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
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Abstract |
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Keywords: allelic exclusion, germline transcription, histone acetylation, rearrangement, TCR enhancer (E
), TCR ß enhancer (Eß)
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Introduction |
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The regulatory machinery which allows the TCR ß gene rearrangement to precede that of the TCR 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
locus also contains one fundamental transcriptional enhancer (E
), which lies just downstream of the TCR
constant region gene (C
) (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),
light chain enhancer (E
) and even by SV40 enhancer in Dß-to-Jß recombination (1618). It is also suggested that E
becomes active later than Eß and promotes the accessibility of the transgenic substrates in the
ß lineage but not in the
lineage (15). Thus, Eß and E
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 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
ß T cell development (19,20). Elimination of E
resulted in dramatic inhibition of J
transcription and TCR
rearrangement, and, as a result, cells were blocked at a stage of development just prior to TCR
expression, although normal numbers of thymocytes were generated (21). More recently, Eß and E
have been suggested to regulate V(D)J recombination through modulation of the histone acetylation status of the corresponding loci at the required timingwith Eß acting early at the DN stage and E
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. 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
is functionally equivalent to Eß in promoting expression of functionally rearranged TCR ß chain genes.
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Methods |
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Antibodies and flow cytometry
Biotinylated, and FITC- and phycoerythrin-conjugated antibodies against mouse CD3 (2C11), CD4 (RM4-5), CD8
(536.7),
ßTCR (H57-597),
TCR (GL3), CD25 (IL2R
) and CD44 (Ly-24) were all purchased from PharMingen (Becton Dickinson, Mountain View, CA). Cells (110x104) were acquired using a FACSCalibur (Becton Dickinson) and analyzed with CellQuest software (Becton Dickinson).
Cytoplasmic staining for TCR ß and TCR
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 15.019.0 of gestation. Each PCR cycle consisted of incubation at 94°C for 60 s, followed by 90 s annealing at 6064°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 TrisHCl (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'V2: 5'-CAGGAGAAACGTGAC-CAGCAGC-3', 3'J
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
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 HpaINcoI Eß fragment and a 530-bp E fragment were used as probes. To detect germline DßJß fragments, the sorted DP cell DNA was digested with HindIII. As probes, a 440-bp BglIXbaI Dß1Jß1 intronic fragment, a 330-bp HpaIEcoRI Dß2Jß2 intronic fragment and a 0.9-kb SacI fragment of the Cß2 region were used. For Northern blot analysis, a 3.2-kb BamHIEcoRI genomic fragment within the Jß1 region, a 1.0-kb ClaIEcoRI 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 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
/
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 5864°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'Vß142: 5'-ATGCTGTACTCTCTCCTTGCCTTTCTCC-3', 3'Vß14: 5'-AGAGTGGCTGAGAAGCAGCTTCTCCGTG-3', 5'Dß12: 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).
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Results |
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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 /
total thymocytes (Fig. 2B
, Exp. 1, lanes 5 and 10), which is consistent with the equivalent DP cell populations between the two mouse lines (Fig. 2A
). These results suggest that the enhancer replacement of Eß with E
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 /
DN and DP cells
Next, to determine whether E 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
/
DN cells with an anti-TCR ß chain mAb. As shown in Fig. 3(A)
, a significant but smaller number of Eß
/
DN cells expressed TCR ß chains at the CD44/lowCD25+ stage.
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In contrast to the low cellularity of TCR ß i.c.+ cells, the absolute number of TCR i.c.+ cells was 2- to 3-fold increased in Eß
/
DN cells compared to that in wild-type cells over the period considered (Fig. 3B
and data not shown). These results support the competitive lineage decision model in which insufficient termination signal for TCR ß recombination via ß/pT
permits further
TCR rearrangement (30). Although the possible involvement of the TCR
chain or a
/pT
complex in DN-to-DP transition should be clarified further (25,3135), it is clear that DP cell production in Eß
/
mice largely depends on E
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 /
DP thymocytes
Next, we analyzed the consequence of enhancer replacement on germline transcription of the TCR ß loci using the Eß /
mice with the RAG2/ background we had established for this experiment. As shown in Fig. 4
, the germline transcription of the DßJßCß loci in Eß
/
RAG2/ DN cells was suppressed to <5% of that in Eß +/+ RAG2/ DN cells (Fig. 4
, Dß1Jß1, Dß2Jß2 and Cß panels, cf. lanes 2 and 3). These results indicate that germline transcription at the DßJß-Cß loci largely depends on Eß activity at the DN stage and that E
can minimally substitute for this Eß function.
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In contrast to the DßJß-Cß loci, germline Vß transcription in Eß /
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. 4
, 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
/
RAG2/ mice (Vß5, Vß8 and Vß14 panels, lanes 4 and 5). These results provide evidence either that E
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 DßJß 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 and Eß may regulate the histone acetylation status of TCR
/
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
regulate the transcription and rearrangements of the corresponding TCR loci at the required time, i.e. Eß at the DN stage and E
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
, we first performed Chip assays in DN cells (Fig. 5A
). In accordance with germline transcription (Fig. 4
), Eß
/
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. 5A
). These results indicate that histone H3 acetylation of the DßJß loci is well correlated with the germline transcription (Fig. 4
) and gene rearrangements (Fig. 2B
) 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
/
mice compared to that of the Dß2 region, indicating that E
is less efficient on Dß1 than Dß2 for modulating the chromatin structure for recombination.
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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. 6). Interestingly, a high proportion of Eß
/
DP cells retained the germline configuration at the DßJß loci (Fig. 6B
), although these loci were transcriptionally active and histone-hyperacetylated by E
(Figs 4
and 5B
). These results may indicate that further chromatin opening of the DßJß loci is not fully associated with the rearrangements of these loci at the DP stage. In wild-type DP cells, the germline DßJß configuration was similarly detected, although the Dß1Jß1 locus was at a lower level than that in Eß
/
DP cells, presumably due to more efficient Vß-to-DJß rearrangements in wild-type thymocytes, which eliminate Dß1Jß1 locus more frequently (Fig. 6A
, Dß1Jß1 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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Discussion |
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Several lines of evidence have previously indicated that Eß and E 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 /
mice established in this study were very useful because Eß
/
thymocytes can develop normally from DN through the DP to SP stages (Fig. 2A
) in more physiological conditions compared to the previous transgenic mouse models. In contrast to the previous predictions, we demonstrated in this study that E
can substitute for Eß function for both transcription and rearrangements of the TCR ß loci at the DN stage, albeit at lower efficiency (Figs 2B
and 4
), resulting in the detectable expression of the TCR ß chains at the CD25+ DN stage (Fig. 3
). 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. 3C
).
Interestingly, the DßJß loci at the DP stage were found to retain the germline configuration at high frequencies (Fig. 6) in spite of fully activated germline transcription (Fig. 4
) and histone hyperacetylation (Fig. 5B
) of the loci irrespective of the associated enhancer. The substantial presence of the germline configuration of the DßJß 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 Dß2Jß2 locus remained in the germline configuration as in wild-type and Eß
/
DP cells (Fig. 6
), even though wild-type DN cells showed more efficient DßJß recombination compared to that in mutant DN cells before the expression of surface TCR ß chains (Fig. 2B
).
At face value, the rearrangement frequencies of the two DßJß loci were distinct between wild-type and Eß /
DP cells (Fig. 6B
). There are several possible explanations for this. First, the rearrangement frequency at the DN stage is critical and the remaining germline DßJß loci in the Eß
/
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
/
mice at the DP stage as well as the DN stage (Figs 2B
, Exp. 2 and 6). This could be explained by a hypothesis that E
on the targeted TCR ß allele differentially regulates these two loci; E
activates more dominantly the Dß2Jß2 locus than the Dß1Jß1 locus as evidenced by the histone acetylation status of these two loci (Fig. 5
). Second, the low frequency of Vß-to-DJß recombination at the DN stage has a lower contribution in eliminating the germline Dß1Jß1 locus in Eß
/
mice, leaving more germline Dß1Jß1 configuration in Eß
/
DP cells. Finally, E
shows a stage-specific activation on the targeted DßJß loci at the DP stage; however, its role has a differential potential for the two DßJß locimore efficient on the Dß2Jß2 locus than the Dß1Jß1 locus, probably due to the relative proximity of these two regions to E
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 DßJß configuration under a fully accessible situation of these loci to the recombinase, by which TCR
loci are rearranged.
We also showed that germline transcription of the Vß segment received regulation distinct from that of the DßJß loci both at the DN and DP stages in Eß /
mice (Fig. 4
). Although the transcriptional activation for DßJß loci in Eß
/
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. 4
). 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 which are activated at the DP stage for transcription but not for recombination of the DßJß 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 DßJß 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 T cells is increased in Eß
/
thymi compared to that of wild-type littermates (Fig. 3B
). Estimated from Southern blot analysis, only ~22% of Eß
/
thymocytes underwent Vß-to-DJß or Dß-to-Jß rearrangement on the second allele (Fig. 6
). This result indicates that the cells, which failed productive V(D)Jß rearrangement on the first allele, may be selectively committed to the
lineage in Eß
/
thymi because of the prolonged duration for
rearrangements before the second V(D)Jß rearrangement takes place. The fact that all of pT
/, Cß/, Eß/ and Eß
/
mice showed similar increments of thymic
T cell numbers (34,36,43 and this study) strongly suggest that abrogated pre-TCR signaling provides a longer duration for the TCR
rearrangements, leading to the increase in
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 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.
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Abbreviations |
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Chip chromatin immunoprecipitation |
DN double negative |
DP double positive |
ES embryonic stem |
i.c. intracellular |
pT![]() ![]() |
SP single positive |
TEA T early ![]() |
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Notes |
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Received 6 June 2000, accepted 3 August 2001.
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References |
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