. 26:3170. 15. Tsoukas, C. D., and J. D. Lambris. 1988. Expression of CR2/EBV receptors on 42. Schubach, W. H., G. Horvath, C. LeVea, and J. Tierney. 1997. Chromatin struc- human thymocytes detected by monoclonal antibodies. Eur. J. Immunol. ture of the lymphocyte Fc⑀ receptor gene (CD23): identification of an upstream 18:1299. transcriptional enhancer. J. Immunol. 158:2228. 16. Fischer, E., C. Delibrias, and M. D. Kazatchkine. 1991. Expression of CR2 (the 43. Wang, F., C. D. Gregory, M. Rowe, A. B. Rickinson, D. Wang, M. Birkenbach, C3dg/EBV receptor, CD21) on normal human peripheral blood T lymphocytes. H. Kikutani, T. Kishimoto, and E. Kieff. 1987. Epstein-Barr virus nuclear antigen J. Immunol. 146:865. 2 specifically induces expression of the B-cell activation antigen CD23. Proc. 17. Levy, E., J. Ambrus, L. Kahl, H. Molina, K. Tung, and V. M. Holers. 1992. T Natl. Acad. Sci. USA 84:3452. lymphocyte expression of complement receptor 2 (CR2/CD21): a role in adhesive 44. Cordier-Bussat, M., M. Billaud, A. Calender, and G. M. Lenoir. 1993. Epstein- cell-cell interactions and dysregulation in a patient with systemic lupus erythem- Barr virus (EBV) nuclear-antigen-2-induced up-regulation of CD21 and CD23 atosus (SLE). Clin. Exp. Immunol. 90:235. molecules is dependent on a permissive cellular context. Int. J. Cancer 53:153. 1278 CELL- AND STAGE-SPECIFIC SILENCING IN THE HUMAN CR2/CD21 GENE

45. Hsieh, J.-D., and S. D. Hayward. 1995. Masking of the CBF1/RBPJ␬ transcrip- 63. McGuire, E. A., C. E. Rintoul, G. M. Sclar, and S. J. Korsmeyer. 1992. Thymic tional repression domain by Epstein-Barr virus EBNA2. Science 268:560. overexpression of Ttg-1 in transgenic mice results in T-cell acute lymphoblastic 46. Ling, P. D., J. J.-D. Hsieh, I. K. Ruf, D. R. Rawlins, and S. D. Hayward. 1994. leukemia/lymphoma. Mol. Cell. Biol. 12:4186. EBNA-2 up-regulation of Epstein-Barr virus latency promoters and the cellular 64. Ghosh, P. K., V. B. Reddy, J. Swinscoe, P. Lebowitz, and S. M. Weissman. 1978. CD23 promoter utilizes a common targeting intermediate, CBF1. J. Virol. 68: Heterogeneity and 5Ј-terminal structures of the late RNAs of simian virus 40. 5375. J. Mol. Biol. 126:813. 47. Henkel, T., P. D. Ling, S. D. Hayward, and M. G. Peterson. 1994. Mediation of 65. Paul, M. S., M. Aegerter, K. Cepek, M. D. Miller, and J. H. Weis. 1990. The Epstein-Barr virus EBNA2 transactivation by recombination signal-binding pro- murine complement receptor gene family. III. The genomic and transcriptional tein J␬. Science 265:92. complexity of the Crry and Crry-ps genes. J. Immunol. 144:1988. 48. Johannsen, E., E. Koh, G. Mosialos, X. Tong, E. Kieff, and S. R. Grossman. 1995. 66. Li, B., C. Sallee, M. Dehoff, S. Foley, H. Molina, and V. M. Holers. 1993. Mouse Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane pro- Crry/p65: characterization of monoclonal antibodies and the tissue distribution of ␬ tein 1 promoter is mediated by J and PU.1. J. Virol. 69:253. a functional homologue of human MCP and DAF. J. Immunol. 151:4295. 49. Tong, X., R. Drapkin, R. Yalamanchili, G. Mosialos, and E. Kieff. 1995. The 67. Korsmeyer, S. J., A. Arnold, A. Bakshi, J. V. Ravetch, U. Siebenlist, P. A. Hieter, Epstein-Barr virus nuclear protein 2 acidic domain forms a complex with a novel S. O. Sharrow, T. W. LeBien, J. H. Kersey, D. G. Poplack, P. Leder, and cellular coactivator that can interact with TFIIE. Mol. Cell. Biol. 15:4735. T. A. Waldmann. 1983. Immunoglobulin gene rearrangement and cell surface 50. McNearney, T. A., C. F. Ebenlichler, M. Totsch, and M. P. Dierich. 1993. Ex- antigen expression in acute lymphocytic leukemias of T cell and B cell precursor pression of complement receptor 2 (CR2) on HTLV-1-infected lymphocytes and origins. J. Clin. Invest. 71:301. transformed cell lines. Eur. J. Immunol. 23:1266. 51. Rayhel, E. J., M. H. Dehoff, and V. M. Holers. 1991. Characterization of the 68. Siminovitch, K. A., J. P. Jensen, A. L. Epstein, and S. J. Korsmeyer. 1986. human complement receptor 2 (CR2, CD21) promoter reveals sequences shared Immunoglobulin gene rearrangements and expression in diffuse histiocytic lym- with regulatory regions of other developmentally restricted B cell proteins. J. Im- phomas reveal cellular lineage, molecular defects, and sites of chromosomal munol. 146:2021. translocation. Blood 67:391. 52. Yang, L., M. Behrens, and J. J. Weis. 1991. Identiﬁcation of 5Ј-regions affecting 69. Ernst, P., and S. T. Smale. 1995. Combinatorial regulation of transcription I: the expression of the human CR2 gene. J. Immunol. 147:2404. general aspects of transcriptional control. Immunity 2:311.

53. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979. 70. Kinoshita, T., J. Takeda, K. Hong, H. Kozono, H. Sakai, and K. Inoue. 1988. Downloaded from Isolation of biologically active ribonucleic acid from sources enriched in ribo- Monoclonal antibodies to mouse complement receptor type 1 (CR1): their use in nuclease. Biochemistry 18:5294. a distribution study showing that mouse erythrocytes and platelets are CR1-neg- 54. Fujisaku, A., J. B. Harley, M. B. Frank, B. A. Gruner, B. Frazier, and ative. J. Immunol. 140:3066. V. M. Holers. 1989. Genomic organization and polymorphisms of the human 71. Holguin, M. H., C. B. Kurtz, C. J. Parker, J. J. Weis, and J. H. Weis. 1990. Loss C3d/Epstein-Barr virus receptor. J. Biol. Chem. 264:2118. of human CR1- and murine Crry-like exons in human CR2 transcripts due to CR2 55. Post, T. W., K. Liszewski, E. M. Adams, I. Tedja, E. A. Miller, and gene mutations. J. Immunol. 145:1776. J. P. Atkinson. 1991. Membrane cofactor protein of the complement system: 72. Sawada, S., J. D. Scarborough, N. Killeen, and D. R. Littman. 1994. A lineage- alternative splicing of serine/threonine/proline-rich exons and cytoplasmic tails specific transcriptional silencer regulates CD4 gene expression during T lympho- produces multiple isoforms that correlate with protein phenotype. J. Exp. Med. cyte development. Cell 77:917. http://www.jimmunol.org/ 174:93. 73. Siu, G., A. L. Wurster, D. D. Duncan, T. M. Soliman, and S. M. Hedrick. 1994. 56. Lublin, D. M., M. K. Liszewski, T. W. Post, M. A. Arce, M. M. LeBeau, A transcriptional silencer controls the developmental expression of the CD4 gene. M. B. Rebentisch, R. S. Lemons, T. Seya, and J. P. Atkinson. 1988. Molecular EMBO J. 13:3570. cloning and chromosomal localization of human membrane cofactor protein 74. Duncan, D. D., M. Adlam, and G. Siu. 1996. Asymmetric redundancy in CD4 (MCP). J. Exp. Med. 168:181. silencer function. Immunity 4:301. 57. Hanson, R. D., N. L. Connolly, D. Burnett, E. J. Campbell, R. M. Senior, and 75. Hu, H., B. K. Martin, J. J. Weis, and J. H. Weis. 1997. Expression of the murine T. J. Ley. 1990. Developmental regulation of the human cathepsin G gene in CD21 gene is regulated by promoter and intronic sequences. J. Immunol. 158: myelomonocytic cells. J. Biol. Chem. 265:1524. 4758. 58. Hourcade, D., A. D. Garcia, T. W. Post, P. Taillon-Miller, V. M. Holers, 76. Forrester, W. C., C. van Genderson, T. Jenuwein, and R. Grosschedl. 1994. De- L. M. Wagner, N. S. Bora, and J. P. Atkinson. 1992. Analysis of the human pendence of enhancer-mediated transcription of the immunoglobulin M gene on regulators of complement activation (RCA) gene cluster with yeast artificial chro- nuclear matrix attachment regions. Science 265:1221. by guest on September 23, 2021 mosomes (YACs). Genomics 12:289. 59. Ulrich, M., and T. J. Ley. 1990. Function of normal and mutated ␥-globin gene 77. Xu, M., R. E. Hammer, V. C. Blasquez, S. L. Jones, and W. T. Garrard. 1989. ␬ promoters in electroporated K562 erythroleukemia cells. Blood 75:990. Immunoglobulin gene expression after stable integration. J. Biol. Chem. 264: 60. Gutkind, J. S., D. C. Link, S. Katamine, P. Lacal, T. Miki, T. J. Ley, and 21190. K. C. Robbins. 1991. A novel c-fgr exon utilized in Epstein-Barr virus-infected 78. Stanhope-Baker, P., M. K. Hudson, L. A. Shaffer, A. Constantinescu, and B lymphocytes but not in normal monocytes. Mol. Cell. Biol. 11:1500. S. M. Schlissel. 1996. Cell type-specific chromatin structure determines the tar- 61. Hanson, R. D., P. A. Hohn, N. C. Popescu, and T. J. Ley. 1990. A cluster of geting of V(D)J recombinase activity in vitro. Cell 85:887. hematopoietic serine protease genes is found on the same chromosomal band as 79. Boulikas, T. 1993. Nature of DNA sequences at the attachment regions of genes the human ␣/␦ T-cell receptor locus. Proc. Natl. Acad. Sci. USA 87:960. to the nuclear matrix. J. Cell. Biochem. 52:14. 62. Saragosti, S., G. Moyne, and M. Yaniv. 1980. Absence of nucleosomes in a 80. Sander, M., and T. Hsieh. 1985. Drosophila topoisomerase II double-strand DNA fraction of SV40 chromatin between the origin of replication and the region cleavage: analysis of DNA sequence homology at the cleavage site. Nucleic Acids coding for the late leader RNA. Cell 20:65. Res. 13:1057.