Proc. Natl. Acad. Sci. USAVol. 92, pp. 5336-5340, June 1995Immunology

Pax5 (BSAP) regulates the murine immunoglobulin 3'ca enhancerby suppressing binding of NF-aP, a protein that controls heavychain transcriptionMARKUS F. NEURATH*, EDWARD E. MAXt, AND WARREN STROBER*t*Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Disease, National Institutes of Health, and tFoodand Drug Administration/Center for Biologics Evaluation and Research, Bethesda, MD 20892-1890

Communicated by Anthony S. Fauci, National Institutes of Health, Bethesda, MD, March 1, 1995 (received for review January 10, 1995)

ABSTRACT The PaxS transcription factor BSAP (B-cell-specific activator protein) is known to bind to and repress theactivity of the immunoglobulin heavy chain 3'a enhancer. Wehave detected an element-designated aP-that lies -50 bpdownstream of the BSAP binding site 1 and is required formaximal enhancer activity. In vitro binding experiments sug-gest that the 40-kDa protein that binds to this element(NF-aP) is a member of the Ets family present in both B-celland plasma-cell nuclei. However, in vivo footprint analysissuggests that the aP site is occupied only in plasma cells,whereas the BSAP site is occupied in B cells but not in plasmacells. When Pax5 binding to the enhancer in B cells wasblocked in vivo by transfection with a triple-helix-formingoligonucleotide, an aP footprint appeared and endogenousimmunoglobulin heavy chain transcripts increased. The tri-ple-helix-forming oligonucleotide also increased enhancer ac-tivity of a transfected construct in B cells, but only when theaP site was intact. Pax5 thus regulates the 3'r enhancer andimmunoglobulin gene transcription by blocking activation byNF-aP.

The mammalian Pax gene family consists of nine proteins thatshare a DNA binding domain homologous to the Drosophilapaired box (1). The Pax5 gene encodes the B-cell-specificactivator protein (BSAP) (2), a homeodomain-class transcrip-tion factor. Pax5 is specifically expressed in the B-cell lineagefrom the pro-B to the mature B-cell stage. However, BSAP isnot found in terminally differentiated plasma cells. Bindingsites for BSAP have been found in a variety of promoters ofB-cell-related genes, including CD19 (3), VpreBl (4), A5 (4), andblk (5), and in the immunoglobulin gene locus (6-13). Func-tional studies suggest that Pax5 participates in controllingB-cell proliferation and isotype switching (14, 15).Although BSAP binding positively regulates transcription

from various promoters, binding of BSAP to sites in the 3'aenhancer has been shown to negatively regulate enhancerfunction (11, 12). In the present study, we have investigatedhow Pax5 acts to inhibit the function of the 3'a enhancer. Wehave found that BSAP regulates this enhancer by affecting thedownstream binding of a 40-kDa protein (NF-aP) that posi-tively affects enhancer activity and immunoglobulin genetranscription.

MATERIALS AND METHODSCell Lines and Culture Conditions. CH12.LX (IgM),

CH12.LX2.1c1.2d10 (IgG2b), and CH12.LX2.5g5.lcl.2e10(IgG3) murine B-cell lymphoma lines were a generous gift ofG. Haughton (16). CH12.LX.A2 and LCD32w cells have beendescribed (14). MOPC-315, MPC-11, EL-4, Raji, Jurkat, and

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HeLa cell lines were obtained from American Type CultureCollection and maintained as described (12).

Ligation-Mediated PCR (LM-PCR). In vivo dimethyl sulfate(DMS) footprint analysis by LM-PCR was carried out as de-scribed (17, 18). Primer sequences for LM-PCR are as follows.BSAP site 1 coding primers: primer 1, 5'-CCCAGTTATCACC-ACTGCTGGAATC-3'; primer 2, 5'-ACCACTGCTGGAATC-TGACCCACCA-3', primer 3, 5'-CTGCTGGAATCTGACCC-ACCAAGAGG-3'. Site 1 noncoding primers: primer 1, 5'-TT-CTGGGATACCCAGGATTTGGAGC-3'; primer 2, 5'-ACC-CAGGATTTGGAGCACACCTACA-3'; primer 3, 5'-TGGA-GCACACCTACAGCCTTCCTGC-3'. BSAP site 2 codingprimers: primer 1, 5'-CCAGAAATAGCATGAGTGACTCCC-3'; primer 2, 5'-AGCATGAGTGACTCCCCCACCTGCA-3';primer 3, 5'-ATGAGTGACTCCCCCACCTGCAGCr-3'. Site 2noncoding primers: primer 1, 5'-TTGCCCATCTCCTGTCAT-GTCCT-3'; 2, 5'-CCTCTTGGTGGGTCAGATTCCAGCA-3';3, 5'-GGTGGGTCAGATTCCAGCAGTGGTG-3'. ds-linker:5'-GCGGTGACCCGGGAGATCTGAATTC-3' and 3'-CTA-GACTTAAG-5'.

Isolation of Nuclear Proteins. Extractions of nuclear pro-teins and BCA protein assays were as described (12).

Electrophoretic Mobility Shift Assays (EMSAs). EMSAswere performed as described (12). Competitor sequences wereas follows: pA (Xf) site, 5'-CAGCAGCTGGCAGGAAG-CAGGTCATGT-3' and 3'-GTCGTCGACCGTCCTTCGTC-CAGTACA-5'; CREB site, 5'-GATTGGCTGACGTCA-GAGAGCT-3' and 3'-CTAACCGACTGCAGTCTCTCGA-5'. For super-shift experiments, a pan-ETS antibody (19) wasadded to the reaction mixtures.

Southwestern Blot Analysis. For Southwestern blot analysis,we used 6M guanidine hydrochloride for protein renaturation.Hybridization was performed with 10,000 cpm of radiolabeledspecific probe.

Triple-Helix-Forming Oligonucleotides (TFOs). TFOs wereobtained from Clontech and synthesized with a propanolaminegroup (5'-R-O-PO2-O-CH(CHOH)-CH2-NH'-3') covalentlyattached to their ends. Triplex formation in vitro and inhibitionof protein binding in the presence of TFOs were assessed byband shift analysis in Mg-containing and Mg-free systems asdescribed (20).Western Blot Analysis. Western blot analysis was performed

by standard procedures using alkaline phosphatase-labeledgoat anti-mouse IgA and color substrate (Promega).

Mutagenesis of Vector Constructs. The 3'a enhancer wascloned by PCR from genomic DNA of CH12.LX B cells byusing specific primers [5'-CTGCCCACCATTCCCATGGTT-CTGGGATACCCAGGAT-3' and 5'-TGACAGGCTGGTA-CAGTTTCTATCCCACAGTCCACAGGCT-3' (21)], sub-

Abbreviations: BSAP, B-cell-specific activator protein; TFO, triple-helix-forming oligonucleotide; DMS, dimethyl sulfate; LM-PCR, li-gation-mediated PCR; EMSA, electrophoretic mobility shift assay; C,constant.ITo whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 92 (1995) 5337

cloned into the pCRII vector, and sequenced. Site-directedmutagenesis was performed by using the U.S.E. mutagenesiskit (Pharmacia), phosphorylated mutagenized NF-aP primer(5 '-GATAACTGTTCCACACGGAATTCAACATCA-3'),and the Spe I selection primer (5'-TGGCGGCCGTACTTA-GTGGATCCGA-3'). Inserts with and without the desiredmutations were cloned between the Sac I and Xho I sites of theK-Luc vector (12) yielding the vectors wt3'e- and mut3'e-Luc.Transfection of Vectors and Synthetic Oligonucleotides.

Electroporation of 2 x 107 CH12.LX or MPC-11 cells with 10,ug of reporter plasmid or 25 ,tg of TFOs was performed inphosphate-buffered sucrose medium (12). Luciferase assayswere performed as described (12).

Northern Blot Hybridization. Total cellular RNA fromtransfected and untransfected cells was isolated by the acidguanidium thiocyanate/phenol/chloroform extractionmethod and Northern blot analysis was performed.

Probes for Northern Blot Hybridization. Northern probeswere generated by reverse transcription-PCR amplificationfrom splenic B-cell cDNA by using specific primers: Ca,5'-TATGAAGGACACTCAACAGC-3' and 5'-ACATGTG-ATGCTGGCATCTG-3'; CD19, 5'-CTACTGAGCCTAAG-CCTTGG-3' and 5'-GGCTCAGGAAGTCCATCATC-3';f3-actin, 5'-TCTAGGCACCAAGGTGTGATG-3' and 5'-GC-AACATAGCACAGCTTCTC-3'; Cg, 5'-CACCTGGAACT-ACCAGAACA-3' and 5'-CTGTGAGTCACAGTACACAC-3'; Cy, 5'-AAGTCACTACCAGAGGTCGGACTCC-3' and5'-ACATGGTACCAGCTTTGCTGGATGC-3'.

RESULTSIn Vivo Footprint Analysis of the 3'a Enhancer Demon-

strates Reciprocal Occupancy of BSAP Binding Site 1 and anAdjacent Binding Site, aP. To explore whether the BSAP sitesin the immunoglobulin 3'a enhancer were occupied in B-celllines, we used DMS in vivo footprint analysis via LM-PCR. Astrong footprint was observed at BSAP binding site 1 on thenoncoding strand in CH12.LX B cells (Fig. 1A). A weakerfootprint was seen on the coding strand but no footprint wasseen at BSAP binding site 2 in CH12.LX B cells.

In contrast, the EL-4 lymphoma T-cell line and the murineplasmacytomas MOPC-315 and MPC-11 did not show a foot-print at BSAP site 1 or 2. However, in both MOPC-315 and

MPC-11 cells, a very strong footprint and hyperreactivitieswere found at a site just downstream of the BSAP site 1 (Fig.1B). No such footprint was seen in EL-4 T cells or CH12.LXB cells. We have termed the protected site aP, as it is locatedin the enhancer 3' of the a chain constant region (Ca) gene andappears to be occupied characteristically in the plasma-celllines that were tested.A Specific Nuclear Factor, NF-aP, Binds to the aP Motif

and Is Expressed in Lymphoid Cell Lines. In initial EMSAstudies, we found a specific retarded complex with nuclearproteins from CH12.LX B cells or MPC-11 plasmacytoma cellsand an end-labeled aP oligonucleotide (Fig. 2A). This complexwas also found with nuclear extracts from splenic B cells, Raji,CH12.LX.A2, CH12.LX2.lcl.2dlO B-lymphoma cells, andMOPC-315 myeloma cells. In contrast, only a weak band wasobtained with extracts from Jurkat and EL4 T cells, and noband was found with extracts from fibroblastic LCD32W cells orHeLa cells (data not shown). The protein in the extractsresponsible for the EMSA bands was termed NF-aP. NF-aPthus is present in both B- and plasma-cell nuclei, though in vivothe aP site is apparently occupied only in plasma cells.

Since the aP motif contains two GGAA motifs, character-istic of binding sites for members of the Ets family of tran-scription factors, we considered the possibility that NF-aPmight be an Ets-like protein. Competition experiments (Fig. 2B and C) showed that the upstream GGAA motif of the aPsequence is essential for binding of NF-aP, whereas mutationsof the downstream GGAA motif actually increased binding. Incontrast, a mutation in the DNA outside the GGAA sites hadlittle effect on NF-aP binding. Addition of a pan-ETS antibodyto the nuclear extracts led to a supershift of NF-aP (Fig. 2D),suggesting that NF-aP is an Ets-like protein. However, no crosscompetition was seen with the ,tA(7r) site of the immunoglob-ulin intron enhancer, suggesting that NF-aP is different fromEts proteins that bind to this site (22, 23). Southwestern blotstudies (Fig. 2E) indicated a molecular mass of -40 kDa forNF-aP.

Site-Directed Mutagenesis of the aP Motif Leads to De-creased Reporter Gene Activity in MPC-11 PlasmacytomaCells. To assess the functional role of NF-aP binding to theenhancer in plasmacytoma-cell lines, we transfected reporterconstructs containing wild-type or aP mutant enhancer se-quences into MPC-11 plasmacytoma cells. The aPM1 muta-

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FIG. 1. In vivo DMS footprint analysis atBSAP binding site 1 (A) and BSAP binding site2 (B) in the 3'a enhancer in CH12.LX B cellsand MPC-11 plasmacytoma cells. Protectedguanine residues (circles) and hyperreactivities(triangles) are indicated. In addition, the LM-PCR ladder in CH12.LX.A2 B cells 4 hr aftertransfection of BSAP-TFO or control TFO isshown (lanes 7 and 8). The fact that the ob-served footprint after BSAP-TFO electropora-tion (arrow) is not identical to the originalfootprint in MPC-11 cells may be due to limi-tations in transfection efficiency of the BSAP-TFO, BSAP-TFO-induced altered DNA con-figuration at the aP site, or the presence ofadditional nuclear proteins in MPC-1 1 cells thatbind close to the aP site.

Immunology: Neurath et at

5338 Immunology: Neurath et aL

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binding. To test this idea, we sought to specifically block Pax5binding to its binding site 1 in the 3'a enhancer in B cells bya site-specific TFO (20). The striking sequence diversity be-tween individual BSAP sites allows design of a TFO that maybe specific for only the intended BSAP binding site. Accord-ingly, we used a 41-nt single-strand TFO (Fig. 3A) antiparallelto the more purine-rich noncoding strand ofBSAP site 1 in theimmunoglobulin 3'a enhancer (BSAP-TFO). We found bysequence analysis that all of the published BSAP binding siteshad at least 29% mismatches for possible triplex formationwith the BSAP-TFO.

A ds-target: BSAP site 1; 41bp, 61% G + C, 71% purine6471

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. FIG. 2. (A) EMSA experiment with nuclear proteins from

CH12.LX or MPC-11 cells and the wild-type aP probe. A retardedband characteristic of NF-aP was detected that could be specificallycompeted by unlabeled aP competitor but not unrelated competitor(CREB site). (B) EMSA analysis of mutated end-labeled aP oligo-nucleotides. (C) Sequences of native and mutagenized aP sites.Dashes, identical nucleotides; triangles, hyperactivities; circles, pro-tected residues in MPC-11 cells in.vivo. (D) Super-shift experimentwith a pan-Ets antibody. A dose-dependent super shift of NF-aP was

seen (arrow). Control rabbit immunoglobulin did not supershift NF-aP. (E) Southwestern blot. End-labeled wild-type aP sequences or

mutated aP sequences were incubated with renatured nuclear proteinsfrom various cell lines that were separated by SDS/PAGE. A singleband at m40 kDa was observed with the wild-type sequence and

proteins from CH12.LX B cells.

tion in the upstream GGAA motif, which abolishes NF-aPbinding, consistently led to a 50% reduction in reporter gene

activity compared with the wild-type enhancer sequence (229* 20.4 times enhancement over enhancerless construct vs. 112

34 times), suggesting that NF-aP binding to the 3'a en-

hancer is required for optimal enhancer function.A TFO Targeted to BSAP Binding Site 1 Prevents Binding

ofBSAP to This Site. The reciprocal binding ofBSAP in B cellsand NF-aP in plasma cells suggested that BSAP inhibition ofenhancer activity might be mediated by inhibition of NF-aP

BSAP

probes: BSAP sitel CD 19 site 1

FIG. 3. (A) Sequences of TFOs and BSAP binding sites in theimmunoglobulin 3'a enhancer and the CD19 promoter. Mismatches ofthe TFOs for possible triplex formation with the relevant regions(boxed areas) of both BSAP sites are indicated by asterisks. Band-shiftdata showed triplex formation with the end-labeled BSAP site 1(Upper) but not the CD19 site 1 (Lower) with the BSAP-TFO. (B)EMSA analysis of the effect of TFOs on the binding of BSAP.End-labeled oligonucleotides were incubated in the absence or pres-ence of TFOs, followed by incubation with crude nuclear extract fromCH12.LX.A2 B cells and gel-shift analysis.

NF-aP

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Proc. Natl. Acad Sd USA 92 (1995)

Proc. NatL Acad ScL USA 92 (1995) 5339

wt3 'e-Luc

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FIG. 4. BSAP-TFO or control TFO was electroporated intoCH12.LX.A2 cells with the wt3'e-Luc or mut3'e-Luc plasmids, andluciferase activity was determined 48 hr after transfection. Values andstandard deviations represent data from three experiments and arereported as the increase in luciferase activity compared with that seenfrom an enhancerless reference vector.

We verified that the BSAP-TFO formed a triplex in aconcentration-dependent manner with the end-labeled dou-ble-stranded BSAP binding site 1 of the 3'a enhancer but notwith a double-stranded oligonucleotide corresponding to themost similar published BSAP binding site, the CD19 site 1(Fig. 3A). Furthermore, a control TFO with identical basecomposition did not induce triplex formation with eitherBSAP site (data not shown). Although we cannot rule out thebinding of the BSAP-TFO to other unknown BSAP sites in thegenome, we conclude that it binds to its intended target sitewith considerable specificity. Additional EMSA studiesshowed that the BSAP-TFO but not the control TFO effec-tively inhibited BSAP binding to binding site 1 in the 3'aenhancer (Fig. 3B). Neither TFO affected BSAP binding at theCD19 site 1.In Vivo Blocking of BSAP Binding Site 1 in CH12.LX.A2 B

Cells Leads to Binding of NF-aP to the Enhancer and In-creased Enhancer Activity. Since the BSAP-TFO preventedbinding of BSAP to binding site 1, we tested the effect of suchinhibition in B cells in vivo. CH12.LX.A2 B cells, which containNF-aP detectable by EMSA but show no evidence of occu-pancy of the aP site in vivo, were transiently transfected withthe BSAP-TFO and control TFO. In vivo DMS footprintanalysis showed a partially protected area at the aP site aftertransfection of the BSAP-TFO (but not of the control TFO)consistent with the idea that the BSAP-TFO-induced inhibi-tion of BSAP binding allowed NF-aP to bind to the aP site(Fig. 1B).

These data are consistent with the model that NF-aPbinding stimulates activity of the 3'a enhancer and that thisprotein is present in B-cell nuclei but prevented from bindingto the aP site by the binding of BSAP to the nearby BSAP site1. To test this model, we performed transient cotransfectionexperiments with the TFOs and wt3'e-Luc or mut3'e-Lucconstructs in CH12.LX.A2 cells (Fig. 4). With wt3'e-Luc,cotransfection of the BSAP-TFO led to an increase in reportergene activity that was not seen with the control TFO. Withmut3'e-Luc, no BSAP-TFO activation of luciferase activitywas observed, suggesting that the increase in enhancer activityachieved by cotransfection of the BSAP-TFO is criticallydependent on an intact aP site.

In Vivo Blocking of BSAP Site 1 in B Cells Results inAugmented Heavy Chain C Region mRNA Synthesis. To assesswhether inhibition of BSAP binding could affect the functionof the enhancer in the endogenous immunoglobulin heavychain locus, we investigated whether transfection of the BSAP-TFO could increase accumulation of immunoglobulin mRNAtranscribed from endogenous heavy chain genes. Thus, wetransfected CH12.LX.A2 B cells (IgA-producing B cells) withBSAP-TFO and control TFO and measured heavy chain Cregion mRNA levels by Northern blot analysis. BSAP-TFO-transfected cells showed an -5-fold greater level of membraneand secretory forms of Ca mRNA compared with cells trans-fected with the control TFO (Fig. 5A). No significant differencesin the level of CD19 expression were found, indicating that theTFO had no general effect on BSAP-regulated transcriptionalcontrol regions. Western blot analysis for IgA expression con-firmed that BSAP-TFO-transfected CH12.LX.A2 cells also ex-hibited higher IgA protein expression.To determine whether the observed effect was specific for

Ca gene transcription or was also observable with other heavychain C region genes lying further upstream from the 3'aenhancer, we explored the effects of the BSAP-TFO onimmunoglobulin expression of several CH12.LX subclonesthat express y or ,u isotypes. Although the BSAP-TFO had lesseffect on CH12.LX cells producing IgM, strikingly increasedimmunoglobulin gene transcription was found in CH12.LXsubclones that produced IgG2b and IgG3 (Fig. SB).

DISCUSSIONEarlier work on the control of immunoglobulin heavy chaingene expression identified the heavy chain variable region genepromoter and the enhancer in the intron between the heavychain joining region and C,u as critical regulatory regions.Recently two additional enhancers have been identified in theimmunoglobulin heavy chain locus: one weak enhancer close

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Cy3 C'y2b cells 8 hr after electroporation of BSAP-TFO orcontrol TFO. Western blot studies 24 hr after trans-fection showed higher levels of IgA expression inBSAP-TFO-transfected CH12.LX.A2 cells. (B)Northern blots for A and y transcripts and f3-actin invarious CH12.LX subclones 8 hr after electropora-

actin tion of BSAP-TFO and control TFO.

Immunology: Neurath et aL

5340 Immunology: Neurath et al

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FIG. 6. Schematic model for BSAP-mediated repression of bindingof NF-aP in vivo. NF-T is identical to the recently described NFABcomplex consisting of Elf-1 and the AP-1 factors Jun-B and c-Fos (28).

to the 3' end of Ca (24) and the other '16 kb downstream ofCa exon 1, which is referred to as the 3'a enhancer (25) andis part of a locus control region that includes two additionaldownstream regulatory elements (26). We show herein thatimmunoglobulin heavy chain gene transcription is controlledby a mechanism in the 3'a enhancer involving BSAP-regulatedbinding of an Ets-like factor (NF-aP) (Fig. 6).The 3'a enhancer was characterized by transfection of

reporter gene constructs into B-lineage cells representative ofdifferent developmental stages and physiological states. Suchstudies demonstrated that this enhancer is inactive in B-celllymphomas and resting B cells (11, 21). However, the enhanceris constitutively active in plasmacytoma-cell lines regardless oftheir isotype, suggesting that it plays an important role in theregulation of high-level immunoglobulin secretion of plasmacells (12).A recent study by Cogne et al. (27) of the 3'a enhancer in

its endogenous chromosomal location (as opposed to trans-fected constructs) has provided insights into the functionalcomplexities of this regulatory region. Chimeric mice contain-ing B cells whose 3'a enhancer had been inactivated by genetargeting showed defects in expression of at least certainisotypes ('y3, y2b, and e). This underlines the importance ofcorrelating results from transfection of exogenous enhancerconstructs with studies on the function of the same sequencesin their endogenous chromosomal context.

In an alternative approach toward the above goal, we haveused a TFO strategy in intact cells to determine the regulatoryfunction of a sequence element in the 3'a enhancer that bindsin vitro to the transcription factor BSAP and represses en-hancer activity in transfection experiments. Although the 3'aenhancer contains two sites that bind to Pax5 in vitro, we foundevidence for occupation of only site 1 in B cells; neither site isoccupied in plasma cells. In addition, we identified a down-stream sequence element that we call aP that has a reciprocalin vivo binding pattern to BSAP binding site 1: it is occupiedin plasma cells but not in B cells. In vitro binding studiessuggested that this element binds a 40-kDa Ets-like nuclearfactor, termed NF-aP, that is present in both B-cell andplasma-cell nuclei. In functional experiments, we then showedthat an intact aP site is necessary for maximal activity of the3'a enhancer in MPC-11 plasmacytoma cells. To directlyaddress the possibility that Pax5 might suppress enhancerfunction in B cells by preventing the activation stimulusconferred by NF-aP binding, we used a TFO strategy to blockBSAP site 1 in B cells and examined functional consequencesof increased NF-aP binding induced by the BSAP-TFO. Wefound an -2.6-fold increase in enhancer activity (vs. control

Proc. Natl. Acad. Sci. USA 92 (1995)

TFO) of a cotransfected enhancer construct containing anintact (but not a mutated) aP sequence, suggesting that thestimulatory effect of the BSAP-TFO and the inhibitory effectof BSAP itself are mediated via the aP site.

Further, we were able to verify that the enhancer, which wasoriginally defined by experiments with transfected constructs,can in fact stimulate transcription of endogenous immuno-globulin genes. We showed that CH12.LX.A2 B cells whoseBSAP binding site 1 had been blocked by the BSAP-TFOcontained increased levels of Ca mRNA and produced in-creased amounts of IgA protein. Studies of Cy-producingsublines of CH12.LX provided similar results. The differentmagnitude of this effect in the CH12.LX sublines tested maybe peculiar to these subclones or may reflect different sensi-tivity of different isotypes to transcriptional regulation by the3'a enhancer. In any case, the data demonstrate clearly thatthe enhancer in its chromosomal context can stimulate immu-noglobulin gene transcription in B cells.The data reported here provide evidence for transcriptional

regulation of heavy chain gene expression by the BSAP/NF-aP axis in the immunoglobulin 3'a enhancer. Since acti-vation of the 3'a enhancer seems to be necessary for high levelimmunoglobulin synthesis, coordinate regulation of BSAP andNF-aP emerges as a key molecular event in secreting B cellsand terminally differentiated plasma cells to obtain high-levelimmunoglobulin synthesis.

We thank Drs. Lou Staudt, David Levens, Eckhard Stiiber, andChad Oh for critical reading of the manuscript and Drs. T. Papas, D.Watson, and M. Busslinger for providing antibodies.

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26.27.

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