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Molecular and Cellular Biology, August 2004, p. 7015-7023, Vol. 24, No. 16
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.16.7015-7023.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Yun Hee Kang,2 Dobrin D. Draganov,1 Minhui Lee,1 Charles E. Whitehurst,1 Hyo Jeong Hong,2 and Jianzhu Chen1*
Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,1 Laboratory of Immunology, Korea Research Institute of Bioscience and Biotechnology, Yusong, Daejon 305-600, Korea2
Received 4 February 2004/ Returned for modification 8 March 2004/ Accepted 19 May 2004
| ABSTRACT |
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| INTRODUCTION |
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Studies have shown that transcriptional regulatory elements, such as promoters and enhancers, play an important role in targeting specific gene segments for recombination. Deletion of the enhancer at any of the TCRß, TCR
, TCR
, IgH, and Ig
loci results in a severe reduction in the level of rearrangement at the respective loci (4, 5, 7, 11, 14, 27, 31, 41, 42). Similarly, deletion of the PDß1 promoter immediately upstream of the Dß1 gene segment at the TCRß locus significantly impairs Dß1 rearrangement (40, 41). To date, most evidence suggests that enhancers and promoters regulate V(D)J recombination by modulating chromatin structures and rendering gene segments accessible to RAG cleavage (13, 24, 32).
Among the various levels of control, the most complex regulation is probably allelic exclusion. Like lineage- and stage-specific regulations, allelic exclusion is apparently also achieved through modulating access of gene segments to RAG cleavage (13, 15, 24, 32). For example, TCRß allelic exclusion is regulated at the Vß-to-DßJß rearrangement step. In CD4 CD8 (DN) thymocytes, where TCRß rearrangement normally occurs, Vß gene segments are transcribed, sensitive to nuclease, and associated with acetylated histones (6, 17, 35). In CD4+ CD8+ (DP) thymocytes, where TCRß allelic exclusion is in effect, Vß transcription, nuclease sensitivity, and association with acetylated histones are markedly reduced. When a Vß gene segment together with 3.6-kb 5' sequences, encompassing the promoter, were inserted 6.8 kb upstream of the Dß1 gene segment, the inserted Vß gene segment rearranged at the same frequency as the natural copy but was no longer subject to allelic exclusion control (29), indicating distinct controls of the frequency and allelic exclusion of Vß gene rearrangement. However, beyond these preliminary observations, little is known about the specific cis elements that modulate variable gene accessibility for 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 junction of coding sequences and RSS (9, 10). The precise cleavages occur probably because RAG proteins interact directly with two RSS to form an enzymatically active complex, known as a synapse (8, 34, 38). Within this synapse, RAG proteins cleave the DNA at the junctions of RSS and coding sequences. Studies have shown that the presence of two intact RSS is required for synapse formation and coupled cleavages (8, 34, 38). However, RAG proteins have also been shown to cleave DNA that contains only a single RSS or RSS-like sequence (19, 20, 36, 37), indicating that RAG proteins are capable of uncoupled and imprecise cleavages. At the endogenous antigen receptor loci, gene segments are packed into chromatin and can be separated by hundreds of kilobase pairs on linear DNA. It is unclear whether RSS alone provide a sufficient cis signal for synapse formation and therefore precise and coupled cleavages. Nor is it known how aberrant cleavages are suppressed at the endogenous antigen receptor loci.
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 separated from Dß, Jß, and Cß by at least 330 kb. The two cis elements, PDß1 promoter and Eß enhancer, which have been shown to regulate Dß-to-Jß rearrangements, do not appear to play a significant role in regulating Vß accessibility and allelic exclusion (17, 18, 40, 41). We have now investigated the role of a variable gene promoter in regulating Vß rearrangement and allelic exclusion by targeted deletion or replacement in the endogenous TCRß locus. By examining the effects of the promoter mutations on specific as well as flanking Vß gene cleavage, joining, allelic exclusion, and transcription, our results show that the normal variable gene promoter is required for promoting local recombination accessibility. Although the promoter is not required for mediating allelic exclusion per se, it appears to suppress aberrant Vß cleavages during allelic exclusion.
| MATERIALS AND METHODS |
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PCR assays for Vß CJ. The semiquantitative nested PCR assays for measuring Vß13-to-DßJß1.1 coding joints (CJ) were performed in a 50-µl reaction mixture containing 0.2 µg of thymocyte DNA, 100 ng of each primer (primer no. 1 and 2), a 0.2 µM concentration of 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 reactions were 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 were transferred from the primary reactions to new tubes for secondary PCRs that were performed under identical conditions, except with nested primers (no. 3 and 4) and 18 cycles of amplification. Quantitative titrations of DNA templates were performed by serially diluting wild-type thymic DNA into RAG2-deficient kidney DNA such that the final amount of DNA remained at 200 ng per reaction mixture. Twenty-five-microliter aliquots of secondary PCR mixtures were electrophoresed on a 1.5% agarose gel, transferred to Zeta-probe membranes (Bio-Rad), and hybridized with 32P-labeled Vß13 cDNA probe or 32P-end-labeled oligonucleotides corresponding to the sequence downstream of Jß1.1 (primer no. 5). Filters were washed at 50°C in 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and subjected to autoradiography. Vß13-to-DßJß2.1 rearrangement was done as described above, except with primers 1 and 6 for the primary reaction and primers 3 and 7 for the secondary reaction. Southern blotting was done with 32P-end-labeled oligonucleotides corresponding to sequence downstream of Jß2.1 (primer no. 8). Vß8.1-to-DßJß1.1 rearrangement was done with primers 9 and 2 for the primary reaction and primers 9 and 4 for the secondary reaction. Vß12-to-DßJß1.1 rearrangement was done with primers 10 and 2 for the primary reaction and primers 10 and 4 for the secondary reaction. Southern blotting for Vß8.1 to DßJß1.1 or Vß12 to DßJß1.1 was done using specific Vß cDNA probes or an oligonucleotide probe corresponding to sequence downstream of Jß1.1 (no. 5). Rearrangement products were quantified by using PhosphorImager analysis software (Fujifilm). PCR products were cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) for sequencing. Semiquantitative JAK3 PCR was done as previously described (40). PCR primer sequences were as follows: primer 1, 5'-CTGCCATGGGCACCAGGCTTCTTG-3'; primer 2, 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 signal ends (SE) were performed as described elsewhere (23, 40) with slight modification. For LM-PCR, 150 ng of ligated DNA was used as template in a 50-µl reaction mixture. In the primary reaction for Vß13, Vß12, and Vß8.1 SE, PCR was carried out for 15 cycles of 45 s at 95°C, 30 s at 63°C, and 45 s at 72°C, followed by a 7-min extension at 72°C. Five microliters was transferred to fresh tubes and amplified in a secondary reaction for 27 cycles with a nested primer and BW-1H using the same conditions. PCR for 5' Dß2 SE was performed using the same conditions as for Vß13 SE, except for 20 cycles in the secondary reaction. For 5' Dß1 SE, 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-min extension at 72°C, and the secondary reaction was 20 cycles using the same condition as the primary reaction. Twenty microliters of 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 rest of the PCR products were cloned into pCR2.1-TOPO (Invitrogen) for sequencing. The specific primers and probes used were as follows: 3'Vß13 distal primer, 5'-GTCGCTTTCAGTTTGGGGTTCTTG-3'; 3'Vß13 proximal primer, 5'-AAAAAATTACTTGGAGTCCCTGAG-3'; 3'Vß13 probe, 5'-CAGAGACCTGGGACTATT-3'; 3'Vß12 distal primer, 5'-AAATCTCTGAACTACCTTCAAGGTC-3'; 3'Vß12 proximal primer, 5'-TTTCTTAATACTCGATTATCTTCTG-3'; 3'Vß12 probe, 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'Dß1 proximal primer, 5'-ACCTATGGGAGGGTCCTTTTTTGTATAAAG-3'; 5'Dß1 probe, 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 Vß13 signal joints (SJ) were performed as described previously with slight modification (40). The primers for Vß13-to-Dß2 SJ were the same as primers for Vß13-to-Dß2 SE. 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 micrograms of thymocyte DNA was used in all PCR mixtures. Both undigested and ApaLI-digested products were detected by Southern hybridization with the same end-labeled Vß13 probe that was used to detect Vß13 SE.
PCR assays for Vß GT. Total RNA was isolated using TRIzol (Invitrogen) from thymocytes of various mice between 6 and 10 weeks of age. To remove residual genomic DNA, 10 µg of the RNA sample was treated with 2 U of amplification-grade DNase I (Invitrogen) for 15 min at 25°C. The reaction was inactivated by the addition of 2 µl of 25 mM EDTA (Invitrogen) and incubation at 65°C for 15 min. First-strand cDNA was synthesized from 50 ng of starting RNA (and 5- and 25-fold dilutions of each of these RNAs) using the Titan One-Tube RT-PCR System (Roche, Indianapolis, Ind.), following the manufacturer's instructions. PCR cycling parameters were 30 min at 50°C, 30 s at 94°C, 45 s at temperatures 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 PCR mixture was directly loaded onto an agarose gel without further amplification. 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. PCR amplification was performed for 30 s at 94°C, 45 s at temperatures specific for each pair of primers, and 1 min at 72°C. Twenty-two to 30 cycles were normally used. PCR products were run on 1% agarose gels and visualized by ethidium bromide staining. Quantification was performed using the software IQ Mac version 1.2. Signals were first normalized to ß-actin and expressed relative to that of the wild type, which was given the value of 1.0.
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| RESULTS |
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In both homozygous P13/ and P13R/R mice, cell numbers in the thymi and CD4 and CD8 staining profiles of thymocytes were indistinguishable from those of wild-type mice (Fig. 1B). In peripheral lymphoid organs, the number of T cells, the relative ratio of CD4+ versus CD8+ T cells, and frequencies of Vß8.1- or Vß12-expressing T cells were also similar between wild-type and promoter-mutant mice (Fig. 1C and data not shown). However, in contrast to wild-type mice, which had an average of 2.7% Vß13-expressing T cells, very few Vß13+ T cells were detected in homozygous P13/ or P13R/R mice (Fig. 1C). In heterozygous P13+/ and P13+/R mice, the percentage of Vß13-positive T cells was reduced by half compared to that in wild-type mice (Fig. 1C and data not shown). Thus, the promoter mutations specifically impaired development 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 on Vß13 rearrangement was examined by semiquantitative PCR assays (Fig. 2A). Compared to levels in wild-type mice, CJ of Vß13 to Dß1Jß1.1 or to DßJß2.1 in total thymocyte DNA were decreased approximately 2-fold in P13+/ mice and 5- to 10-fold in P13/ mice (Fig. 2B, left panel). In P13R/R mice, the levels of Vß13 CJ were reduced approximately twofold. Similar results were also obtained with DNA from purified DN thymocytes (Fig. 2B, right panel), in which TCRß rearrangement usually occurs and TCRß-expressing thymocytes have not yet undergone ß-selection (16). In contrast, there was no significant difference in the levels of Vß8.1 and Vß12 CJ between wild-type and mutant mice. Sequence analysis of the CJ products revealed that the frequencies and lengths of nucleotide deletions and additions at the Vß13-Dß1Jß1 junction were indistinguishable among wild-type, P13/, and P13R/R mice (unpublished data). Despite the substantial Vß13 rearrangements, both P13/ and P13R/R mice had virtually no Vß13-expressing T cells (Fig. 1C), indicating that the variable gene promoter is required for efficient rearrangement as well as expression of the rearranged products of specific variable genes.
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and ß chains of a TCR called 2C (28), were introduced into the promoter-mutant mice. As shown for other TCRs, expression of the 2C TCR reduced the fraction of DP thymocytes slightly and promoted T-cell development into either the CD4 or CD8 lineage (28). However, the number and CD4 and CD8 staining profiles of thymocytes were similar in 2C TCR transgenic mice on either the wild-type or the promoter-mutant background (data not shown). As expected, expression of the TCR transgene blocked Vß8.1 and Vß12 CJ formation in wild-type, P13+/, P13/, P13+/R, and P13R/R mice (Fig. 2C). Similarly, expression of the TCR transgene also blocked the CJ formation of both the wild-type and the mutant Vß13 alleles in wild-type, P13+/, P13/, P13+/R, and P13R/R mice. When a TCRß transgene alone was introduced into P13+/ mice, rearrangement of both the wild-type and the mutant Vß13 allele was also inhibited (data not shown). Thus, in the absence of the normal variable gene promoter, the Vß13 gene segment appears to undergo allelic 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-mutant mice is expected to be accompanied by a corresponding decrease in Vß13 SJ. In wild-type mice, SJ resulting from Vß13-to-Dß1 or -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 presence of the TCR transgene, only a low level of Vß13-to-Dß1 SJ was detected (lanes 11 and 12), consistent with inhibition of Vß13 rearrangement under allelic exclusion. In both P13/ and P13R/R mice, Vß13 (to Dß1 or to Dß2) SJ were detected, but the levels were reduced approximately nine- and threefold, respectively, compared to those in wild-type mice (Fig. 3). As in wild-type mice, the majority of these SJ were cleaved by ApaL1. In the presence of the TCR transgene, no Vß13 SJ were detected in the promoter-mutant mice (Fig. 3A, lanes 3, 4, 7, and 8). Thus, diminished Vß13 rearrangement in promoter-mutant mice is associated with a corresponding reduction in the level of Vß13 SJ, in either the presence or the absence of a TCR transgene.
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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 (Table 3), 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. These results indicate that the majority of Vß13 cleavages in wild-type thymocytes occur precisely at the junction between RSS and coding sequences and only a small fraction of the cleavages is imprecise (13%). As in wild-type mice, most of Vß13 SE in P13/ mice were derived from precise cleavages at the Vß13-RSS junction, and a similar fraction of the cleavages was aberrant (18%). In contrast, in the presence of the TCR transgene, all 30 Vß13 cleavage products in P13/ mice were derived from imprecise cleavages either within the Vß13 coding sequences or the RSS. Similarly, in 2C+ P13R/R mice, all 24 Vß13 cleavage products sequenced were derived from imprecise cleavages within either the Vß13 coding sequences or the RSS. Furthermore, in P13R/R mice in the absence of the TCR transgene, among 19 Vß13 SE products sequenced 9 were derived from aberrant cleavages (47%).
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Effect of promoter mutations on Vß13 GT. To examine the effect of the promoter mutations on Vß13 transcription, we measured the levels of Vß13 GT in thymocytes by RT-PCR. Vß13 GT was readily detected in wild-type thymocytes (Fig. 5A, lanes 17 to 19). In the presence of the 2C TCR transgene, the level was decreased approximately 10- to 20-fold (lanes 2 to 4), consistent with previous observations (6, 26). In P13/ thymocytes, the levels of Vß13 GT were about fivefold lower than in wild-type mice (lanes 5 to 7). In the presence of the TCR transgene, the level was further reduced two- to threefold (lanes 8 to 10). Similarly, the level of Vß13 GT was about fivefold lower in P13R/R mice than in wild-type mice (lanes 11 to 13). However, expression of the TCR transgene in P13R/R mice did not result in any further reduction in the levels of Vß13 GT (lanes 14 to 16). As controls, similar levels of Vß12 and Vß8.1 GT were detected in thymocytes of wild-type, P13/, and P13R/R mice. Expression of the TCR transgene resulted in a 10- to 20-fold decrease in the levels of Vß12 and Vß8.1 GT in wild-type and P13/ mice, but somewhat less in P13R/R mice. Thus, germ line Vß13 is transcribed in the absence of the normal promoter, and most of the residual transcription is inhibited by the expression of a TCR transgene in P13/, but not in P13R/R, mice.
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| DISCUSSION |
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9-,
3-, and
5-fold, respectively. The smaller-than-expected reduction in the levels of Vß13 SE could be due to the limitations of semiquantitative LM-PCR assays for steady-state levels of SE. Furthermore, the residual Vß13 rearrangements in P13/ mice had normal levels of nucleotide deletions and additions, indicating that the promoter deletion did not affect the recombination reaction per se. Together, these findings suggest that the variable gene promoter regulates Vß rearrangement by promoting access of its associated Vß gene segment to RAG-mediated cleavage.
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Regulation of local recombination accessibility by the variable gene promoter is analogous to the PDß1 promoter that regulates access and rearrangement of the proximal Dß1 and Jß1, but not the distal Dß2 and Jß2, gene segments (22, 40, 41). Thus, a general mechanism of promoter control of V(D)J recombination is probably by regulating local accessibility. The local control of accessibility may also contribute to the observed differences in usages and recombination efficiencies among different Vß gene segments (21). Different Vß promoters share conserved sequence motifs but also exhibit significant sequence differences (2, 12). The conserved cis elements may contribute to the overall regulation, and the different cis elements may underlie the differences among gene segments. As in the TCRß locus, each variable gene segment in other antigen receptor loci is associated with its own promoter. It is possible that these promoters also regulate their associated variable gene rearrangement by a similar mechanism (3).
A critical role of the variable gene promoter in mediating Vß cleavage, joining, and allelic exclusion is further supported by the differences observed between the deletion and replacement mutations of the Vß13 promoter. Higher levels of Vß13 CJ and SE were observed in P13R/R mice than in P13/ mice (Table 4), indicating that the inserted SV40 minimal promoter and/or Gal4 sequences can partly compensate for the loss of the normal promoter. However, no significant difference in the level of Vß13 SE was detected between P13R/R and wild-type mice. Several nonexclusive mechanisms could account for this discrepancy. First, the LM-PCR assay may not be sufficiently sensitive to detect differences in SE corresponding to twofold differences in Vß13 rearrangement. Consistent with this possibility, reduction of Vß13 SE in P13/ mice was also less than expected from the reduction in the levels of Vß13 CJ and SJ. Second, Vß13 SJ could be recleaved, resulting in an elevated level of SE. Third, some of the Vß13 cleavages may not lead to CJ formation either because the normal variable gene promoter is required for efficient CJ formation or because the cleavages were aberrant. In support of the latter possibility, a significantly higher fraction of Vß13 cleavages in P13R/R mice was imprecise, and these cleavages did not contribute to CJ formation as indicated by PCR amplification and sequencing (unpublished data). Regardless of the precise mechanisms, the observed differences in Vß13 cleavage and rearrangement in P13/ and P13R/R mice suggest a critical influence of the sequences in the promoter region on Vß accessibility and cleavage.
The variable gene promoter is not required for allelic exclusion.
One of the remarkable regulations of antigen receptor gene assembly is allelic exclusion. TCRß allelic exclusion is controlled at the step of Vß gene rearrangement and is initiated by expression of the pre-TCR complex, consisting of TCRß, pT
, and CD3 proteins (39). Studies have shown that TCRß allelic exclusion is associated with changes in Vß chromatin structures and accessibility to nuclease (6, 17, 26, 35). However, cis elements that mediate allelic exclusion are not known. When the Vß13 gene segment together with the promoter were inserted upstream of the Dß1 gene segment, the inserted Vß13 gene segment continued to rearrange in the presence of a TCR transgene expression (29), indicating that the promoter alone is not sufficient to mediate allelic exclusion. Complementary to this observation, we have now shown that in the absence of the normal promoter in both P13/ and P13R/R mice, Vß13 rearrangement was excluded by the expression of a TCR transgene. These findings suggest that the normal variable gene promoter is dispensable for allelic exclusion.
Unexpectedly, Vß13 cleavage products were detected in both 2C+ P13/ and 2C+ P13R/R mice (Fig. 4). Although the levels of cleavage were variable and low, especially in 2C+ P13/ mice, Vß8.1 and Vß12 cleavage products were never detected in the same DNA samples. The more-abundant cleavages in 2C+ P13R/R mice seem to correlate with a higher level of germ line Vß13 transcription and a lack of inhibition of this transcription by the TCR transgene. Although RAG dependent, all Vß13 cleavages in 2C+ P13/ and 2C+ P13R/R mice took place within Vß13 coding sequence and RSS and did not result in CJ or SJ formation, suggesting aberrant cleavage of the Vß13 gene segment alone. In addition, we found that initiation sites of Vß13 germ line transcription were altered by the expression of the TCR transgene in RAG2/ mice as well as in P13/ and P13R/R mice. Because of the promoter mutations, the initiation sites and their alterations following TCR transgene expression were different between wild-type and mutant mice. These differences might underlie the occurrence of aberrant Vß13 cleavages in promoter-mutant mice. Consistent with this hypothesis, a recent study showed that Dß1 accessibility in a recombination substrate is determined by position and orientation of the PDß1 promoter but not by histone acetylation (30).
Because of the low frequency and variability, the implication of the observed aberrant Vß cleavages on the role of the variable gene promoter in V(D)J recombination is unclear at the present time. Findings reported here suggest a need to further investigate whether the variable gene promoter suppresses aberrant Vß cleavage during allelic exclusion. Importantly, our findings, for the first time, demonstrate that the normal variable gene promoter is required for efficient cleavage, rearrangement, and transcription of its associated Vß gene segment, but not for allelic exclusion.
| ACKNOWLEDGMENTS |
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This work was supported in part by Public Health Service grants CA100875 and AI40146 (to J.C.) and a grant from the Ministry of Health & Welfare, Republic of Korea (02-PJ1-PG3-20908-0002 to C.J.R.). B.B.H. was partly supported by a Postdoctoral Fellowship from the American Cancer Society.
| FOOTNOTES |
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Present address: Department of Internal Medicine, Inje University College of Medicine, Ilsan Paik Hospital, Ilsan-gu, Goyang 411-706, South Korea. ![]()
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