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Increase of TCR V Accessibility within E Regulatory Region Influences its Recombination Frequency But Not Allelic Exclusion

Makoto Senoo^1,*, Lili Wang^*, Daisuke Suzuki^*, Naoki Takeda, Yoichi Shinkai and Sonoko Habu^2,*

^* Department of Immunology, Tokai University School of Medicine, Isehara, Kanagawa, Japan; Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan; and Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Seventy percent of the murine TCR locus (475 kb) was deleted to generate a large deleted TCR (^LD) allele to investigate a possible linkage between germline transcription, recombination frequency, and allelic exclusion of the TCR V genes. In these ^LD/LD mice, the TCR gene locus contained only four V genes at the 5' side of the locus, and consequently, the V10 gene was located in the original D1-J1cluster within the E regulatory region. We showed that the frequency of recombination and expression of the V genes are strongly biased to V10 in these mutant mice even though the proximity of the other three 5'V genes was also greatly shortened toward the D-J cluster and the E enhancer. Accordingly, the germline transcription of the V10 gene in ^LD/LD mice was exceptionally enhanced in immature double negative thymocytes compared with that in wild-type mice. During double negative-to-double positive transition of thymocytes, the level of V10 germline transcription was prominently increased in ^LD/LD recombination activating gene 2-deficient mice receiving anti-CD3 Ab in vivo. Interestingly, however, despite the increased accessibility of the V10 gene in terms of transcription, allelic exclusion of this V gene was strictly maintained in ^LD/LD mice. These results provide strong evidence that increase of V accessibility influences frequency but not allelic exclusion of the TCR V rearrangement if the V gene is located in the E regulatory region.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
During early development of T cells, the TCR gene is rearranged and expressed at the CD4^-CD8^- double-negative (DN) ³ stage, and subsequently the TCR gene is rearranged and expressed at the CD4⁺CD8⁺ double-positive (DP) stage. In the TCR gene locus, which is composed of variable (V), diversity (D), and joining (J) segments, V(D)J recombination occurs in a highly-ordered and tightly-regulated fashion in two sequential steps at the DN stage: first D-to-J joining and then V-to-DJ assembly (1). The V(D)J recombination is considered to be regulated through the control of gene accessibility and cis-acting regulatory elements such as enhancers (2). In the TCR gene, several lines of evidence suggest that the 5'V regions and the 3'D-J clusters discretely regulated in regard to accessibility to the recombinase. In DN thymocytes of both E-deficient mice and E-to-E replaced mice, histone acetylation and transcription were dramatically reduced in the 3'D-J clusters but not in the 5'V genes as we and others have reported (3, 4). In these mice, the rearrangement of the D-J clusters was substantially reduced. It has been estimated that the 5'-end of the E regulatory region is limited to 25 kb upstream of E, which covers the entire D-J clusters plus 5 kb upstream of this region (3). These results imply that the E enhancer might regulate accessibility of the D-J clusters but not further 5'V genes.

Another striking feature of the V(D)J recombination process is the phenomenon termed TCR allelic exclusion. D-to-J rearrangement is assumed to take place concurrently on both alleles. However, functional V(D)J recombination on one allele inhibits V-to-DJ joining on the second allele by a feedback mechanism, ensuring all T cells express a single TCR polypeptide on the cell surface (5). This inhibitory effect on the opposite allele is mediated by signaling through a pre-TCR complex, which consists of the TCR, pT, and CD3 components (6, 7, 8), and by the activity of a protein tyrosine kinase lck (9, 10). The pre-TCR signal is also thought to drive immature thymocytes from the DN stage to the DP stage (11, 12). During DN-to-DP transition, accessibility of the V genes is selectively down-regulated in terms of histone acetylation status (13, 14, 15). These reports have allowed us to speculate that down-regulation of V gene accessibility may play a pivotal role on TCR allelic exclusion.

The germline TCR locus spans 700 kb. At the 3'-end of the locus, two D-J clusters are located within a relatively small (15 kb) region. The E enhancer is located at the more 3'-end of the TCR locus flanked 5.9 kb upstream by the D2-J2 cluster. In contrast, the V genes are located at the 5' side of the TCR locus except V14, which lies 9.4 kb downstream of the D-J clusters, and are separated by a large intron expanding 250 kb from the 3'D-J clusters (16). The long distance between the V and the E enhancer may enable us to predict the distinctive regulation between the 5'V and 3'D-J cluster in the control of V(D)J recombination and accessibility. However, very limited studies have addressed the effects of such a large intron on recombination and accesibility of the TCR locus. Recently, Sieh et al. (17) showed that a certain V gene is not strikingly altered in its recombination frequency when it was inserted 6.8 kb upstream of the D1-J1 cluster, which was estimated to be outside of the E regulatory region. Using recombination activating gene (RAG)2^-/- mice, Chen et al.(18) showed that expression of the V germline transcription before rearrangement does not correlate with proximity to E enhancer. Therefore, it is possible to speculate that a recombination frequency of the V genes may not be solely determined by the proximity to the D-J clusters and the E enhancer. Alternatively, a particular region within the apparent limit of E activity may be important for the V genes to be accessible for further recombination and transcription.

To examine how the regulation of recombination frequency, accessibility and allelic exclusion of the TCR V genes are influenced when it is located under the control of E, we have deleted 70% of the TCR locus (475 kb) in mice including the 3' half of the 5'V region (220 kb), a central large intron of the locus (250 kb) and the first D-J cluster. Consequently, these mutant mice contain one V gene (V10), which is located within the original J1 region in the wild-type allele. Using this mouse model, we show evidence that alteration of V accessibility under the control of E influences recombination frequency but not allelic exclusion of the TCR V genes.

	Materials and Methods

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Targeting constructs and homologous recombination

The first targeting vector consisted of a floxed phosphoglycerate kinase (PGK) promoter-driven neomycin resistance gene (neo^r) that was flanked upstream by a 0.6 kb KpnI-EcoRI homologous fragment and flanked downstream by a 5.0 kb KpnI homologous fragment. A PGK promoter-driven thymidine kinase gene was inserted at the 3'-end. After transfection into TT2 embryonic stem (ES) cells (19), G418- and ganciclovir-resistant clones were selected by PCR and confirmed by Southern blotting (data not shown). Homologous recombinants were then transiently infected with Cre-expressing adenovirus, AxCANCre (20). The second targeting vector was similarly flanked upstream by a 1.4 kb NcoI-SacI fragment and downstream by a 5.2 kb SacI fragment and was transfected into the neo^r gene-deleted homologous recombinants. The G418- and ganciclovir-resistant clones were then screened by PCR and by Southern blotting (data not shown). By infection with AxCANCre, ES clones completely deleted of the 475 kb of the TCR locus, were obtained and subsequently used to make chimeric mice that were bred to generate germline mutant mice.

Southern and Northern blot analyses

Southern and Northern blot analyses were conducted as described previously (14). For Southern blotting, filters were hybridized with the following [³²P]-dCTP-labeled probes: a 0.8-kb BglII-NcoI intronic fragment between V10 and V1 (Fig. 1, probe A), a 3.5-kb HindIII-NcoI intronic fragment upstream of V5 (Fig. 1, probe B), a 2.0-kb KpnI-HindIII C1 probe (Fig. 1, probe C), or a 0.6-kb HpaI-NcoI E probe (Fig. 1, probe D). For Northern blotting, filters were hybridized with the following probes: V2, V4, V16, and V10 exon probes that were excised from the constructs generated by inserting the PCR-amplified V exon fragments into the EcoRV site of SKII (Stratagene, La Jolla, CA). The probes for V14, C2 and -actin were described previously (4).

View larger version (44K): [in this window] [in a new window]   FIGURE 1. Targeted deletion of the large intron within the murine TCR locus by two-step homologous recombination. A, Schematic diagrams of the 3' TCR locus (not to scale), first targeting vector and the targeted allele after Cre-mediated deletion of the PGK-neo (neo). B, Schematic diagrams of the 5' TCR locus (not to scale), second targeting vector and the targeted allele before Cre-mediated deletion of the PGK-neo. C, Schematic diagrams of the entire TCR locus (not to scale) in wild-type mice (WT) and LD/LD mice (LD). Large triangles flanking PGK-neo and small open triangles represent loxP sites and HindIII sites, respectively. Details about probes A-to-D are described in Materials and Methods. D, Southern blot analysis for identifying wild-type mice (lanes 1, 4, 6, 9), heterozygous (+/LD) mice (lanes 2 and 7) and homozygous (LD/LD) mice (lanes 3, 5, 8, 10). The purified tail DNA was digested with HindIII and the blots were hybridized with the indicated probe. Note that probe C (C1 probe) also detects a 3 kb fragment of the C2 region.

Biotinylated, and FITC- and PE-conjugated Abs against mouse CD3 (2C11), CD4 (RM4-5), CD8 (53-6.7), CD25 (7D4), CD44 (Ly-24), V2 (B20.6), V4 (KT4), V10 (B21.5), V8.1/8.2 (MR5-2), V7 (TR310), and V14 (14-2) were all purchased from BD PharMingen (San Diego, CA). The stained cells were acquired using a FACSCalibur (BD Biosciences, Mountain View, CA) and analyzed with CellQuest software (BD Biosciences). Enrichment of the DN cells was performed as described previously (4).

Hybridoma analysis

Hybridoma clones were produced by fusion of Con A-stimulated spleen T cells with the thymoma cell line BW-1100.129.237 deficient in TCR and TCR proteins (21). DNA samples were digested with HindIII and subjected to Southern blot analysis by hybridization of the membrane with a C1 fragment (Fig. 1, probe C), which cross hybridizes with a 3-kb C2 fragment. Loss of hybridization with this probe of 2 kb indicates the rearrangement status of VD2J2/VD2J2 or VD2/VD2J2. Further rearrangement status and the usage of all the possible V and J gene segments were examined by PCR with the primer pairs listed in the previous literature (22). Samples which underwent V(D)J rearrangements on both alleles with the same usage of the V and J gene segments were further processed for sequencing analysis to confirm that they contained two distinct V(D)J fragments. Samples with evidence of more than two alleles were excluded from analysis.

Analysis of V(D)J rearrangement

PCR analysis was conducted as described previously (4). Briefly, each PCR cycle consisted of an incubation at 94°C for 60 s, followed by 90 s annealing at 60°C, and extension for 90 s at 72°C. Before the first cycle, a 10 min 94°C denaturation and Taq activation step was included, and after 31 cycles the extension at 72°C was prolonged to 5 min. The sequences of primers and probes used in this study were as follows:

5'RAG2: 5'-TTAATTCAACCAGGCTTCTCACTT-3', 3'RAG2: 5'-GCCTGCTTATTGTCTCCTGGTATG-3', RAG2 probe: 5'-CTCGACTATACACCACGTCAATG-3', 5'D2: 5'-GTAGGCACCTGTGGGGAAGAAACT-3', 5'V2: 5'-CCAACATGAGCCAGGGCAGA-3', 5'V4: 5'-ATGGGCTCCATTTTCCTCAG-3', 5'V16: 5'-ATGGGATATCTGGCTTCTAGG-3',5'V10: 5'-GCGCTTCTCACCTCAGTCTTCA-3',5'V14: 5'-AGAGTCGGTGGTGCAACTGAACCT-3',3'J2.7: 5'-TGAGAGCTGTCTCCTACTATCGATT-3', and 3'J2.7 probe: 5'-TTTCC CTCCCGGAGATTCCCTAA-3'.

For sequence analysis, the amplified fragments were cloned into pCR2.1 vector (Invitrogen, San Diego, CA) and were directly sequenced with a M13 primer.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of mutant mice with large deleted TCR alleles by two-step homologous recombination

The large deleted TCR (^LD) allele was generated by two-step homologous recombination as depicted in Fig. 1. The first targeting vector was designed to replace the D1-J1 cluster with a neo^r gene that was flanked by loxP sites at both ends (Fig. 1A). The targeted ES cells were then infected with AxCANCre (20). Subsequently, the clones sensitive to G418 were processed to Southern blot analysis to confirm proper targeting (data not shown). After re-targeting the appropriate clones by the second targeting vector that was designed to insert a loxP-flanked neo^r gene into the V1 exon (Fig. 1B), the clones resistant to G418 were infected with AxCANCre to produce the ^LD allele (Fig. 1C). As a result, only five intact V genes were left in the ^LD allele. V10 lies 6.9 kb upstream of the D2 gene in the ^LD allele, corresponding to the J1 region in the wild-type TCR allele (Fig. 1C). Unlike V10, four other V genes were located considerably upstream of the wild-type J1 region, although their proximity was shortened to 190 kb from the E enhancer for V2 and 10 kb from the original D1 gene for V4 and V16, which were separated by a small intron (230 bp) (Fig. 1C). The position of V14 was not changed. The multiple ES clones thus obtained were used for generating chimeric mice to produce heterozygous LD mice (^+/LD), which were intercrossed to generate homozygotes (^LD/LD) (Fig. 1D). Two independent ^LD/LD mouse lines were established but were not distinguished in this study since they showed equivalent phenotypes in the analyses.

Flowcytometric analysis of ^LD/LD mice showed that thymocyte development based on the CD4^- and CD8^- expression is almost equivalent to that of wild-type mice, although the total cell number was slightly reduced in ^LD/LD mice (Fig. 2A). The purified DN cells showed that CD25 vs CD44 profile is almost equivalent between the two mouse lines (Fig. 2B). These data suggest that 475 kb deletion of the TCR locus did not significantly alter the overall TCR rearrangements and thymocyte development.

View larger version (52K): [in this window] [in a new window]   FIGURE 2. Flow cytometry analysis of LD/LD thymocytes. A, CD4 vs CD8 profile of total thymocytes. Thymocytes were doubly stained with PE-conjugated anti-CD8 and Cy-Chrome-conjugated anti-CD4 Abs. The relative percentages of the DN, DP, and single positive cells present per thymus are indicated in the corner of each quadrant. The total cell number present per thymus is indicated above each panel (mean ± SD). Representative data are shown. B, CD25 vs CD44 profile of the DN cells. The magnetically purified DN cells (>99% purity) were stained with FITC-conjugated anti-CD25 and Cy-Chrome-conjugated anti-CD44 Abs. The relative percentages of each quadrant are indicated in the corners. The data shown are representative of three independent experiments with similar results.

Predominant increase of V10 usage in ^LD/LD mice

To investigate whether the V gene usage is biased to the particular segments in the ^LD/LD mice, we first examined the proportion of T cells expressing each V gene. For this purpose, the cells from the thymus and lymph nodes were stained with the Abs to all the possible V gene products except for V16 since an anti-V16 Ab was not available. The results clearly showed that the proportion of V10⁺ cells among CD3⁺ population is extremely high (>60%) compared with the other V-expressing cells (5–10%) in both thymus and lymph node in ^LD/LD mice (Fig. 3A, top and middle panels).

View larger version (29K): [in this window] [in a new window]   FIGURE 3. TCR V repertoire in LD/LD mice. A, Thymocytes (upper panel) and lymph node cells (middle panel) from 6-wk-old WT (solid bars) and LD/LD (open bars) mice were stained with anti-CD3 mAb and each TCR V-specific mAb, and analyzed by flow cytometry. The data shown are gated on the CD3+ fractions. Spleen T cell hybridomas (lower panel) were also analyzed by PCR for the usage of the V genes involved in V(D)J rearrangements (for details, see Materials and Methods). *, ND. B, Semiquantitative PCR of V(D)J rearrangements of thymocytes. RAG2 was also amplified to serve as a control. The histograms on the right side indicate the relative intensities of the bands.

Next, to determine whether the V10 gene is preferentially rearranged due to its unique location within the original J1 region, we performed semiquantitative PCR analysis. As shown in Fig. 3B, the levels of V2-, V4-, V16-, and V14-to-DJ2 rearrangement in thymocytes were almost equivalent between wild-type and ^LD/LD mice or only slightly increased in ^LD/LD mice. In sharp contrast, the frequency of V10-to-DJ2 rearrangement was robustly enhanced in ^LD/LD mice than that in wild-type mice (Fig. 3B). One may argue that V10, but not the other V genes, has a tendency to be rearranged preferentially to D2, leading to an exceptional enhancement of V10(D)J rearrangement in ^LD/LD mice. To investigate this possibility, we have compared the frequency of the D gene usage in V(D)J recombination in wild-type mice. The results showed that usage between D1 and D2 are roughly equal for all the V genes we examined (Table I), suggesting that the V10 gene does not have a biased tendency to be recombined to D2. In addition, the rearrangement profiles of V gene segments were comparable between ^LD/LD and wild-type T cells (Table II), suggesting that T cells in ^LD/LD mice do not undergo excess V(D)J rearrangements. It is of note that V10-to-D2 rearrangement without D2-to-J2 joining was observed in ^LD/LD mice, although the significance of this finding should be further studied.

View this table: [in this window] [in a new window]   Table I. Frequency of the D usage in the V(D)J rearrangement in wild-type T cellsa

View this table: [in this window] [in a new window]   Table II. V(D)J rearrangement status in wild-type and LD/LD hybridoma T cell clonesa

Germline V transcription in ^LD/LD thymocytes

In many Ag receptor loci and recombination substrates, initiation of germline transcription precedes their rearrangements (23, 24). Therefore, we queried whether the high frequency of the V10 usage in ^LD/LD mice is also associated with increased germline V10 transcription over the other four V genes at the DN stage. In an attempt to examine germline V transcription, thymocytes from RAG2^-/- background mice are useful because thymocyte development of the mutant mouse are arrested at a specific stage just before the TCR gene rearrangements (CD25⁺CD44^-DN stage) (25). Therefore, ^LD/LD mice were crossed with RAG2^-/- mice to generate ^LD/LD RAG2^-/- double-knockout mice. Total RNA was extracted from thymocytes in ^LD/LD RAG2^-/- mice (LD/R) as well as the parent RAG2^-/- mice (WT/R) and Northern blot analysis was performed with the V2, V4, V16, V10, and V14 probes (Fig. 4).

View larger version (41K): [in this window] [in a new window]   FIGURE 4. Germline transcription of the TCR V genes in LD/LD thymocytes. The total RNAs prepared from RAG2-/- (WT/R, lanes 1 and 3) and LD/LD RAG2-/- (LD/R, lanes 2 and 4) thymocytes before (lanes 1 and 2) and after (lanes 3 and 4) 2C11 treatment were subjected to Northern blot analysis using V2, V4, V16, V10, V14, and C2 specific probes. Flow cytometric analysis showed that >98% and >90% of the cells were arrested at the DN, and DP stages before and after 2C11 treatment, respectively, in both mouse lines (data not shown). The histograms on the right side indicate the relative intensities of the bands determined by a densitometer. In this analysis, the intensities of WT/R at the DN stage (lane 1) were set as 1.0 after normalizing with those of -actin (top panel). The total RNA purified from LD/LD mice was also included for comparison (lane 5).

At the DP stage driven by anti-CD3 Ab (2C11) treatment, germline V transcription was entirely repressed in control RAG2^-/- mice. However, V10 germline transcription was prominently up-regulated by 2C11 treatment in the ^LD/LD RAG2^-/- mice (Fig. 4, V10 panel, lanes 2 and 4), reaching 50 times higher levels than that of control RAG2^-/- thymocytes (V10 panel, lanes 3 and 4). In contrast to the V10, germline transcription of the other V genes in these mutant mice was maintained at an equivalent level in the DN stage (for V4 and V16) or was suppressed as well as in control RAG2^-/- mice (for V2). As reported previously, germline transcription of the V14 is exceptionally up-regulated at the DP stages of control RAG2^-/- mice (14). In ^LD/LD RAG2^-/- mice in which V14 location has not changed, V14 germline transcription was also up-regulated as equally as that of control RAG2^-/- mice (Fig. 4).

TCR allelic exclusion in ^LD/LD mice

Our data suggest that the V10 gene in TCR LD-allele seems to be highly accessible, at least in terms of its germline transcription, even at the DP stage where V genes have normally completed its rearrangement. Therefore, we aimed to evaluate the allelic exclusion status of the V10 gene in ^LD/LD mice.

To address this issue, the lymph node cells in wild-type and ^LD/LD mice were stained with PE-conjugated V10 Ab and a mixture of biotinylated-V14 and -V4 Abs revealed by Cy-Chrome-conjugated stretavidin as shown in Fig. 5A. The result showed that double V-bearing T cells are not significantly detectable in ^LD/LD mice, as is the case in wild-type mice, suggesting that TCR allelic exclusion is intact in ^LD/LD mice. To confirm that V10 expression is allelically excluded, we crossed ^LD/LD mice with TCR transgenic mice (26) to generate ^+/LD mice harboring or missing the V8 type TCR transgene. Consistent with Fig. 5A, no double bearing cells were found by staining with anti-V8 and anti-V10 Abs in ^+/LD:Tg mice (Fig. 5B). These results suggest that TCR allelic exclusion is tightly maintained in ^LD/LD mice.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Analyses of TCR allelic exclusion by flow cytometry. Lymph node cells were stained with PE-conjugated anti-V10 Ab and a mixture of biotinylated anti-V4 and anti-V14 Abs. The numbers indicate the percentage of the cells in each quadrant. Representative data from three independent experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In our ^LD/LD mice, 475 kb-deletion of the TCR locus has left only five intact V genes out of 22 segments (as a reference, see GenBank accession no. AE000663-AE000665) (Fig. 1). Because each V gene has its own recombination frequency, one may predict that the frequency of V(D)J recombination in the V genes in ^LD/LD mice should be equally increased 4-fold in each V gene compared with those of wild-type mice. However, our data clearly showed that a proportion of V10⁺ cells in ^LD/LD mice is extremely up-regulated 10-fold compared with those in wild mice (Fig. 3A), and that the ratios of other V expressing cells are almost unchanged despite the shortened distance to the D2-J2 cluster and the E enhancer (i.e., 30 kb from E at the V16 which is the second proximal segment in the TCR LD-allele). In accordance with protein expression, only the V10 gene showed a large increase of rearrangement (Fig. 3B). These results are consistent with recent reports showing that an inserted V gene 6.8 kb upstream of the D1 gene and 29 kb upstream of the E enhancer, which may lie outside the putative boundary of E activity, is rearranged at a similar frequency as the natural copy located 470 kb upstream (17) and that the frequency of the V usage is independent upon simple proximity of the V gene segments to the D-J cluster (27). Thus, a highly increased rearrangement of the V10 gene in ^LD/LD mice must be accomplished by its unique location on the locus, which is within the E regulatory region, but not simply closer to the D-J cluster and the E enhancer compared with the other V genes.

Previous studies have demonstrated that abolishing E activity, either by deletion or by replacement to E, causes a reduction in both germline transcription and accessibility to the recombinase in the D-J clusters but not in the V genes including the most 3' proximal V14 (3, 4). More precise mapping by chromosomal accessibility has demonstrated that the 5'-end of the E regulatory region is limited to an area 2–5 kb upstream of the D1 gene at the DN stage (3). The V10 gene in TCR LD-allele presumably lies within the E regulatory region, but the other V gene segments are beyond that region. Consistent with the above findings, the V10 gene in ^LD/LD mice became exceptionally accessible for transcription as much as the D2-J2 cluster. (Fig. 4 and Ref. 4) As shown in Fig. 4, germline transcription of the V10 gene was largely increased by the CD3-mediated signal in ^LD/LD RAG2^-/- mice (LD/R) during the DN-to-DP transition but was reduced in control RAG2^-/- mice (WT/R). In comparison to the V10, germline transcription of the V16 and V4 genes was not enhanced in ^LD/LD RAG2^-/- mice during the DN-to-DP transition but was not reduced as much as those in control RAG2^-/- mice. Collectively, these results suggest that the E enhancer may be equally effective in inducing the accessibility of both the D2-J2 cluster and the V genes during DN-to-DP transition if they are located within its regulatory region. The E regulatory region contains a particular region immediate upstream of the D1 that may be responsible for accessibility—rearrangement and germline transcription—in the D1-J1 cluster but not the D2-J2 (28). It is notable that despite of lacking the upstream region of D1 as well as the D1-J1 cluster, ^LD/LD mice showed the increased accessibility of the V10 located in the original D1-J1 region, In this sense, enhanced transcription of the V10 gene during DN-to-DP transition in our mutant mice may reflect a physiological event during TCR rearrangement in which a V gene moves into the E regulatory region by recombining with the DJ segment, leading to sufficient production of the rearranged TCR-chain gene.

Little is known whether cis-acting effects of the E enhancer are limited at the 5' side or covers 3' region of it. The V14 is located closely to the E enhancer at the 3' side and its germline transcription was exceptionally up-regulated in DP cells of normal mice as reported previously (14). In ^LD/LD mice in which V14 location has not been changed, V14 germline transcription was also up-regulated as equally as that of wild-type mice (Fig. 4), indicating that structural alteration of the 5' E does not result in a particular effect on germline transcription of the 3' V gene segment. Mathieu et al.(3) demonstrated that the V gene germline transcription is intact in the E-deficient mice. Thus, the increased germline transcription of V14 at the DP stage may be governed by unknown factors other than simple distance between the V14 and E enhancer as suggested.

It has been reported that during the V(D)J rearrangement, the D-J cluster seemed to remain accessible to the recombinase on both alleles even at the DP stage (28). However, after the first functional V(D)J rearrangement, further V-to-DJ rearrangement on the second allele is inhibited by allelic exclusion, which is mediated by a signaling via functionally rearranged TCR-chain. Although this mechanism is not fully understood, one might predict from the present results that the V10 gene in ^LD/LD mice can be rearranged in both alleles because the V10 gene is located in the E regulatory region. However, we showed that V10-positive T cells do not express any other V gene products on their surface in ^LD/LD mice (Fig. 5). Furthermore, in ^LD/LD mice crossed with TCR transgenic mice, the transgene-derived TCR-chain fully suppressed the appearance of other V gene products including V10 on the same cell surface. Thus, it can be concluded that V-to-DJ rearrangement is allelically exclusive even if the V10 gene is highly accessible under the control of the E enhancer. From a different perspective, our results also suggest that the large intron from the 5'V genes to the D2-J2 cluster does not have any critical cis-element for completing TCR allelic exclusion. In ^LD/LD mice, we also found that the V10 gene in the TCR LD-allele is occasionally rearranged to D2 without D2-to-J2 rearrangement (Table II). This rearranged V10-D2 may further recombine with the J2 as seen in the TCR genes, which recombine in this order. If that is a case, the resultant V(D)J rearrangement on the second allele would be allelically excluded in TCR genes, whereas TCR genes can be allelically included (29).

What are the possible factors regulating TCR allelic exclusion? Very recently, several studies have implicated important features concerning the recombinase activity and the recombination signal sequences (RSS) of the substrate. First, two independent groups demonstrated that the recombination between V-23RSS and D1-12RSS but not between 3'D1-23RSS and J1-12RSS is impaired in the knockin mice with a mutant truncated-RAG2 protein lacking the C-terminal region (30, 31). This suggests that enzymatic activity of the RAG protein might be differentially regulated during the sequential recombination steps. Second, it has been shown that the frequency of the V14⁺ cells was increased when the corresponding 23RSS was replaced with the 3'D1-23RSS (32), suggesting that RSS also regulates accessibility to the recombinase. These intrinsic characteristics may help to understand a molecular mechanism of allelic exclusion of the TCR gene locus as well as the ordered rearrangement between D-to-J and V-to-DJ. For this point, ^LD/LD mice showing the properties described in the present study would be useful for further analysis of epigenetic alterations during TCR-chain gene rearrangement.

	Acknowledgments

 
We thank Dr. Eun-Kyung Suh for reading the manuscript.

	Footnotes

 
¹ Current address: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115.

² Address correspondence and reprint requests to Dr. Sonoko Habu, Department of Immunology, Bohseidai, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan. E-mail address: sonoko{at}is.icc.u-tokai.ac.jp

³ Abbreviations used in this paper used in this paper: DN, double negative; DP, double positive; PGK, floxed phosphoglycerate kinase; E, TCR gene enhancer; ES, embryonic stem; RAG, recombination-activating gene; neo^r, neomycin resistance gene; RSS, recombination signal sequence.

Received for publication May 28, 2002. Accepted for publication May 6, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Sleckman, B. P., C. H. Bassing, C. G. Bardon, A. Okada, B. Khor, J. C. Bories, R. Monroe, F. W. Alt. 1998. Accessibility control of variable region gene assembly during T-cell development. Immunol. Rev. 165:121.[Medline]
2. Khor, B., B. P. Sleckman. 2002. Allelic exclusion at the TCR locus. Curr. Opin. Immunol. 14:230.[Medline]
3. Mathieu, N., W. M. Hempel, S. Spicuglia, C. Verthuy, P. Ferrier. 2000. Chromatin remodeling by the T cell receptor (TCR)- gene enhancer during early T cell development: implications for the control of TCR locus recombination. J. Exp. Med. 192:625.[Abstract/Free Full Text]
4. Senoo, M., N. Mochida, L. Wang, Y. Matsumura, D. Suzuki, N. Takeda, Y. Shinkai, S. Habu. 2001. Limited effect of chromatin remodeling on D-to-J recombination in CD4⁺CD8⁺ thymocyte: implications for new aspect in the regulation of TCR gene recombination. Int. Immunol. 13:1405.[Abstract/Free Full Text]
5. Bergman, Y.. 1999. Allelic exclusion in B and T lymphopoiesis. Semin. Immunol. 11:319.[Medline]
6. Groettrup, M., K. Ungewiss, O. Azogui, R. Palacios, M. J. Owen, A. C. Hayday, H. von Boehmer. 1993. A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor -chain and a 33 kd glycoprotein. Cell 75:283.[Medline]
7. Saint-Ruf, C., K. Ungewiss, M. Groettrup. 1994. Analysis and expression of a cloned pre-T cell receptor gene. Science 266:1208.[Abstract/Free Full Text]
8. Fehling, H. J., A. Krotkova, C. Saint-Ruf, H. von Boehmer. 1995. Crucial role of the pre-T-cell receptor gene in development of but not T cells. Nature 375:795.[Medline]
9. Anderson, S. J., K. M. Abraham, T. Nakayama, A. Singer, R. M. Perlmutter. 1992. Inhibition of T-cell receptor -chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56^lck. EMBO J. 11:4877.[Medline]
10. Anderson, S. J., S. D. Levin, R. M. Perlmutter. 1993. Protein tyrosine kinase p56^lck controls allelic exclusion of T-cell receptor -chain genes. Nature 365:552.[Medline]
11. Shinkai, Y., F. W. Alt. 1994. CD3-mediated signals rescue the development of CD4⁺CD8⁺ thymocytes in RAG2^-/- mice in the absence of TCR-chain gene expression. Int. Immunol. 6:995.[Abstract/Free Full Text]
12. Jacobs, H., D. Vandeputte, L. Tolkamp, E. de Vries, J. Borst, A. Berns. 1994. CD3 component at the surface of pro-T cells can mediate pre-T cell development in vivo. Eur. J. Immunol. 24:934.[Medline]
13. Tripathi, R., A. Jackson, M. S. Krangel. 2002. A Change in the Structure of V chromatin associated with TCR allelic exclusion. J. Immunol. 168:2316.[Abstract/Free Full Text]
14. Senoo, M., Y. Shinkai. 1998. Regulation of V germline transcription in RAG-deficient mice by the CD3-mediated signals: implication of V transcriptional regulation in TCR allelic exclusion. Int. Immunol. 10:553.[Abstract/Free Full Text]
15. Chattopadhyay, S., C. E. Whitehurst, F. Schwenk, J. Chen. 1998. Biochemical and functional analyses of chromatin changes at the TCR gene locus during CD4^-CD8^- to CD4⁺CD8⁺ thymocyte differentiation. J. Immunol. 160:1256.[Abstract/Free Full Text]
16. Hood, L., L. Rowen, B. F. Koop. 1995. Human and mouse T-cell receptor loci: genomics, evolution, diversity, and serendipity. Ann. NY Acad. Sci. 758:390.[Medline]
17. Sieh, P., J. Chen. 2001. Distinct control of the frequency and allelic exclusion of the V gene rearrangement at the TCR locus. J. Immunol. 167:2121.[Abstract/Free Full Text]
18. Chen, F., L. Rowen, L. Hood, E. V. Rothenberg. 2001. Differential transcription regulation of individual TCR V segments before gene rearrangement. J. Immunol. 166:1771.[Abstract/Free Full Text]
19. Yagi, T, T. Tokunaga, Y. Furuta, S. Nada, M. Yoshida, T. Tsukada, Y. Saga, N. Takeda, Y. Ikawa, S. Aizawa. 1993. A novel ES cell line, TT2, with high germline-differentiating potency. Anal. Biochem. 214:70.[Medline]
20. Kanegae, Y., G. Lee, Y. Sato, M. Tanaka, M. Nakai, T. Sakaki, S. Sugano, I. Saito. 1995. Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res. 23:3816.[Abstract/Free Full Text]
21. White, J., M. Blackman, J. Bill, J. Kappler, P. Marrack, D. P. Gold, W. Born. 1989. Two better cell lines for making hybridomas expressing specific T cell receptors. J. Immunol. 143:1822.[Abstract]
22. Gartner, F., F. W. Alt, R. Monroe, M. Chu, B. P. Sleckman, L. Davidson, W. Swat. 1999. Immature thymocytes use distinct signaling pathways for allelic exclusion versus differentiation and expansion. Immunity 10:537.[Medline]
23. Lansford, R., A. Okada, J. Chen, E. M. Oltz, T. K. Blackwell, F. W. Alt, G. Rathbun. 1996. Mechanism and control of immunoglobulin gene rearrangement. B. D. Hames, and D. M. Glover, eds. Molecular Immunology 1. Oxford IRL Press, Oxford.
24. Sleckman, B. P., J. R. Gorman, F. W. Alt. 1996. Accessibility control of Ag-receptor variable-region gene assembly: role of cis-acting elements. Annu. Rev. Immunol. 14:459.[Medline]
25. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A. M. Stall, F. W. Alt. 1992. RAG2-deficient mice lack mature lymphocyte owing to inability to initiate V(D)J rearrangement. Cell 68:855.[Medline]
26. Uematsu, Y., S. Ryser, Z. Dembic, P. Borgulya, P. Krimpenfort, A. Berns, H. von Boehmer, M. Steinmetz. 1988. In transgenic mice the induced functional T cell receptor gene prevents expression of endogenous genes. Cell 52:831.[Medline]
27. Wilson, A., C. Maréchal, H. R. MacDonald. 2001. Biased V Usage in immature thymocytes is independent of DJ proximity and pT pairing. J. Immunol. 166:51.[Abstract/Free Full Text]
28. Whitehurst, C. E., S. Chattopadhyay, J. Chen. 1999. Control of V(D)J recombinational accessibility of the D1 gene segment at the TCR locus by a germline promoter. Immunity 10:313.[Medline]
29. Sleckman, B. P., B. Khor, R. Monroe, F. W. Alt. 1998. Assembly of productive T cell receptor variable region genes exibits allelic inclusion. J. Exp. Med. 188:1465.[Abstract/Free Full Text]
30. Liang, H., L. Hsu, D. Cado, L. G. Cowell, G. Kelose, M. S. Schlissel. 2002. The "dispensable" portion of RAG2 is necessary for efficient V-to-DJ rearrangement during B and T Cell development. Immunity 17:639.[Medline]
31. Akamatsu, Y., R. Monroe, D. D. Dudley, S. K. Elkin, F. Gartner, A. R. Talukder, Y. Takahama, F. W. Alt, C. H. Bassing, M. A. Oettinger. 2003. Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice. Proc. Natl. Acad. Sci. USA 100:1209.[Abstract/Free Full Text]
32. Wu, C., C. H. Bassing, D. Jung, B. B. Woodman, D. Foy, F. W. Alt. 2003. Dramatically increased rearrangement and peripheral representation of V14 driven by the 3'D1 recombination signal sequence. Immunity 18:75.[Medline]

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