The T-Cell Receptor {beta} Variable Gene Promoter Is Required for Efficient V{beta} Rearrangement but Not Allelic Exclusion

To investigate the role of promoters in regulating variablegene rearrangement and allelic exclusion, we constructed mutantmice in which a 1.2-kb region of the Vß13 promoterwas either deleted (P13^–/–) or replaced with thesimian virus 40 minimal promoter plus five copies of Gal4 DNAsequences (P13^R/R). In P13^–/– mice, cleavage, rearrangement,and transcription of Vß13, but not the flanking Vßgene segments, were significantly inhibited. In P13^R/R mice,inhibition of Vß13 rearrangement was less severe andwas not associated with any apparent reduction in Vß13cleavage. Expression of a T-cell receptor (TCR) transgene blockedcleavages at the normal Vß13-recombination signalsequence junction and Vß13 coding joint formationof both wild-type and mutant Vß13 alleles. However,a low level of aberrant Vß13 cleavage was consistentlydetected, especially in TCR transgenic P13^R/R mice. These findingssuggest that the variable gene promoter is required for promotinglocal recombination accessibility of the associated Vßgene segment. Although the promoter is dispensable for allelicexclusion, it appears to suppress aberrant Vß cleavagesduring allelic exclusion.

INTRODUCTION

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Introduction

The variable regions of immunoglobulin (Ig) and T cell receptor(TCR) genes are assembled from variable (V), diversity (D),and joining (J) gene segments. V(D)J recombination is tightlyregulated in the context of lymphocyte development, exhibitinglineage, developmental stage, and allele specificity (1, 13,24, 32). Although recombination at different TCR and Ig lociis mediated by the same recombinase complex and conserved recombinationsignal sequences (RSS), complete rearrangements of TCR genesare limited to T cells, while complete rearrangements of Iggenes are limited to B cells. Within the appropriate cell lineage,recombination is regulated temporally and in a stage-specificmanner. In addition, in a given lymphocyte, only one of twoalleles of antigen receptor loci usually undergoes functionalrearrangement, a process known as allelic exclusion.

Studies have shown that transcriptional regulatory elements,such as promoters and enhancers, play an important role in targetingspecific gene segments for recombination. Deletion of the enhancerat any of the TCRß, TCR ${delta}$ , TCR ${alpha}$ , IgH, and Ig ${kappa}$ loci resultsin a severe reduction in the level of rearrangement at the respectiveloci (4, 5, 7, 11, 14, 27, 31, 41, 42). Similarly, deletionof the PDß1 promoter immediately upstream of the Dß1gene segment at the TCRß locus significantly impairsDß1 rearrangement (40, 41). To date, most evidencesuggests that enhancers and promoters regulate V(D)J recombinationby modulating chromatin structures and rendering gene segmentsaccessible to RAG cleavage (13, 24, 32).

Among the various levels of control, the most complex regulationis probably allelic exclusion. Like lineage- and stage-specificregulations, allelic exclusion is apparently also achieved throughmodulating access of gene segments to RAG cleavage (13, 15,24, 32). For example, TCRß allelic exclusion is regulatedat the Vß-to-DßJß rearrangementstep. In CD4^– CD8^– (DN) thymocytes, where TCRßrearrangement normally occurs, Vß gene segments aretranscribed, sensitive to nuclease, and associated with acetylatedhistones (6, 17, 35). In CD4⁺ CD8⁺ (DP) thymocytes, where TCRßallelic exclusion is in effect, Vß transcription,nuclease sensitivity, and association with acetylated histonesare markedly reduced. When a Vß gene segment togetherwith 3.6-kb 5' sequences, encompassing the promoter, were inserted6.8 kb upstream of the Dß1 gene segment, the insertedVß gene segment rearranged at the same frequency asthe natural copy but was no longer subject to allelic exclusioncontrol (29), indicating distinct controls of the frequencyand allelic exclusion of Vß gene rearrangement. However,beyond these preliminary observations, little is known aboutthe specific cis elements that modulate variable gene accessibilityfor rearrangement initially and then for allelic exclusion.

Another cardinal feature of V(D)J recombination is site specificity,which is achieved by precise cleavage of DNA at the junctionof coding sequences and RSS (9, 10). The precise cleavages occurprobably because RAG proteins interact directly with two RSSto form an enzymatically active complex, known as a synapse(8, 34, 38). Within this synapse, RAG proteins cleave the DNAat the junctions of RSS and coding sequences. Studies have shownthat the presence of two intact RSS is required for synapseformation and coupled cleavages (8, 34, 38). However, RAG proteinshave also been shown to cleave DNA that contains only a singleRSS or RSS-like sequence (19, 20, 36, 37), indicating that RAGproteins are capable of uncoupled and imprecise cleavages. Atthe endogenous antigen receptor loci, gene segments are packedinto chromatin and can be separated by hundreds of kilobasepairs on linear DNA. It is unclear whether RSS alone providea sufficient cis signal for synapse formation and thereforeprecise and coupled cleavages. Nor is it known how aberrantcleavages are suppressed at the endogenous antigen receptorloci.

In mice, the TCRß locus spans approximately 600 kb(22). All Vß gene segments, except Vß14,are clustered together at the 5' end of the locus and separatedfrom Dß, Jß, and Cß by at least330 kb. The two cis elements, PDß1 promoter and Eßenhancer, which have been shown to regulate Dß-to-Jßrearrangements, do not appear to play a significant role inregulating Vß accessibility and allelic exclusion(17, 18, 40, 41). We have now investigated the role of a variablegene promoter in regulating Vß rearrangement and allelicexclusion by targeted deletion or replacement in the endogenousTCRß locus. By examining the effects of the promotermutations on specific as well as flanking Vß genecleavage, joining, allelic exclusion, and transcription, ourresults show that the normal variable gene promoter is requiredfor promoting local recombination accessibility. Although thepromoter is not required for mediating allelic exclusion perse, it appears to suppress aberrant Vß cleavages duringallelic exclusion.

MATERIALS AND METHODS

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Materials and Methods

Targeting vectors and mice. The targeting vector used for electroporation into J1 embryonicstem cells consisted of a floxed phosphoglycerate kinase (PGK)promoter-driven neomycin resistance gene (neo) flanked upstreamby a 2.4-kb PstI-NdeI fragment and downstream by a 7.6-kb NcoI-ClaIfragment (Fig. 1A). The targeting vector for the replacementmutation contained an additional 113-bp concatemer of five copiesof Gal4 sequences and the 111-bp simian virus 40(SV40) minimalpromoter downstream of neo. A PGK promoter-driven thymidinekinase gene (tk) was inserted downstream of the 7.6-kb fragment.Embryonic stem cell clones with proper deletion of the Vß13promoter were identified by Southern blotting and were injectedinto C57BL/6 blastocysts to generate chimeric mice, which werebred with deleter mice to remove the floxed neo (25). Heterozygousmutant mice were bred with each other to generate homozygousmutant mice. A single loxP site was left in place of the Vß13promoter in the final P13^–/– mutant mice, whilea single loxP site, Gal4 sequences, and the SV40 minimal promoterwere left in place of the Vß13 promoter in the finalP13^R/R mutant mice. Mutant mice were analyzed in mixed 129 xC57BL/6 backgrounds and maintained under specific-pathogen-freeconditions. Routine genotype analyses of the Vß13promoter mutations were performed by PCR of tail DNA using primers5'-GGTCAAGCATCTACTTATTGTTC-3' and 5'-AGCCAAGAAGCCTGGTGCCCAT-3'.All promoter mutationswere confirmed by another round of PCRusing the wild-type Vß13 promoter-specific primers5'-GGACTGTGCTAAGACTGATTC-3' and 5'-GGACTGCATATCTGGGAGACTG-3'.TCR transgenic mice expressing the 2C TCR (28) were assayedby flow cytometry using an antibody specific for the 2C TCR.

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FIG. 1. Inactivation of the normal Vß13 promoter specifically blocks development of Vß13-expressing T cells. (A) Schematic diagrams of the TCRß locus, Vß13 region, targeting vectors, and targeted alleles. Gene segments are shown as vertical lines or filled boxes. Vß promoters are shown as open ovals. Triangles indicate loxP sequences. Open circles represent five copies of Gal4 sequences plus the SV40 minimal promoter. Selectable neo marker was deleted from the targeted alleles in mutant mice by Cre/loxP-mediated recombination. As a result, the 1.2-kb Nde1-Nco1 fragment, encompassing the Vß13 promoter plus transcriptional initiation sites, but not the start codon, was replaced by one copy of the loxP sequence in deletion mutant mice (P13^–/–). In replacement mutant mice (P13^R/R), the promoter was replaced by loxP followed by Gal4 sequences and the SV40 minimal promoter. (B) CD4 versus CD8 staining profiles of thymocytes from 4- to 6-week-old wild-type (+/+), P13^–/–, and P13^R/R mice. Numbers outside the plots indicate the average numbers of thymocytes from four to six mice. Numbers inside the plots indicate percentages of cells in each quadrant. (C) Flow cytometry analysis of Vß13-expressing T cells. Cells from lymph nodes of +/+, P13^+/–, P13^–/–, and P13^R/R mice were stained with antibodies specific for the pan-T-cell marker Thy-1.2 plus Vß13, Vß8.1, or Vß12. Expression of Thy1.2 versus Vß is shown for live cells. Numbers indicate the percentages of Thy-1.2⁺ cells that are also Vß⁺. Representative data from one mouse of each genotype are shown.

Flow cytometry. Antibodies specific for CD4, CD8, Thy-1.2, Vß13, Vß8.1,and Vß12 were direct conjugates from PharMingen (SanDiego, Calif.). Flow cytometry was performed on a FACSCaliburapparatus (Becton Dickinson), and data were analyzed using CellQuestsoftware. CD4^– CD8^– thymocytes were purified bycomplement-mediated lysis of CD4⁺ and/or CD8⁺ cells followedby cell sorting. The sorted cell populations were more than97% pure.

PCR assays for Vß CJ. The semiquantitative nested PCR assays for measuring Vß13-to-DßJß1.1coding joints (CJ) were performed in a 50-µl reactionmixture containing 0.2 µg of thymocyte DNA, 100 ng ofeach primer (primer no. 1 and 2), a 0.2 µM concentrationof each deoxynucleoside triphosphate, 3.5 mM MgCl₂, 10 mM Tris-HCl(pH 8.3), 50 mM KCl, and 2.5 U of Taq polymerase. Primary reactionswere run for 12 cycles of 30 s at 95°C, 30 s at 61°C,and 2 min at 72°C. Two-microliter reaction mixtures weretransferred from the primary reactions to new tubes for secondaryPCRs that were performed under identical conditions, exceptwith nested primers (no. 3 and 4) and 18 cycles of amplification.Quantitative titrations of DNA templates were performed by seriallydiluting wild-type thymic DNA into RAG2-deficient kidney DNAsuch that the final amount of DNA remained at 200 ng per reactionmixture. Twenty-five-microliter aliquots of secondary PCR mixtureswere electrophoresed on a 1.5% agarose gel, transferred to Zeta-probemembranes (Bio-Rad), and hybridized with ³²P-labeled Vß13cDNA probe or ³²P-end-labeled oligonucleotides correspondingto the sequence downstream of Jß1.1 (primer no. 5).Filters were washed at 50°C in 5x SSC (1x SSC is 0.15 MNaCl plus 0.015 M sodium citrate) and subjected to autoradiography.Vß13-to-DßJß2.1 rearrangementwas done as described above, except with primers 1 and 6 forthe primary reaction and primers 3 and 7 for the secondary reaction.Southern blotting was done with ³²P-end-labeled oligonucleotidescorresponding to sequence downstream of Jß2.1 (primerno. 8). Vß8.1-to-DßJß1.1 rearrangementwas done with primers 9 and 2 for the primary reaction and primers9 and 4 for the secondary reaction. Vß12-to-DßJß1.1rearrangement was done with primers 10 and 2 for the primaryreaction and primers 10 and 4 for the secondary reaction. Southernblotting for Vß8.1 to DßJß1.1or Vß12 to DßJß1.1 was done usingspecific Vß cDNA probes or an oligonucleotide probecorresponding to sequence downstream of Jß1.1 (no.5). Rearrangement products were quantified by using PhosphorImageranalysis software (Fujifilm). PCR products were cloned intopCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) for sequencing. SemiquantitativeJAK3 PCR was done as previously described (40). PCR primer sequenceswere as follows: primer 1, 5'-CTGCCATGGGCACCAGGCTTCTTG-3'; primer2, 5'-AGATACTCGAATATGGACACGGAG-3'; primer 3, 5'-GGCACCAGGCTTCTTGGCTGGGCAG-3';primer 4, 5'-TGGACACGGAGGACATGCTTTGTC-3'; primer 5, 5'-AGAGAGACCTGGAAATTTACCTG-3';primer 6, 5'-GGTTTTCTGCTCCGGGGGTCTTTG-3'; primer 7, 5'-GGTCTTTGTGGCTGACTGTCCTAC-3';primer 8, 5'-TCTCTCCCACCTGTATGGCCTCTG-3'; primer 9, 5'-ACTCTTCTTTGTGGTTTTGATT-3';primer 10, 5'-GCTGGAGTTACCCAGACACCC-3'.

PCR assay for SE. Linker-mediated PCR (LM-PCR) assays for Vß13, Vß12,Vß8.1, 5' Dß1, or 5' Dß2 signalends (SE) were performed as described elsewhere (23, 40) withslight modification. For LM-PCR, 150 ng of ligated DNA was usedas template in a 50-µl reaction mixture. In the primaryreaction for Vß13, Vß12, and Vß8.1SE, PCR was carried out for 15 cycles of 45 s at 95°C, 30s at 63°C, and 45 s at 72°C, followed by a 7-min extensionat 72°C. Five microliters was transferred to fresh tubesand amplified in a secondary reaction for 27 cycles with a nestedprimer and BW-1H using the same conditions. PCR for 5' Dß2SE was performed using the same conditions as for Vß13SE, except for 20 cycles in the secondary reaction. For 5' Dß1SE, the primary reaction was 15 cycles of 30 s at 95°C,30 s at 62°C, and 30 s at 72°C, followed by a 7-minextension at 72°C, and the secondary reaction was 20 cyclesusing the same condition as the primary reaction. Twenty microlitersof the secondary reaction mixtures was electrophoresed on 1.6%agarose gels, transferred to nylon N⁺ membranes (Amersham),and probed with end-labeled oligonucleotide probes. The restof the PCR products were cloned into pCR2.1-TOPO (Invitrogen)for sequencing. The specific primers and probes used were asfollows: 3'Vß13 distal primer, 5'-GTCGCTTTCAGTTTGGGGTTCTTG-3';3'Vß13 proximal primer, 5'-AAAAAATTACTTGGAGTCCCTGAG-3';3'Vß13 probe, 5'-CAGAGACCTGGGACTATT-3'; 3'Vß12distal primer, 5'-AAATCTCTGAACTACCTTCAAGGTC-3'; 3'Vß12proximal primer, 5'-TTTCTTAATACTCGATTATCTTCTG-3'; 3'Vß12probe, 5'-AGGATGCCCTGCCTGTGC-3'; 3'Vß8.1 distal primer,5'-AAATCTGTCAGAATGACCTTAGTA-3'; 3'Vß8.1 proximal primer,5'-GTAAGGATGAGACTCATGCTGTGT-3'; 3'Vß8.1 probe, 5'-TGGCTTCCTTCACTCTGC-3';5'Dß1 distal primer, 5'-GGTAGACCTATGGGAGGGTC-3'; 5'Dß1proximal primer, 5'-ACCTATGGGAGGGTCCTTTTTTGTATAAAG-3'; 5'Dß1probe, 5'-TGTAACATTGTGGAATTC-3'; 5'Dß2 distal primer,5'-GATTTACCCAGCTTGAGACTTTTTCC-3'; 5'Dß2 proximal primer,5'-CAGCCCCTCTCAGTCAGACAAACC-3'; 5'Dß2 probe, 5'-TGCCACCTGGTCTCCCTGCCCCTG-3'.

PCR assays for SJ. The PCRs and ApaLI digestion used to measure Vß13signal joints (SJ) were performed as described previously withslight modification (40). The primers for Vß13-to-Dß2SJ were the same as primers for Vß13-to-Dß2SE. The same upstream primers and two downstream primers, 5Dß1A(5'-GAACAGGGGGTAAAGAGGAAACCC-3') and 5Dß1B (5'-CATTAGCTCGCATCTTACCACCAC-3'),were used to assay Vß13-to-Dß1 SJ. Two microgramsof thymocyte DNA was used in all PCR mixtures. Both undigestedand ApaLI-digested products were detected by Southern hybridizationwith the same end-labeled Vß13 probe that was usedto detect Vß13 SE.

PCR assays for Vß GT. Total RNA was isolated using TRIzol (Invitrogen) from thymocytesof various mice between 6 and 10 weeks of age. To remove residualgenomic DNA, 10 µg of the RNA sample was treated with2 U of amplification-grade DNase I (Invitrogen) for 15 min at25°C. The reaction was inactivated by the addition of 2µl of 25 mM EDTA (Invitrogen) and incubation at 65°Cfor 15 min. First-strand cDNA was synthesized from 50 ng ofstarting RNA (and 5- and 25-fold dilutions of each of theseRNAs) using the Titan One-Tube RT-PCR System (Roche, Indianapolis,Ind.), following the manufacturer's instructions. PCR cyclingparameters were 30 min at 50°C, 30 s at 94°C, 45 s attemperatures specific for each primer pair (Tables 1 and 2),and 1 min at 72°C for 15 (Vß genes) or 24 (ß-actin)cycles. Twenty microliters of ß-actin primary PCRmixture was directly loaded onto an agarose gel without furtheramplification. To amplify Vß germ line transcripts(GT), 5 µl of cDNA from the primary reverse transcription-PCR(RT-PCR) mixture was used as template for seminested PCR. PCRamplification was performed for 30 s at 94°C, 45 s at temperaturesspecific for each pair of primers, and 1 min at 72°C. Twenty-twoto 30 cycles were normally used. PCR products were run on 1%agarose gels and visualized by ethidium bromide staining. Quantificationwas performed using the software IQ Mac version 1.2. Signalswere first normalized to ß-actin and expressed relativeto that of the wild type, which was given the value of 1.0.

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TABLE 1. PCR primers for assaying Vß GT

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TABLE 2. PCR conditions for assaying Vß GT

Rapid amplification of 5' cDNA ends (5' RACE). One microgram of DNase I-treated total thymic RNA from variousmice was reverse transcribed with random hexamers by using theSMART RACE cDNA amplification kit (Clontech, Palo Alto, Calif.),according to the manufacturer's instructions. cDNA was amplifiedaccording to the instructions for primary PCRs, with 2.5 µlof cDNA, UPM, and V13-5 (5'-CTGCTGGCACAGAGATAGGTGGCTGTGTC-3')primers for 20 cycles. Five microliters of the primary reactionmixture (diluted 1:50 in Tris-EDTA buffer) was used in secondaryPCRs with NUP and V13-4 (5'-CTGCTTTGCAGACTGGATCTTGAGAGTGGA-3')primers at 94°C for 5 s, 68°C for 10 s, and 72°Cfor 3 min for 20 cycles. Twenty microliters of PCR product waselectrophoresed on a 1% agarose gel, followed by hybridizationwith a Vß13-specific probe.

RESULTS

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Results

Effect of promoter mutations on Vß13-expressing T-cell development. We constructed mutant mice in which a 1.2-kb region, encompassingthe known promoter and transcription initiation site, but notthe translation start codon, of the Vß13 gene segment,was deleted (P13^–/–) (Fig. 1A). The same promoterregion was also replaced with the SV40 minimal promoter plusfive copies of the Gal4 DNA sequences (P13^R/R) in order to inactivatethe promoter and potentially afford future opportunities totarget heterologous proteins to the site. The effects of thepromoter mutations on Vß13 rearrangement and expressionwere examined, using the flanking Vß8.1 and Vß12gene segments, each of which has its own independent promoter,as controls.

In both homozygous P13^–/– and P13^R/R mice, cellnumbers in the thymi and CD4 and CD8 staining profiles of thymocyteswere indistinguishable from those of wild-type mice (Fig. 1B).In peripheral lymphoid organs, the number of T cells, the relativeratio of CD4⁺ versus CD8⁺ T cells, and frequencies of Vß8.1-or Vß12-expressing T cells were also similar betweenwild-type and promoter-mutant mice (Fig. 1C and data not shown).However, in contrast to wild-type mice, which had an averageof 2.7% Vß13-expressing T cells, very few Vß13⁺T cells were detected in homozygous P13^–/– or P13^R/Rmice (Fig. 1C). In heterozygous P13^+/– and P13^+/R mice,the percentage of Vß13-positive T cells was reducedby half compared to that in wild-type mice (Fig. 1C and datanot shown). Thus, the promoter mutations specifically impaireddevelopment of Vß13-expressing T cells.

Effect of promoter mutations on Vß13 rearrangement. To investigate the mechanisms underlying the diminished Vß13⁺T-cell development, the effect of the promoter mutations onVß13 rearrangement was examined by semiquantitativePCR assays (Fig. 2A). Compared to levels in wild-type mice,CJ of Vß13 to Dß1Jß1.1 or to DßJß2.1in total thymocyte DNA were decreased approximately 2-fold inP13^+/– mice and 5- to 10-fold in P13^–/– mice(Fig. 2B, left panel). In P13^R/R mice, the levels of Vß13CJ were reduced approximately twofold. Similar results werealso obtained with DNA from purified DN thymocytes (Fig. 2B,right panel), in which TCRß rearrangement usuallyoccurs and TCRß-expressing thymocytes have not yetundergone ß-selection (16). In contrast, there wasno significant difference in the levels of Vß8.1 andVß12 CJ between wild-type and mutant mice. Sequenceanalysis of the CJ products revealed that the frequencies andlengths of nucleotide deletions and additions at the Vß13-Dß1Jß1junction were indistinguishable among wild-type, P13^–/–,and P13^R/R mice (unpublished data). Despite the substantialVß13 rearrangements, both P13^–/– and P13^R/Rmice had virtually no Vß13-expressing T cells (Fig.1C), indicating that the variable gene promoter is requiredfor efficient rearrangement as well as expression of the rearrangedproducts of specific variable genes.

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FIG. 2. Promoter mutations diminish Vß13 rearrangement but not allelic exclusion. (A) Schematic diagram of recombination reaction and assays for CJ, SE, and SJ. Gene segments are shown as open boxes. RSS are shown as open or filled triangles. Arrows indicate the position and direction of PCR primers. (B) Vß13 CJ formation is inhibited in promoter-mutant mice. Vß13, Vß8.1, and Vß12 CJ were amplified by PCR in DNA from total or purified DN thymocytes of +/+, P13^+/–, P13^–/–, and P13^R/R mice. PCR products were separated on agarose gels and then transferred and hybridized with specific Vß probes. DNA from total or DN thymocytes of wild-type mice was serially diluted into RAG2^–/– kidney DNA and then amplified to determine the linear range of the PCR assay. JAK3 was amplified to verify DNA quality and relative amount. Numbers indicate the band intensities normalized to that of JAK3. The level of rearrangement in wild-type DNA was arbitrarily defined as 1. PCR assays were performed five times with two independently isolated total thymocyte DNA samples and three times with DN thymocyte DNA. Representative results from one set of the PCR assays are shown. (C) Vß13 CJ formation is inhibited by expression of a TCR transgene. Vß13-to-Dß1Jß1 CJ of the wild-type (wt) or the mutant (mt) Vß13 allele were assayed separately using primers specific for the promoter sequences in the wild-type allele or the loxP sequences in the mutant allele, respectively. PCR assays for Vß8.1 and Vß12 rearrangements and for JAK3 were the same as in panel B. Representative results from three independent assays are shown.

Effect of promoter mutations on Vß13 allelic exclusion. To investigate the effect of the promoter mutations on allelicexclusion, functionally assembled TCR transgenes, encoding boththe ${alpha}$ and ß chains of a TCR called 2C (28), were introducedinto the promoter-mutant mice. As shown for other TCRs, expressionof the 2C TCR reduced the fraction of DP thymocytes slightlyand promoted T-cell development into either the CD4 or CD8 lineage(28). However, the number and CD4 and CD8 staining profilesof thymocytes were similar in 2C TCR transgenic mice on eitherthe wild-type or the promoter-mutant background (data not shown).As expected, expression of the TCR transgene blocked Vß8.1and Vß12 CJ formation in wild-type, P13^+/–,P13^–/–, P13^+/R, and P13^R/R mice (Fig. 2C). Similarly,expression of the TCR transgene also blocked the CJ formationof both the wild-type and the mutant Vß13 allelesin wild-type, P13^+/–, P13^–/–, P13^+/R, andP13^R/R mice. When a TCRß transgene alone was introducedinto P13^+/– mice, rearrangement of both the wild-typeand the mutant Vß13 allele was also inhibited (datanot shown). Thus, in the absence of the normal variable genepromoter, the Vß13 gene segment appears to undergoallelic exclusion.

Effect of promoter mutations on Vß13 SJ formation. During V(D)J recombination, SE are joined to form SJ (Fig. 2A).The diminished Vß13 rearrangement in promoter-mutantmice is expected to be accompanied by a corresponding decreasein Vß13 SJ. In wild-type mice, SJ resulting from Vß13-to-Dß1or -to-Dß2 rearrangements were readily detected (Fig.3A, lanes 14 to 17). Most of these SJ were cleaved by ApaL1(lane 13), indicating precise joining of two SE. In the presenceof the TCR transgene, only a low level of Vß13-to-Dß1SJ was detected (lanes 11 and 12), consistent with inhibitionof Vß13 rearrangement under allelic exclusion. Inboth P13^–/– and P13^R/R mice, Vß13 (toDß1 or to Dß2) SJ were detected, but thelevels were reduced approximately nine- and threefold, respectively,compared to those in wild-type mice (Fig. 3). As in wild-typemice, the majority of these SJ were cleaved by ApaL1. In thepresence of the TCR transgene, no Vß13 SJ were detectedin the promoter-mutant mice (Fig. 3A, lanes 3, 4, 7, and 8).Thus, diminished Vß13 rearrangement in promoter-mutantmice is associated with a corresponding reduction in the levelof Vß13 SJ, in either the presence or the absenceof a TCR transgene.

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FIG. 3. Expression of a TCR transgene abolishes Vß13 SJ formation in promoter-mutant mice. (A) Analysis of Vß13 SJ in various types of mice. Vß13-to-Dß1 or -to-Dß2 SJ were assayed by PCR in thymocyte DNA from RAG2^–/– mice or +/+, P13^–/–, and P13^R/R mice in the presence or absence of the 2C TCR transgene. Half of the PCR products were digested with ApaLI before separation on agarose gels. PCR products were hybridized with a Vß13-specific oligonucleotide probe. Shown are representative data from three experiments. (B) Comparison of the levels of Vß13 SJ in wild-type and promoter-mutant mice. DNA samples were serially diluted and then used to assay for Vß13-to-Dß1 or -to-Dß2 SJ. Numbers indicate the band intensities normalized to that of JAK3. The level of Vß13 SJ in wild-type DNA was arbitrarily defined as 1. Shown are representative data from three experiments.

Effect of promoter mutations on Vß13 cleavages. To examine whether reduced Vß13 CJ and SJ formationin promoter-mutant mice is associated with diminished Vß13cleavage, we measured the levels of Vß13 SE in thymocyteDNA using LM-PCR (Fig. 2A). Compared to that in wild-type mice,a similar level of Vß13 SE was detected in P13^R/Rmice, whereas the level of Vß13 SE in P13^–/–mice was reduced approximately threefold (Fig. 4A). In general,the same relative levels of Vß13 SE were detectedin wild-type, P13^–/–, and P13^R/R mice using independentlyisolated DNA samples, although there were variations in specificamounts of SE detected in different experiments. The smaller-than-expectedreduction in SE in P13^–/– and in P13^R/R mice couldbe because the LM-PCR assay for SE is not as sensitive as PCRassays for CJ and SJ. As a result, the assay may not be sufficientlysensitive to detect the difference in Vß13 SE correspondingto a twofold difference in Vß13 rearrangement in P13^R/Rmice. It is also possible that in P13^R/R mice the introducedGal4 sequences and SV40 minimal promoter may have impaired Vß13CJ formation without significantly affecting Vß13cleavage. Nevertheless, in P13^–/– mice, the reducedVß13 rearrangement is correlated in large part witha reduced level of Vß13 cleavage (accessibility).

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FIG. 4. Expression of a TCR transgene fails to abolish Vß13 cleavages in promoter-mutant mice. (A and B) Comparison of the levels of Vß13 SE in wild-type and promoter-mutant mice. The levels of Vß13 SE were assayed by LM-PCR in thymocyte DNA from +/+, P13^–/–, and P13^R/R mice with or without the TCR transgene. DNA was either undiluted (undil) or serially diluted (every threefold) and then amplified. PCR products were separated on agarose gels and hybridized with an oligonucleotide probe. JAK3 was amplified as in Fig. 2B and then hybridized with an oligonucleotide probe. Numbers indicate the band intensities normalized to that of JAK3. The level of Vß13 SE in wild-type DNA was arbitrarily defined as 1. Shown are representative data using two sets of independently isolated DNA samples of each genotype. (C) Vß13 cleavages in TCR transgenic promoter-mutant mice are RAG1 dependent. Vß13 SE were assayed by LM-PCR in thymocyte DNA of RAG2^–/– mice, +/+, P13^+/–, and P13^–/– mice with or without the 2C TCR transgene and 2C⁺ P13^–/– RAG1^–/– and 2C⁺ P13^+/– RAG1^–/– mice. Lanes 13 and 14 are DNA from purified DN thymocytes of 2C⁺ P13^–/– and P13^–/– mice, respectively. One set of PCR assays was carried out for 27 cycles, and the other set was carried out for 40 cycles. Shown are representative data from three experiments.

Vß13 cleavages under allelic exclusion. Allelic exclusion is associated with an inhibition of variablegene cleavage (33). Corresponding to the block of CJ formation,no Vß13 SE was detected in thymocyte DNA of TCR transgenicmice on the wild-type background (Fig. 4B and C). In 2C⁺ P13^–/–mice, although a Vß13 cleavage product was consistentlydetected, the level was reduced approximately 15- to 20-foldand another 3-fold compared to those in P13^–/– andwild-type mice, respectively. Noticeably, the size of the cleavageproducts was altered compared to that in P13^–/–mice (Fig. 4A and C). Similarly, Vß13 cleavage productswere detected in 2C⁺ P13^R/R mice, but the levels varied considerablyin independent experiments using thymocyte DNA from differentmice, despite our best efforts (Fig. 4A and data not shown).Because the sizes of PCR products varied in some of the assayswith different DNA, the PCR product may represent amplificationof rare cleavage products. Nevertheless, in all 2C⁺ P13^–/–and 2C⁺ P13^R/R samples analyzed (>5 for each), Vß13,but never Vß8.1 or Vß12, cleavage productswere always detected (Fig. 4 and unpublished data). When a differentTCR transgene was expressed in P13^–/– mice, a lowlevel of Vß13 cleavage products was also detected(data not shown).

Vß13 cleavage products were cloned and sequenced.In wild-type mice, among 31 Vß13 SE products sequenced,26 started with the heptamer (CACAGTA) at the 5' end (Table3), 3 started with ACTCAGA 8 nucleotides downstream of the heptamer(the first C of the heptamer is counted as nucleotide –1),and 2 started within the Vß13 coding sequences. Theseresults indicate that the majority of Vß13 cleavagesin wild-type thymocytes occur precisely at the junction betweenRSS and coding sequences and only a small fraction of the cleavagesis imprecise (13%). As in wild-type mice, most of Vß13SE in P13^–/– mice were derived from precise cleavagesat the Vß13-RSS junction, and a similar fraction ofthe cleavages was aberrant (18%). In contrast, in the presenceof the TCR transgene, all 30 Vß13 cleavage productsin P13^–/– mice were derived from imprecise cleavageseither within the Vß13 coding sequences or the RSS.Similarly, in 2C⁺ P13^R/R mice, all 24 Vß13 cleavageproducts sequenced were derived from imprecise cleavages withineither the Vß13 coding sequences or the RSS. Furthermore,in P13^R/R mice in the absence of the TCR transgene, among 19Vß13 SE products sequenced 9 were derived from aberrantcleavages (47%).

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TABLE 3. Comparison of Vß13 cleavage sites in wild-type and promoter-mutant mice

Although cleavage products that have ends starting within theRSS might have been generated by nuclease processing of SE derivedfrom cleavage at the normal Vß13-RSS junction, thepostcleavage processing of normal SE does not generate productsthat have ends starting within Vß13 coding sequences.The aberrant Vß13 cleavages observed in TCR transgenicpromoter-mutant mice are RAG dependent, because in the absenceof RAG1 no Vß13 cleavage products were detected inthymocytes of 2C⁺ P13^–/– or 2C⁺ P13^+/– mice,even when PCRs were carried out for 40 cycles (Fig. 4C, lanes7 to 11). In addition, the aberrant Vß13 cleavageproducts were detected in both DN and DP thymocytes of 2C⁺ P13^–/–and 2C⁺ P13^R/R mice (Fig. 4C, lanes 13 and 14, and data notshown). Thus, the observed Vß13 SE in TCR transgenicpromoter-mutant mice most likely reflects rare aberrant cleavagesof Vß13, consistent with a lack of Vß13CJ and SJ formation.

Effect of promoter mutations on Vß13 GT. To examine the effect of the promoter mutations on Vß13transcription, we measured the levels of Vß13 GT inthymocytes by RT-PCR. Vß13 GT was readily detectedin wild-type thymocytes (Fig. 5A, lanes 17 to 19). In the presenceof the 2C TCR transgene, the level was decreased approximately10- to 20-fold (lanes 2 to 4), consistent with previous observations(6, 26). In P13^–/– thymocytes, the levels of Vß13GT were about fivefold lower than in wild-type mice (lanes 5to 7). In the presence of the TCR transgene, the level was furtherreduced two- to threefold (lanes 8 to 10). Similarly, the levelof Vß13 GT was about fivefold lower in P13^R/R micethan in wild-type mice (lanes 11 to 13). However, expressionof the TCR transgene in P13^R/R mice did not result in any furtherreduction in the levels of Vß13 GT (lanes 14 to 16).As controls, similar levels of Vß12 and Vß8.1GT were detected in thymocytes of wild-type, P13^–/–,and P13^R/R mice. Expression of the TCR transgene resulted ina 10- to 20-fold decrease in the levels of Vß12 andVß8.1 GT in wild-type and P13^–/– mice,but somewhat less in P13^R/R mice. Thus, germ line Vß13is transcribed in the absence of the normal promoter, and mostof the residual transcription is inhibited by the expressionof a TCR transgene in P13^–/–, but not in P13^R/R,mice.

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FIG. 5. Initiation sites of Vß13 germ line transcription are altered by the expression of a TCR transgene. (A) Vß13 germ line transcription occurs in promoter-mutant mice. GT of Vß13, Vß12, and Vß8.1 were assayed by nested RT-PCR in thymocyte RNA from +/+, P13^–/–, and P13^R/R mice with or without the 2C TCR transgene. cDNA was serially diluted before the PCR assay. The levels of ß-actin transcript were assayed to verify RNA quality and relative amounts. Numbers indicate the band intensities normalized to that of ß-actin. The level of Vß13 GT in wild-type thymocytes was arbitrarily defined as 1. Data from two separate experiments are shown. (B) Initiation sites of Vß13 germ line transcription are altered by the expression of a TCR transgene. RNA from +/+, P13^–/–, P13^R/R, and RAG2^–/– mice with or without the 2C TCR transgene was reverse transcribed into cDNA. The initiation sites were then determined by RACE-PCR using the cDNA samples. PCR products were separated on agarose gels and hybridized with a Vß13-specific probe. The sizes of the marker DNA are indicated. Shown are representative data from three experiments. (C) Schematic diagrams of initiation sites of Vß13 GT in various mice. PCR products from the experiment shown in panel B were cloned and sequenced (results not shown). The initiation sites were determined based on sequencing and the sizes of the PCR products in panel B. The leader and Vß13 exon are depicted. Triangles indicate loxP sequences. Open circles represent five copies of Gal4 sequences plus the SV40 minimal promoter. The higher arrows indicate the major initiation sites.

In promoter-mutant mice, a 1.2-kb region including the knownpromoter and transcription initiation site was deleted or replaced.Vß13 GT in P13^R/R mice might have been initiated fromthe SV40 minimal promoter that was used to replace the endogenouspromoter. However, Vß13 GT in P13^–/– micemust have been initiated from previously unknown sites upstreamof the Vß13 coding sequence. To map the transcriptioninitiation sites in the absence of the normal promoter, we carriedout RACE assays. In RAG2^–/– thymocytes, most Vß13transcription was initiated from one major site (Fig. 5B, lane8), which corresponds to the correct promoter and is consistentwith a previous report (2). In P13^–/– and P13^R/Rthymocytes, however, two major RACE products were detected (lanes4 and 6), suggesting that Vß13 transcription is initiatedfrom two major sites. Noticeably, in the presence of the TCRtransgene, the sizes of the RACE products were dramaticallyaltered in RAG2^–/–, P13^–/–, and P13^R/Rmice. Based on the sizes and sequences of the RACE products(data not shown), most Vß13 transcription initiationsites were mapped to the normal promoter region in RAG2^–/–and wild-type mice (Fig. 5C) and to a 1.5-kb region immediatelyupstream of the normal promoter in P13^–/– and P13^R/Rmice.

DISCUSSION

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Discussion

The variable gene promoter is required for efficient Vß cleavage, rearrangement, and transcription. In mice, the Vß13 gene segment resides approximatelyin the middle of the Vß cluster and is closely flankedby Vß8.1 and Vß12 gene segments (22). Inthe promoter-mutant mice, Vß8.1 and Vß12rearrangements were not significantly affected, as indicatedby their normal cleavage, joining, allelic exclusion, and expression.In contrast, deletion of the Vß13 promoter resultedin a significant reduction of Vß13 CJ formation (5-to 10-fold) (Table 4). Correspondingly, the levels of Vß13SJ, SE, and GT were reduced $~$ 9-, $~$ 3-, and $~$ 5-fold, respectively.The smaller-than-expected reduction in the levels of Vß13SE could be due to the limitations of semiquantitative LM-PCRassays for steady-state levels of SE. Furthermore, the residualVß13 rearrangements in P13^–/– mice hadnormal levels of nucleotide deletions and additions, indicatingthat the promoter deletion did not affect the recombinationreaction per se. Together, these findings suggest that the variablegene promoter regulates Vß rearrangement by promotingaccess of its associated Vß gene segment to RAG-mediatedcleavage.

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TABLE 4. Reduction of Vß13 CJ, SJ, SE, and GT in P13^–/– and P13^R/R mice compared to wild-type mice

Studies have shown that the Vß gene segments themselvesas well as the sequences between gene segments are associatedwith acetylated histones in DN thymocytes (35). Based on thesedata, the presence of a global regulator has been proposed tocontrol the accessibility of the entire Vß cluster(35). While the putative global regulator might have contributedto the residual Vß13 accessibility and rearrangementobserved in P13^–/– mice, our results strongly suggestthat the individual promoter also plays a critical role in targetingspecific Vß gene for rearrangement. Previously, ourinvestigators have shown that when the Vß13 gene segmenttogether with the promoter were inserted upstream of the Dß1gene segment, the inserted Vß13 gene segment rearrangedat the same frequency as the natural copy (29). Together, thesefindings strongly suggest that the variable gene promoter playsan essential role in regulating local access of gene segmentsfor rearrangement.

Regulation of local recombination accessibility by the variablegene promoter is analogous to the PDß1 promoter thatregulates access and rearrangement of the proximal Dß1and Jß1, but not the distal Dß2 and Jß2,gene segments (22, 40, 41). Thus, a general mechanism of promotercontrol of V(D)J recombination is probably by regulating localaccessibility. The local control of accessibility may also contributeto the observed differences in usages and recombination efficienciesamong different Vß gene segments (21). Different Vßpromoters share conserved sequence motifs but also exhibit significantsequence differences (2, 12). The conserved cis elements maycontribute to the overall regulation, and the different ciselements may underlie the differences among gene segments. Asin the TCRß locus, each variable gene segment in otherantigen receptor loci is associated with its own promoter. Itis possible that these promoters also regulate their associatedvariable gene rearrangement by a similar mechanism (3).

A critical role of the variable gene promoter in mediating Vßcleavage, joining, and allelic exclusion is further supportedby the differences observed between the deletion and replacementmutations of the Vß13 promoter. Higher levels of Vß13CJ and SE were observed in P13^R/R mice than in P13^–/–mice (Table 4), indicating that the inserted SV40 minimal promoterand/or Gal4 sequences can partly compensate for the loss ofthe normal promoter. However, no significant difference in thelevel of Vß13 SE was detected between P13^R/R and wild-typemice. Several nonexclusive mechanisms could account for thisdiscrepancy. First, the LM-PCR assay may not be sufficientlysensitive to detect differences in SE corresponding to twofolddifferences in Vß13 rearrangement. Consistent withthis possibility, reduction of Vß13 SE in P13^–/–mice was also less than expected from the reduction in the levelsof Vß13 CJ and SJ. Second, Vß13 SJ couldbe recleaved, resulting in an elevated level of SE. Third, someof the Vß13 cleavages may not lead to CJ formationeither because the normal variable gene promoter is requiredfor efficient CJ formation or because the cleavages were aberrant.In support of the latter possibility, a significantly higherfraction of Vß13 cleavages in P13^R/R mice was imprecise,and these cleavages did not contribute to CJ formation as indicatedby PCR amplification and sequencing (unpublished data). Regardlessof the precise mechanisms, the observed differences in Vß13cleavage and rearrangement in P13^–/– and P13^R/Rmice suggest a critical influence of the sequences in the promoterregion on Vß accessibility and cleavage.

The variable gene promoter is not required for allelic exclusion. One of the remarkable regulations of antigen receptor gene assemblyis allelic exclusion. TCRß allelic exclusion is controlledat the step of Vß gene rearrangement and is initiatedby expression of the pre-TCR complex, consisting of TCRß,pT ${alpha}$ , and CD3 proteins (39). Studies have shown that TCRßallelic exclusion is associated with changes in Vßchromatin structures and accessibility to nuclease (6, 17, 26,35). However, cis elements that mediate allelic exclusion arenot known. When the Vß13 gene segment together withthe promoter were inserted upstream of the Dß1 genesegment, the inserted Vß13 gene segment continuedto rearrange in the presence of a TCR transgene expression (29),indicating that the promoter alone is not sufficient to mediateallelic exclusion. Complementary to this observation, we havenow shown that in the absence of the normal promoter in bothP13^–/– and P13^R/R mice, Vß13 rearrangementwas excluded by the expression of a TCR transgene. These findingssuggest that the normal variable gene promoter is dispensablefor allelic exclusion.

Unexpectedly, Vß13 cleavage products were detectedin both 2C⁺ P13^–/– and 2C⁺ P13^R/R mice (Fig. 4).Although the levels of cleavage were variable and low, especiallyin 2C⁺ P13^–/– mice, Vß8.1 and Vß12cleavage products were never detected in the same DNA samples.The more-abundant cleavages in 2C⁺ P13^R/R mice seem to correlatewith a higher level of germ line Vß13 transcriptionand a lack of inhibition of this transcription by the TCR transgene.Although RAG dependent, all Vß13 cleavages in 2C⁺P13^–/– and 2C⁺ P13^R/R mice took place within Vß13coding sequence and RSS and did not result in CJ or SJ formation,suggesting aberrant cleavage of the Vß13 gene segmentalone. In addition, we found that initiation sites of Vß13germ line transcription were altered by the expression of theTCR transgene in RAG2^–/– mice as well as in P13^–/–and P13^R/R mice. Because of the promoter mutations, the initiationsites and their alterations following TCR transgene expressionwere different between wild-type and mutant mice. These differencesmight underlie the occurrence of aberrant Vß13 cleavagesin promoter-mutant mice. Consistent with this hypothesis, arecent study showed that Dß1 accessibility in a recombinationsubstrate is determined by position and orientation of the PDß1promoter but not by histone acetylation (30).

Because of the low frequency and variability, the implicationof the observed aberrant Vß cleavages on the roleof the variable gene promoter in V(D)J recombination is unclearat the present time. Findings reported here suggest a need tofurther investigate whether the variable gene promoter suppressesaberrant Vß cleavage during allelic exclusion. Importantly,our findings, for the first time, demonstrate that the normalvariable gene promoter is required for efficient cleavage, rearrangement,and transcription of its associated Vß gene segment,but not for allelic exclusion.

ACKNOWLEDGMENTS

We thank Tara Schmidt and Maria Fragoso for technical assistance,Herman N. Eisen, Tyler Jacks, Tania Baker, and Phillip Sharpfor critical review of the manuscript, and members of the Chenlaboratory for helpful discussions.

This work was supported in part by Public Health Service grantsCA100875 and AI40146 (to J.C.) and a grant from the Ministryof Health & Welfare, Republic of Korea (02-PJ1-PG3-20908-0002to C.J.R.). B.B.H. was partly supported by a Postdoctoral Fellowshipfrom the American Cancer Society.

FOOTNOTES

* Corresponding author. Mailing address: Center for Cancer Research, E17-131, Massachusetts Institute for Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 258-6173. Fax: (617) 258-6172. E-mail: jchen{at}mit.edu.

Present address: Department of Internal Medicine, Inje UniversityCollege of Medicine, Ilsan Paik Hospital, Ilsan-gu, Goyang 411-706,South Korea.

REFERENCES

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