Assessing the Role of the T Cell Receptor beta  Gene Enhancer in Regulating Coding Joint Formation during V(D)J Recombination*

Noëlle MathieuDagger, Salvatore Spicuglia, Sophie Gorbatch§, Olivier Cabaud, Corinne Fernex, Christophe Verthuy, William M. Hempel, Anne-Odile Hueber||, and Pierre Ferrier**

From the Centre d'Immunologie de Marseille-Luminy, INSERM, CNRS, Université de la Méditerranée, 13288 Marseille, France

Received for publication, December 11, 2002, and in revised form, February 4, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To assess the role of the T cell receptor (TCR) beta  gene enhancer (Ebeta ) in regulating the processing of VDJ recombinase-generated coding ends, we assayed TCRbeta rearrangement of Ebeta -deleted (Delta Ebeta ) thymocytes in which cell death is inhibited via expression of a Bcl-2 transgene. Compared with Delta Ebeta , Delta Ebeta Bcl-2 thymocytes show a small accumulation of TCRbeta standard recombination products, including coding ends, that involves the proximal Dbeta -Jbeta and Vbeta 14 loci but not the distal 5' Vbeta genes. These effects are detectable in double negative pro-T cells, predominate in double positive pre-T cells, and correlate with regional changes in chromosomal structure during double negative-to-double positive differentiation. We propose that Ebeta , by driving long range nucleoprotein interactions and the control of locus expression and chromatin structure, indirectly contributes to the stabilization of coding ends within the recombination processing complexes. The results also illustrate Ebeta -dependent and -independent changes in chromosomal structure, suggesting distinct modes of regulation of TCRbeta allelic exclusion depending on the position within the locus.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

V(D)J recombination, one example of developmentally regulated DNA rearrangement known to occur in higher eukaryotes, is required for T cell receptor (TCR)1 and Ig gene assembly and for T and B lymphocyte differentiation. This process is mediated by an enzymatic complex (the VDJ recombinase) whose targets (the recombination signal sequences or RSSs) flank dispersed V, D, and J gene segments and consist of conserved seven- and nine-nucleotide sequences (the heptamer and nonamer) separated by a non-conserved 12- or 23-nucleotide spacer (1). The recombination-activating-gene (RAG)-1 and -2 products constitute the core components of the recombinase (2). The RAG genes (possibly together with the original RSSs) are thought to have been transferred, in the form of a composite transposon, from the prokaryotic world to the germline of a common ancestor of the jawed vertebrates (3).

V(D)J recombination has been divided into two phases, based on in vitro recombination studies and the biochemical characterization of rearrangement products (4). In the first phase, the RAG factors (assisted by architectural factors; i.e. the high mobility group-1 and -2 proteins) initiate recombination by binding to, and introducing DNA cleavage at, two RSSs with spacers of dissimilar lengths. Ensuing DNA double strand breaks (DSBs) yield two pairs of products that consist of 5'-phosphorylated, blunt-ended RSSs (called signal ends, SEs), and the hairpinned, adjacent coding sequences (called coding ends, CEs). In a second phase, which depends on the coordinated action of the RAG and DNA repair non-homologous end-joining (NHEJ) factors (including Ku70/86, DNA-PK/Artemis, XRCC4, and DNA ligase IV), the two CEs are rapidly processed (this involves opening of the hairpins and, often, deletion and/or addition of nucleotides) and ligated to form a coding joint (CJ). With slower kinetics, the SEs are precisely joined to form a signal joint (SJ). As in the case of various recombination systems in prokaryotes (5), synaptic complexes of V(D)J recombination have been characterized that contain all or part of the aforementioned nucleotide sequences and catalytic factors (reviewed in Ref. 6).

V(D)J recombination is confined to immature lymphocytes because of the restricted expression of the RAG genes. In addition, it is tightly controlled with respect to lymphoid cell lineage and within a given lineage to the developmental stage and possibly also the TCR/Ig allele used. For example, the TCRbeta and TCRalpha genes (assembled from Vbeta , Dbeta , and Jbeta and from Valpha and Jalpha gene segments, respectively) are, with a few exceptions, rearranged exclusively in the T cell lineage, with TCRbeta gene rearrangement in double negative (DN) pro-T cells preceding that of TCRalpha in double positive (DP) pre-T cells. Moreover, at the TCRbeta locus, Dbeta -to-Jbeta rearrangement occurs first in CD44+CD25+ DN thymocytes and, presumably, simultaneously on both alleles, followed by complete Vbeta -to-DJbeta assembly in more mature CD44-/loCD25+ cells, possibly with no allele synchronicity. Formation of a productive Vbeta -to-DJbeta joint (i.e. that maintains an open reading frame within the TCRbeta gene) and expression of a TCRbeta chain (conditional for alpha beta lineage normal development past the CD44-/loCD25+ DN stage) result in the arrest of further Vbeta rearrangement to mediate TCRbeta allelic exclusion (7). To a large extent, these controls are thought to involve the regulated modulation of RSS accessibility to the recombinase (8). The findings, using transgenic and knock-out mice, that transcriptional regulatory elements (enhancers/promoters) modulate cis-rearrangement and chromatin structure at TCR/Ig gene segments and/or loci gave a first hint toward an understanding of how recombinational accessibility is achieved (4, 9).

In the mouse germline, the ~500-kb TCRbeta locus consists of ~35 distinct Vbeta genes that, for the most part, are spread over a large DNA region extending from 200-450 kb upstream of the duplicated Dbeta 1-Jbeta 1-Cbeta 1/Dbeta 2-Jbeta 2-Cbeta 2 clusters, except for one (Vbeta 14), which lies, in opposite orientation, ~10 kb downstream (10). A single TCRbeta gene enhancer (Ebeta ) has been described that is located within the Cbeta 2-Vbeta 14 intervening region (11). Targeted deletion of Ebeta has revealed a striking phenotype. In the T cell lineage, Dbeta -to-Jbeta CJs are drastically reduced at the targeted TCRbeta allele(s) (>50-100-fold compared with the wild-type (wt)), with an even more severe defect in Vbeta -to-DJbeta CJs. In homozygously deleted (Ebeta -/-, hereafter Delta Ebeta ) mice, no TCRbeta chains are made, and no alpha beta T cells can develop (12-14). Moreover, comparative analysis of molecular markers for chromatin structure in developmentally arrested DN, CD25+ pro-T cells from either Rag-/- (hereafter Rag) or combinatorial (Rag-/- × Delta Ebeta ; Rag Delta Ebeta ) mice provided compelling evidence for a primary function of Ebeta in regulating chromatin opening within a limited (~25 kb) upstream domain comprised of the Dbeta -Jbeta -Cbeta clusters, with a minor effect on the 5' distal Vbeta genes or 3' proximal Vbeta 14 (15). However, RAG-mediated SEs at Dbeta and Jbeta gene segments (as well as the corresponding SJs) can readily be detected at Ebeta -deleted alleles, although at a level 10-30-fold lower compared with the wt (16). The facts that TCRbeta rearrangement was initiated in Delta Ebeta thymocytes, but that formation of CJs may be more severely impaired, suggested an additional function for Ebeta in CE processing. Here, using Delta Ebeta mice expressing an anti-apoptotic Bcl-2 transgene, we attempt to better delineate the actual impact of Ebeta in enhancing DNA repair/CJ formation during TCRbeta locus recombination, relative to its effects on chromosomal accessibility. Our findings are consistent with a model in which Ebeta impinges on the stabilization of CEs within the post-cleavage synaptic complex, in addition to its primary functions in regulating chromosomal access and locus expression.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mice-- Single knockout, RAG-1-deficient (Rag), Ebeta -deleted (Delta Ebeta ) mice, and double knockout (Rag Delta Ebeta ) mice, as well as mouse housing and analyzing conditions, were as described previously (16). wt C57BL/6J and CB17 SCID (Scid) mice, transgenic Eµ-Bcl-2 (B2) and P14 TCRbeta /Vbeta 8.1-DJbeta 2.4 (p14) mice (17, 18), and combinatorial knock-out and transgenic (Delta Ebeta B2) mice were handled similarly.

Thymocyte Preparation and Cell Culture Conditions-- Thymocyte preparation and cell culture have been described previously (15). 150 µg of anti-CD3-epsilon (2C11; Pharmingen) monoclonal antibody were utilized for intraperitoneal injection of 4-week-old animals.

Flow Cytofluorometry Analyses and Cell Sorting-- Cell-staining conditions, flow cytometric analyses, and cell purification by cell sorting were carried out as described by Leduc et al. (14).

Molecular Analyses of V(D)J Recombination Products and Chromatin Structure at the TCRbeta Locus-- Nucleic acid extraction, assays for SE, SJ, CJ, and hybrid joints (HJ) products, sequencing, and RT-PCR analyses, as well as ligation-mediated (LM)-PCR analysis of restriction enzyme accessibility and chromatin immunoprecipitation (ChIP)-PCR analysis of histone H3 acetylation were performed as described previously (13, 15, 16). Assays for CE products were performed according to Zhu et al. (19), with PCR products for CEs/SEs being separated through polyacrylamide gels (instead of agarose gels, as for the analysis of the amplified products in all other PCR assays). All PCR experiments were performed at least twice with consistent results. A list of oligonucleotide primers used in these experiments is available upon request.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production of the Delta Ebeta B2 Mice and Characterization of Their TCRbeta Gene Recombination Profile-- A defect in resolving RAG-mediated DSBs that form at Ebeta -deleted (Ebeta -) alleles must result in cell death of the particular thymocytes. This could mask the actual levels of TCRbeta recombination products (e.g. SEs) in Delta Ebeta thymi and the role played by Ebeta in promoting RSS accessibility versus post-cleavage assembly in vivo. To overcome this problem, we analyzed TCRbeta gene recombination at Ebeta - alleles in the situation where cell death is inhibited; Delta Ebeta mice were bred with mice that express an anti-apoptotic human Bcl-2 transgene (Tg Bcl-2) in T lineage cells (B2 mice) (17). Cell counting and flow cytometric analysis indicated that constitutive Bcl-2 expression results in a slightly reduced proportion of DN cells in Delta Ebeta B2 versus Delta Ebeta thymi (from ~33 to 25.6%) and an increase in that of DP cells (from ~58 to 69%) although TCRbeta + and genuine single positive cells are still missing (Table I). Moreover, Bcl-2 expression was found to prolong cell survival of Ebeta -deleted DN and DP thymocytes without rescuing the CD44-/loCD25+ DN developmental block and accompanying cell proliferation defect (data not shown). These findings are in agreement with those from earlier studies demonstrating that Tg Bcl-2 expression inhibits cell death without substituting for major selection processes in the developing T cells such as pre-TCR-based beta -selection or TCRalpha beta -based positive selection (17, 20-22).


                              
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Table I
Absolute numbers and percentages of thymocytes in wt, Delta Ebeta , and Delta Ebeta B2 mice
Average cell numbers (upper lanes) and percentages (bottom lanes) for the individual cell subsets were calculated from the indicated number (n) of mice for each strain. CD4+ and CD8+ cell stainings within the Delta Ebeta and Delta Ebeta B2 single positive (SP) subsets were mostly of low to intermediate intensities, as opposed to those within the wt SP subsets that demonstrated mostly CD4high and CD8high cells. Three color flow cytometric analysis further demonstrated that CD4+ CD8- and CD4- CD8+ cells within the Delta Ebeta B2 thymus do not express cell-surface TCRbeta indicating that genuine SP thymocytes are still missing in this strain.

We started our evaluation of TCRbeta gene rearrangement in Tg Bcl-2-expressing thymocytes using well defined, semi-quantitative PCR assays to test for SE, SJ, and CJ products at the Dbeta 2-Jbeta 2 cluster (Fig. 1A). In agreement with earlier findings (16), both 3' Dbeta 2 SEs and Dbeta 2-to-Jbeta 2.6 SJs were detected in Delta Ebeta thymocytes, at decreased levels compared with the wt (Fig. 1, B (lanes 1-5) and C, lanes 1-12), whereas Dbeta 2-to-Jbeta 2.1/Jbeta 2.6 CJs appeared to be more severely reduced (Fig. 1D, lanes 1-5). According to PhosphorImager scanning and densitometric analysis (Fig. 1E), SEs and SJs without Ebeta were reduced to ~16-20% of those in wt thymi whereas CJs were reduced to ~7%, arguing that Ebeta deletion indeed impacts on Dbeta /Jbeta RSS cleavage with an additional, weaker effect on CE resolution. Significantly, all the three types of recombination products were amplified at increased levels in Delta Ebeta B2 compared with Delta Ebeta thymocytes, including Dbeta -to-Jbeta CJs (Fig. 1, B, C, and D, lanes 5-7, 9-20, and 5-7, respectively). By densitometric comparison of Delta Ebeta and Delta Ebeta B2 thymocytes (Fig. 1E), we estimated that SEs and SJs increased from, respectively, ~16 to 29% (~1.8×) and ~20 to 26% (~1.3×) relative to those in wt cells, whereas CJs showed a greater rescue, from 7 to 30% (~4.3×). Overall, the effect of Bcl-2 in rescuing Dbeta -to-Jbeta CJs may thus result from a small accumulation of intermediate SE/CE products combined to a specific, additional enhancement of CE processing (formally, because the CJ effect should be the product of SE/CE formation (or abundance) and CE resolution, the apparent effect on CE resolution in this case could be ~2.4× (4.3/1.8) only). Consistent with this, the germline fragment containing Dbeta 2/Jbeta 2 gene segments was detected at high levels in both Delta Ebeta and Delta Ebeta B2 but not wt thymocytes (Fig. 1D). Also, as judged from similar SE and CJ assays, recombinase activity in Delta Ebeta B2 cells generally appears less marked within the Dbeta 1-Jbeta 1 cluster compared with Dbeta 2-Jbeta 2 (data not shown).


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Fig. 1.   Dbeta 2-to-Jbeta 2 SE, SJ, and CJ products in Delta Ebeta B2 thymocytes. A, schematic representation of the PCR assays used to analyze SE, SJ, and CJ products within the Dbeta 2-Jbeta 2 gene cluster. RSSs of 23- or 12-nucleotide spacer are figured by shaded and open triangles, respectively. Oligonucleotide primers and the linker used in LM-PCR are schematized by horizontal arrows and asymmetric pairs of bold lines. B, genomic DNA from thymocytes of the indicated mice (including two Delta Ebeta B2 individuals) was analyzed by LM-PCR for SEs 3' of Dbeta 2 (Dbeta SE). Lanes 1-4, serial dilution analysis using linker-ligated DNA from a wt thymus and/or kidney (lane 1, undiluted thymus; lanes 2 and 3, thymus/kidney, 1/5 and 1/25 dilutions; lane 4, undiluted kidney). PCR amplifications for a Cbeta 2-containing DNA fragment (Cbeta ) were carried out in parallel to control for sample loading. C, DNA samples were analyzed by PCR for Dbeta 2-to-Jbeta 2.1 SJs (DJbeta SJ). Digestion of amplified DNAs by the restriction enzyme ApaLI (arrows) was performed to check the accuracy of RSS ligation to form the SJs. D, PCR analysis of Dbeta 2-to-Jbeta 2.1/2.6 CJs (DJbeta CJ; top panels) and control Cbeta 2 amplifications (Cbeta ; bottom panels). Gl, Dbeta 2/Jbeta 2-containing germline fragment. The asterisk indicates a nonspecific band that was also amplified from kidney DNA. Serial dilution was as in B except that genomic (non-ligated) DNAs were used; dilution in lanes 2 and 3 was 1/4 and 1/8, respectively. E, quantitation of SE, SJ, and CJ products. PhosphorImager signals for the recombination products were quantified by densitometric scanning and corrected according to the signal from the Cbeta control. Graphic representation for each product is shown, relative to a 100% value as defined from the corresponding signals in wt thymocytes.

The small accumulation of Dbeta 2-to-Jbeta 2 recombination products in Delta Ebeta B2 thymocytes prompted us to also check for the presence of Vbeta -to-DJbeta CJs. Surprisingly, Vbeta 14-to-DJbeta 2 CJs were found in thymocytes from the Delta beta B2 mice (at levels varying from ~12 to 48% of those in the wt) whereas these products were routinely not detected in cells from Delta Ebeta littermates (e.g. Fig. 2). Analysis of SEs at Vbeta 14 once again argued for a specific effect of Bcl-2 expression on the rescue of CEs compared with SEs (see below). However, Vbeta -to-DJbeta 2 assembly in Delta Ebeta B2 thymocytes seems to be restricted to Vbeta 14 only (i.e. the single Vbeta gene located on the 3' end of the TCRbeta locus), as no accumulation of CJs was detected for several 5' Vbeta s, including members located in the proximal (Vbeta 18), median (Vbeta 20, Vbeta 11, and Vbeta 5), or distal (Vbeta 4) parts of the 5' Vbeta gene cluster (Fig. 2) (data not shown).


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Fig. 2.   Vbeta 14-to-DJbeta 2 CJ products in Delta Ebeta B2 thymocytes. Long range PCR analyses of Vbeta 14- and Vbeta 5-to-(D)Jbeta 2.1/2.6 CJs (Vbeta 14 CJ and Vbeta 5 CJ, respectively) in the indicated thymocytes are shown as outlined in the legend to Fig. 1. Quantitation of Vbeta 14 CJs in Delta Ebeta B2 thymocytes was performed by densitometric scanning of PhosphorImager signals from each amplified fragment, yielding 48% (lane 7) and 17% (lane 8) of those in wt thymocytes. The preferential amplifications of low sized Vbeta 14DJbeta 2.5/2.6-containing fragments in Delta Ebeta B2 compared with control thymocytes underlies the lower level of Vbeta 14 CJs in the former cells.

Sequence analysis of Dbeta 2-to-Jbeta 2.5/2.6 CJs from Delta Ebeta B2 thymocytes generally showed the hallmarks of normal CE processing prior to joining, including occasional P and/or N nucleotide additions and short deletions; one Dbeta 2-to-Jbeta 2.5 CJ showed an unusually long (11-bp) N region (data not shown) (also, in Fig. 1C, SE resolution in Delta Ebeta B2 T cells appears to mostly result in standard SJs as they were digested by restriction enzyme ApaLI that cleaves the perfect fusion of two heptamers). Parallel analysis of Vbeta 14-to-DJbeta 2 rearrangement similarly revealed canonical CJ/SJ features and, as expected, joining by DNA inversion.

Analysis of Delta Ebeta B2 Thymocytes for CE and HJ Products-- The above data indicate that constitutive Bcl-2 expression effects a small accumulation of TCRbeta SE, SJ, and CJ products in Ebeta -deleted thymocytes that is confined to gene segments located proximal to the Ebeta deletion, including the Dbeta -Jbeta upstream segments and downstream Vbeta 14 gene. We next investigated whether this effect also impacts on the accumulation of other forms of V(D)J recombination products within this domain, namely CEs and HJ products.

Although SEs 3' of Dbeta gene segments can be found in thymocytes from the Delta Ebeta mice (e.g. Fig. 1), the corresponding CEs could not be detected by LM-PCR of genomic DNA treated with mung bean nuclease (to open the hairpin structures) and T4 DNA polymerase (to blunt occasional DNA overhang extremities)2 (for details on this strategy, see Ref. 19). However, CEs are difficult to detect, even in wt thymocytes, because of their rapid processing and resolution into CJs. Conversely, CEs readily accumulate in developmentally arrested lymphocytes from NHEJ-deficient mice such as the Scid (DNA-PK-deficient) mice (19). Using the aforementioned strategy, CEs 3' of Dbeta 2 were indeed observed in thymocytes from a Scid mouse (together with SEs 5' of Dbeta 2), but not in those from a Delta Ebeta B2 mouse (Fig. 3, upper panels; in Delta Ebeta B2 DNA, the faint signal at a size close to that of CEs was not reproducibly observed), arguing that there is no accumulation of CEs at Ebeta - alleles, even under the condition of constitutive Bcl-2 expression.


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Fig. 3.   Analysis of CE and HJ products in Delta Ebeta B2 thymocytes. Thymocyte DNA was analyzed by LM-PCR for the presence of 3' Dbeta 2 CEs (upper panels: M, 190-bp size marker) and by PCR for the presence of Dbeta 2-to-Jbeta 2.6 HJs (bottom panels); DNA from Rag-/- (Rag) and Scid or wt mice were used as negative and positive controls, respectively. The diagram schematizes the two assays and the region of the TCRbeta locus that was analyzed (outlined as in Fig. 1A), emphasizing the Dbeta 2/Jbeta 2 cleavage/recombination sites that were investigated. The vertical arrow indicates the position of 5' Dbeta 2 SEs that, in the LM-PCR assay, can be amplified simultaneously with 3' Dbeta 2 CEs. Horizontal arrows indicate the relative location of oligonucleotide primers used for PCR amplifications. The size of the amplified fragments is indicated.

HJs are non-standard V(D)J rearranged products that result from the attack of a hairpinned CE by the SE liberated from the opposite gene segment participating to the recombination complex (schematized in Fig. 3). The reaction, which is mechanistically similar to a transposition, was partially reproduced in vitro using purified, truncated forms of RAG proteins (catalytically active core RAGs), in the absence of the NHEJ factors (23). In vivo, HJs are found at a low level in wt lymphocytes. In line with the in vitro results, it is widely accepted that HJs predominate in developing lymphocytes with NHEJ deficiencies. However, a recent study (24) suggests a more complex situation, as the non-core regions in full-length RAGs seem to down-modulate HJ levels in the absence of NHEJ. Intriguingly, HJs involving CEs 3' of Dbeta 2 and Jbeta 2 SEs could not be detected in Delta Ebeta or Delta Ebeta B2 thymocytes (Fig. 3, bottom panels). Thus, Tg Bcl-2 expression in Ebeta - thymocytes results in an accumulation of SE/SJ/CJ products of Dbeta -to-Jbeta rearrangement, but it has a negligible impact on parallel accumulation of CEs and HJs. Overall, these data do not support a role for Ebeta in the recruitment of NHEJ factors to the recombination complex (see "Discussion"). However, they do not exclude an indirect effect of Ebeta on CJ formation; e.g. the stabilization of CE intermediates within the PCS complex.

TCRbeta Gene Expression in Ebeta -Deleted Thymocytes-- At TCR/Ig loci, activation of regional transcription and V(D)J recombination frequently (but not always) correlate (4). Earlier studies have shown that transcription of the unrearranged (germline) Dbeta -Jbeta loci is strongly inhibited at Ebeta - compared with Ebeta + alleles in early developing T cells, whereas that of Vbeta genes is not significantly altered (15). We have analyzed TCRbeta gene expression in Delta Ebeta B2 versus Delta Ebeta thymocytes, using RT-PCR assays to study transcription through either germline Jbeta or Vbeta gene segments (Jbeta Gl or Vbeta Gl) or through partially (DJbeta ) or completely (Vbeta DJbeta ) rearranged products (DJbeta Rg or Vbeta Rg). We found no Jbeta Gl transcription in Delta Ebeta B2 thymocytes at the Dbeta 1-Jbeta 1 or Dbeta 2-Jbeta 2 loci; also, DJbeta Rg transcription was negative in these cells (Fig. 4A, upper two panels) (data not shown). Therefore, despite the evidence of Dbeta -to-Jbeta recombination at Ebeta - alleles, there is no evidence of transcription through these loci (including in the Tg Bcl-2 expressing cells). This is yet another example of differential activation of the two processes. In addition, we found Vbeta Gl transcription for Vbeta 5, Vbeta 11, and Vbeta 14 at Ebeta - alleles, but no Vbeta Rg transcription, including for Vbeta 14 and the Delta Ebeta B2 thymocytes (Fig. 4A) (data not shown), in agreement with the lack of TCRbeta + cells (and beta -selection) in the Delta Ebeta /Delta Ebeta B2 mice, as evidenced by flow cytometry. Whereas Vbeta Gl transcription of Vbeta 5 (and Vbeta 11) appeared to be reduced in Delta Ebeta B2 compared with Delta Ebeta thymocytes, that of Vbeta 14 was unchanged (or increased slightly; e.g. see Fig. 4A). These differential profiles likely result from a reduced DN/DP cell ratio in Delta Ebeta B2 versus Delta Ebeta thymi, coupled with Ebeta -independent developmental changes in Vbeta Gl transcription (rather than from intrinsic differences between the two types of Delta Ebeta B2 and Delta Ebeta cells). Indeed, we found a dramatic down-regulation of Vbeta 5 and Vbeta 11 Gl transcripts in purified DP versus DN Delta Ebeta B2 thymocytes and steady-state (or slightly increased) levels of Vbeta 14 Gl transcripts (Fig. 4B) (data not shown). Therefore, Vbeta Gl transcription profiles in DP Delta Ebeta B2 cells (5' Vbeta Gl-/Vbeta 14 Gl+) correlate with those of Vbeta -to-DJbeta CJs in Delta Ebeta B2 thymi. This lead us to investigate whether, in this situation, TCRbeta recombination also depends on DN-to-DP development and, potentially, Ebeta -independent changes in chromosomal access.


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Fig. 4.   TCRbeta gene expression in Ebeta -deleted thymocytes. A, transcripts initiating upstream of the unrearranged Jbeta 2 gene segment (Jbeta Gl), rearranged Dbeta 2-Jbeta 2 CJs (DJbeta Rg), and unrearranged or Vbeta -to-(D)Jbeta rearranged Vbeta 5 and Vbeta 14 genes (Vbeta Gl or Vbeta Rg) in thymocytes from the indicated mice were analyzed by RT-PCR. Thymocyte (T) and kidney (K) RNA from a Rag mouse and thymocyte RNA from a wt mouse were used as controls. RT-PCR products for beta -actin are shown. Note the differential Jbeta Gl versus DJbeta Rg expression profiles in Rag but not wt or B2 thymocytes, confirming the nature of the analyzed transcripts. B, same as in A, exemplifying Vbeta 5 Gl (but not Vbeta 14 Gl) drop in expression in DP versus DN Delta Ebeta B2 thymocytes.

Developmentally Regulated Activity of the VDJ Recombinase May Be Compromised at Ebeta -Deleted Alleles-- To investigate whether the predominance of TCRbeta SE, SJ, and CJ products in Delta Ebeta B2 versus Delta Ebeta thymi also correlates with their differences in cell distribution, we purified DN and DP thymocytes from both types of mice and analyzed the rearrangement of their TCRbeta locus, as described above. We first focused on Dbeta 2-to-Jbeta 2 rearrangement. Remarkably, we found SEs 3' of Dbeta 2 and Dbeta 2-to-Jbeta 2-6 SJs predominantly in DP thymocytes from both Delta Ebeta and Delta Ebeta B2 mice and at higher levels in Tg Bcl-2 expressing cells; however, the effect of Bcl-2 on the accumulation of rearrangement products was also visible in DN pro-T cells (Fig. 5, A (lanes 3-6) and B (lanes 5-12)). Likewise, in Ebeta -deleted animals, we detected Dbeta 2-to-Jbeta 2 CJs predominantly in Delta Ebeta B2 DP thymocytes; Bcl-2 also had an effect on CJ accumulation in DN cells (Fig. 5C). Notably, CJs in Delta Ebeta B2 DN cells were detected at a slightly higher level (~1.2×) compared with those in Delta Ebeta DP cells despite a bias for SEs (~2×) in favor of the latter (Fig. 5, C and A, lanes 4 and 5, respectively) (data not shown). Further quantitation analysis (Fig. 5D) indicated that SEs without Ebeta are reduced to ~7% (DN) and ~26% (DP) of those in the corresponding wt cells, whereas CJs are reduced to ~4% (DN) and ~10% (DP). By comparison, in Delta Ebeta B2 thymocytes, SEs increased slightly to, respectively, ~12% (DN; ~1.7×) and ~42% (DP; ~1.6×) whereas CJs increased to 24% (DN; ~6×) and ~36% (DP; ~3.6×). These data confirm our previous results of a preferential, although limited effect of Bcl-2 on Dbeta /Jbeta CE resolution at Ebeta - alleles, which is apparent in both DN (~3.5× (6/1.7)) and DP (~2.2× (3.6/1.6)) cells. They further suggest that, in most Ebeta -deleted thymocytes, recombinase activity at the Dbeta /Jbeta RSSs is extended/delayed to cells that have developed to the DP stage. Significantly, we also found high levels of SEs 3' of Dbeta 2 and Dbeta 2-to-Jbeta 2-6 SJs in both DN and DP wt thymocytes (Fig. 5, A (lanes 1 and 2) and B (lanes 1-4)), in agreement with similar findings at the Dbeta 1-Jbeta 1 gene cluster (25). Because unresolved DSBs generated in DN cells would arrest cell proliferation during DN-to-DP cell differentiation, the detection of SEs 3' of Dbeta in DP cells from wt mice must reflect cell autonomous activity of the RAG factors at unrearranged Dbeta /Jbeta loci (25) and/or at Dbeta -to-Jbeta SJs within extrachromosomal circles (26).


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Fig. 5.   Dbeta 2-to-Jbeta 2 SE, SJ, and CJ products in Ebeta -deleted DN and DP thymocytes. A-C, genomic DNA was prepared from sorted CD4-CD8- (DN) and CD4+CD8+ (DP) thymocytes in the indicated mouse lines. PCR assays for the presence of SEs 3' of Dbeta 2 (A), Dbeta 2-Jbeta 2.6 SJs (B), or Dbeta 2-Jbeta 2.1/6 CJs (C), as well as quantitations of SE and CJ products (D) are shown as outlined in the legend to Fig. 1. Sample loadings for experiments in A and B were identical.

Ebeta -Independent Changes in Chromatin Structure at the TCRbeta Locus during T Cell Development-- The extended lifespan conferred by a Bcl-2 transgene is likely to provide an extended time window per cell for V(D)J recombination (27), thus accounting for one aspect of our results (i.e. the rescue of CJ formation; see below). However, other mechanisms must account for the accumulation of SEs on a large scale in DP cells (including DP cells that develop in the absence of beta -selection) (14) and for the profile of Vbeta gene recombination in Delta Ebeta B2 thymocytes. One possibility would be that, upon DP development, accessibility to the RAG factors is established along the Dbeta /Jbeta loci in an Ebeta -independent manner, whereas a repressive structure invades the 5' Vbeta genes but not Vbeta 14. This would impact on the controls of both TCRbeta gene recombination and allelic exclusion during T cell differentiation.

We have tested this model and analyzed chromatin structure at discrete TCRbeta regions using nuclei from Rag and Rag Delta Ebeta thymocytes (Ebeta + and Ebeta - DN cells, respectively) and enzyme restriction/LM-PCR chromosomal accessibility assays, as described previously (15). Thymocytes from Rag and Rag Delta Ebeta mice treated by intraperitoneal injection of anti-CD3-epsilon monoclonal antibody (to mimic pre-TCR signaling) (28) were used as a source of DP-enriched nuclei. Fibroblasts were used as a non-lymphoid control. As predicted, we found that the Jbeta 1 region is more likely to be cleaved in Ebeta - DP (Rag Delta Ebeta CD3) and in Ebeta - DN (Rag) or DP (Rag CD3) thymic nuclei, compared with Ebeta - DN (Rag Delta Ebeta ) thymic or to fibroblastic nuclei (Fig. 6A, top panels; consistent results were also obtained at the Jbeta 2 locus). Furthermore, we found Vbeta 5 to be more resistant to cleavage in both Ebeta + and Ebeta - DP (Rag CD3 and Rag Delta Ebeta CD3) nuclei relative to Ebeta + or Ebeta - DN (Rag and Rag Delta Ebeta ) nuclei (Fig. 6A, middle upper panels; similar results were found at Vbeta 11) (data not shown). Finally, we found Vbeta 14 to be cleaved in T cell nuclei, independent of the developmental (DN or DP) stage and the presence of Ebeta (Fig. 6A, middle lower panels).


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Fig. 6.   Ebeta -independent changes in TCRbeta chromatin structure in DP versus DN thymocytes. A, thymocyte nuclei from Rag-/- (Rag) and Rag-/- Ebeta -/- (Rag Delta Ebeta ) mice and from littermates that have been injected with anti-CD3-epsilon monoclonal antibody (Rag CD3 and Rag Delta Ebeta CD3) or nuclei from 3T3 fibroblasts (fibro.) were treated with increasing amounts of the restriction enzyme RsaI and analyzed by LM-PCR for enzyme cleavage in various parts of the TCRbeta locus, as described previously (15). The bottom panels show Cbeta 2 PCR amplifications (Cbeta ) using RsaI-restricted, linker-ligated DNA samples. The Jbeta 1 cleavage profile, with three visible bands (instead of the two in Ref. 15), is because of a longer gel migration and better separation of the RsaI restricted fragments. The results shown are representative of three independent experiments. B, histone H3 acetylation at Jbeta 1-, Dbeta 1-, Dbeta 2-, Vbeta 5-, and Vbeta 14-associated sequences was investigated in Rag Delta Ebeta and Rag Delta Ebeta CD3 thymocytes by ChIP, in parallel with that at the hyperacetylated TCRdelta gene enhancer (Edelta ) and the hypoacetylated Oct-2 (Oct) gene. ChIP was performed using either an anti-AcH3 anti-serum (alpha AcH3) or no antibody (control). Two concentrations (dilution 1/0 and 1/3) are shown for amplification of the alpha AcH3-precipitated fractions and input materials. The graphics represent the alpha AcH3-precipitated/input material ratios, determined after PhosphorImager scanning of the hybridizing images. The 100% value was attributed to the acetylated Edelta control.

Histone acetylation has emerged as an important regulator of chromatin structure (29) and of locus accessibility for V(D)J recombination in vivo (30). We used ChIP-PCR to compare histone H3 acetylation at Dbeta -Jbeta and Vbeta loci in Rag Delta Ebeta CD3 (DP) versus Rag Delta Ebeta (DN) thymocytes. We found that, during the course of anti-CD3-epsilon -induced DN-to-DP differentiation, H3 acetylation of Ebeta - thymocytes (i) increases slightly at Jbeta 1 and Dbeta 1 and, more readily, at Dbeta 2; (ii) decreases (by ~2-fold) at Vbeta 5; and (iii) is maintained at a steady state level at Vbeta 14 (Fig. 6B). Overall, these data support our model of Ebeta -independent, DN-to-DP regulated changes in chromosomal organization (including histone H3 acetylation) at distinct regions throughout the TCRbeta locus.

Ebeta -Independent DSB Cleavage at Vbeta 14 in DP Thymocytes-- The drop in accessibility of 5' Vbeta genes in CD3-epsilon triggered Rag thymocytes likely mimics a physiological mechanism involved in the feedback inhibition of Vbeta gene rearrangement in response to pre-TCR-induced signaling (i.e. allelic exclusion). In this context, persistent accessibility of the Dbeta -Jbeta and Vbeta 14 locus regions potentially threatens allelic exclusion so that this process must be regulated differently at the 3' end of the TCRbeta locus. The finding of Vbeta 14-to-DJbeta CJs in Delta Ebeta B2 thymocytes also raises the question as to whether Ebeta , in conjunction with cell death control, could participate in this regulation. To address these issues, we tested Ebeta + and Ebeta - thymocytes for the presence of SEs at both Vbeta 5 and Vbeta 14 and of Vbeta 5-/Vbeta 14-to-DJbeta 2 CJs. As a source of Ebeta + or Ebeta - thymocytes, we used wt mice and TCRbeta transgenic mice (p14, a model for TCRbeta allelic exclusion (18)) or the Delta Ebeta and Delta Ebeta B2 mice, respectively.

As expected, we detected SEs at Vbeta 5 predominantly in DN thymocytes from the Ebeta + animals and at a reduced level in p14 compared with wt cells (a >6-fold decrease as judged from densitometric analysis of PhosphorImager signals) whereas, in agreement with previous findings, these products were hardly visible in Ebeta - (Delta Ebeta and Delta Ebeta B2) thymocytes (Fig. 7A, top panel). In contrast, we found Vbeta 14 SEs to predominate in DP cells from the p14, Delta Ebeta , and Delta Ebeta B2 mice (Fig. 7A, middle panel; Vbeta 14 SEs were occasionally detected, at a lower level, in DN and/or DP cells from wt mice) (data not shown). Yet both Vbeta 5 and Vbeta 14 CJs were normally found in wt thymocytes and were strongly reduced in p14 cells (although, possibly, to a lesser extend for the Vbeta 14 CJs); as expected, CJs were not detected at Ebeta - alleles except for Vbeta 14 and the Delta Ebeta B2 cells (Fig. 7B). The latter findings, coupled to those of Vbeta 14 SEs in Delta Ebeta DP thymocytes (Fig. 7A, lane 7), are consistent with a specific effect of Bcl-2 on CE processing also at Vbeta 14. Two other elements should also be considered. First, a 6-fold decrease of Vbeta 5 SEs between wt and p14 DN cells (LM-PCR assays of Fig. 7A, lanes 2 and 4) can account for the drop of the corresponding CJs (PCR assays of Fig. 7B, lanes 4 and 6; also see lanes 2 and 3), implying that exclusion of Vbeta 5 rearrangement is likely to be regulated primarily at the level of chromosomal access. In p14 thymocytes, Vbeta 5 SEs may correspond to normally rearranging alleles; e.g. in a few DN cells that do not express the beta  transgene. We cannot exclude, however, that inhibition of Vbeta 5 rearrangement is regulated beyond the step of DSB cleavage in a small population of TCRbeta rearranging cells. Second, Vbeta 5 and Vbeta 14 CJs look similar in wt thymocytes (Fig. 7B, lanes 4 and 5). It is thus reasonable to assume that, similar to Vbeta 5, most of the Vbeta 14 CJs detected in wt DP cells are indeed generated in DN cells and then expanded by beta -selection. The failure to detect Vbeta 14 SEs in wt DN thymocytes may be because of a specific feature(s) in the processing of these products, linked to the mode of Vbeta 14 rearrangement by DNA inversion and the constraint to preserve chromosome integrity at the site of SJ formation (31). Conversely, increased accumulation of Vbeta 14 SEs in DP thymocytes from p14 and Ebeta -deleted mice may reveal a disorder of the latter control in these cells and, indeed, a unique mode of allelic exclusion at the 3' end of the TCRbeta locus, evidenced here by the high frequency of attempted Vbeta 14 rearrangement. Given the accessibility of the Vbeta 14/DJbeta loci, the level at which Vbeta 14 23-nucleotide RSS cleavage can be observed in total DP cells is predicted to depend on the proportion of complementary 5' Dbeta 12-necleotide RSS left available for synapsis (4, 32, 33).


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Fig. 7.   Analysis of Vbeta SE and CJ products in DN and DP thymocytes from Ebeta + and Ebeta - mice. A and C, lower panels, genomic DNA from total and/or DN- and DP-sorted thymocytes in the indicated mouse lines was analyzed by LM-PCR for the presence of Vbeta 5 or Vbeta 14 SEs (Vbeta 5 SE and Vbeta 14 SE, respectively) and by long range PCR for the presence of Vbeta 5-(D)Jbeta 2 or Vbeta 14-(D)Jbeta 2 CJs (Vbeta 5 CJ and Vbeta 14 CJ, respectively; B and C, upper panels). In A, thymus DNA from a Rag mouse was used as a negative control. Serial dilution analyses of wt thymus DNA shown in B and C were as in Fig. 1D. C, PhosphorImager scanning analysis of recombination products in p14 B2 versus p14 thymocytes gave the following results (after normalization to Cbeta controls): CJs, 2.53/0.88; SEs, 2.2/1.8, respectively. Because control Cbeta 2 amplifications (Cbeta ) used oligonucleotide primers located, respectively, upstream of (5' primer) and within (3' primer) exon I of Cbeta 2, the p14 transgene was not detected in this assay.

Processing of Vbeta 14 SEs into CJs at Ebeta - alleles can be rescued, to some extent, by constitutive Bcl-2 expression. To check whether this also occurs in the presence of Ebeta , we analyzed thymocytes from p14 B2 double transgenic mice. Indeed, we found increased levels (~2.9×) of Vbeta 14 CJs in p14 B2 compared with p14 thymocytes, whereas Vbeta 14 SEs were roughly equivalent in DP cells from both types of mice (Fig. 7C). Altogether, the above data strongly suggest that exclusion of Vbeta 14 rearrangement proceeds through a unique mechanism, one aspect of which could be a specific defect in the resolution of discrete DNA DSBs and, most likely, the induction of cell death. Ebeta does not appear to interfere with these processes, including the initial steps of the recombination reaction (synapsis and RAG-mediated DNA cleavage) during attempted Vbeta 14-to-DJbeta rearrangement.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A Critical Function for Ebeta in Promoting Access to the Dbeta -Jbeta Domains in pro-T Cells-- We have analyzed TCRbeta gene rearrangement in Delta Ebeta B2 thymocytes to better assess Ebeta function in the regulation of CJ formation during V(D)J recombination. Compared with Delta Ebeta , Delta Ebeta B2 thymocytes show a small accumulation of TCRbeta standard recombination products, most notably CJs, that is confined to proximal Dbeta -Jbeta and Vbeta 14 loci. Although detectable in DN CD25+ pro-T cells, these effects predominate in cells that have differentiated to the DP stage. Developmental cell selection is unlikely to account for these findings, as no TCRbeta rearranged products could be detected at either the RNA or protein levels. Instead, evidence for VDJ recombinase activity at these loci also in Ebeta + DP thymocytes, and the fact that the rearrangement profiles at distinct TCRbeta loci in DN and DP Delta Ebeta thymocytes correlate with their level of chromosomal accessibility in the particular cell subset, argue for delayed access of the recombinase to Ebeta - alleles. Besides pointing to a possible, likely indirect effect of Ebeta in assisting CJ formation, these data most notably emphasize this critical function of the enhancer for chromatin opening within the Dbeta -Jbeta domains, which, unexpectedly, appears also limited to an early window of T cell development only (i.e. anterior to the CD44-/loCD25+ DN cell stage).

A Mechanism of Enhancement of Dbeta -to-Jbeta CJ Formation by Ebeta ?-- Resolution of Rag-mediated DNA breaks that can happen in Ebeta -deleted thymocytes is improved by Tg Bcl-2 expression, apparently with a slightly greater impact on CJ compared with SJ formation (e.g. Fig. 2). May this tell us something about a putative role of Ebeta in enhancing CE processing? First, the effects conferred by Bcl-2 transgenes on developmental processes in lymphoid cells (including V(D)J recombination) have generally been attributed to cell extended lifespan (27, 34), although a non-conventional role of Bcl2 and incidental effect(s) on the processing of injured DNA cannot be formally ruled out (35). Second, the resolution of CE and SE products is thought to proceed along two different pathways, involving distinct requirements and kinetics (4). Thus, although a deficiency in any of the factors of the NHEJ apparatus results in a CJ defect, some proceed with unaltered SJ formation (e.g. the SCID defect resulting from DNA-PK deficiency). Also, whereas SEs accumulate in lymphoid cells undergoing V(D)J recombination (and are eventually resolved after RAG expression is down-regulated), CEs are difficult to detect in the wild-type situation (they accumulate in DNA-PK-deficient lymphocytes), indicating that CJ formation must be tightly linked to RSS cleavage. In possible relation to these distinct behaviors, in vitro studies have suggested diametrically opposed stability of V(D)J recombination post-cleavage complexes depending on product content; complexes consisting of the two CEs and two SEs appear to be highly unstable whereas those consisting of the two SEs bound by the RAG factors are resistant to dissociation challenges (reviewed in Ref. 6). We believe that a direct effect of Ebeta in mediating the recruitment of DNA-PK (or a DNA-PK/Artemis complex (36)) is unlikely, based on the CJ sequences from Delta Ebeta and Delta Ebeta B2 thymocytes that do not show the typical abnormalities associated with the SCID defect (i.e. extensive deletions, frequent usage of short sequence homologies, long palindromic junctional inserts), along with the seeming lack of CEs and HJs at Ebeta - loci in Tg Bcl-2 expressing T cells (16) (this study). However, an effect of Ebeta on the stabilization of post-cleavage synaptic complexes would be compatible with the small accumulation of CJs observed in Delta Ebeta B2 thymocytes; unstable CEs (especially at Ebeta - alleles) would have more chances of being resolved when cell survival is extended. As for HJ formation (not found in Delta Ebeta or Delta Ebeta B2 thymocytes), it could be especially sensitive to suboptimal conformation and/or stability of the post-cleavage synaptic complexes (24).

We stress that the effect of Ebeta on chromatin structure at the Dbeta -Jbeta region and an incidental effect on CJ formation may not be mutually exclusive. First, the maintenance of an open structure may be required for NHEJ repair. Second, Ebeta modulation of chromosomal accessibility almost certainly involves nucleo-protein interactions including additional cis-regulatory element(s) (e.g. the Dbeta 1 upstream promoter (25)). Within such structures, recombination synaptic complexes (Dbeta -to-Jbeta and, subsequently, Vbeta -to-DJbeta ) could be optimally organized for RAG cleavage and/or the processing of the cleaved extremities. As an integral component, Ebeta (and bound factors) may thereby contribute to optimizing V(D)J repair processes, for example through the anchorage/tethering of loose extremities within interacting distances or, in a more sophisticated way, by favoring catalytic transitions within the synaptic complexes. As the efficiency of CJ formation is likely influenced by the efficiency with which loose CEs are recaptured by the post-cleavage complex (37), DNA tethering should improve this process. A model of post-synaptic conformational isomerization of a recombination complex has also been described during phage Mu DNA transposition (38). Importantly, enhancers at other TCR/Ig loci may share the dual functions of Ebeta on coupled regulation of chromatin modulation and CJ formation, which could be revealed once the two effects can be distinguished.

Implications for T Cell Differentiation and the Control of Allelic Exclusion at the TCRbeta Locus-- Allelic exclusion at the TCRbeta locus is regulated at the level of Vbeta -to-DJbeta joining. In this context, evidence of chromatin compaction at 5' Vbeta genes during the DN-to-DP cell transition, including reduced histone acetylation, has accumulated (39-41) (this study). This change in organization likely contributes to lock in allelic exclusion, at least within alleles carrying a germline 5' Vbeta cluster (because we used Rag mice in our analyses, it is still unclear whether unrearranged Vbeta genes upstream of a Vbeta DJbeta CJ unit behave similarly). We now demonstrate that developmentally regulated re-organization of the 5' Vbeta genes does not require Ebeta , in line with their previously reported Ebeta -independent transcription in earlier DN thymocytes. Although long range accessibility within antigen receptor loci has been inferred to depend on matrix attachment regions (42), targeted deletion of matrix attachment region beta  (located ~400 bp upstream of Ebeta ) does not change TCRbeta transcription and V(D)J recombination in developing T cells (43). Likewise, targeted deletion of Jbeta 2-Cbeta 2 intronic cis-elements does not alter these processes (44). Therefore, the re-organization at 5' Vbeta genes could rather involve as yet undefined regulatory sequences within the upstream part of the TCRbeta locus. Assuming that the promoters of 5' Vbeta genes and of Vbeta 14 behave similarly (45), our finding that Vbeta 14 is spared by the repressive effect makes the individual 5' Vbeta promoters unlikely candidates. Indeed, a 5' Vbeta gene was no longer under allelic exclusion control when inserted, together with associated promoter sequences, in the region upstream of Dbeta 1 (46).

In DP thymocytes, the 3' end of the TCRbeta locus containing the Dbeta -Jbeta -Cbeta and Vbeta 14 domains maintains (or gains in the case of Ebeta - alleles and the Dbeta -Jbeta -Cbeta clusters) chromosomal accessibility. This might involve the regulated activation of additional cis elements that normally act redundantly with Ebeta within these loci (40). At this stage of development and in the wild-type situation, fully derepressed chromatin may be required to ensure high levels of expression of a rearranged Vbeta DJbeta -Cbeta unit. The ensuing drawback is cleavage by the RAG machinery past the beta -selection checkpoint, with consequences such as the specific accumulation of Dbeta and Vbeta 14 SEs and DJbeta CJs in DP thymocytes (see Fig. 5, A and C and see Fig. 7A) (25). Nevertheless, the fact that levels of Vbeta 14 CJs remain extremely low in p14 DP cells (Fig. 7B) indicates that these consequences on TCRbeta allelic exclusion are minimized, possibly involving regulated cell death (Fig. 7C). Recent results (47) suggest that programmed cell death may be a parameter that also limits ongoing rearrangements along the TCRJalpha locus in DP thymocytes. In this context, the particular situation of Vbeta 14 and inversional mode of rearrangement could concur to the surprisingly opposite outcomes of Vbeta 14-to-DJbeta versus Dbeta -to-Jbeta attempted recombination in DP cells. Notably, intrachromosomal Vbeta 14-to-Dbeta SJs within this accessible part of the locus could be ideal targets for DSB formation by a RAG-mediated nick-nick mechanism (26). It is, however, important to stress that the actual levels of V(D)J recombination at Dbeta -Jbeta and Vbeta 14 loci in DP thymocytes is unclear and could be quite low given the large amounts of germline-sized Dbeta -Jbeta containing fragments detected by PCR and Southern assays of DNA from Ebeta - (Delta Ebeta B2) and Ebeta + (p14) thymocytes.3 The reasons for these paradoxical effects of combining chromosomal accessibility and low levels of recombination within RAG expressing cells (48) warrant further investigating efforts.

Based on in vitro studies, cleaved SEs are thought to form excellent substrates for RAG-mediated transposition, with the risk to compromise genomic stability in lymphocytes leading possibly to translocation and leukemia (26). One would predict that Delta Ebeta B2 mice may be subject to these effects. However, neoplasia does not significantly affect these animals (but translocation into an enhancerless TCRbeta locus may not result in oncogene-deregulated expression in the developing lymphocytes), and we did not find evidence of TCRbeta genomic instability using FISH analysis and PCR assays (including assays to search for TCRbeta -to-TCRalpha /delta interlocus recombination). Specific pathways might exist to counteract such events in DP thymocytes involving, for example, a role of the recombination machinery in the prevention of transposition (49, 50) and/or sensors of injured DNA (e.g. p53, ATM) acting to uncover and eliminate such damages and/or the damaged cells (51). The latter possibility is supported by the finding that introduction of an Ebeta mutation onto a p53-deficient background accelerates tumor development in T cells.4

Our findings indicate that Ebeta is not involved in the sophisticated controls that, following beta -selection, secure allelic exclusion at the TCRbeta locus. However, allele asynchronicity of Vbeta gene assembly in earlier DN pro-T cells is a prerequisite for allelic exclusion, as this should leave cells enough time to test for Vbeta -to-DJbeta productivity before Vbeta gene rearrangement proceeds on the other allele (7). The molecular basis for this phenomenon are still unclear. It may involve a recombination machinery that incidentally operates at suboptimal efficiency or structural features to ensure that a single allele rearrange at a time, including specific properties of Dbeta -flanking RSS (4) and/or an epigenetic mark(s) established at antigen receptor genes early in development (52). We stress that Ebeta could be involved in any of these controls (except may be for the latter), as evidenced by the differential effect exerted on activation of the Dbeta -Jbeta - versus the Vbeta -containing chromosomal domains and associated regulatory elements (15). Our current results of a rigorous control by Ebeta in regulating chromosomal access in early pro-T cells further sustain this hypothesis. This may explain the evolutionary constrain to maintain a function of Ebeta that is limited both spatially (the Dbeta -Jbeta domains) and temporally (prior to the CD44-/loCD25+ DN cell stage).

    ACKNOWLEDGEMENT

This manuscript was improved by the helpful criticisms of an anonymous reviewer.

    FOOTNOTES

* This work was funded by institutional grants from INSERM and the CNRS and by specific grants from the Association pour la Recherche sur le Cancer, the Commission of the European Communities, and the Fondation Princesse Grace de Monaco.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: CEA, Centre d'Etudes Nucléaires, 60/68 Avenue du Général Leclerc, BP6, 92 265 Fontenay-aux-Roses Cedex, France.

§ Present address: Immunologie Rétrovirale et Moléculaire, IRD CNRS/CTS, 240 Avenue Pr. Emile Jeanbraud, 34090 Montpellier, France.

Present address: Département de Biologie Cellulaire et Moléculaire, Pfizer Global Research & Development/Fresnes Laboratories, 3-9 rue de la Loge, 94265 Fresnes Cedex, France.

|| Present address: Institut de Signalisation, Biologie du Développement et Cancer, CNRS-UMR 6543, Centre Antoine Lacassagne, 33, Avenue Valombrose, 06189 Nice, France.

** To whom correspondence should be addressed: CIML, Case 906, 13288 Marseille Cedex 9, France. Tel.: 33-491-269435; Fax: 33-491-269430; E-mail: ferrier@ciml.univ-mrs.fr.

Published, JBC Papers in Press, March 14, 2003, DOI 10.1074/jbc.M212647200

2 W. M. Hempel and N. Mathieu, unpublished results.

3 N. Mathieu, unpublished results.

4 J. Chen, personal communication.

    ABBREVIATIONS

The abbreviations used are: TCR, T cell receptor; DNA-PK, DNA-dependent protein kinase; DN, double negative; DP, double positive; wt, wild-type; LM, ligation-mediated; RT, reverse-transcribed; CE, coding end; SE, signal end; CJ, coding joint; SJ, signal joint; HJ, hybrid joint; DSB, double strand break; RSS, recombination signal sequence; RAG, recombination-activating-gene; NHEJ, non-homologous end-joining; ChIP, chromatin immunoprecipitation; Gl, germ line.

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ABSTRACT
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RESULTS
DISCUSSION
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