Although it is generally agreed that lymphoid enhancers may participate in chromatin remodeling 1011, the molecular mechanisms responsible are still poorly understood and other possibilities also exist to explain the effect of these elements in regulating V(D)J recombination 11. Moreover, the discrete domains that an individual enhancer can influence with respect to chromatin structure have not been precisely defined. Here we show that, in early DN T cells, the effect of Eβ deletion on chromatin structure exhibits an unanticipated degree of plasticity depending on the TCR-β region considered, with the influence of Eβ in chromatin remodeling being primarily restricted to the Dβ-Jβ gene clusters. We discuss these results with respect to Eβ function in the regulation of Dβ-to-Jβ versus Vβ-to-DJβ recombination.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice.
The mice used in this study, as well as mouse housing and analyzing conditions, were as described previously 11.

Thymocyte Preparation and Cell Cultures.
Thymocytes were separated from the stroma according to standard procedures. NIH-3T3 fibroblasts were maintained in DMEM (GIBCO BRL) supplemented with 10% FCS. Fibroblasts that were growing in log phase were used to prepare nuclei.

For treatment with the specific histone deacetylase inhibitor trichostatin A (TSA), thymic lobes were excised from fetal mice at day 14.5 postcoitum and placed on cellulose ester filters (Millipore) in 6-well plates containing DMEM supplemented with 10% FCS, penicillin, streptomycin, L-glutamine, and sodium pyruvate, without or with TSA (3 ng/ml; Sigma-Aldrich). The culture was carried out for 7 h at 37°C in a humidified, 10% CO₂ atmosphere.

Restriction Endonuclease Accessibility Assays.
Cell nuclei were prepared as described previously 911. For restriction enzyme digests, 10⁵ nuclei were treated with increasing concentrations of restriction endonuclease in digestion buffer consisting of 10 mM Tris, pH 7.9, 50 mM NaCl, 10 mM MgCl₂, 4% glycerol. Digestion was performed on ice for 1 h. Nuclei were lysed in extraction buffer (10 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% SDS) and treated with proteinase K (final concentration of 0.4 mg/ml) at 56°C for 2 h. DNA was resuspended in 25 µl of H₂O. Linker ligation, using 10 µl of the above preparation, and detection of the generated blunt ends by ligation-mediated (LM)-PCR were carried out as described previously 11. Quantification was performed by use of a PhosphorImager^® (BAS 1000; Molecular Dynamics) and MacBAS software. The sequences of the TCR-β–specific oligonucleotide primers used in LM-PCR reactions are available upon request.

Reverse Transcription PCR.
Total RNA and cDNA preparation as well as analysis of T cell–specific transcripts by reverse transcription (RT)-PCR, using cDNA templates and locus-specific oligonucleotide primers, were performed as described (7; primer sequences available upon request).

Southern Blot Assay.
Preparation of genomic DNA, restriction enzyme digests, gel electrophoresis, Southern blotting, and hybridization procedures were performed as described previously 12. Hybridization signals were detected by autoradiography. The hybridizing probes used for hybridization were prepared by PCR amplification using genomic DNA from Rag^–/– kidney as a template.

Bisulfite Sequencing.
Bisulfite treatment of genomic DNA was performed according to Zeschnigk et al. 13 with the following modifications. DNA was mixed with 2 vol of 2% LMP agarose (GIBCO BRL) dissolved in H₂O, and the agarose/DNA mix was pipetted into chilled mineral oil to form agarose beads, which were then incubated overnight at 56°C in the presence of NaHSO₃ (Sigma-Aldrich). The reaction was stopped by washing of the beads with 10 mM Tris, pH 8.0, 1 mM EDTA (TE; six times for 15 min) followed by the addition of 500 µl of 0.2 N NaOH (twice for 15 min), and then 1/5 vol of 1 M HCl. The beads were washed with 1 ml H₂O (twice for 15 min) and used directly for PCR. Primers were designed to be complementary to the deaminated DNA strands within sequences that do not include CG dinucleotides. 5 µl of the PCR product was cloned into the vector PGEM-T (Promega). After bacterial transformation, DNA from individual clones was sequenced using the ABI PRISM^TM 310 Genetic Analyzer (PerkinElmer).

Chromatin Immunoprecipitation PCR.
Chromatin fixation and immunoprecipitation were performed using the Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit (Upstate Biotechnology, Inc.) as recommended by the manufacturer. The input fraction corresponded to 10% of the chromatin solution before immunoprecipitation. After DNA purification, the input, unbound, and bound fractions were resuspended in 100 µl of TE, and analyzed by 25 cycles of PCR (40 s at 94°C, 40 s at 55°C, 40 s at 72°C) in 25-µl reactions. A twofold dilution series was typically used for PCR reactions with all TCR-β–specific primers (primer sequences available upon request). Oct-2– and TCR- enhancer (E)-specific primers were designed according to McMurry et al. 14.

PCR Analysis of Dβ2-to-Jβ2 Rearrangements.
Oligonucleotide primers, PCR conditions, and analysis of the amplified products were as described previously 7.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential endonuclease access to chromosomal DNA and the level of transcription and DNA methylation are molecular parameters widely used to monitor changes in chromatin architecture associated with the activation of gene expression 1516. To study the effects of Eβ deletion on these parameters, we applied several molecular assays to comparatively analyze specific regions of the TCR-β locus (e.g., the Dβ-Jβ clusters and Vβ gene regions) in thymocytes from single knockout RAG-1 (Rag^–/–) versus double knockout Rag^–/–Eβ^–/– mice. Due to a lack of V(D)J recombination, Rag^–/– animals demonstrate no thymocyte development beyond the CD44^–/loCD25⁺ DN cell stage 1. Thus, utilization of Rag^–/– (and Rag^–/– Eβ^–/–) thymocytes offers two advantages: (a) the analyzed TCR-β locus is maintained in the germline configuration; and (b) the analyzed cells are enriched in thymocytes that are arrested at the developmental stage where TCR-β gene recombination normally takes place. Using CD4/CD8 and DN CD44/CD25 cell surface stainings and flow cytofluorometry, we have verified that the cell developmental block in Rag^–/–Eβ^–/– and Rag^–/– thymi can be superimposed (data not shown).

Differential Effects of the Eβ Deletion on Accessibility of the Dβ-Jβ and Vβ Regions.
To examine the in vivo effects of Eβ deletion on the chromatin structure throughout TCR-β regions, we used a restriction enzyme accessibility assay. This involved incubating isolated cell nuclei with increasing concentrations of a restriction enzyme, then lysing them to prepare genomic DNA. Cleavage at the sites of interest was assayed by LM-PCR followed by Southern blotting (Fig. 1A–C). To assess their quality and the variability of sample loading, the linker-ligated DNAs were tested in parallel for amplification of a fragment within the Cβ2 constant region gene. By comparing amplified products from Rag^–/– versus Rag^–/–Eβ^–/– thymic nuclei, the relative level of restriction enzyme cleavage at a given site can thus be evaluated. As a nonlymphoid control, we used nuclei from NIH-3T3 fibroblasts.

Fig. 1B–E, shows the results from the analysis of selected sites within the Dβ-Jβ regions. Whereas two RsaI sites located within the intronic sequences between the Jβ1.3 and Jβ1.4 gene segments (sites #1^R and #2^R; Fig. 1 B) and one RsaI site located within the Jβ2.6 gene segment (3^R; Fig. 1 C) were readily cleaved at a low enzyme concentration in Rag^–/– thymic nuclei, those in Rag^–/–Eβ^–/– nuclei were more resistant to cleavage, showing profiles largely comparable to those in fibroblast nuclei. After PhosphorImager^® scanning of ³²P emission from the hybridizing bands and correction for differences in sample loading, based on Cβ2 amplifications, we estimated that the levels of site #1^R, #2^R, and #3^R cleavage in Rag^–/–Eβ^–/– thymocytes and normal fibroblasts were 3.2 and 2%, 20 and 12%, and 60 and 55%, respectively, of those in Rag^–/– thymocytes (Fig. 1 F). Related percentages were obtained in two separate experiments. These results strongly suggest that the Dβ-Jβ loci demonstrate reduced levels of chromatin disruption in DN cells from the Eβ mutant mice (on average, <5% of those in Rag^–/– thymocytes, once cleavage background in fibroblasts is taken into account) and, consequently, that Eβ critically impacts on the chromatin structure of these regions in early developing T cells. Reduced chromatin accessibility in the Rag^–/–Eβ^–/– thymocytes was consistently observed when testing two HaeIII sites (#1^H and #2^H) located 5' of Jβ1.6 and within Dβ2, respectively (Fig. 1E and Fig. F). It is noteworthy that, in these instances, the cleavage assays also demonstrated larger differences between the Eβ mutant and fibroblast nuclei (percentages of 45 and 23%, and 64 and 38%, respectively), implying that Eβ may not be the only determinant of TCR-β gene accessibility in early T cells and/or that the action of Eβ may not be uniform across the entire length of the Dβ-Jβ clusters. Finally, a difference in chromosomal access depending on the presence or absence of Eβ was also observed when analyzing an RsaI site (#4^R) located 540 bp 5' of the Dβ1 gene segment (Fig. 1 D, top). However, the difference was attenuated when analyzing two such sites (#5^R and #6^R) located 2 and 2.15 kb, respectively, further upstream, with roughly equivalent cleavage levels observed in both Rag^–/– and Rag^–/–Eβ^–/– thymic nuclei (Fig. 1 D, middle). These latter data suggest that, in DN thymocytes, the upper limit of Eβ control on chromosomal access may fall within the 2-kb sequences that separate these oppositely regulated sites.

Similar analyses of Vβ gene–containing regions demonstrated a lack of Eβ influence on chromosomal access. Thus, pairs of RsaI sites located in either the 3' region of Vβ5.2 and downstream of this element (#7^R and #8^R; toward the 5' end of the TCR-β locus) or within the leader exon and promoter region of Vβ14 (#9^R and #10^R; 3' proximal to Eβ) exhibited equivalent levels of accessibility in Rag^–/– and Rag^–/–Eβ^–/– thymic nuclei (Fig. 2). In these last two cases, enzyme cleavage in nuclei derived from thymocytes appeared to be significantly increased compared with those from fibroblasts, implying tissue-specific control of the underlying chromosomal structure. Similar results were obtained upon analysis of restriction sites within Vβ11, Vβ20, and Vβ18, three Vβ genes scattered further 3' of Vβ5.2 (data not shown). Altogether, the above results demonstrate unexpected regional consequences of Eβ deletion on chromatin remodeling in early developing T cells, consisting of reduced accessibility of the upstream Dβ-Jβ clusters, whereas the Vβ gene regions still develop an accessible structure irrespective of their distance from this deletion.

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Accessibility to restriction enzyme cleavage within the Vβ5.2/Vβ8.3- and Vβ14-containing regions. Representative LM-POR experiments and accessibility level quantifications are shown, as outlined in the legend to Fig. 1B–F. The DNAs used in this experiment were the same as those used for analysis of the #3R site shown in Fig. 1 C.

As expected, germline transcripts (GTs) initiating upstream of the Jβ1 and/or the Jβ2 gene clusters (the Jβ1 GT and Jβ2 GT) were found to be expressed in Rag^–/– thymocytes (Fig. 3 A, top two panels, lane 1; note the heterogeneity in size of the Jβ1 GT, as reported previously [17, 18]). In striking contrast, neither transcript was detected using RNA from Rag^–/–Eβ^–/–, Eβ^–/–, or TCR-^–/–Eβ^–/– thymocytes (Fig. 3 A, lanes 5–7). Using RAG-deficient blastocyst complementation and embryonic stem cells carrying both a wt and an Eβ-deleted TCR-β allele to generate chimeric mice, Alt and colleagues have reported a lack of detectable Jβ1 GT in the resulting thymocytes 6, a finding that is reproduced here using thymocytes from Eβ^+/– mice carrying the germline-inherited Eβ deletion (Fig. 3 A, top, lane 4). In Eβ^+/– thymocytes, the Dβ1-to-Jβ1 intervening sequences (to which the RT-PCR forward Jβ1 primer hybridizes) are present on the unrearranged mutant allele but are removed by several types of rearrangements on the wt allele (i.e., by Dβ1-to-Jβ1 joining and by any rearrangement involving an upstream Vβ and the downstream Dβ2-Jβ2 cluster). Altogether, these data strongly suggest that the Eβ deletion results in the cis-inhibition of germline transcription throughout the Jβ1 gene cluster. Presumably, the Jβ2 GT that can be found (at reduced levels) in Eβ^+/– thymocytes (Fig. 3 A, middle, lane 4) are produced from wt alleles harboring a productive Vβ-to-DJβ rearrangement that involves the Dβ1 and Jβ1 gene segments. In agreement with this interpretation, Jβ2 GTs were not detected in Eβ^+/– thymocytes derived from (Eβ^–/– x NZW) mouse intercrosses (data not shown; NZW TCR-β alleles have a deletion covering the Dβ2 and Jβ2 elements 19). We conclude that, as a result of the Eβ deletion, germline transcription across the Dβ-Jβ loci is severely inhibited in early developing T cells.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 3 TCR-β locus expression in thymocytes from mutant mice. Total RNA from thymocytes of the indicated mouse lines was assayed by RT-PCR to analyze: (A), GTs initiated upstream of the unrearranged Jβ1 or Jβ2 clusters (Jβ1 GT and Jβ2 GT); or (B), Vβ5.2, Vβ11, and Vβ14 rearranged (Vβ RgT) and/or germline (Vβ GT) transcripts, as schematized above the corresponding panels. Arrowheads indicate the relative location of the primers used in the RT-PCR assays. Lanes 1–3 show a titration of Rag–/– thymocyte cDNA. Input cDNA was kept constant; lane 1: thymocyte cDNA undiluted; lanes 2 and 3: thymocyte cDNA diluted 1:3 and 1:30 with kidney cDNA. Amplified products from β-actin controls are shown.

View larger version (53K): [in this window] [in a new window] [Download PPT slide]   Figure 4 CpG methylation analyses within the Dβ-Jβ loci. (A) The relative location of the HpaII/MspI or HhaI restriction sites that have been analyzed is indicated. The relevant HindIII, XbaI, BglI, and NcoI restriction sites used in Southern blot analyses are also shown. The bottom part of the diagram summarizes the results of these assays. Eβ+ and Eβ–: results obtained when using Rag–/– and Rag–/–Eβ–/– thymocyte DNA, respectively. A filled or open oval underneath the corresponding site indicates hyper- or hypomethylation, respectively; partial methylation is indicated by the partially filled ovals. (B) Representative results of Southern blot analyses at specific HpaII/MspI sites, using genomic DNA from thymocytes of either one Rag–/– (T) or two Rag–/–Eβ–/– (T#1 and T#2) mice and appropriate single and double restriction enzyme digestion, as indicated. Kidney DNA (K) from a Rag–/– mouse was used as a nonlymphoid control. The size of the hybridizing fragments is indicated; the relative location of each probe used for hybridization of the individual blot is shown by a horizontal bar within the upper diagram. The 1.4-, 1.5-, and 1.6-kb hybridizing fragments seen in the D/Jβ2 (#6H/M) analyses correspond to additional HpaII/MspI restriction sites located in between sites #5H/M and #6H/M. (C) Genomic sequencing of a 215-bp DNA fragment overlapping with the Jβ2.6 gene segment. The sequences of untreated (–Bisulph.) and bisulfite-treated (+Bisulph.) DNAs are shown. The methylated cytosines that, within the Eβ– DNA, are resistant to thymidine conversion are indicated (arrowheads). The diagram on the right summarizes the results of these analyses (representing 4 [Eβ+] and 11 [Eβ–] sequencing reactions, respectively). Only the sequences that contain CG dinucleotides (horizontal bars) are shown; the asterisk indicates the central CG within the site #6H/M. The percentage of methylated residues which was found in bisulfite-converted DNA at six out of the seven analyzed CGs is indicated.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 5 CpG methylation analyses within the Vβ5.2/Vβ8.3 and Vβ14 gene–containing regions. A summary of the results and representative Southern blot experiments at the indicated HpaII/MspI sites are shown, as outlined in the legend of Fig. 4A and Fig. B.

Strikingly, whereas H3 acetylation was comparable for Vβ5.2 and Vβ14 (as well as for E control) in both Rag^–/– and Rag^–/–Eβ^–/– thymocytes, that for Dβ1 and Dβ2 sequences markedly differed depending on mouse origin, with lower levels specifically observed in Rag^–/–Eβ^–/– thymocytes (Fig. 6). Moreover, within a given sample, acetylation levels differed depending on the site analyzed, with significantly higher levels observed for the Dβ loci. Altogether, these results strongly suggest that, in early developing thymocytes, Eβ deletion predominantly affects histone acetylation within the Dβ-containing loci, with essentially no effect on acetylation at Vβ-containing loci. These differential effects are reminiscent of those observed in analyses for restriction enzyme access and germline transcription, as reported above. Moreover, Dβ versus Vβ histone H3 overacetylation at Eβ-containing alleles inversely correlates with our findings of opposite hypo- and hypermethylation profiles at these gene segments, in line with previous observations at various genomic loci. Finally, the residual amplification of transcriptionally inactive Dβ sequences in the AcH3-bound fraction from Rag^–/–Eβ^–/– thymocytes (compared with the Oct-2 negative control; Fig. 6 B) indicates that Eβ activity is not the only determinant of histone acetylation at these sites. Indeed, tissue-specific genes may be acetylated in the cell type in which they are potentially active, independent of their transcriptional status 25.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Histone H3 acetylation within TCR Vβ and Dβ locus regions. (A) H3 acetylation level was investigated in total thymocytes from the indicated mouse lines by chromatin immunoprecipitation (CHIP)-PCR assays. CHIP was performed with anti-AcH3 (AcH3) antiserum or no antibody (control). Serial twofold dilutions of input, AcH3-bound (B), and -unbound (U) fractions were analyzed by PCR. (B) H3 acetylation was quantified after PhosphorImager® scanning of Southern blots and plotting of B/input measurements.

View larger version (59K): [in this window] [in a new window] [Download PPT slide]   Figure 7 (A) Rescue of Jβ1/Jβ2 GTs upon TSA treatment of Eβ-deleted thymocytes. Thymocytes were prepared from either Rag–/– (Eβ+) or Eβ–/– (Eβ–) fetal thymic lobes (day 14.5 postcoitum) that have been cultured in the presence (+) or absence (–) of TSA. Jβ1 GTs and Jβ2 GTs were assayed by RT-PCR, as indicated in the legend to Fig. 3. Lanes 2–4 correspond to three individual Eβ–/– littermates. (B) Presence of DJβ2 transcripts (DJβ2T) in total RNA from TSA-treated Eβ–/– fetal thymocytes. Experimental procedures were as in A except that RNA from wt thymocytes was included as a positive control. (C) Dβ2-to-Jβ2 rearrangements (Dβ2-JβR) in genomic DNA isolated from the indicated fetal thymocytes were analyzed by PCR. NR, unrearranged Dβ2-Jβ2–containing fragments. Amplification of a Cβ2-containing fragment was carried out to control for sample loading.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Dβ-Jβ Locus Recombination and Chromatin Opening by Eβ.
A function for Eβ in opening heterochromatinized Dβ-Jβ loci can explain, to a large extent, the defect in Dβ-to-Jβ rearrangement observed at Eβ-deleted alleles. Accordingly, using thymocyte DNA from Eβ-deleted, DN developmental knockout mouse models (such as the TCR-^–/–Eβ^–/– mouse), we have found that V(D)J recombinase–mediated DSB cleavages at the recombination signal sequences (RSS) flanking Dβ and Jβ gene segments are reduced up to 1/10 to 1/30 the level observed in thymi with an unaltered TCR-β locus 11. Significantly, our estimations on decrease in chromosomal accessibility at the Dβ-Jβ clusters in Rag^–/–Eβ^–/– thymocytes, based on quantification data from restriction enzyme cleavage assays, were mostly comprised within the same range. Based on a similar approach to study DSB cleavage at a transgenic minilocus, local chromatin remodeling by the TCR- gene enhancer has also been proposed to control D-to-J joining 10. Assuming that Eβ regulates Dβ-to-Jβ recombination in a similar fashion, it is, however, noteworthy that: (a) in vitro–generated DSBs can be produced at Dβ/Jβ RSS within chromosomal DNA from both Rag^–/– and Rag^–/– Eβ^–/– thymi 11; and (b) in vivo–generated DSB intermediates at the Dβ and Jβ RSS are consistently found at a higher frequency than the DJβ rearranged products (11; Mathieu, N., unpublished data). Based on all of the available data, we favor the possibility that Eβ, in addition to a regulatory function via chromatin decondensation, may also play a role during the course of the Dβ-to-Jβ joining reaction (e.g., the stabilization and/or processing of recombination synaptic complexes; for discussion, see reference 11).

How does Eβ act to mediate this regional control of chromatin unfolding? In light of our estimation of the 5' limit of Eβ action proximal to the Dβ1 gene segment, it is significant that specific defects in rearrangement and accessibility of this segment have recently been described after the genomic deletion of a 3-kb upstream DNA fragment 27. The deleted region, encompassing three DNase I hypersensitive sites (HS, usually associated with occupied regulatory sequences) present in DN cells 21, contains a minimal promoter adjacent to Dβ1 which is most active in T cell lines when linked with Eβ 1718. Although our results cannot formally exclude a model in which Eβ and the Dβ1 upstream sites independently delimit and organize the intervening region by unidirectional activity, the prevailing view on the mechanisms of long-distance gene activation by enhancers 528, and the lower nuclease sensitivity that we observe for the region immediately 5' of Dβ1 in the absence of Eβ (Fig. 1, site #4^R), would argue rather that Eβ acts by providing a crucial activity to the Dβ1 promoter. In the simplest scheme, this may occur via interactions between transcription factors bound to each element (and to a putative promoter element associated with the Dβ2-Jβ2 cluster) which, as described in other systems, could themselves nucleate further contacts with chromatin modifying and/or remodeling activities (e.g., SWI/SNF-like complexes and/or histone acetyltransferases) and also bind to components of the basal transcription machinery. The resulting nucleoprotein structures, by favoring the propagation of chromatin changes across the intervening sequences, would ultimately permit their accessibility. The observed differential histone H3 acetylation profiles at Dβ loci of Eβ-containing versus Eβ-deleted alleles, and release of the regional block in Dβ/Jβ gene expression and recombination upon TSA treatment of Eβ^–/– thymocytes (Fig. 6 and Fig. 7), are both compatible with this scenario. Importantly, the aforementioned model could accommodate an intrinsic boundary (or insulator) function for the Dβ promoter–associated sequences as well, analogous to the recently described property of certain promoters in Drosophila 29.

Our data do not indicate the exact function(s) of Eβ in promoting chromatin remodeling at the Dβ-Jβ loci. These may include an opening activity 30, a protective role against the spreading of repressive structures 31, and/or an influence on subnuclear localization 32. Also, the localized promotion of germline transcription, demethylation, and/or histone hyperacetylation may be involved since these activities are not separable in Rag^–/– thymi and are downmodulated in Rag^–/–Eβ^–/– thymi (Fig. 3 and Fig. 4; methylation appears less strictly regulated at Jβ2 sequences, however). Recently, based on results from cell transfection experiments using an enhancerless construct that carries an inducible promoter, germline transcription has been proposed to be sufficient for heterochromatin disruption and rearrangement at Dβ and Jβ gene segments 33. However, a direct role for transcription remains to be demonstrated given all the examples in which, upon factor binding, chromatin remodeling occurs independently of RNA polymerase movement 34, a situation that could also be relevant to the control of V(D)J recombination 35.

A Putative Function for Eβ in Vβ Gene Recombination?
An unusual profile consisting of nuclease sensitivity, germline transcription, methylation, and intermediate level of histone acetylation was observed at the Vβ genes in both Rag^–/– and Rag^–/–Eβ^–/– thymocytes. Thus, different from the Dβ-Jβ loci, establishment of a specific chromosomal structure at these regions depends on regulatory sequences distinct from Eβ, possibly involving the T cell–specific, Vβ promoter–associated elements 36. How does this structure impact on Vβ-to-DJβ rearrangement, and do these findings preclude any role for Eβ in regulating these recombination events? Contrary to Dβ-to-Jβ, Vβ-to-DJβ rearrangements are not found at Eβ-deleted alleles even when using sensitive PCR assays 67. Provocatively, DSBs at Vβ gene–flanking RSS are difficult to detect at Eβ-deleted and wt loci (Hempel, W.M., unpublished data), in striking contrast to endonuclease-mediated cleavage (e.g., Fig. 2). These findings may reflect a lower efficiency for Vβ-to-DJβ joining (relative to Dβ-to-Jβ joining) and/or, because V(D)J recombination involves coupled cleavage of pairs of gene segments (e.g., reference 10), the inaccessibility of partner RSS at the downstream Dβ-Jβ loci. Keeping in mind the differences between Dβ-to-Jβ and Vβ-to-DJβ assembly (see Introduction), it is therefore tempting to speculate on how these same factors may impact on the control of Vβ rearrangement in the wt situation where Eβ is active. First, the inherent inefficiency of V gene rearrangement, operating through epigenetic control, has been hypothesized to accommodate allelic exclusion at given antigen receptor genes 37. In this respect, the unusual profile (e.g., accessible/transcribed sequences are classically undermethylated) which we (this study) and others 20 have described at Vβ genes is intriguing. CpG methylation, in particular, could underlie a "unique" chromosomal structure to yield a system in which Vβ gene activation for recombination becomes stochastic and infrequent, and, ultimately, monoallelic. This would be consistent with data suggesting that, at individual Ig/TCR loci, V gene rearrangement in general, and allelic exclusion in particular, correlate with demethylation but not with transcription 4. Second, the interaction between Eβ- and Dβ-associated promoters may insulate the intervening, fully accessible, Dβ-to-Jβ recombining domains (see above). Thus, Vβ-to-DJβ joining would have to proceed through the casual relaxation of these boundary structures at either allele. This, for example, could involve competition for Eβ between the Vβ and Dβ promoters, similar to a model that has been proposed to explain developmental gene activation at multigene loci 28. The specialized structure at the Vβ genes would strongly disfavor the Vβ promoters in this competition, accounting for both the delay and allele specificity of Vβ gene recombination (i.e., relative to Dβ-to-Jβ assembly).

Methylation of Vβ-containing sequences may yet serve an additional function. It has been shown that methylated recombination substrates become highly resistant to V(D)J rearrangement after undergoing DNA replication, presumably by acquiring a fully repressed chromosomal structure 38. The intense cellular proliferation that is triggered by β selection 1 may similarly drive the unrearranged, methylated Vβ genes into a heterochromatin structure, thus terminating Vβ recombination and locking in TCR-β allelic exclusion as thymocytes develop towards the DP cell stage.

Apparently, in early T cells, the downstream Vβ14 gene presents the same profile of Eβ-independent regulation as that of the 5' Vβ genes (Fig. 2, Fig. 3 B, 5, and 6). This is quite remarkable, given their differences in position relative to Eβ. Therefore, Vβ14 gene recombination may also be subject to gene competition as proposed above. Conceivably, at this location, the system may be further refined by the cis-acting, possibly blocking, activity of a regulatory element(s) located between Eβ and the Vβ14 promoter. Interestingly, T cell–specific hypersensitive (HS) sites have been described within the region that links Eβ to the Vβ14 gene 21 and, within a transgenic substrate, intron sequences in this region have been reported to function in regulating lineage-specific accessibility 3. However, it remains possible that, as thymocytes develop beyond the DN cell stage, Vβ14 may be regulated differently from the 5' Vβ genes, as suggested by reports indicating that DP cell development results in augmented expression instead of an inhibition of this element 2021. Additional studies will be necessary to further clarify this issue.