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Distinct Control of the Frequency and Allelic Exclusion of the V Gene Rearrangement at the TCR Locus¹

Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ag receptor gene loci contain many V gene segments, each of which is recombined and expressed at a different frequency and is subject to allelic exclusion. To probe the parameters that mediate the different levels of regulation of V gene rearrangement, a V gene segment together with 3.6-kb 5' and 0.7-kb 3' flanking sequences was inserted 6.8 kb upstream of the D1 gene segment in the murine TCR locus. Despite its proximity to the D gene segments and the E enhancer, the inserted V segment underwent VDJ recombination at the same frequency as the natural copy located 470 kb upstream. However, the inserted V segment was no longer under allelic exclusion control as it recombined at a similar frequency in the presence of a TCR transgene. These results suggest that while the inserted fragment contains the necessary cis-regulatory elements for determining the frequency of V rearrangement, additional cis-regulatory elements are required for mediating V allelic exclusion. Interestingly, most of the inserted V rearrangements were not transcribed and expressed in the presence of a TCR transgene, suggesting that TCR allelic exclusion can also be achieved by blocking the transcription of the rearranged gene segments. These findings provide strong evidence for distinct control of the frequency and allelic exclusion of V gene rearrangement.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recombination of V(D)J, by which the primary Ag receptor diversity is generated, is a highly regulated process. At the TCR locus, recombination occurs at the CD4^-CD8^-CD44^-CD25⁺ stage of thymocyte development (1). In general, D to J rearrangement precedes V to DJ rearrangement and occurs on both alleles, whereas V to DJ rearrangement appears to occur on one allele at a time (2). If the initial VDJ rearrangement is nonproductive, then V to DJ rearrangement can proceed on the other allele. If the first VDJ rearrangement is productive, V to DJ joining on the second allele is inhibited. Thus, an individual T cell expresses a single TCR-chain, a phenomenon known as allelic exclusion. In addition, both human and mouse TCR loci contain multiple V gene segments. Although individual Vs are rearranged and expressed at different frequencies, a given V gene segment is recombined and expressed at a constant frequency as most clearly shown in different mice of the same strain (3). This report investigates the parameters that regulate the frequency, timing, order, and allelic exclusion of V gene rearrangement.

V(D)J recombination at different Ag receptor gene loci is mediated by the same recombinase machinery and conserved recombination signal sequences (RSS).⁴ The various levels of regulation of V(D)J recombination are thought to be mediated through the control of accessibility of substrate gene segments (4, 5). Studies have shown that cis-regulatory elements that activate transcription of germline gene segments also promote their recombination accessibility (reviewed in Refs. 6 and 7). For example, deletion of the transcriptional enhancer E from the endogenous TCR locus in mice impairs both germline transcription and rearrangement of D and J gene segments (8, 9, 10). Conversely, inclusion of the E in recombination substrates promotes germline transcription as well as rearrangements of both D to J and V to DJ in transgenic mice (11, 12). In contrast to the long-range effect of E, deletion of the germline promoter PD1, located immediately upstream of the D1 gene segment (13, 14), impairs germline transcription and rearrangement of the D1 without affecting germline transcription and rearrangement of D2, J2, and V gene segments (15, 16). Similarly, E is required for rearrangement of all J gene segments at the TCR locus, while T early promoter is required for the rearrangement of only proximal J gene segments (17, 18). Although the precise role of transcription in recombination accessibility has yet to be elucidated, these findings suggest that promoter-enhancer interaction may be a general mechanism for targeting recombination accessibility of specific gene segment.

Assuming that accessibility control applies to the V gene rearrangement, at least three types of regulation could be envisioned. First, the various levels of regulation of V rearrangement could be mediated by a single master cis-regulatory element. This possibility is not likely because it cannot account for the different frequencies of V rearrangements. Second, individual Vs could be regulated separately, for example by their own specific promoters. Although this kind of regulation can readily account for the different frequencies of V rearrangements, it may not be most efficient for achieving the ordered, stage- and allele-specific V rearrangement. The third possibility, a combination of the two extremes, appears to be more likely. In this scenario, rearrangement of each V is regulated by an individual cis element, such as a promoter, as well as one or more common cis elements, such as enhancers, analogous to the accessibility control of the D1 by both PD1 promoter and E enhancer. The individual elements may determine the frequencies of V rearrangements, while the common element may control the order, timing, and allelic exclusion of V gene rearrangements.

Although transcriptional activation of V often precedes their rearrangement and the occurrence of allelic exclusion is associated with a down-regulation of V germline transcription (19, 20), studies that examine the role of V promoter and E enhancer in V rearrangement have so far been inconclusive. In experiments using a TCR minilocus in transgenic mice, a V was shown to recombine in the absence of germline transcription (21), although the observed rearrangement may have been promoted by a V promoter present 2.5 kb upstream in the same construct. It is also possible that different cis-regulatory elements in the promoter may function in transcription and recombination as observed for enhancers (22, 23). Similarly, the dependence on E for V to DJ rearrangement in recombination substrates may have been complicated by integration site influences, concatenation of substrate, and changes in distance and configuration of V to D and E in the substrate (24). In E^-/- mice, the impaired V to DJ rearrangement could be a result of a block in accessibility of V, or D, or both. In the absence of E, the D-J region, but not the V region, was silent in germline transcription and resistant to endonuclease treatment (10), indicating that E may not directly regulate V transcription or recombination accessibility. Whether there are long-range acting cis-regulatory elements for regulating V recombination is not known.

To probe the parameters that regulate the different levels of V rearrangement, we inserted a V gene segment, together with its endogenous promoter and RSS, just 6.8 kb upstream of the D1 gene segment at the TCR locus in mice. We found that the inserted V gene segment recombined at the same frequency as the natural copy but its rearrangement was no longer inhibited by the presence of a functional TCR transgene. These findings support a regulatory mechanism by which distinct cis-regulatory elements control the frequency and allelic exclusion of V rearrangement.

	Materials and Methods

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Targeting construct and chimeric and germline mutant mice

Homologous recombination in embryonic stem (ES) cells followed by Cre/loxP-mediated deletion was used to insert a copy of the V13 gene segment at the SpeI site 6.8 kb upstream of the D1 gene segment (see Fig. 1A). Previously, we showed that the three DNase I-hypersensitive sites within a 3-kb region upstream of D1 were required for efficient D1 rearrangement (15). The insertion was 4 kb upstream of the most 5' DNase I-hypersensitive site and was not expected to affect D1 rearrangement. The inserted 4.8-kb PstI-SnaBI DNA fragment contains V13 and 3.6-kb and 0.7-kb flanking sequences at 5' and 3', respectively, to ensure the inclusion of the endogenous V13 promoter and RSS. The targeting vector contained, in the following order, a 5-kb SpeI fragment as the 5' homologous sequences, the 4.8-kb V13 fragment, a phosphoglycerate kinase (PGK) promoter-driven neomycin (neo) resistance gene flanked by loxP sites, and a 8.4-kb SphI-SmaI fragment as the 3' homologous sequences (see Fig. 1A). A PGK promoter-driven thymidine kinase (tk) gene was inserted upstream of the 5' homologous sequences. J1 ES cells were transfected with the targeting construct, and G418 and gancyclovir double-resistant clones were analyzed by Southern blot to identify homologous recombinants (see Fig. 1B). Heterozygous mutant ES cell clones were grown in high G418 concentrations to select for homozygous mutant ES cell clones (25). The PGK-neo was deleted by transient expression of Cre in multiple heterozygous and homozygous mutant ES cell clones. Homozygous ES cells with or without the inserted PGK-neo were then transfected with a functionally assembled TCR vector (26).

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Targeted insertion of the V13 gene segment. A, Schematic diagrams of the murine TCR locus (not to scale), targeting vector, and targeted allele before (neo) and after (neo) Cre-mediated deletion of the PGK-neo. Arrows flanking PGK-neo represent loxP sites. Transcriptional orientations of V13 and PGK-neo are indicated. Probe A is a 1.5-kb HindIII-EcoRI fragment; probe B is a 450-bp cDNA containing V13. P, PvuII; S, SpeI; Sh, SphI; Sm, SmaI; B, BamHI. B, Southern blot analysis for identifying homologous recombinant ES cell clones before and after PGK-neo deletion. DNA from ES cell clones was digested with PvuII and hybridized with probe A. The wild-type allele gives rise to a 11.5-kb fragment, and the targeted allele gives rise to a 9.6-kb fragment. Probe A also hybridized with a 1.8-kb fragment from both wild-type and targeted alleles (data not shown). After Cre-mediated deletion, DNA from mutant ES cell clones was digested with BamHI and hybridized with probe B. The targeted allele with PGK-neo gives rise to a 8.6-kb fragment, and the targeted allele with deleted PGK-neo gives rise to a 10.6-kb fragment.

Abs specific to CD3, CD4, CD8, Thy-1.2, V2, 4, 6, 7, 8.1/8.2, 9, 10, 12, 13, and 14 were direct conjugates from BD PharMingen (San Diego, CA). Single-cell suspensions were prepared from lymph nodes and thymi. A total of 5 x 10⁵ cells were stained with the appropriate combination of Abs, and 10,000–20,000 live cells (propidium iodide negative) were collected for each sample using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). Data were analyzed using CellQuest software (BD Biosciences).

CD44⁺CD4^-CD8^- thymocytes were purified by complement-mediated lysis followed by cell sorting. A single-cell suspension of whole thymi was washed twice with PBS and resuspended in medium (HEPES-buffered RPMI 1640, supplemented with 5% FCS, L-glutamate, 2-ME, penicillin, and streptomycin) at 2 x 10⁷ cells/ml. Cells were stained with Abs specific for CD4 and CD8 at 2.5 µg/ml for 1 h on ice, pelleted and washed with medium, and resuspended again at 2 x 10⁷ cells/ml. Low-Tox-M Rabbit Complement (CL3051; Cedarlane Laboratories, Hornby, Ontario, Canada) was added at a 1/10 dilution, and the mixture was incubated for 1 h at 37°C with frequent mixing. Cells were then pelleted and washed with medium, and live cells were collected by Ficoll-Paque centrifugation. Cells were then stained with FITC-CD44 and PE-CD25 Abs, and CD44⁺ cells were sorted by FACS.

Southern and Northern blot analyses

Following overnight digestion with proteinase K, DNA from tails and single-cell suspensions of lymph nodes or thymi were isolated by phenol/chloroform extraction and ethanol precipitation. For Southern blotting analysis, 10 µg of DNA was digested with the appropriate enzymes, fractionated on a 0.8% agarose gel, and transferred to -probe filters. Filters were hybridized with ³²P-labeled probes and exposed to phosphorimaging screens. Total RNA was isolated from thymi using RNazol (Biotecx, Houston, TX), as per the manufacturer’s instructions. For Northern blotting analysis, 10 µg of total RNA was fractionated on a 1.2% formaldehyde agarose gel and transferred to -probe filters. The filters were hybridized with ³²P-labeled probes and exposed to phosphorimaging screens. Images were analyzed by ImageQuant (Molecular Dynamics, Sunnyvale, CA).

PCR and RT-PCR

Nested PCR for measuring V13 to D1J1.1 rearrangement were performed in a 50-µl reaction containing 0.6 µg of lymph node DNA, 100 ng of each primer (1 and 4), 0.2 µM of each dNTP, 3.5 mM MgCl₂, 10 mM Tris-HCl, 50 mM KCl (pH 8.3 at 20°C), and 1 U Taq polymerase. Primary reactions were run for 12 cycles of 15 s at 94°C, 30 s at 61°C, and 2 min at 72°C. Next, 2 µl were transferred from the primary reactions to new tubes for secondary PCR that were performed with identical conditions, except with nested primers (2 and 5) and 18 cycles of amplification. Then, 25 µl of secondary PCR were electrophoresed on a 1.5% agarose gel, transferred to -probe membranes, hybridized with ³²P-labeled V13 cDNA probe, and exposed to phosphorimaging screens.

Seminested PCR for amplifying V12 to D1J1.1 rearrangements were performed in a 50-µl reaction using the same buffers as above, except that primers 3 and 4 were used. Primary reactions were run for 12 cycles of 15 s at 94°C, 30 s at 61°C, and 2 min at 72°C. Next, 2 µl is transferred from the primary reactions to new tubes for secondary reactions that were performed with identical conditions, except with seminested primers (3 and 5) and 16 cycles of amplification. Then, 25 µl of secondary PCR were analyzed by Southern blotting as above using a V12 cDNA probe.

Seminested PCR for amplifying V13 to D1 rearrangements were performed in a 50-µl reaction using 1.2 µg, DNA, primers 1 and 6, and the same buffer conditions as described above. Primary reactions were 25 cycles of 30 s at 94°C, 30 s at 61°C, 2 min at 72°C. Secondary reactions were done using 2 µl of the primary reactions and the same conditions as above except seminested primers (2 and 6) and 20 cycles of amplification. Then 10 µl of the secondary reactions were analyzed by Southern blotting as above using a V13 cDNA probe.

Semiquantitative JAK3 PCR were done as previously described (15). Briefly, reactions were performed in a 50-µl reaction containing 50 ng of DNA, 100 ng of each primer (7 and 8), 0.2 µM of each dNTP, 2 mM MgCl₂, 10 mM Tris-HCl, 50 mM KCl (pH 8.3 at 20°C), and 1 U Taq polymerase. Primary reactions were run for 12 cycles of 15 s at 94°C, 30 s at 61°C, and 2 min at 72°C. Next, 2 µl were transferred from the primary reactions to new tubes for secondary PCR that were performed with identical conditions, except with seminested primers 7 and 9, and 25 cycles of amplification. Then, 25 µl of the secondary amplification were loaded on a 1.5% agarose gel and stained with ethidium bromide.

PCR for specifically amplifying Vi to D1J1.1 rearrangements from thymic and lymph node DNA were performed using the Expand High Fidelity PCR System kit (Boehringer Mannheim, Mannheim, Germany) as per the manufacturer’s instructions. A 50-µl PCR contained 0.6 µg DNA, 100 ng of primers 14 and 5, 0.4 µM of each dNTP, 4.5 mM MgCl₂, 10 mM Tris-HCl, 50 mM KCl (pH 8.3 at 20°C), and 1 U of enzyme. An initial heating of reaction mixture at 94°C for 3 min was followed by 10 cycles of 30 s at 94°C, 30 s at 63°C, and 3 min 45 s at 68°C, immediately followed by 20 cycles of 30 s at 94°C, 30 s at 63°C, and (3 min 45 s plus 20 s/cycle) at 68°C. Then 25 µl of PCR were analyzed by Southern blotting as above using a V13 cDNA probe.

RT-PCR for assaying germline V13 transcripts were performed using total RNA from thymi. Reactions were performed in 50 µl using the One-Tube Titan kit (Boehringer Mannheim) as per the manufacturer’s instructions. A cDNA synthesis reaction was performed at 50°C for 30 min using 30 ng total RNA, 1 ng of each primer (10 and 11), and 3.5 mM MgCl₂. Amplification of cDNA was done in the same reaction tube, using the following conditions: 30 s at 94°C, 30 s at 60°C, 2 min at 72°C, for 22 cycles. Then, 25 µl of reactions were analyzed by Southern blotting as above using a V13 cDNA probe.

RT-PCR for assaying immature -actin transcripts (29) were performed on total RNA isolated from thymi using the same reaction conditions as above except using primers 12 and 13. Then 25 µl of reactions were electrophoresed on a 1.0% gel and stained with ethidium bromide.

Primer sequences are as follows: 1, 5'-CTGCCATGGGCACCAGGCTTCTTG; 2, 5'-GGCACCAGGCTTCTTGGCTGGGCAG; 3, 5'-GCTGGAGTTACCCAGACACCC; 4, 5'-AGATACTCGAATATGGACACGGAG; 5, 5'-TGGACACGGAGGACATGCTTTGTC; 6, 5'-CAATCTTGGCCTAGCAGGCTGCAG; 7, 5'-CCTCTCAGACCCCACACCTGGCATC; 8, 5'-CCATAGCTGACTCCCCGGTACTTG; 9, 5'-ACGATGAAGTCGCTGTGCAGAGCCTTA; 10, 5'-TCCTTGACACAGTACTGTCTGAAGC; 11, 5'-CTCTGGATACACGCAGCATGGCCT; 12, 5'-CCTAAGGCCAACCGTGAAAAG; 13, 5'-TCTTCATGGTGCTAGGAGCCA; 14, 5'-CACTCGCTGCATCCTACACATAGCGCTC.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of mice with targeted insertion of V13 gene segment

At the murine TCR locus, D and J gene segments are clustered together and are within 18 kb of the E enhancer. In contrast, V gene segments are dispersed over 250 kb and are at least 340 kb away from D and E, with the exception of V14, which is only 5 kb away from the E at the 3' end of the locus (Fig. 1) (30, 31). We inserted a copy of the V13 gene segment, called Vi, together with 3.6-kb 5' and 0.7-kb 3' flanking sequences 6.8 kb upstream of the D1 at the TCR locus through homologous recombination in ES cells (Fig. 1 and Materials and Methods). Compared with the natural V13 gene segment, referred to as Ve, which is 470 kb 5' of D1, Vi is 70 times closer to D and E than Ve on the linear chromosomal DNA. Heterozygous and homozygous mutant ES cells in the presence or absence of the PGK-neo cassette were differentiated into mature T cells by RAG-2-deficient blastocyst complementation (27). Germline mutant mice with or without the PGK-neo were also derived. There was no difference between the results obtained from chimeric mice derived from targeted ES cells and germline mutant mice. Therefore, no distinction is made whether analyzed mice were chimeras or germline mutant mice, unless necessary. A partial list of generated mice and their genotypes is detailed in Materials and Methods.

Frequency of Vi rearrangement

The relative usage of Vi in recombination was estimated by comparing the percentages of V13-expressing T cells between wild-type and mutant mice. Lymph node cells were stained with an Ab specific to the pan-T cell marker Thy-1.2 and Abs to V13, V6, or V14, then analyzed by flow cytometry. In wild-type (+/+) mice, an average of 2.3% of Thy-1.2⁺ T cells expressed V13 (Fig. 2 and Table II). In homozygous mutant mice without the PGK-neo (V/V(neo)), 4.7% T cells were V13 positive. The frequencies of V6, V14, and other V-expressing T cells were similar between wild-type and mutant mice (Fig. 2 and data not shown), indicating that Vi did not significantly affect the rearrangement and expression of the endogenous V gene segments. Because T cells from V/V(neo) mice had four copies of the V13 gene segment compared with two in wild-type T cells, the increase in percentages of V13-expressing T cells correlated with the copy numbers of the V13 gene segment. Thus, despite its proximity to D and E, Vi appears to be used at the same frequency as Ve in mature T cells.

View larger version (62K): [in this window] [in a new window]   FIGURE 2. Flow cytometry analysis of V expression on lymph node T cells. Lymph node cells from wild-type and mutant mice were stained with anti-Thy-1.2 and anti-V Abs, followed by flow cytometry. The numbers indicate the percentages of T cells (Thy-1.2+) that express the specific V.

View this table: [in this window] [in a new window]   Table II. Percentages of V13+ T cells in lymph nodes of various types of mice1

View larger version (40K): [in this window] [in a new window]   FIGURE 4. Analysis of allelic exclusion by flow cytometry. A, Flow cytometry analysis of V expression on lymph node T cells. Lymph node cells were stained with anti-CD3 and anti-V8.1/8.2, or anti-Thy-1.2 and anti-V6 or V13 Abs, followed by flow cytometry. The numbers indicate the percentages of T cells (CD3+ or Thy-1.2+) that express the specific V. In the FACS plots showing double staining of V8.1/8.2 and V13, the numbers indicate the percentages of V13 cells that are also V8.1/8.2+. B, Summary of percentages of V-expressing T cells in lymph nodes of various types of mice. Lymph node cells were assayed as in A. TCR transgenes, tg-1 and tg-4, were expressed on 95.5 and 99% of T cells, respectively. Each symbol represents an individual mouse.

View larger version (56K): [in this window] [in a new window]   FIGURE 3. PCR assays for V rearrangements. A, Comparison of the levels of V to D1J1.1 rearrangements among various types of mice. PCR products representing V13 or V12 to D1J1.1 rearrangements were detected with a V13 or a V12 cDNA probe, respectively. Lanes 11–13 are PCR using 0.9, 0.3, and 0.1 µg, respectively, of input DNA from +/V(neo) mice to show the linearity of the PCR. PCR amplification of JAK3 shows the relative amount and quality of DNA for each sample. For germline mutant mice, either with or without the TCR transgene (lanes 1–3 and 7–9), similar results were obtained from at least two independent mice assayed in two different PCR. For chimeric transgenic mice (lanes 4–6), similar results were obtained from two independent PCR. B, PCR analysis of V13 to D1J1.1 rearrangements in thymic DNA of various types of mice. C, PCR analysis of Vi to D1J1.1 rearrangements in thymic (lanes 7–12) and lymph node (lanes 1–6) DNA of various types of mice.

Allelic exclusion of the Vi gene segment

To investigate Vi allelic exclusion, we examined the frequencies of V13-expressing T cells in the presence of a functionally assembled TCR transgene. The TCR transgene expressed V8.2, and the extent of its expression on mature T cells was monitored by flow cytometry using anti-CD3 and anti-V8.1/8.2 Abs. In wild-type (+/+) mice, 18% of CD3⁺ T cells expressed V8.1/8.2. In TCR (tg-5)-transgenic mice on the +/+, +/V(neo), or +/V(neo) background, over 99% of T cells expressed the TCR transgene (Fig. 4A). In these same mice, the percentage of V13-expressing T cells was decreased at least 18-fold (Fig. 4, A and B). Similar folds of decrease in V4-, V6-, or V12-expressing T cells were also observed. Thus, the expression of the TCR (tg-5) transgene appears to exclude the expression of the endogenous Vs as well as Vi.

The same TCR transgene was also transfected into V/V(neo) and V/V(neo) ES cells, and allelic exclusion of Vi expression was analyzed in chimeras derived from ES cell clones. Like tg-5-transgenic mice, V13-expressing T cells were reduced 18-fold (from 4.7 to 0.26%) in chimeras generated with V/V(neo);tg-2 and with V/V(neo);tg-1 ES cell clones (Fig. 4, A and B). However, Vi exclusion was leaky in chimeras generated with V/V(neo);tg-3 and V/V(neo);tg-4 ES cell clones (in the presence of the neo cassette). For example, in V/V(neo);tg-3 chimeras the TCR transgene was expressed on 97% of T cells and V6-expressing T cells were decreased 9-fold, but V13-expressing T cells were decreased only 3-fold (from 14 to 4.3%). In fact, 3.7% of T cells expressed both V13 and the V8.2 transgene (Fig. 4A). The leaky Vi exclusion could be caused by the presence of PGK-neo and/or by different levels of TCR transgene expression due to different copies of the transgene that were integrated at different sites. Nevertheless, even in the presence of PGK-neo, Vi expression is excluded in over 95% of total T cells by the TCR transgene.

To verify Vi allelic exclusion at the DNA level, V13 rearrangement was assayed by PCR in lymph node DNA of various transgenic mutant mice. In +/+;tg-5 mice, PCR product corresponding to V13 to D1J1.1 rearrangement was barely detectable (Fig. 3A, lane 9), indicating allelic exclusion of the endogenous V13 and correlating with the results obtained by flow cytometry. Unexpectedly, a slightly higher levels of V13 rearrangement were detected in +/V(neo);tg-5 mice than in wild-type or +/V(neo) mice (Fig. 3A, lanes 1, 3, and 8), although the mutant mice had <0.13% of V13-expressing T cells as compared with 2.3% in wild-type mice and 3.5% in +/V(neo) mice (Table II). Similar levels of V13 rearrangement were also detected in thymocyte DNA from the three types of mice (Fig. 3B). The loss of exclusion was specific for Vi (V13) because rearrangement of V12 was undetectable in the same DNA from +/V(neo);tg-5 mice (Fig. 3A, lanes 8 and 9). In the presence of PGK-neo as in +/V(neo);tg-5 mice, even higher levels of V13 rearrangement were detected (Fig. 3A, lanes 1 and 7–9). Particularly, in V/V(neo);tg-3 and V/V(neo);tg-4 chimeras, where allelic exclusion was leaky as shown by flow cytometry (Fig. 4) and by the presence of V12 rearrangement at the DNA level (Fig. 3A), the levels of V13 rearrangement were 10- to 20-fold higher than those in wild-type mice (Fig. 3A, lanes 1, 5, and 6). Together, these data show that while rearrangements of endogenous V are inhibited by the presence of a TCR transgene, Vi continues to rearrange at a similar level (3–5%) as if in the absence of the transgene. However, the majority of the rearranged Vi is not expressed on the cell surface.

To probe the discrepancy between the levels of V13 rearrangement detected by PCR and by cell surface V13 staining, we assayed for V13-containing transcripts in total thymic RNA by Northern blotting analysis. The 1.3-kb mature transcript was readily detected in wild-type, +/V(neo), and +/V(neo) mice (Fig. 5A, lanes 1–3), with levels of the transcript corresponding to the percentages of T cells that expressed V13 in these mice. As expected, in the presence of the TCR transgene (tg-5), no mature transcript was detected in wild-type or mutant mice either in the presence or absence of PGK-neo (Fig. 5A, lanes 4–6). Germline V13 transcript, which is around 1.0 kb, was not detected on the Northern blot, but was readily detected by RT-PCR, even in the presence of the TCR transgene (Fig. 5B). These results show that, in the presence of the TCR transgene, most rearranged Vi is not highly transcribed and therefore not expressed on the cell surface.

View larger version (46K): [in this window] [in a new window]   FIGURE 5. V13 transcription in thymocytes. A, Northern blot analysis of V13 transcripts. Ten micrograms of total RNA from thymocytes of wild-type and mutant mice were fractionated, and the filter was hybridized with a V13 cDNA probe. The filter was stripped and rehybridized with a GAPDH probe for monitoring the relative amount of RNA in each sample. The position of 18S RNA is indicated. B, RT-PCR analysis of germline V13 transcripts. Total RNA from thymocytes was assayed for V13 germline transcripts by RT-PCR. PCR product was verified by hybridization with a V13 cDNA probe. Lanes 8–10 are PCR using different dilutions of input RNA from +/V(neo) mice to show the linearity of PCR. RT-PCR for -actin transcripts was used to estimate the relative amount of RNA in each sample.

Timing of Vi rearrangement

As shown above, in the absence of PGK-neo and the TCR transgene, Vi rearranges and expresses like Ve; however, Vi rearrangement is not inhibited to the same extent as the endogenous Vs by the TCR transgene. To determine whether the leaky Vi allelic exclusion results from an earlier onset of Vi rearrangement, we assayed the levels V13 rearrangement in CD4^-CD8^-CD44⁺ thymocytes. Normally, CD4^-CD8^- (double negative or DN) thymocytes progress from CD44⁺CD25^- to CD44⁺CD25⁺, then to CD44^-CD25⁺, and finally to CD44^-CD25^- phenotype. TCR gene rearrangement occurs predominantly at the CD44^-CD25⁺ stage. If the onset of Vi rearrangement occurs earlier, one would expect to detect higher levels of V13 rearrangement in CD44⁺ fraction of DN thymocytes from +/V(neo) mice as compared with wild-type mice. Thus, CD44⁺ DN thymocytes were purified from wild-type and +/V(neo) mice (97% CD44⁺), and the levels of V13 to D1J1.1 rearrangement were assayed by semiquantitative PCR (Fig. 6A). Quantification of the intensity of V13 PCR product and normalization of the input DNA by JAK3 PCR amplification showed that 10% more V13 rearrangement was detected in +/V(neo) DNA than in wild-type DNA (Fig. 6A, lanes 5 and 6). Compared with the levels of V13 rearrangements in total wild-type thymus, the level of V13 rearrangement in the CD44⁺ fraction was 8- to 10-fold lower (Fig. 6A, lanes 1–6). Because the level of V13 rearrangement in wild-type thymus is 2.3% of the total V rearrangement, the overall level of V13 rearrangement in the CD44⁺ fraction of +/V(neo) thymus is low. Together, these results demonstrate that Vi is not significantly recombined earlier during T cell development.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Analysis of the timing and the order of V13 rearrangements. A, Comparison of V13 to D1J1.1 rearrangements in total and purified CD44+ DN thymocytes. CD44+ DN thymocytes from wild-type and +/V(neo) mice were purified by complement-mediated lysis of CD4+, CD8+, and CD4+CD8+ cells followed by cell sorting for CD44+ cells (>97% CD44+). DNA (1.0 µg) isolated from purified CD44+ DN thymocytes was assayed for V13 to D1J1.1 rearrangement (lanes 5 and 6) and JAK3 as in Fig. 3A. Different amounts of total thymic DNA (1.0, 0.1, and 0.05 µg) from a wild-type mouse was assayed (lanes 1–3) to estimate the level of rearrangements in CD44+ cells. B, Comparison of the levels of V13 to D1 rearrangement in various types of mice. DNA from thymocytes (1.2 µg) was amplified for V13 to D1 rearrangements by two rounds of PCR. Different amounts of thymocyte DNA from J1M2/ mice were amplified to estimate the level of V13 to D1 rearrangements (lanes 7–9). Specific PCR product was detected by hybridization with a V13 probe. PCR amplification of JAK3 shows the relative amount of DNA for each sample. C, Sequence analysis of V13-D1 rearrangements. V13-D1 rearrangements from +/V(neo) mice were subcloned and sequenced. Overlapping nucleotides that could be encoded by either germline segment or P nucleotides are underlined.

To quantify the level of V13D1 rearrangement, we used as comparison the total thymic DNA from J1^M2/ mutant mice, in which the D2-J2-C2 region of the TCR locus was deleted (32). In J1^M2/ mice, the allele undergoes both D1-J1 and V-D1J1 rearrangements, whereas the M2 allele undergoes only V-D1 rearrangement because the 3' D1 RSS was mutated. It was previously shown that the M2 allele undergoes diverse V to D1 rearrangement at a frequency of 21% (32). Assuming that usage of V13 in J1^M2/ mice is comparable to that in wild-type mice (2.3%), because the level of V13D1 rearrangement in our mutant mice is approximately one-third the level of V13D1 rearrangement observed in J1^M2/ mice, the percentage of Vi allele undergoing V13D1 rearrangement is 0.16% [(0.21)(0.023)(0.33) x 100]. Thus, the steady-state level of ViD1 rearrangement in total thymocytes is at least 10 time lower than V13DJ rearrangement in wild-type mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Position vs distance in V(D)J recombination

Recombination of V gene segments is controlled in terms of frequency, timing, order, and allelic exclusion. Insertion of the V13 gene segment 6.8 kb upstream of the D1 not only dramatically shortens the distance between the V and the D1 gene segment but also may have removed the V from regulation by cis-regulatory elements naturally present in the V region (position effect). A role of distance between gene segments and/or their position in the locus on V(D)J recombination was initially suggested by observations at the murine IgH locus. It was found that initial DJ_H rearrangements preferentially use the D segments proximal to J_H while the rearrangements of the more distal D segments occur through secondary rearrangements (33, 34). Similarly, V_H segments that are proximal to J_H are preferentially used in VDJ recombination during fetal B cell development (35, 36, 37) and tend to escape allelic exclusion in Igµ-transgenic mice (38). Recently, it was found that IL-7R was preferentially required for the rearrangement of the J_H-distal but not the J_H-proximal V_H gene segments (39). Because IL-7R-mediated signaling probably promotes V_H rearrangement by modulating recombination accessibility (40, 41), this finding implies that different cis-regulatory elements may control recombination accessibility of the J_H-distal and J_H-proximal V_H gene segments (position effect).

If distance between gene segments affects their frequency of recombination, one would expect that Vi would be preferentially used in TCR recombination in our mutant mice. However, our results clearly show that, in the absence of PGK-neo, Vi is rearranged at the same frequency as the endogenous copy (Figs. 2 and 3 and Table II). The failure to detect higher levels of Vi rearrangement and expression is not because an initial Vi rearrangement is deleted by a subsequent rearrangement of an upstream V to a downstream DJ or because there is an intrinsic selection against V13-expressing T cells. Much higher levels of V13 rearrangement and expression were detected if PGK-neo was left at the insertion site. Our results are consistent with observations that recombination frequencies of V gene segments are not correlated with their distances to the Ds in the TCR locus (3). For example, the V14 gene segment is within 25 kb of the D gene segments but is not most frequently recombined and expressed (3). In contrast, the V8.2 gene segment, which is used with the highest frequency in TCR rearrangement, is not the most proximal to the D-J region (30, 31). V8.2 is highly transcribed during early T lymphocyte development (42, 43), suggesting that local accessibility of V gene segments is probably more important in regulating recombination frequency (see below). In our targeted insertion, although we cannot exclude unequivocally a role of distance between Vi and D in Vi rearrangement, our results are consistent with the interpretation that position of V gene segments in the locus, and therefore their control by cis-regulatory elements, influences the V gene rearrangement.

Local regulation of V recombination frequency

The fact that Vi recombines at the same frequency as the endogenous copy suggests that the inserted DNA fragment contains the necessary cis-regulatory elements for regulating the frequency of V13 rearrangement. A potential cis-regulatory element in the inserted fragment is the V promoter. Although V promoters have not been shown to promote V rearrangement, cis elements upstream of the V gene segment, corresponding to the likely promoter, control the timing of V rearrangement during development (44). Consistent with the promoter control of V recombination frequency, the presence of the PGK-neo transcriptional cassette at the insertion site resulted in a 5- to 7-fold increase in Vi usage in recombination (Fig. 3 and Table II). The stimulating effect of PGK-neo on Vi rearrangement is in contrast to the inhibitory effect of the neo cassette on recombination when inserted into IgH and Ig loci. Insertion of a PMC1neo downstream of Ig intronic enhancer severely blocked V to J rearrangement upstream (45). Insertion of PGK-neo in between the 3' IgH enhancer and constant region exons blocks class switch recombination to the constant genes 5' of the neo cassette (46). As PGK-neo does not have an intrinsic property in promoting recombination, its effect on Vi rearrangement likely reflects the mechanisms by which V gene segments are normally targeted for recombination. The neo transcription is driven by a constitutively active PGK promoter, and the entire cassette is deleted after Vi recombination. Recent studies have shown that transcriptional coactivators possess histone acetyltransferase activity (47, 48, 49) and histone acetylation is tightly correlated with V(D)J recombination accessibility (50). The transcription process itself also leads to changes in DNA-nucleosome interaction (51). Together, these findings suggest that chromatin changes, mediated by local cis-regulatory elements, probably determine V recombination frequency.

Why is Vi not recombined more frequently as its promoter is much closer to E? An explanation is suggested by recent findings on the extent of E and PD1 in controlling the chromatin structure of the TCR locus. We have shown previously that three DNase I-hypersensitive sites are present within 3 kb of D1 gene segment (19) and the site immediately upstream of D1 corresponds to the PD1 promoter (13, 14). In the presence of PD1 promoter, the promoter region as well as the downstream D1-J1 region are hypomethylated in developing T cells, whereas the upstream region including at least one DNase I-hypersensitive site is hypermethylated (16). Deletion of PD1 alone or plus the two upstream hypersensitive sites results in the invasion of hypermethylation into the downstream D1-J1 region. These findings suggest that there is probably a boundary between the inaccessible upstream region and the accessible D1-J1 region. Consistent with this interpretation, deletion of the E resulted in the hypermethylation, histone hypoacetylation, resistance to endonuclease treatment, and transcriptional silencing of the D-J region, whereas no significant effect was observed in the V region or 5 kb upstream of the D1 gene segment (10). Thus, the chromosomal domain regulated by E appears to be limited to the D-J region. Although Vi is much closer to the D1 gene segment and E enhancer, it probably still resides within a relatively inaccessible region and outside the E-regulatory domain, accounting for the absence of a higher level of Vi rearrangement.

Long-range regulation of V allelic exclusion

We found that Vi was rearranged at a similar level in the absence or presence of a TCR transgene (Fig. 3), indicating that Vi is no longer subject to allelic exclusion. The lack of Vi allelic exclusion cannot be attributed significantly to an earlier onset of Vi rearrangement because there is only a slight increase of V13 rearrangement at the CD44⁺ DN stage (Fig. 6). However, the presence of V to D rearrangement before D-J rearrangement may have contributed to the lack of Vi allelic exclusion (Fig. 6). Because the D-J region remains accessible to the recombinase in double-positive thymocytes (15), VD rearrangements likely undergo further VD-J rearrangements when the recombinase is re-expressed in double-positive thymocytes for TCR rearrangement. Although 0.16% of VD rearrangement is relatively low as compared with 2–5% Vi rearrangement in the presence of a TCR transgene, the amount represents the steady-state level but does not take into account the rate of VD generation and the rate of conversion to VDJ. Based on these considerations, VD rearrangement before DJ rearrangement probably has contributed to the Vi rearrangement in the presence of a TCR transgene, but whether it can account for all Vi rearrangement is not clear.

As discussed above, the inserted DNA fragment contains cis-regulatory elements for determining the frequency of V13 rearrangement. The similar level of Vi rearrangement in the presence of a TCR transgene suggests that the same cis elements are insufficient for mediating Vi allelic exclusion. There are likely other cis-regulatory element in the V region that normally mediate V allelic exclusion, and insertion of the V13 gene segment in the proximity of D-J region may have moved the Vi outside the regulatory range of these additional cis-regulatory elements. It is possible that each V has its own cis elements for mediating allelic exclusion and these elements happen not to be included in the inserted fragment. However, it seems more likely that a few Vs or all Vs share common cis elements for allelic exclusion (see Introduction). While the nature and location of these cis-regulatory elements are unknown, the continuous Vi rearrangement at a similar frequency in the presence of a TCR transgene strongly suggests that the frequency and allelic exclusion of V rearrangement is regulated by distinct cis-regulatory elements.

Interestingly, most of the rearranged Vi was not expressed on the cell surface in the presence of a TCR transgene (Figs. 4 and 5). T cells have been shown to be capable of expressing two different TCR-chains simultaneously (52). Similarly, in our mutant mice, some T cells expressed both V13 and V8.2 (Fig. 4), suggesting that T cells expressing V13 and V8.2 are not intrinsically selected against. The lack of cell surface expression of the rearranged Vi in the presence of a TCR transgene is because the rearranged Vi is not highly transcribed (Fig. 5). During normal thymocyte development, transcription of the rearranged allele is up-regulated following recombination (53). This apparently did not occur in most of the developing T cells in which Vi underwent rearrangement in the presence of a TCR transgene. While further studies are required to elucidate the mechanism of this regulation, the present findings clearly show that allelic exclusion can be mediated functionally by inhibiting transcription of a rearranged gene segment.

Regulation of the timing and order of V rearrangement

In addition, we found a low level of Vi to D rearrangement occurring before D-J rearrangement and a small increase of V rearrangement in CD44⁺ DN thymocytes in the mutant mice as compared with the wild-type mice. Recently, it was shown that the 5' RSS of D1 plays an important role in the ordered DJ before VDJ rearrangements (54). Although our findings could result from the close proximity of Vi to the D-J region, we favor the notion that the Vi is removed from the regulation by additional cis-regulatory elements normally present in the V region. In this scenario, the significant increase of Vi to D joining in mutant mice would suggest that these additional cis-regulatory elements may also contribute to the ordered TCR rearrangement.

	Acknowledgments

 
We thank Tara Schmidt for blastocyst injection and for generating chimeric and germline mutant mice; Glenn Paradis for help with flow cytometry and sorting; members of the laboratory, especially Charles Whitehurst, for discussion and help; Dr. Chris Nelson for plasmids containing various V genomic fragments; Drs. Barry Sleckman and Fred Alt for TCR-transgenic mice and thymic DNA from J1^M2/ mice; Dr. Yoichi Shinkai for the TCR transgene construct; and Drs. Jim Haber, Herman Eisen, and Marjorie Oettinger for critical reading of the manuscript.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grant AI40146 (to J.C.).

² Current address: Department of Molecular Biology, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114.

³ Address correspondence and reprint requests to Dr. Jianzhu Chen, Center for Cancer Research, Massachusetts Institute of Technology, E17-128, 40 Ames Street, Cambridge, MA 02139. E-mail address: jchen{at}mit.edu

⁴ Abbreviations used in this paper: RSS, recombination signal sequence; ES, embryonic stem; PGK, phosphoglycerate kinase; neo, neomycin; RAG, recombination-activating gene; DN, double negative.

Received for publication April 27, 2001. Accepted for publication June 5, 2001.