Limited effect of chromatin remodeling on Dß-to-Jß recombination in CD4+CD8+ thymocyte: implications for a new aspect in the regulation of TCR ß gene recombination

Makoto Senoo1,2, Naoko Mochida1, Lili Wang1, Yasuko Matsumura1, Daisuke Suzuki1, Naoki Takeda3, Yoichi Shinkai4 and Sonoko Habu1,2

1 Department of Immunology, Tokai University School of Medicine, Bouseidai, Isehara, Kanagawa 259-1193, Japan
2 Core Research for Evolution Science and Technology (CREST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
3 Center of Animal Research and Development, Kumamoto University, 4-21-1 Kuhonji, Kumamoto 862-0976, Japan
4 Department of Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin, Kawara-cho, Sakyo-ku, Kyoto 606-8507, Japan

Correspondence to: Y. Shinkai and S. Habu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have generated mutant mice in which TCR ß chain enhancer (Eß) was replaced with the TCR {alpha} chain enhancer (E{alpha}). Using this mouse model, we analyzed (i) recombination status of the TCR ß chain genes after functional V(D)J rearrangements occurred in the first allele during double-negative (DN)-to-double-positive (DP) transition and (ii) involvement of Eß for the expression of rearranged TCR ß chain genes. Our data show that E{alpha} substituted for Eß function to express a similar extent of TCR ß chains exactly at the same time as did Eß (CD25+CD44 DN stage), although the proportion of TCR ß+ cells at this stage was low in mutant mice. At the DP stage, germline transcription and histone acetylation of Dß–Jß loci were detectable at a high degree in both mutant and wild-type mice. However, DP cells in mutant mice retained the germline Dß–Jß configuration at a higher frequency than that of wild-type mice, whereas both DP cells expressed TCR ß chains to a similar extent. These data suggest that chromatin opening has a limited impact on Dß-to-Jß recombination at the DP stage and that E{alpha} is functionally equivalent to Eß in promoting expression of functionally rearranged TCR ß chain genes through DN-to-DP transition.

Keywords: allelic exclusion, germline transcription, histone acetylation, rearrangement, TCR {alpha} enhancer (E{alpha}), TCR ß enhancer (Eß)


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During thymocyte development, TCR ß, {gamma} and {delta} genes are rearranged and expressed early at the CD25+ double- negative (DN) stage (14), whereas the TCR {alpha} gene is not rearranged and expressed until the double-positive (DP) stage (5,6).

The regulatory machinery which allows the TCR ß gene rearrangement to precede that of the TCR {alpha} gene is poorly understood, although transcriptional enhancers on the corresponding loci have been considered to be preferable candidates for explaining these sequential rearrangements. A single transcriptional enhancer on the TCR ß locus (Eß) has been found downstream of Cß2 exons (710). The TCR {alpha} locus also contains one fundamental transcriptional enhancer (E{alpha}), which lies just downstream of the TCR {alpha} constant region gene (C{alpha}) (11,12). Previous studies using transgenic mice with artificial TCR ß miniloci have demonstrated that Eß activates V(D)J recombination as well as the corresponding germline transcription in a T cell-specific and stage-specific manner (1316), although these enhancer activities were complemented partially by Ig heavy chain enhancer (Eµ), {kappa} light chain enhancer (E{kappa}) and even by SV40 enhancer in Dß-to-Jß recombination (1618). It is also suggested that E{alpha} becomes active later than Eß and promotes the accessibility of the transgenic substrates in the {alpha}ß lineage but not in the {gamma}{delta} lineage (15). Thus, Eß and E{alpha} have now been considered to be potent developmental regulators, as they impart lineage- and developmental stage-specific control to the V(D)J recombination, at least in the transgenic substrates.

Recently, gene targeting was applied to examine the importance of Eß and E{alpha} in TCR gene recombination during thymocyte development. Targeted deletion of Eß in mice showed a drastic inhibition of TCR ß rearrangements, indicating that Eß is absolutely necessary for V(D)Jß recombination and consequently for {alpha}ß T cell development (19,20). Elimination of E{alpha} resulted in dramatic inhibition of J{alpha} transcription and TCR {alpha} rearrangement, and, as a result, cells were blocked at a stage of development just prior to TCR {alpha} expression, although normal numbers of thymocytes were generated (21). More recently, Eß and E{alpha} have been suggested to regulate V(D)J recombination through modulation of the histone acetylation status of the corresponding loci at the required timing—with Eß acting early at the DN stage and E{alpha} acting later on, at the DP stage (22,23). However, the correlation between histone acetylation and gene recombination of the TCR ß loci beyond the DN stage is completely unknown.

Here, in the present study, we produced mice in which Eß was replaced with E{alpha}. Using this mouse model, we provide evidence that chromatin remodeling (i.e. opening) has a limited impact on Dß-to-Jß recombination at the DP stage and that E{alpha} is functionally equivalent to Eß in promoting expression of functionally rearranged TCR ß chain genes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of mutant mice
To produce a targeted replacement of Eß with E{alpha}, we constructed a replacement vector containing a 6.7-kb homologous DNA fragment, the E{alpha} element (15) attached with the PGK-neor cassette flanked by loxP sites and the PGK-tk cassette at the 3' end of the replacement vector. TT2 embryonic stem (ES) cells were transfected with the linearized vector by electroporation, and clones resistant to both G418 (0.3mg/ml) and gancyclovir (1.5 µM) were isolated (referred to as Eß +/{alpha}N clones). Multiple clones showed the expected pattern of homologous recombination revealed by Southern blot analysis with Vß14 probe (data not shown). To delete the neor gene, the Eß +/{alpha}N ES cell clones were infected by the recombinant adenovirus expressing Cre enzyme (24) during their culture. Subsequently, multiple independent ES cell clones (Eß +/{alpha}) were injected into eight-cell-stage embryos from ICR mice. The chimeric mice were crossed with C57BL/6 mice and mice heterozygous for the mutation (Eß +/{alpha}) were intercrossed to generate homozygotes (Eß {alpha}/{alpha}). For genotyping of mice, the following primer pairs were used: 5'Eß: 5'-GTTAACCAGGCACAGTAGGACC-3' and 3'Eß: 5'-CCATGGTGCATACTGAAGGC-3' for a wild-type allele, and 3'Eß1: 5'-GTTCTGCTGCCATAACTGTTTC-3' and 3'E{alpha}R2: 5'-CTATGTCCAGTCCAGCTTGTG-3' for a mutant allele.

Antibodies and flow cytometry
Biotinylated, and FITC- and phycoerythrin-conjugated antibodies against mouse CD3{varepsilon} (2C11), CD4 (RM4-5), CD8{alpha} (53–6.7), {alpha}ßTCR (H57-597), {gamma}{delta}TCR (GL3), CD25 (IL2R{alpha}) and CD44 (Ly-24) were all purchased from PharMingen (Becton Dickinson, Mountain View, CA). Cells (1–10x104) were acquired using a FACSCalibur (Becton Dickinson) and analyzed with CellQuest software (Becton Dickinson).

Cytoplasmic staining for TCR ß and TCR {gamma}{delta}
Extracellular/intracellular (i.c.) double staining was performed as described previously (25). For enrichment of the DN cells, 1x108 thymocytes stained with biotinylated anti-mouse CD4 plus CD8 were treated with streptavidin-conjugated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and negatively selected through VS+ columns (Miltenyi Biotec) according to the manufacturer's instructions. Purity was confirmed to be >99% by FACS re-analysis (data not shown).

PCR analysis of TCR gene rearrangements
DNA was prepared from fetal thymocytes at days 15.0–19.0 of gestation. Each PCR cycle consisted of incubation at 94°C for 60 s, followed by 90 s annealing at 60–64°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 30 cycles the extension at 72°C was prolonged to 5 min. The 25-µl reaction mixture included 100 ng (or less, as indicated) genomic DNA templates, 10 pmol primers, 0.2 mM dNTPs, 50 mM KCl, 10 mM Tris–HCl (pH 8.0), 2.5 mM MgCl2, 0.01% gelatin and 0.1 U of Gold Taq polymerase (Takara, Tokyo, Japan). Samples were electrophoresed in 1.0% agarose gel, blotted onto nylon membranes (Pall, Port Washington, NY) and probed with end-labeled oligonucleotide probes. The sequences of primers and probes used in this study were as follows. 5'Vß2: 5'-ATGTGGCAGTTTTGCATTCTGTGCC-3', 5'Vß8: 5'-AACACATGGAGGCTGCAGTCACCCAAA-3', 5'Dß1: 5'-GAGGAGCAGCTTATCTGGTGGTTT-3', 5'Dß2: 5'-GTAGGCACCTGTGGGGAAGAAACT-3', 3'Jß1.7: 5'-CACAACCCTTCCAGTCAGAAATG-3', 3'Jß2.7: 5'-GATTCCCTAACCCTTGGTCTACTCCAAAC-3', 5'V{alpha}2: 5'-CAGGAGAAACGTGAC-CAGCAGC-3', 3'J{alpha}32: 5'-TTCTGTTCAGAATCGAGGGACC-3', 5'RAG2: 5'-TTAATTCAACCAGGCTTCTCACTT-3', 3'RAG2: 5'-GCCTGCTTATTGTCTCCTGGTATG-3', Jß1.7 probe: 5'-ATA-CCTGTCACAGTGAGCC-3', Jß2.7 probe: 5'-GGGACCGAAGTACTGTTCATAGG-3', J{alpha}32 probe: 5'-TTAGCCTCTGCCATCTTGATCA-3' and RAG2 probe: 5'-CTCGACTATACACCACGTCAATG-3'.

Southern and Northern blot analyses
Southern and Northern blot analyses were carried out as described previously (28). For verification of proper enhancer replacement in mice, a 560-bp HpaI–NcoI Eß fragment and a 530-bp E{alpha} fragment were used as probes. To detect germline Dß–Jß fragments, the sorted DP cell DNA was digested with HindIII. As probes, a 440-bp BglI–XbaI Dß1–Jß1 intronic fragment, a 330-bp HpaI–EcoRI Dß2–Jß2 intronic fragment and a 0.9-kb SacI fragment of the Cß2 region were used. For Northern blot analysis, a 3.2-kb BamHI–EcoRI genomic fragment within the Jß1 region, a 1.0-kb ClaI–EcoRI genomic fragment downstream of the Jß2 region and ß-actin cDNA (Clontech, Palo Alto, CA) were used. Vß5, Vß8 and Vß14 probes were described previously (28).

In vivo 2C11 treatment of RAG2–/– background mice
Four-week-old mice were injected i.p. with 150 µg of purified anti-CD3{varepsilon} antibody 145-2C11 (2C11). On day 7 post-treatment, the mice were sacrificed to prepare thymocytes; >90% of the cells were at the DP stage (data not shown). RNA from either Eß +/+ RAG2–/– or Eß {alpha}/{alpha} RAG2–/– mice was subjected to Northern blot analysis as described previously (28).

Chromatin immunoprecipitation (Chip) assay
Mononucleosomes were prepared as described (22). The input fraction corresponded to 10% of the chromatin solution and Chip was performed with antibody to diacetylated histone H3 (ARTKQTAR[ACK]STGG[ACK]APRKQL-C)-purified rabbit IgG (Upstate Biotechnology, Lake Placid, NY) or control rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Serial 3-fold dilutions of bound and input DNA fractions were analyzed by 30 cycles of PCR (40 s at 94°C, 40 s at 58–64°C and 60 s at 72°C). The primer sequences are as follows: V5.2LF: 5'-CCAAGAGTACAGAGAGCCCA-3', V5.2LR: 5'-CATGCTTCTTCTCAGGATGC-3', V8.1LF: 5'-GAGAAGTGGTGGAGTGTCTT-3', V8.1LR: 5'-CATCTCAGAACTAAGGCAGG-3', 5'Vß14–2: 5'-ATGCTGTACTCTCTCCTTGCCTTTCTCC-3', 3'Vß14: 5'-AGAGTGGCTGAGAAGCAGCTTCTCCGTG-3', 5'Dß1–2: 5'-AACCCTGCATTAGCTCGCATC-3', 3'Dß1: 5'-CTGCAGAGGTGACGTG AAAGC-3', 3'Dß2: 5'-TTCGTAATTTCCCATGCATGTACGG-3', 5'Cß2F: 5'-AGAGACTCTCATGGTCACAC-3' and 5'Cß2R: 5'-CTGATAACTGTCTGGATCAG-3'. Quantification was performed by ATTO Densito Graph 4.1 software (Atto, Tokyo, Japan).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of mice with targeted replacement of Eß with E{alpha} by homologous recombination
We replaced Eß with E{alpha} by use of the Cre–loxP strategy (Fig. 1AGo). The construct was designed to replace the entire 560-bp HpaI–NcoI Eß fragment with a 530-bp E{alpha} element, both of which have been shown to be functionally minimum (1012,15,19,20). Several targeted TT2 ES cell clones (Eß +/{alpha}N) were isolated from each targeting experiment and used in subsequent analyses. Eß +/{alpha}N ES cell clones were transiently infected by the Cre-expressing recombinant adenovirus AxCANCre (24). Cells with Cre-mediated deletion of the PGK-neor gene were isolated (Eß +/{alpha}). Multiple Eß +/{alpha} TT2 ES cell clones were used to generate chimeric mice for germline transmission, and the offspring were bred to generate Eß +/+, Eß +/{alpha} and Eß {alpha}/{alpha} mice (Fig. 1BGo). To verify the targeting replacement of Eß with E{alpha}, Southern blot analysis was carried out with E{alpha}- and Eß-specific probes (Fig. 1CGo and DGo). As shown in Fig. 1(C)Go, the exogenously introduced E{alpha} element was detected on one allele (Eß +/{alpha}) and on both alleles (Eß {alpha}/{alpha}) at 2 kb as indicated by the increasing hybridizing intensity, including the endogenous 6-kb E{alpha} fragments. On the other hand, Eß +/{alpha} and Eß {alpha}/{alpha} mice were found to lack the Eß element at 10 kb from one allele and from both alleles respectively (Fig. 1DGo). Two independent Eß {alpha}/{alpha} mouse lines generated in this study had no significant differences with respect to measured parameters and, therefore, will not be distinguished in the following sections.



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Fig. 1. Targeted replacement of Eß with E{alpha} by homologous recombination. (A) Schematic diagram of the TCR ß locus (not to scale), targeting vector and targeted allele before and after Cre-mediated deletion of the PGK-neor gene. The targeting vector is composed of 6.7-kb homologous sequences, E{alpha} element, PGK-neor gene flanked by loxP sites (indicated by solid diamonds) and PGK-tk cassette at the 3' end. EcoRI (RI), HindIII (H) and NcoI (N) sites are indicated. (B) PCR analysis for identifying Eß +/+, Eß +/{alpha} and Eß {alpha}/{alpha} mice with primer pairs 5'Eß/3'Eß (indicated as 1/2) and 3'Eß1/3'E{alpha}R2 (indicated as 3/4) for a wild-type allele and a replaced allele respectively. (C and D) Southern blot analysis carried out with the Eß and E{alpha} probes. Tail DNA was digested with EcoRI, and hybridized sequentially first with the E{alpha} probe (C) and then with the Eß probe (D).

 
Ontogenical delay of {alpha}ß T cell development in Eß {alpha}/{alpha} mice
To evaluate the effect of enhancer replacement of Eß with E{alpha} on T cell development, flow cytometric analysis was performed on thymocytes at different ages (Fig. 2AGo). The results demonstrated (i) a striking reduction of the absolute DP cell number in younger Eß {alpha}/{alpha} mice compared to wild-type littermates, and (ii) a normal profile of DP and SP cell subpopulations >4 weeks after birth in Eß {alpha}/{alpha} mice (Fig. 2AGo, 4 weeks). Interestingly, the thymic cellularity ~5 weeks after birth reached a maximum in Eß {alpha}/{alpha} mice (1.6 ± 0.2x108, data not shown), ~1 week later than that in wild-type littermates. These results indicate that enhancer replacement of Eß in the TCR ß locus with E{alpha} caused inefficient thymocyte development.



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Fig. 2. Delayed thymocyte development in Eß {alpha}/{alpha} mice. (A) Flow cytometric analysis of total thymocytes at different time points on a logarithmic scale of fluorescence intensity. Thymocytes (n = 10) were doubly stained with phycoerythrin-conjugated anti-CD8 and CyChrome-conjugated anti-CD4, and representative data are shown. Note that the first obvious appearance of DP cells in wild-type and Eß {alpha}/{alpha} mice was E16.0 and the day of birth respectively. The total cell number and the relative DP cell percentage present per thymus are indicated below each panel and in the upper right corner of the quadrants respectively. (B) Semi-quantitative PCR showing replacement of Eß with E{alpha} impairs ontogenical TCR ß gene rearrangements in fetal thymocytes. The DNA used was from total thymocytes of Eß +/+ or Eß {alpha}/{alpha} mice on days 15.0–19.0 of gestation as well as at 8 weeks old. RAG2 amplification was used for normalizing the DNA concentration and all the PCR analyses were performed in the logarithmic phase. The arrows indicate the Dß2–Jß2 region in the germline configuration. In experiment 2 (Exp. 2), wild-type thymocyte DNA was diluted into wild-type kidney DNA (lanes 1–3).

 
Eß {alpha}/{alpha} mice exhibit a significant but still insufficient level of TCR ß gene rearrangements at the DN stage
The developmental pathway along the DN-to-DP stage is regulated via TCR ß-mediated signals (ß-selection). Therefore, inefficient thymocyte development in Eß {alpha}/{alpha} mice may result from insufficient and/or delayed expression of the TCR ß chains. To address this issue, we examined the initiation of TCR ß gene rearrangements along with the developing stages of fetal thymocytes. For this purpose, we carried out DNA PCR assays with thymocytes at different gestation days so as to obtain semi-quantitative information about gene rearrangements (Fig. 2BGo). As expected, on gestation day 15.0, a significant level of Dß-to-Jß rearrangements was observed in wild-type mice, while Eß {alpha}/{alpha} mice showed reduced Dß-to-Jß rearrangements (Fig. 2BGo, Dß1–Jß1, Dß2–Jß2 and Dß1–Jß2 panels), in spite of the fact that both thymocytes were all composed of DN cells (data not shown). Of particular interest was the observation that among these three rearrangements within Dß–Jß loci, Dß1-to-Jß1 rearrangement was most severely impaired in Eß {alpha}/{alpha} mice compared to the other two (Fig. 2BGo, Exp. 2, lanes 4 and 8). More strikingly, subsequent rearrangements of Vß-to-DJß and V{alpha}-to-J{alpha} were undetectable in Eß {alpha}/{alpha} mice at least until birth (Fig. 2BGo, Vß2-DJß2, Vß8-DJß2 and V{alpha}2–J{alpha}32 panels).

In adult mice, however, the difference in the rearrangement statuses of the TCR ß loci seemed to be less significant between wild-type and Eß {alpha}/{alpha} total thymocytes (Fig. 2BGo, Exp. 1, lanes 5 and 10), which is consistent with the equivalent DP cell populations between the two mouse lines (Fig. 2AGo). These results suggest that the enhancer replacement of Eß with E{alpha} caused inefficient thymocyte development due to delayed initiation and/or lower efficiency of the Dß-to-Jß rearrangements, mainly at the Dß1-to-Jß1 locus, at the DN stage.

The expression of functionally rearranged TCR ß chain genes in Eß {alpha}/{alpha} DN and DP cells
Next, to determine whether E{alpha} on the TCR ß loci is involved in promoting the expression of functionally rearranged TCR ß genes and in the consequent DN-to-DP transition, we performed i.c. staining of the purified Eß {alpha}/{alpha} DN cells with an anti-TCR ß chain mAb. As shown in Fig. 3(A)Go, a significant but smaller number of Eß {alpha}/{alpha} DN cells expressed TCR ß chains at the CD44–/lowCD25+ stage.



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Fig. 3. Expression of the TCR ß chains in Eß {alpha}/{alpha} mice. (A) The purified DN cells (>99% purity) from wild-type and Eß {alpha}/{alpha} mice and RAG2–/– thymocytes were stained with phycoerythrin-conjugated mAb to i.c. TCR ß protein after surface staining with FITC-conjugated anti-CD25 and CyChrome-conjugated anti-CD44, fixation and permeabilization with saponin. The relative percentages of the cells are indicated in each quadrant. Intracellular TCR ß expression in CD44low/–CD25+ cells (as gated by open boxes) is shown by histograms on a logarithmic scale of fluorescence intensity. (B) The purified DN cells from 4-week-old wild-type and Eß {alpha}/{alpha} mice were stained with phycoerythrin-conjugated mAb to i.c. TCR ß protein and CyChrome-conjugated mAb to i.c. TCR {gamma}{delta} protein after surface staining with FITC-conjugated anti-CD25. The total DN cell number per thymus is indicated above each panel. The relative percentages of the TCR ß i.c.+, TCR {gamma}{delta} i.c.+ and TCR ß/TCR {gamma}{delta} i.c.+ cells are indicated in each quadrant. CD25 expressions among TCR {gamma}{delta} i.c.+ and TCR ß i.c.+ cells are indicated by histograms on a logarithmic scale of fluorescence intensity. In the lower histogram panels, the thin and the bold lines indicate TCR ß/TCR {gamma}{delta} i.c. (CD25high control) and TCR ß i.c.+ cells respectively. (C) Intracellular TCR ß expression among CD25+CD44 DN cells, CD25CD44 DN cells and CD4+CD8+ DP cells is indicated by histograms on a logarithmic scale of fluorescence intensity. The bold and thin lines indicate Eß {alpha}/{alpha} cells and wild-type cells respectively. Arrowheads indicate the peaks of fluorescence intensity of i.c. TCR ß expression. The percentages of TCR ß i.c.+ cells among CD25+CD44 DN cells and CD25CD44 DN cells were 17.2 and 51.6% in wild-type mice respectively, and 1.0 and 5.1% in Eß {alpha}/{alpha} mice respectively. Representative data from at least three independent experiments.

 
In wild-type mice, CD25+ DN cells develop into the DP stage through CD25lowCD44 and CD25CD44 stages via signaling through the TCR ß chains on their surface (29). To examine whether Eß {alpha}/{alpha} DN cells undergo the same process, we performed double i.c. staining of the DN cells with antibodies against TCR ß and TCR {gamma}{delta} chains in combination with surface staining for CD25. As shown in Fig. 3(B)Go, the proportions of TCR ß i.c.+ cells in wild-type and Eß {alpha}/{alpha} mice were found to be similar at the CD25+, CD25low and CD25 stages respectively (lower histograms). This result strongly indicates that Eß {alpha}/{alpha} DN cells generate TCR ß chains at exactly the same development stage (CD25+CD44 DN stage) as wild-type DN cells do. It is notable that the fluorescence intensity of the TCR ß chains among TCR ß i.c.+ cells was very similar between wild-type and Eß {alpha}/{alpha} thymocytes at both DN and DP stages (Fig. 3CGo). These results suggest that E{alpha} is functionally equivalent to Eß in promoting expression of functionally rearranged TCR ß chain genes. Furthermore, DP cells in Eß {alpha}/{alpha} mice were all TCR ß i.c.+ (Fig. 3CGo, DP). From these results, we conclude that the development of thymocytes depends on a limited number of DN cells expressing TCR ß chains at the CD25+CD44 stage, resulting in a delayed accumulation of DP cells in Eß {alpha}/{alpha} thymi (Fig. 2AGo).

In contrast to the low cellularity of TCR ß i.c.+ cells, the absolute number of TCR {gamma}{delta} i.c.+ cells was 2- to 3-fold increased in Eß {alpha}/{alpha} DN cells compared to that in wild-type cells over the period considered (Fig. 3BGo and data not shown). These results support the competitive lineage decision model in which insufficient termination signal for TCR ß recombination via ß/pT{alpha} permits further {gamma}{delta}TCR rearrangement (30). Although the possible involvement of the TCR {gamma} chain or a {gamma}/pT{alpha} complex in DN-to-DP transition should be clarified further (25,3135), it is clear that DP cell production in Eß {alpha}/{alpha} mice largely depends on E{alpha} function on the TCR ß locus, since Eß–/– mice generate only a few DP cells (20,36).

Germline transcription of the TCR ß loci in Eß {alpha}/{alpha} DP thymocytes
Next, we analyzed the consequence of enhancer replacement on germline transcription of the TCR ß loci using the Eß {alpha}/{alpha} mice with the RAG2–/– background we had established for this experiment. As shown in Fig. 4Go, the germline transcription of the Dß–Jß–Cß loci in Eß {alpha}/{alpha} RAG2–/– DN cells was suppressed to <5% of that in Eß +/+ RAG2–/– DN cells (Fig. 4Go, Dß1–Jß1, Dß2–Jß2 and Cß panels, cf. lanes 2 and 3). These results indicate that germline transcription at the Dß–Jß-Cß loci largely depends on Eß activity at the DN stage and that E{alpha} can minimally substitute for this Eß function.



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Fig. 4. Germline transcription of the TCR ß loci in Eß {alpha}/{alpha} thymocytes. Total RNAs prepared from Eß +/+ RAG2–/– and Eß {alpha}/{alpha} RAG2–/– thymocytes before (lanes 2 and 3) and after (lanes 4 and 5) 2C11 treatment were subjected to Northern blot analysis using Vß5-, Vß8-, Vß14- Dß1–Jß1-, Dß2–Jß2- and Cß-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). Total RNA purified from C57BL/6 mice was also included for comparison (lane 1). Quality and equal loading amount of RNA were verified by hybridization with a ß-actin cDNA probe (bottom). The positions of 18S and 28S are indicated on the right side of each panel.

 
Treatment of RAG2–/– thymocytes with 2C11 results in the transition of DN cells into the DP stage via signaling through the CD3 complex without a TCR ß chain (37,38). Therefore, we utilized this R2CD3 mouse system for Eß {alpha}/{alpha} RAG2–/– mice to determine the germline transcription of TCR ß loci at the DP stage. At this stage, germline transcription of the Dß–Jß-Cß loci in Eß {alpha}/{alpha} RAG2–/– mice was found to reach wild-type levels (Fig. 4Go, Dß1–Jß1, Dß2–Jß2 and Cß panels, cf. lanes 4 and 5). This result indicates that E{alpha} can fully stimulate the transcription machinery in these loci specifically at the DP stage, although we cannot neglect the possibility that transcription at these loci in this particular situation may be enhancer independent.

In contrast to the Dß–Jß-Cß loci, germline Vß transcription in Eß {alpha}/{alpha} RAG2–/– DN cells was equal to that in Eß +/+ RAG2–/– DN cells; it was highly activated in Vß5 and Vß8 regions, and exceptionally silenced in the Vß14 region (Fig. 4Go, Vß5, Vß8 and Vß14 panels, lanes 2 and 3). In DP cells induced in the R2CD3 mouse system, germline transcription of the Vß regions became suppressed (Vß5 and Vß8) and activated (Vß14) equally between Eß +/+ RAG2–/– and Eß {alpha}/{alpha} RAG2–/– mice (Vß5, Vß8 and Vß14 panels, lanes 4 and 5). These results provide evidence either that E{alpha} on the targeted TCR ß chain locus is functionally equivalent to endogenous Eß in the regulation of germline Vß transcription or that germline Vß transcription is autonomously regulated in an enhancer-independent manner.

Enhancement of histone H3 acetylation in the Dß–Jß loci at the DP stage
Histone acetylation is considered to participate in a common mechanism regulating gene expression by alteration of the chromatin structure, as suggested by increased sensitivity to endonucleases (39) and increased binding of transcription factors (40,41). Recently, two independent studies have shown that E{alpha} and Eß may regulate the histone acetylation status of TCR {alpha}/{delta} loci at the DP stage and TCR ß loci at the DN stage respectively (22,23). These results are consistent with the scenario in which Eß and E{alpha} regulate the transcription and rearrangements of the corresponding TCR loci at the required time, i.e. Eß at the DN stage and E{alpha} at the DP stage, by modulation of the chromatin structure. To further evaluate the stage-specific modulation of the histone acetylation status by Eß and E{alpha}, we first performed Chip assays in DN cells (Fig. 5AGo). In accordance with germline transcription (Fig. 4Go), Eß {alpha}/{alpha} RAG2–/– thymocytes showed remarkably lower acetylation within Dß-containing regions compared to that of Eß +/+ RAG2–/– thymocytes, but it was equal in the Vß regions between the two mouse lines (Fig. 5AGo). These results indicate that histone H3 acetylation of the Dß–Jß loci is well correlated with the germline transcription (Fig. 4Go) and gene rearrangements (Fig. 2BGo) at the DN stage in both mouse lines. In addition, among two Dß regions, the acetylation status of the Dß1 region was more severely impaired in Eß {alpha}/{alpha} mice compared to that of the Dß2 region, indicating that E{alpha} is less efficient on Dß1 than Dß2 for modulating the chromatin structure for recombination.



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Fig. 5. Histone H3 acetylation within TCR ß loci at the DN and DP stages. (A) H3 acetylation was investigated in thymocytes from Eß +/+ RAG2–/– and Eß {alpha}/{alpha} RAG2–/– by Chip assays. Chip was performed with anti-AcH3 or normal rabbit IgG (control), and serial 3-fold dilutions of bound and input fractions were analyzed by PCR. H3 acetylation was quantified by an image analyzer and plotted as acetylation index, which was calculated using the formula: [(binding to {alpha}AcH3 antibody) – (binding to control IgG)]/(input). (B) H3 acetylation was measured as in (A) in the sorted DP thymocytes from wild-type and Eß {alpha}/{alpha} mice. TEA was included as hyperacetylated control at the DP stage (22).

 
Next, we analyzed the acetylation status of the Dß–Jß loci in Eß {alpha}/{alpha} DP cells (Fig. 5BGo). To distinguish between rearranged and non-rearranged Dß regions, we designed primer pairs within intron regions 5' and 3' to Dß loci, which are deleted when recombination occurs. In contrast to the DN stage, both Dß1 and Dß2 regions of the sorted Eß {alpha}/{alpha} DP cells were significantly hyperacetylated, a consistent finding with activated germline transcription of these loci at the DP stage in R2CD3 mice system (Fig. 4Go, Dß1–Jß1 and Dß2–Jß2 panels). These results indicate that E{alpha} rendered chromatin opening of the Dß–Jß loci in a stage-specific manner at the DP stage. To obtain definitive conclusions about the stage-specificity of Eß and E{alpha} on chromatin remodeling, we have to await analysis of cells of matched surface phenotype. However, considering the transcription and histone acetylation status of the Dß–Jß loci (Figs 4Go and 5BGo), it is assumed that these loci are not heterochromatinized, but rather that they are accessible to the recombinase at the DP stage.

Limited impact of chromatin remodeling on Dß-to-Jß recombination at the DP stage
We then asked whether or not Dß-to-Jß rearrangements successfully associate with histone hyperacetylation at the DP stage. To evaluate this issue, we performed Southern blot analysis using the sorted DP cells (Fig. 6Go). Interestingly, a high proportion of Eß {alpha}/{alpha} DP cells retained the germline configuration at the Dß–Jß loci (Fig. 6BGo), although these loci were transcriptionally active and histone-hyperacetylated by E{alpha} (Figs 4Go and 5BGo). These results may indicate that further chromatin opening of the Dß–Jß loci is not fully associated with the rearrangements of these loci at the DP stage. In wild-type DP cells, the germline Dß–Jß configuration was similarly detected, although the Dß1–Jß1 locus was at a lower level than that in Eß {alpha}/{alpha} DP cells, presumably due to more efficient Vß-to-DJß rearrangements in wild-type thymocytes, which eliminate Dß1–Jß1 locus more frequently (Fig. 6AGo, Dß1–Jß1 panel and B). These data imply that down-regulation of Dß-to-Jß recombination may also physiologically function to prevent further Dß-to-Jß rearrangements in wild-type DP cells.



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Fig. 6. Southern blot analysis of Dß-to-Jß rearrangements. (A) The sorted DP cell DNA from 4-week-old Eß +/+ (lanes 2–6) and Eß {alpha}/{alpha} mice (lanes 7–11) was digested with HindIII (indicated as H) to examine Dß1-to-Jß1 and Dß2-to-Jß2 rearrangements. The positions of the probes are indicated by solid bars. The Southern blot was sequentially hybridized with Dß1–Jß1 intronic probe (upper panel) and then Dß2–Jß2 intronic probe (middle panel). The Cß2 fragment was used for normalization of the hybridizing intensity (lower panel). C57BL/6 kidney DNA (K) was loaded for non-rearranging control (lane 1). The intensity of the germline hybridization signal relative to the total genomic DNA content (Rel. hybri. %) is indicated below each panel. (B) Phosphorimager quantification of germline Dß–Jß loci in Eß +/+ and Eß {alpha}/{alpha} DP cells from the panels in (A).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
To clarify the regulatory mechanism of TCR ß gene recombination beyond the DN stage under modified enhancer activity, we generated mutant mice with targeted replacement of Eß with E{alpha} on the TCR ß locus. By analyzing these enhancer-replaced mice, we have provided evidence that Dß–Jß loci are retained in the germline configuration at the DP stage, leaving these loci accessible to the recombinase.

Several lines of evidence have previously indicated that Eß and E{alpha} are required for immature thymocytes to develop beyond the DN and DP stage respectively, presumably through chromatin remodeling of the corresponding loci (1923). However, it was not clarified how the DNA status of the TCR ß loci is modulated beyond the DN stage after functional V(D)J rearrangements occur on the first allele.

To assess this issue, Eß {alpha}/{alpha} mice established in this study were very useful because Eß {alpha}/{alpha} thymocytes can develop normally from DN through the DP to SP stages (Fig. 2AGo) in more physiological conditions compared to the previous transgenic mouse models. In contrast to the previous predictions, we demonstrated in this study that E{alpha} can substitute for Eß function for both transcription and rearrangements of the TCR ß loci at the DN stage, albeit at lower efficiency (Figs 2BGo and 4Go), resulting in the detectable expression of the TCR ß chains at the CD25+ DN stage (Fig. 3Go). Although the proportion of TCR ß+ CD25+ DN cells was smaller in mutant mice, they must selectively develop into the DP stage via ß-selection, because all mutant DP cells expressed TCR ß as high as wild-type cells (Fig. 3CGo).

Interestingly, the Dß–Jß loci at the DP stage were found to retain the germline configuration at high frequencies (Fig. 6Go) in spite of fully activated germline transcription (Fig. 4Go) and histone hyperacetylation (Fig. 5BGo) of the loci irrespective of the associated enhancer. The substantial presence of the germline configuration of the Dß–Jß loci in DP cells implies that DN cells with functional Vß-to-DJß rearrangement on the first allele are immediately driven into the DP stage where germline or the DJß segments may not be subjected to further Dß-to-Jß or Vß-to-DJß rearrangements. Although it is still unclear how the Dß-to-Jß recombination is limited under fully accessible situation at the DP stage, this mechanism may be fundamentally related to TCR ß allelic exclusion. In fact, the Dß2–Jß2 locus remained in the germline configuration as in wild-type and Eß {alpha}/{alpha} DP cells (Fig. 6Go), even though wild-type DN cells showed more efficient Dß–Jß recombination compared to that in mutant DN cells before the expression of surface TCR ß chains (Fig. 2BGo).

At face value, the rearrangement frequencies of the two Dß–Jß loci were distinct between wild-type and Eß {alpha}/{alpha} DP cells (Fig. 6BGo). There are several possible explanations for this. First, the rearrangement frequency at the DN stage is critical and the remaining germline Dß–Jß loci in the Eß {alpha}/{alpha} DP cells may simply reflect the character of DN cells, in which further rearrangements were down-regulated. Indeed, Dß1-to-Jß1 rearrangement was more severely impaired than that of Dß2-to-Jß2 rearrangement in Eß {alpha}/{alpha} mice at the DP stage as well as the DN stage (Figs 2BGo, Exp. 2 and 6). This could be explained by a hypothesis that E{alpha} on the targeted TCR ß allele differentially regulates these two loci; E{alpha} activates more dominantly the Dß2–Jß2 locus than the Dß1–Jß1 locus as evidenced by the histone acetylation status of these two loci (Fig. 5Go). Second, the low frequency of Vß-to-DJß recombination at the DN stage has a lower contribution in eliminating the germline Dß1–Jß1 locus in Eß {alpha}/{alpha} mice, leaving more germline Dß1–Jß1 configuration in Eß {alpha}/{alpha} DP cells. Finally, E{alpha} shows a stage-specific activation on the targeted Dß–Jß loci at the DP stage; however, its role has a differential potential for the two Dß–Jß loci—more efficient on the Dß2–Jß2 locus than the Dß1–Jß1 locus, probably due to the relative proximity of these two regions to E{alpha} on the targeted TCR ß allele. In either case, it is clear from the present study that a certain population of the DP cells retains the germline Dß–Jß configuration under a fully accessible situation of these loci to the recombinase, by which TCR {alpha} loci are rearranged.

We also showed that germline transcription of the Vß segment received regulation distinct from that of the Dß–Jß loci both at the DN and DP stages in Eß {alpha}/{alpha} mice (Fig. 4Go). Although the transcriptional activation for Dß–Jß loci in Eß{alpha}/{alpha} mice was drastically accelerated up to a level equivalent to that in wild-type mice at the DP stage, Vß transcription, except for Vß14, was high at the DN stage but very low at the DP stage, and this suppression of Vß transcription along with DN-to-DP transition was not affected at all by enhancer replacement (Fig. 4Go). Thus, a reasonable interpretation may be that Vß transcription is independent of enhancer function and is a matter of autonomous regulation at the corresponding loci through development from the DN-to-DP stages. This speculation is consistent with the current report that Vß transcription does not depend on Eß, although their analysis was restricted to the DN stage using Eß–/– RAG–/– thymocytes (23).

A very recent report suggested the existence of a possible 20-bp fragment in the Eß element that regulates germline transcription within the transgenic array without recombination activity (42). This result allows us to speculate on the existence of similar sequences within E{alpha} which are activated at the DP stage for transcription but not for recombination of the Dß–Jß loci. In addition, it is also possible to hypothesize that DNA substrates targeted by RAG during thymocyte development are changed in a stage-specific manner. This model would also explain why Dß–Jß loci are not fully rearranged at the DP stage in spite of being accessible to the recombinase.

In the present work, we clearly showed that the absolute number of {gamma}{delta} T cells is increased in Eß {alpha}/{alpha} thymi compared to that of wild-type littermates (Fig. 3BGo). Estimated from Southern blot analysis, only ~22% of Eß {alpha}/{alpha} thymocytes underwent Vß-to-DJß or Dß-to-Jß rearrangement on the second allele (Fig. 6Go). This result indicates that the cells, which failed productive V(D)Jß rearrangement on the first allele, may be selectively committed to the {gamma}{delta} lineage in Eß {alpha}/{alpha} thymi because of the prolonged duration for {gamma}{delta} rearrangements before the second V(D)Jß rearrangement takes place. The fact that all of pT{alpha}–/–, Cß–/–, Eß–/– and Eß {alpha}/{alpha} mice showed similar increments of thymic {gamma}{delta} T cell numbers (34,36,43 and this study) strongly suggest that abrogated pre-TCR signaling provides a longer duration for the TCR {gamma}{delta} rearrangements, leading to the increase in {gamma}{delta} T cell number.

In summary, we have analyzed TCR ß gene activities during early thymocyte development under modified enhancer activity. Our data demonstrated that E{alpha} can substitute for Eß function exactly at the same CD25+ DN stage and that Eß was not critical for the expression of functionally rearranged TCR ß chain genes through development into the DP stage. In addition, our data imply that there exists a novel, presumably Eß-independent mechanism that down-regulates further Dß-to-Jß recombination during the transition into the DP stage. Clarifying this issue will provide us with a better understanding of the precise mechanisms of the V(D)J recombination machinery.


    Abbreviations
 
Chip chromatin immunoprecipitation
DN double negative
DP double positive
ES embryonic stem
i.c. intracellular
pT{alpha} pre-TCR {alpha}
SP single positive
TEA T early {alpha}

    Notes
 
Transmitting editor: T. Watanabe

Received 6 June 2000, accepted 3 August 2001.


    References
 Top
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 Introduction
 Methods
 Results
 Discussion
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