The positional effect of Eß on Vß genes of TCRß chain in the ordered rearrangement and allelic exclusion

Daisuke Suzuki1, Lili Wang1, Makoto Senoo2 and Sonoko Habu1

1 Department of Immunology, Tokai University School of Medicine, Bouseidai, Isehara, Kanagawa 259-1193, Japan
2 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA

Correspondence to: S. Habu; E-mail: sonoko{at}is.icc.u-tokai.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the TCRß gene locus, the Vß, Dß and Jß gene segments are assembled in a tightly ordered manner. To investigate the positional effects of TCRß enhancer (Eß) on the recombination processes of the Vß genes, we utilized ßLD mice lacking 70% of the TCRß locus, leaving four Vß genes at the 5' side and, consequently, the Vß10 gene moves into the Eß regulatory region. In this mutant mouse, the Vß10 gene showed direct Vß-to-Dß and Vß-to-Jß recombination, although the Dß-to-Jß joining was still predominant. Interestingly, these two aberrant recombination processes were barely suppressed when ßLD mice were crossed with TCRß transgenic mice, whereas V(D)J recombination of the Vß10 gene was sufficiently suppressed. These results suggest that the positional effects of Eß on the Vß genes may enable the recombination potential to increase prior to Dß-to-Jß joining and that such aberrant recombination may be free from allelic suppression.

Keywords: allelic exclusion, gene rearrangement, T cell receptors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the TCRß gene that is composed of variable (V), diversity (D) and joining (J) gene segments, V(D)J recombination takes place at the CD4CD8 double-negative (DN) stage during early thymocyte development (1). The TCRß recombination is initiated by the complex of lymphocyte-specific recombinase proteins, RAG1 and RAG2, which targets the recombination signal sequences (RSSs) comprising of relatively conserved heptamer and nonamer elements separated by 12 bp (12RSS) and 23 bp (23RSS) spacers, respectively. The assembly among V, D and J segments occurs in a restricted manner between 12RSS and 23RSS, referred to as the 12/23 rule (2). At the targeted RSS, DNA double-strand breaks are generated by the RAG1/2 complex, followed by a general DNA-repairing machinery to join the cleaved gene segments.

In V(D)J recombination of the TCRß gene, one interesting feature is the ordered rearrangement. The V(D)J recombination in the TCRß gene occurs in a tightly regulated fashion in two sequential steps—first Dß-to-Jß joining and then Vß-to-DJß assembly (35). It has also been shown that direct Vß-to-Jß assembly without involving Dß is precluded (6, 7). According to the 12/23 rule, direct assembly of Vß-to-Jß is theoretically possible because the Vß and Jß genes are flanked by 23RSS and 12RSS, respectively. However, such skipping assembly has not been actually detected. Regarding this discrepancy, a new principle termed ‘Beyond the 12/23 rule’ (B12/23 rule) has been proposed (8). This B12/23 rule is based on the detailed analysis of the mutant mice in which in addition to the deletion of Dß, 5'12RSS of Jß1.2 is replaced with that of Dß1 (8) and prescribes that 3'23RSS of Vß preferentially utilizes 5'12RSS of Dß but not 5'12RSS of Jß in a sequence-dependent manner (912).

Recently, a tight linkage between histone modifications and accessibility of TCRß locus to recombinase has been demonstrated (1318). For instance, the Dß-Jß region but not the Vß region holds highly acetylated histones at the early stage of thymocyte development (17, 18). This selective hyper-acetylation status of the Dß-Jß region might be regulated by the TCRß enhancer (Eß), which is located at the 3'side of Dß-Jß clusters and covers ~25 kb upstream (19, 20). These differential controls between Vß and Dß-Jß regions might be involved in the sequential V(D)J recombination.

Another striking aspect of TCRß recombination is the phenomenon termed allelic exclusion (21, 22). Dß-to-Jß joining is assumed to take place on both alleles in the TCRß gene. However, functional Vß-to-DJß assembly is permissive in only one allele due to a feedback inhibition of allelic exclusion signaling which results from pre-TCR expression, ensuring that all T cells have single specificity of TCRß chain on the cell surface (6). During DN to double-positive transition, the accessibility of Vß genes is selectively down-regulated in terms of histone acetylation and germ line transcription (17, 23, 24). This down-regulation of the Vß gene accessibility might play a pivotal role in TCRß allelic exclusion, although underlying mechanisms are not well understood.

In the study for the regulation of TCRß recombination, we have been interested in the structure of the germ line TCRß locus spanning ~700 kb. At the 5' side of the locus, there are 22 functional Vß genes spanning over 300 kb. At ~250 kb downstream of Vß region, there are two Dß-Jß clusters within a relatively small (15 kb) region. Eß is located at the 3' side of the locus, flanked 5.9 kb upstream by the Dß2-Jß2 cluster. At the most 3' end is the Vß14 gene, situated 9.4 kb downstream of the Dß2-Jß2 cluster. In our previous report, we have established a mutant mouse, designated ßLD mouse, in which 70% of the TCRß locus, including the 3' half of the 5'Vß region, a large central region (250 kb) and the Dß1-Jß1 cluster, was deleted and the Vß10 gene moved to the position in the Eß regulatory region (25). Using this mutant mouse model, we showed that the position of the Vß genes in relation to Eß plays a role in the regulation of Vß recombination frequency. In the present study, we sought to determine how the Vß genes located in the Eß regulatory region are affected in the process of ordered rearrangement and allelic exclusion of the TCRß gene.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and cell preparation
All animals used in this study, including wild-type (WT) mice, ßLD mice (25), RAG2–/– mice and TCRß transgenic (ßTg) mice (6), had a C57BL/6 background and were maintained in a specific pathogen-free breeding facility. For the enrichment of DN cells, thymocytes harvested from these mice were stained with biotinylated antibodies against mouse CD4 (RM4-5) and CD8 (53-6.7), treated with anti-biotin Microbeads (Miltenyi Biotec, Auburn, CA, USA) and the negative fraction was isolated by AUTOMACS (Miltenyi Biotec). For isolation of the DN3 fraction, the enriched DN cells were stained with FITC-conjugated anti-CD44 (IM7) and PE-conjugated anti-CD25 (PC61) mAbs (eBioscience, San Diego, CA, USA), and the CD25+CD44 fraction was sorted by FACStar plus (BD Biosciences, San Diego, CA, USA). The purity of the sorted cells was >98% by FACS re-analysis (data not shown).

Chromatin immunoprecipitation assay
Chromatin fixation and chromatin immunoprecipitation (ChIP) were performed according to the instruction manual of the Acetyl Histone H3 Immunoprecipitation Assay Kit (Upstate Biotechnology, Inc., Saranac Lake, NY, USA). Briefly, soluble chromatin was prepared from 1 x 105 to 10 x 105 cells fixed with formaldehyde. For immunoprecipitation, chromatin solution was incubated with 4 µg of anti-acetyl histone H3 antibody (UBI 06-599) or 4 µg of normal rabbit IgG (UBI 12-370) as control. After elution of the chromatin fraction bound to Protein A agarose and the reversal of histone–DNA cross-linking, the DNA fraction was purified by standard phenol/chloroform extraction and ethanol precipitation. An aliquot of the sonicated chromatin was applied for the input DNA. Serial 4-fold dilutions of bound and input DNA fractions were analyzed by PCR amplification. PCR products were electrophoresed in agarose gel, blotted onto nylon membranes (Pall, Port Washington, NY, USA) and hybridized with 32P-end-labeled oligonucleotide probes. Quantification was performed by FLA-2000 (Fuji Film, Tokyo, Japan) and plotted as acetylation index, which was calculated using the formula: [(binding to {alpha}AcH3 antibody) – (binding to control IgG)]/(input). Oligonucleotide sequences of primers and probes used in this study are shown in Table 1, which includes the previously described one (25, 26).


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Table 1. Oligonucleotide sequences

 


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Fig. 4. Estimation of the relative frequency of Dß-to-Jß, Vß-to-Dß and Vß-to-Jß rearrangements among Vß10, Dß2 and Jß2. The plasmid templates, harboring the target sequences for detection (Dß2-Jß2.1, Vß10-Dß2 and Vß10-Jß2.7SJ), were serially diluted into 0.1 µg µl–1 genomic DNA purified from RAG2–/– mouse kidney and used as standard (lanes 1–7). Genomic DNA from the DN3 thymocytes (0.1 and 0.01 µg as indicated) was amplified with 32 cycles for Dß-to-Jß rearrangements and with 36 cycles for Vß-to-Dß rearrangements and Vß-to-Jß SJs. The data shown are representative of three independent experiments with similar results.

 
Semi-quantitative PCR
PCR was carried out as described previously (25). Briefly, each PCR cycle consisted of incubation at 95°C for 30 s, followed by 45–120 s annealing at 56–64°C and extension for 45–120 s at 72°C. Before the first cycle, a 15-min denaturation and Taq activation step at 94°C was included, and after 24–38 cycles the extension at 72°C was prolonged for 5 min. PCR products were electrophoresed in agarose gel, blotted onto nylon membranes and hybridized with 32P-end-labeled oligonucleotide probes. Quantification was performed by detecting the radioactivity of hybridized signals, and the relative intensity was compared after normalizing with rag2 amplification. Oligonucleotide sequences of primers and probes used are shown in Table 1. In some experiments, the plasmids containing the amplified fragment (germ line or rearranged Dß2-Jß2.1, rearranged Vß10-Dß2 and rearranged Vß10-Jß2.7SJ) were used as standard templates.

Sequence analysis
The fragments amplified by PCR were cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) and sequenced using M13 reverse and forward primers with ABI prism autosequencer (Applied Biosystems, Foster City, CA, USA).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have previously established ßLD mice, in which 70% of the TCRß locus was deleted by gene targeting (25). In this mutant mouse, there are only four Vß genes remaining at the 5' side of the TCRß locus (Fig. 1A). In these four 5'Vß genes, the most proximal Vß gene, Vß10, moved 20 kb upstream of Eß, corresponding to the original Jß1 in the WT TCRß locus, which is located within the Eß regulatory region (20). The Vß4 and Vß16 genes, separated by an original intervening sequence, is situated 30 kb upstream of Eß, which is slightly out of the tentative Eß regulatory region (20). The most distal Vß2 gene is located 190 kb away from Eß in the ßLD TCRß locus.



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Fig. 1. Histone H3-acetylation status in the TCRß locus. (A) Schematic diagram of the TCRß locus in WT and ßLD mice (not to scale). The distance between Eß and the most proximal 5'Vß is 360 kb and 20 kb in the WT allele and the ßLD allele, respectively. The Vß4 and Vß16 genes are separated by a small region (230 bp) and located 10 kb upstream of Vß10. The most distant 5'Vß on the locus, Vß2, is located 170 kb upstream of Vß10. The position of Vß14 is identical between WT and ßLD alleles. The solid circles indicate the tested positions in the locus. (B) Histone H3-acetylation status in the various regions of the TCRß locus. Genomic DNA was prepared from WT and ßLD thymocytes in the RAG2-deficient background, and applied for ChIP assay. Precipitated DNA was serially 4-fold diluted and analyzed by PCR. The Eß locus and J{kappa} locus were used as positive and negative control, respectively, in this experimental condition (18, 26). (C) Histone H3-acetylation index. The amplified fragments shown in (B) were quantified by an image analyzer and plotted as acetylation index. The data shown are representative of three independent experiments with similar results.

 
Eß is known to regulate chromatin remodeling of Dß-Jß clusters but not of Vß genes in terms of histone acetylation, DNA methylation, transcription and recombination (19, 20). We have previously shown that in ßLD mice, germ line transcription and recombination frequency of the Vß10 gene are enhanced compared with those in WT mice (25). To further characterize the chromatin status of Vß genes in ßLD mice, we investigated their histone H3-acetylation status by ChIP assay (Fig. 1A). In this assay, we used the thymocytes obtained from RAG2–/–ßLD double-mutant mice, which are arrested at the CD25+CD44 stage (DN3) and contain germ line configuration of the TCRß gene, allowing quantitative analysis independently of the recombination status. As shown in Fig. 1(B and C), we found that histone H3 is significantly hyper-acetylated specifically at the Vß10 gene in ßLD mice compared with WT mice (ßLD panel, RAG2–/–ßLD; WT panel, RAG2–/–). In contrast, other Vß genes did not show any significant difference between ßLD and WT mice. These results implied that the chromatin status of the Vß10 gene in ßLD mice is relatively accessible compared with that in the original position, presumably due to the positional effects of Eß. This raised the question of whether the Vß genes located in the Eß regulatory region undergo different regulation in V(D)J recombination. Therefore, we further analyzed the process of TCRß recombination in ßLD mice.

Aberrant rearrangements, Vß-to-Dß and Vß-to-Jß, at the Vß10 gene in ßLD mice
V(D)J recombination in the TCRß gene takes place in two sequential steps, first Dß-to-Jß and then Vß-to-DJß. However, it is not well understood what limits Vß-to-Dß assembly, if it takes place, before Dß-to-Jß joining. Based on the results showing enhanced chromatin status of the Vß10 gene in ßLD mice, it might be possible that the Vß10 gene assembles to Dß before Dß-to-Jß joining takes place. To investigate this possibility, the level of Vß-to-Dß rearrangement was measured by semi-quantitative PCR using the genomic DNA purified from the DN3 thymocytes. To detect direct Vß-to-Dß rearrangements, the 3' primers and oligonucleotide probes were designed at the flanking sequence downstream of Dß2 (Fig. 2A), which is excised by Dß-to-Jß joining. By this strategy, Vß-to-Dß rearrangements in WT mice were detectable in all Vß genes tested (Fig. 2A and C). However, such rearrangements could not be visualized until a highly sensitive test with high-amplification cycles was performed. Thus, it was shown that the basal level of Vß-to-Dß rearrangements, which may be physiologically generated, is at extremely low frequency, if they take place at all.



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Fig. 2. Semi-quantitative PCR analysis of Vß-to-Dß rearrangements. (A) Detection of Vß10-to-Dß2 rearrangements. Genomic DNA (0.1 µg) isolated from the DN3 thymocytes was amplified by PCR for the indicated number of cycles. The rag2 gene (RAG2) was amplified using the same DNA samples to serve as an internal control; arrows, primers for amplification; solid bars, sequence-specific probes for Southern hybridization. (B) Sequence analysis of Vß10-to-Dß2 rearrangement in ßLD thymocytes. (C) Detection of Vß-to-Dß rearrangements with Dß2 in multiple Vß genes. Genomic DNA used in (A) was serially 4-fold diluted and amplified for 36 cycles. (D) Histograms showing relative intensity of Vß-to-Dß rearrangements. The intensity in WT sample was set as 1.0, and the relative intensity was plotted over logarithmic scale after normalizing with rag2 amplification; asterisks, not detected.

 
In ßLD mice, however, Vß10-to-Dß2 rearrangement was clearly detectable (Fig. 2A). In addition, Vß-to-Dß rearrangements in Vß4 and Vß16 genes were slightly increased in ßLD mice compared with those in WT mice in a proximity-dependent manner, whereas the Vß2 gene did not show any significant difference between the WT and the ßLD mice (Fig. 2C and D). These Vß-to-Dß rearrangements were submitted to sequence analysis, revealing that the patterns of nucleotide deletions and additions in the V–D junction did not differ from those of standard V(D)J rearrangements (Fig. 2B for ßLD mice). The frequency of Vß10-to-Dß2 rearrangement in ßLD mice was ~100-fold higher than that in WT mice (Fig. 2D). Accordingly, this high Vß10-to-Dß2 rearrangement will not simply depend on the fact that the number of Vß genes in ßLD mice was reduced to 22.7% (5 from standard 22) in ßLD mice. Thus, these results suggest that the Vß10 gene in ßLD mice is susceptible to recombination to Dß prior to Dß-to-Jß joining under the positional effects of Eß.

Next, we sought to determine whether the Vß10 gene in the Eß regulatory region assembled directly to Jß. To address this issue, we analyzed the presence of signal joints (SJs) (27). If SJs between Vß and Jß were detectable, they would be evidence of direct Vß-to-Jß assembly, as their formation of SJs is not generated until each of the RSSs-flanking Vß, Dß and Jß segments forms mutual pairs in accompanying the formation of coding joints (Fig. 3A). Using this SJ assay, SJs between Dß2 and Jß2 and between Vß and Dß2 were distinctly detectable in both WT and ßLD mice in the context of ordinary recombination (Fig. 3B). Surprisingly, SJs were also detectable between Vß10 and Jß2 in ßLD mice (Fig. 3C, Vß10 panel). The presence of ladder bands indicated that the Vß10 gene has rearranged directly with multiple segments of Jß2.1-2.7 in direct Vß-to-Jß assembly. In contrast to the Vß10 gene, none of the other Vß genes showed SJ formation to Jß in either WT or ßLD mice, at least with the several primer sets tested (Fig. 3C). These results suggest that the Vß10 gene in ßLD mice is susceptible to direct Vß-to-Jß assembly as well as Vß-to-Dß assembly.



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Fig. 3. Detection of SJs for direct Vß-to-Jß recombination. (A) Schematic diagram of the strategy to detect SJs in Vß-to-Jß, Dß-to-Jß and Vß-to-Dß assemblies at the DN3 stage; arrows A–D, primers for amplification; solid bars P1 and P2, sequence-specific probes for Southern hybridization. (B) Detection of SJs in Dß2-to-Jß2 and Vß-to-Dß2 assemblies. Note that Vß-to-Dß SJs are generated by both direct Vß-to-Dß rearrangements and ordinary Vß-to-DJß assembly. Purified genomic DNA was serially 4-fold diluted and was amplified for 36 cycles of PCR. (C) Detection of SJs in Vß-to-Jß2 assembly. Amplification was done for 38 cycles of PCR. The data shown are representative of three independent experiments with similar results.

 
Comparative frequencies of Dß-to-Jß, Vß-to-Dß and Vß-to-Jß rearrangements
The next question is whether or not Vß10-to-Dß and Vß10-to-Jß recombinations routinely occur as often as Vß10-to-DJß in ßLD mice. To address this issue, we measured the relative frequency of Vß10-to-Dß and Vß10-to-Jß rearrangements in relation to Dß-to-Jß rearrangements. For this purpose, the plasmids harboring PCR fragments of the corresponding rearrangements were serially diluted into the genomic DNA purified from RAG2–/– mouse kidney and were used as a relative standard (Fig. 4). Using these serially diluted templates, we measured the level of each rearrangement in 0.1 µg of genomic DNA prepared from FACS-sorted DN3 thymocytes. By simple arithmetic, the copy number of the TCRß genes/alleles per 0.1 µg of genomic DNA was estimated as equivalent to 0.25 pg of a plasmid template.

As shown in Fig. 4, the detectable level of Dß2-to-Jß2 rearrangement was of the order equivalent to 10–2 pg of the plasmid template in WT and ßLD mice (Dß2-to-Jß2.1, DJ panel). By contrast, the Vß10-to-Jß2 rearrangement was estimated as being equivalent to 10–6 pg of the plasmid template in ßLD mice (Vß10-to-Jß2.7, VJ SJ panel). This result indicated that the level of Vß10-to-Jß2 rearrangement is 104-fold lower than that of Dß2-to-Jß2 rearrangement in ßLD mice. In the Vß10-to-Dß2 rearrangement, the level in ßLD mice was approximately equivalent to 10–4 pg of the plasmid template, while the level in WT mice was equivalent to 10–6 pg (VD panel). This means that the level of the Vß10-to-Dß2 rearrangement in ßLD mice was ~100-fold lower than that of the Dß2-to-Jß2 rearrangement. Collectively from these results, the relative ratio of Dß-to-Jß:Vß10-to-Dß:Vß10-to-Jß rearrangements is estimated as 1:10–2:10–4 in ßLD mice. These results suggest that the Dß-to-Jß joining is still predominant in the process of TCRß recombination in ßLD mice. In addition, the frequency of direct Vß-to-Jß assembly was extremely lower than that of Vß-to-Dß assembly (~100-fold) in ßLD mice.

Escape from TCRß-mediated allelic exclusion signaling of direct Vß10-to-Dß and Vß10-to-Jß rearrangements in ßLD locus
As allelic exclusion signaling is believed to inhibit further recombination at the process of Vß-to-DJß, of interest was whether allelic suppression in ßLD mice strictly affects the assembly process of Vß-to-Dß and Vß-to-Jß in the Vß10 gene. To address this issue, we crossed ßLD mice with ßTg mice (6) to investigate the rearrangement status of the endogenous TCRß locus under allelic exclusion signaling. Genomic DNA was isolated from total thymocytes, and TCRß rearrangements were analyzed by semi-quantitative PCR.

Interestingly, the assembly process of Vß-to-Dß in the Vß10 gene in ßLD mice was not sufficiently suppressed in the presence of transgenic TCRß chain (Fig. 5A, ßLD panel). In the upstream Vß4 and Vß16 genes, Vß-to-Dß rearrangements were gradually suppressed in reverse correlation with the proximity to Eß and were suppressed below the detection level in the Vß2 gene in ßLD mice (Fig. 5A, ßLD panel). But, in WT mice, Vß-to-Dß rearranged bands had mostly disappeared in all Vß genes (Fig. 5A, WT panel). In the context of direct Vß10-to-Jß assembly, we could not detect any decrease in the level of SJ formation in the presence of transgenic TCRß chain in ßLD mice (Fig. 5B). These results suggest that both the assembly processes of direct Vß-to-Dß and Vß-to-Jß in ßLD mice are not sufficiently targeted by the transgenic TCRß chain-mediated signaling, at least in a dependent manner on the position of the Vß genes in relation to the Eß regulatory region. In contrast, overall V(D)J rearrangements in the Vß10 gene in the presence of transgenic TCRß chain, as well as the other Vß genes, were mostly suppressed in ßLD mice as efficiently as in WT mice; detectable rearranged bands were ~5% or less of those in the absence of transgenic TCRß chain (Fig. 5C). Therefore, overall allelic exclusion was basically maintained during the process of V(D)J recombination even at the Vß10 gene in ßLD mice.



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Fig. 5. Recombination status of the endogenous TCRß locus in the presence of transgenic TCRß chain. (A) Direct Vß-to-Dß2 rearrangements. Total thymocyte DNA was isolated from WT and ßLD mice with or without harboring a TCRß transgene (ßTg) and was serially 4-fold diluted. Direct Vß-to-Dß2 rearrangements were amplified for 36 cycles of PCR. (B) Detection of SJs of Dß-to-Jß and direct Vß-to-Jß. Dß2-to-Jß2 SJ products were amplified for 36 cycles of PCR (top), while Vß10-to-Jß2 SJ products were amplified for 38 cycles (bottom). (C) V(D)J rearrangements. Vß-to-DJß2 rearrangements were amplified for 33 cycles of PCR. (D) Dß-to-Jß rearrangements. Dß2-to-Jß2 rearrangements were amplified for 28 cycles of PCR. The rag2 gene was also amplified for 28 cycles and served as an internal control. The histograms indicate the relative intensity of the signal normalized by RAG2 amplicon. Open bars, non-ßTg mice; solid bars, ßTg mice. Shown are the representative data from three independent experiments with similar results; n.d., not detected.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Using ßLD mice that have a large deletion of TCRß genes and resultant movement of the Vß10 gene within the Eß regulatory region, we investigated the effect of Eß on the Vß genes in the process of the V(D)J recombination order and allelic exclusion. We showed that, although the preference of Dß-to-Jß joining is still maintained in V(D)J recombination in our mutant mice, extraordinary rearrangements such as direct Vß10-to-Dß and Vß10-to-Jß significantly increased. Moreover, these aberrant assembly processes did not seem to experience allelic suppression by transgenic TCRß chain in ßLD mice.

Past studies have demonstrated that V(D)J recombination occurs in the tightly regulated sequential steps—Dß-to-Jß and subsequent Vß-to-DJß (35). In this study, we first demonstrated that when the Vß gene is located in the Eß regulatory region, the Vß-to-Dß assembly can significantly take place without the Jß-joining process (Fig. 2). In fact, the Vß10 gene located in the original Jß region with intact flanking sequences containing RSS showed a significant increase of Vß-to-Dß rearrangements with enhanced histone acetylation (Fig. 1C) and germ line transcription (25). Therefore, this suggests that the first step in the sequential recombination of the TCRß genes is not limited to between Dß and Jß but remains potential for any combination among Vß, Dß and Jß segments if they are in the status similar to the Dß-Jß clusters that are under the Eß regulation. In addition to the Vß10 gene, Vß-to-Dß assembly of the other Vß genes in ßLD mice, such as Vß4 and Vß16, which were slightly upstream of the Eß regulatory region, was slightly enhanced in a proximity-dependent manner from the Eß regulatory region (Fig. 2D), although the frequencies were low. Sieh et al. previously found direct Vß-to-Dß rearrangements in their knock-in mouse harboring one Vß gene with partial flanking sequences that was inserted around the position of the Vß16 gene in ßLD mice (28). Presumably, the effects of Eß may be more widely influential than previously reported (20).

It is notable, however, that the frequency of Vß-to-Dß rearrangements was still much lower than that of Dß-to-Jß rearrangements (~1–100) even at the Vß10 gene in ßLD mice (Fig. 4). These observations imply that Eß effects on Vß recombination such as Vß-to-Dß and/or Vß-to-Jß are less efficient in comparison with Dß-to-Jß joining even if the Vß gene is located in the Eß regulatory region. In other words, the Vß recombination seems to undergo a certain restriction beyond Eß regulation. As a result of highly efficient Dß-to-Jß joining, the majority of the Vß genes is retained to join to the rearranged-DJß even if the Vß10 gene has the potential to assemble directly to Dß, resulting in the sequential order of recombination, Dß-to-Jß and then Vß-to-DJß. In fact, in RSS mutant mice in which Dß-to-Jß joining in the TCRß gene is defective due to the manipulated 3'23RSS of Dß, direct Vß-to-Dß rearrangements are detectable at relatively high frequency (9). Then, what restricts the recombination efficiency of the Vß10 gene beyond the Eß effects? RSSs flanking the respective TCRß gene segments might be the most likely cis-element for such restriction. Previous in vitro experiments showed that 3'23RSS of Dß possesses a higher recombination potential than that of Vß (1012). On the other hand, a recent in vivo study using RSS-replaced mice showed that high Vß-to-Dß rearrangement or recombination disorder was not detectable in the Vß gene flanked with 3'23RSS of Dß (11). Collectively, the cis-elements limiting Vß recombination beyond Eß effects are still controversial and waiting to be resolved. As such cis-elements regulate Vß recombination and Dß-to-Jß assembly in a different way, the promoter region of each TCRß gene segment may be a candidate. Previous reports may support this notion, namely, when the promoter is deleted in a certain Vß segment or Dß segment, the respective gene rearrangement is undetectable in a segment-specific manner (29, 30).

In a two stepwise process of TCRß recombination, the first Dß-to-Jß joining occurs simultaneously on both alleles, but then the second Vß-to-DJß assembly occurs in either single allele (6, 22). The latter asynchronous recombination results in the allelic exclusion; pre-TCR generated by the gene product of the firstly rearranged Vß-to-DJß on a single allele provides suppressive signaling for further Vß-to-DJß assembly on the other allele but not for Dß-to-Jß joining. In light of the fact that the Vß10 gene is located in the Eß regulatory region as is the Dß-Jß cluster, Vß10 recombination might escape from allelic exclusion in ßLD mice. Expectedly, the process of the direct assembly of Vß-to-Dß and Vß-to-Jß in the Vß10 gene is insufficiently suppressed in ßLD mice by allelic exclusion signaling (Fig. 5A and B) as similar as Dß-to-Jß joining (Fig. 5D), while the overall V(D)J rearrangement of the Vß10 gene was suppressed as strongly as in WT mice (Fig. 5C).

One may offer the argument that, if direct Vß-to-Jß recombination can escape allelic suppression and if the aberrant Vß-to-Dß rearrangements proceed to further VDß-to-Jß assembly beyond allelic suppression, the rearranged bands of V(D)J derived from such recombination process should be detectable in the presence of transgenic TCRß chain in ßLD mice. In our present study, however, the bands of V(D)J rearrangement at the Vß10 gene were only faintly detectable in the presence of transgenic TCRß chain in ßLD mice when high-amplification cycles were performed in PCR analysis (Fig. 5C). Thus, it seemed likely that Vß-to-Jß and VDß-to-Jß via Vß-to-Dß rearrangements, if any, may not significantly contribute to any bands of V(D)J rearrangements as allelic inclusion, presumably because of their low frequency. We would not rule out that Vß-to-Dß rearrangements may proceed to further VDß-to-Jß assembly in ßLD mice as suggested by Chen and colleagues (28, 29), but it is also conceivable that ßLD mice, which carry only the Dß2-to-Jß2 cluster, may be more suppressive of the VDß-to-Jß recombination because Dß2-to-Jß2 joining is more susceptible to allelic suppression than Dß1-to-Jß1 joining (6, 16, 31).

Despite the intact allelic exclusion for V(D)J recombination in ßLD mice, aberrant Vß-to-Dß assembly escaped from TCRß-mediated suppression signaling. One could presume that, if the recombination process of Vß-to-Dß in the Vß10 gene occurs at the same stage as Dß-to-Jß joining, it could not be a target of allelic exclusion signaling because TCRß-mediated signaling is provided at least after the Dß-to-Jß-joining stage (around DN2). In fact, Vß10-to-Dß2 rearrangement in ßLD mice was significantly detectable from the DN2 stage (data not shown). Therefore, it may be possible to speculate that the assembly process of Vß10-to-Dß2 in ßLD mice could not be targeted by allelic exclusion signaling because it precedes TCRß signaling. However, our finding obtained from ßLD mice crossed with ßTg mice may not always rule out another possibility that the assembly process of Vß10-to-Dß2 in ßLD mice is resistant to the suppressive effect by TCRß signaling as well as Dß-to-Jß joining is, because TCRß transgene has been already expressed at the pre-DN2 stage, where most of the Vß10-to-Dß2 assembly may not yet occur. In this case, these results might predict that the Vß genes directly assembling to Dß independently from that certain restriction of the recombination order due to the effects of Eß are free from allelic suppression, although the mechanism is unclear. Alternatively, in the Vß-to-DJß recombination that is susceptible to allelic suppression, Vß generally assembles to the rearranged-DJß, in which the intervening region between Dß and Jß including each RSS has already been deleted. This epigenetic difference of the cis-region and probably the subsequent chromatin remodeling between the assembly process of Vß10-to-DJß and aberrant Vß10-to-Dß may affect the susceptibility to allelic exclusion signaling over the Eß regulation in the assembly process of the Vß10 gene.

Collectively, our present study using mutant mice indicates that Eß may possess the potential for Vß-to-Dß assembly without Jß joining in its regulatory region but may be less effective in overcoming the Vß recombination with a certain restriction that is operative beyond the enhancer effects of Eß.


    Acknowledgements
 
The authors thank Y. Shinkai, K. Hozumi, Y. Kametani and T. Sato for comments on the manuscript.


    Abbreviations
 
ChIP   chromatin immunoprecipitation
DN   double negative
   TCRß enhancer
RAG   recombination activating genes
RSS   recombination signal sequence
SJ   signal joint
ßTg   TCRß transgenic
WT   wild type

    Notes
 
Transmitting editor: K. Okumura

Received 2 September 2005, accepted 14 September 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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