Allelic exclusion of the murine Tcrb locus is imposed at the level of recombination and restricts each cell to produce one functional VDJβ rearrangement. Allelic exclusion is achieved through asynchronous Vβ to DJβ recombination as well as feedback inhibition that terminates recombination once a functional rearrangement has occurred. Because the accessibility of Vβ gene segment chromatin is diminished as thymocytes undergo allelic exclusion at the CD4CD8 (double-negative) to CD4+CD8+ (double-positive) transition, chromatin regulation was thought to be an important component of the feedback inhibition process. However, previous studies of chromatin regulation addressed the status of Tcrb alleles using genetic models in which both alleles remained in a germline configuration. Under physiological conditions, developing thymocytes would undergo Vβ to DJβ recombination on one or both alleles before the enforcement of feedback. On rearranged alleles, Vβ gene segments that in germline configuration are regulated independently of the Tcrb enhancer are now brought into its proximity. We show in this study that in contrast to Vβ segments on a nonrearranged allele, those situated upstream of a functionally rearranged Vβ segment are contained in active chromatin as judged by histone H3 acetylation, histone H3 lysine 4 (K4) methylation, and germline transcription. Nevertheless, these Vβ gene segments remain refractory to recombination in double-positive thymocytes. These results suggest that a unique feedback mechanism may operate independent of chromatin structure to inhibit Vβ to DJβ recombination after the double-negative stage of thymocyte development.

A diverse repertoire of Ag receptors is used by the adaptive immune system to provide protection from foreign pathogens. This diversity is achieved through somatic recombination of Ig loci in B lymphocytes and TCR loci in T lymphocytes. Combinatorial rearrangement of V, D, and J gene segments within these loci is highly regulated and occurs during discrete stages of lymphocyte development (1, 2, 3). The Ig and Tcrb loci are also restricted to the expression of Ag receptor proteins from a single allele. This form of regulation is termed allelic exclusion and is strictly enforced in Ig and Tcrb loci at the level of rearrangement (4, 5, 6). Allelic exclusion ensures that each lymphocyte expresses Ag receptors of a single specificity.

In both T and B lymphocytes, each V, D, and J gene segment is flanked by a recombination signal sequence (RSS)3 that is recognized and cleaved by the recombinase proteins RAG1 and RAG2 (RAG) (7). RAG proteins assemble a synaptic complex between appropriate RSSs and produce double-strand breaks between the RSSs and coding regions. These breaks are then repaired by members of the nonhomologous end joining pathway to form coding joints and signal joints.

The use of a common recombinase and conserved RSSs throughout T and B cell development implies that the recombinase must be targeted to specific receptor loci during defined developmental stages. Chromatin structure is thought to play a crucial role in controlling this targeting, analogous to its role in transcriptional regulation (3, 8, 9, 10). Promoters and enhancers within Ag receptor loci orchestrate cell-specific and developmentally regulated changes in chromatin structure, which allow the recombinase machinery access to RSSs (2, 3, 11). Rearrangement-permissive chromatin is characterized by active germline transcription, by increased nuclease sensitivity, and by histone modifications such as H3 hyperacetylation and H3 K4 methylation (12, 13, 14, 15, 16).

The Tcrb locus spans ∼700 kb and can be divided into two main functional domains (Fig. 1). The 3′ end of the locus contains two DJCβ gene clusters whose accessibility and rearrangement are regulated by Tcrb enhancer (Eβ) (13). The Vβ gene segments are located further 5′ and are separated from the DJCβ segments by a 250-kb region containing trypsinogen genes that are inactive in T cells. The exception is Vβ14, which is situated on the 3′ edge of the locus. The Vβ gene segments are associated with their own promoters and are regulated independently of Eβ before rearrangement (13, 17). However, VDJβ recombination places the promoter of the rearranged Vβ within the regulatory influence of Eβ, where together they coordinate transcription of the mature Ag receptor gene (18).

FIGURE 1.

Tcrb locus alleles. Schematic of the wild-type (C57BL/6 or 129), SJL, and M4 Tcrb loci showing V, D, J, and C gene segments (▪), promoters (bent arrows), and Eβ (•). The SJL Tcrb locus lacks an 80-kb region spanning Vβ5.2 to Vβ9. The M4 Tcrb locus lacks the Dβ2-Jβ2 gene cluster and includes a mutation in the 5′Dβ1 RSS that renders it nonfunctional (25 ).

FIGURE 1.

Tcrb locus alleles. Schematic of the wild-type (C57BL/6 or 129), SJL, and M4 Tcrb loci showing V, D, J, and C gene segments (▪), promoters (bent arrows), and Eβ (•). The SJL Tcrb locus lacks an 80-kb region spanning Vβ5.2 to Vβ9. The M4 Tcrb locus lacks the Dβ2-Jβ2 gene cluster and includes a mutation in the 5′Dβ1 RSS that renders it nonfunctional (25 ).

Close modal

RAG is first expressed in CD4CD8 double-negative (DN) thymocytes, allowing Dβ to Jβ rearrangement on both alleles. Vβ to DJβ rearrangement then follows on a single allele. Asynchronous Vβ to DJβ rearrangement allows the cell to test for a functional rearrangement on the first allele before initiating Vβ to DJβ rearrangement on the second allele. The production of a TCRβ protein, expressed at the cell surface in the form of a pre-TCR, results in signals that promote differentiation, proliferation, a temporary down-regulation of RAG expression, and a halt to Vβ to DJβ rearrangement (19). Transition into the CD4+CD8+ double-positive (DP) compartment is accompanied by the re-expression of RAG and rearrangement of the Tcra locus. Thus, allelic exclusion of the Tcrb locus is dependent upon both asynchronous Vβ to DJβ rearrangement and feedback inhibition once a functional VDJβ rearrangement has been formed (4, 5).

Although progress has been made in understanding the requirements for activating VDJβ rearrangement, the mechanisms underlying feedback inhibition of the Tcrb locus are not well understood. The observed loss of Vβ chromatin accessibility within the 5′ Vβ gene cluster during DN to DP differentiation suggested that decreased Vβ accessibility may prevent further rearrangements in the DP compartment (17, 20, 21, 22). However, we recently showed that forced activation of the distal Vβ gene segments in DP thymocytes was not sufficient to overcome the block to recombination (23). This implies that another mechanism, distinct from accessibility, may constrain Vβ rearrangement in DP thymocytes.

Why might there be a need for regulation at a level distinct from accessibility? Various studies demonstrating a loss in accessibility and transcription of Vβ segments have addressed the status of Vβ segments in circumstances in which they remain in a germline configuration and far from Eβ (17, 20, 21, 22). However, the status of Vβ segments upstream of a rearranged Vβ in physiological T cell populations has not previously been addressed. These Vβ segments are much closer to Eβ and may as a consequence be accessible for secondary Vβ to DJβ2 rearrangements when RAG is re-expressed in DP thymocytes. This study addresses the accessibility status of these Vβ segments and the need for additional constraints on Vβ rearrangement to enforce allelic exclusion.

Rag2−/− mice, Rag2−/− × Tcrb transgenic mice (R × β) (24), C57BL/6 (B6)/SJL F1 hybrid mice, M4/SJL F1 hybrid mice, 129/M4 F1 hybrid mice, and Tcrb−/−Tcrd−/− mice were housed at the Duke University Vivarium. M4 mice were a gift of B. Sleckman (Washington University, St. Louis, MO) (25). All mice were used in accordance with protocols approved by the Duke University Institutional Animal Care and Use Committee.

Anti-CD16 and CD32 (clone 2.4G2), anti-Vβ11 (clone RR3-15), anti-Vβ9 (clone MR10-2), anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), anti-CD3ε (clone 145-2C11), anti-CD24 (cloneM1/69), anti-CD25 (clone 7D4), and anti-CD44 (clone 1M7) were purchased from BD Pharmingen. Cell staining was performed on ice according to standard procedures, unless otherwise noted.

To isolate peripheral T cells for analysis of the nonrearranged allele, 100 × 106 LN T cells were harvested from M4/SJL mice and passed through a nylon wool column to remove adherent cells. Nonadherent cells were eluted with 20 ml of cold RPMI 1640 medium containing 10% FCS, centrifuged at 500 × g for 10 min at 4°C, and resuspended in 2 ml of fresh medium. T cell purity was determined by flow cytometry on a sample stained with FITC-conjugated anti-CD3ε.

To isolate peripheral T cells expressing TCRs using Vβ9 or Vβ11, 200–300 × 106 LN T cells from B6/SJL mice were harvested in RPMI 1640 medium containing 10% FCS and incubated for 60 min at 37°C on a nylon wool column (Polysciences) to remove adherent cells. The T cells were adjusted to a concentration of 25 × 106 per ml and were incubated with a mAb specific for CD16 and CD32 to reduce nonspecific staining.

For chromatin immunoprecipitation (ChIP) analysis, T cells were then incubated on ice with biotin-conjugated mAbs specific for Vβ11 and Vβ9, washed twice, and incubated with anti-biotin magnetic beads (Miltenyi Biotec). Approximately 10 × 106 Vβ9+Vβ11+ LN T cells were recovered using the POSSELD program on an autoMACS (Miltenyi Biotec). Purity was determined by flow cytometry on a sample stained with Texas Red-conjugated streptavidin (BD Pharmingen).

For germline transcription analysis, T cells were stained on ice using PE-conjugated anti-CD3ε, FITC-conjugated anti-Vβ11 and anti-Vβ9, and the exclusion dye 7-aminoactinomycin D. Cell sorting and analysis were conducted using a using a FACSVantage SE (BD Biosciences) and CellQuest software. T cells were collected from a 7-aminoactinomycin D CD3ε+ Vβ11+Vβ9+ gate and sorted a second time using the same parameters.

To isolate DP thymocytes for ChIP analysis of the nonrearranged allele, 30 × 106 thymocytes were harvested from M4/SJL mice. After incubation with a mAb specific for CD16 and CD32, the cells were stained on ice with PE-conjugated anti-CD8 and FITC-conjugated anti-CD4. Ten million DP thymocytes were isolated using a FACStarPlus (BD Biosciences) and CellQuest software.

Populations of DN3 and DP thymocytes were isolated from 129/M4 mice, as previously described (26), for analysis of signal end intermediates. To analyze signal ends in a DP population enriched for Vβ11 surface expression, 100 × 106 thymocytes were incubated with a mAb specific for CD16 and CD32. Staining was performed on ice with PE-Cy5-conjugated anti-CD8, FITC-conjugated anti-CD4, and PE-conjugated anti-Vβ11. Cell sorting and analysis were conducted using a FACVantage SE and CellQuest software. CD4+CD8+ thymocytes were collected with and without a Vβ11low gate.

Cells were incubated with formaldehyde to cross-link protein-DNA complexes, and cross-linked chromatin was sheared to an average size of 100–500 bp and subjected to immunoprecipitation, as previously described (27). Immunoprecipitation was performed using 5 μg each of antidiacetylated histone H3, antidimethylated histone H3 K4, and control rabbit IgG (Upstate Biotechnology). The bound and input fractions were quantified using SYBR green real-time PCR (Roche). Ratios of bound/input were calculated and were normalized to those for carbamoyl transferase dihydrorotase (Cad) in each sample. Vβ8.1 RSS primers were: 5′-GCTTCCCTTTCTCAGACAGCTG-3′ and 5′-CTTCCTGGGGTACACAGAGAGC-3′. Vβ11P and T4-T5 primers (22); Vβ12, Vβ13, and Cad primers (23); and Eβ primers (16) were previously described.

RNA was extracted from cells using TRIzol (Invitrogen Life Technologies), according to the manufacturer’s instructions. Contaminating genomic DNA was removed with DNA-free (Ambion), according to the manufacturer’s instructions. cDNA synthesis was performed with Transcriptor (Roche) and random hexamer primers (Roche), according to the Roche protocol. PCR was performed on 3-fold serial dilutions of cDNA using a touchdown PCR strategy: 5 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at annealing temperature, and 1 min at 72°C, and a 10-min extension at 72°C. Annealing temperature was held at 68°C, 65°C, and 62°C for 5 cycles each, and at 58°C for 17 cycles. Amplicons were electrophoresed through agarose gels and analyzed by Southern blot using 32P-labeled oligonucleotide probes. Forward and reverse Vβ primers were positioned in the leader sequence and downstream of the RSS, respectively. Vβ8.1 primers were 5′-GCGAACCTGCCTTAGTTCTG-3′ and 5′-CTTCCTGGGGTACACAGAGAGC-3′. Vβ12, Vβ13, and Actb primers were previously described (23). The Vβ8.1 probe was 5′-GCTTCCCTTTCTCAGACAGCTG-3′, and the Actb probe was 5′-GTCATCACTATTGGCAACGAG-3′.

To analyze signal end intermediates, thymocyte genomic DNA was extracted and linker ligation was performed, as previously described (28). Linker-ligated DNA was then used to amplify signal end intermediates by touchdown PCR (see above). Cd14 was amplified by PCR, as follows: 94°C for 5 min, followed by 20 cycles of 94°C for 30 s, 62°C for 30 s, 72°C for 1 min, and a 10-min extension at 72°C. Amplicons were electrophoresed through agarose gels and analyzed by Southern blot using 32P-labeled oligonucleotide probes. The primer and probe for Jα42 were 5′-GAGGATGCTCTAAGCCTTCCC-3′ and 5′-GGGAAGATGATGTCGCTTTTC-3′, respectively. Primers and probes for Vβ12, Vβ13, and Cd14 (23) and the linker and linker primer (29) were described previously.

To examine the chromatin structure of Vβ gene segments on alleles that had previously undergone a Vβ to DJβ rearrangement, we analyzed B6/SJL F1 hybrid mice. The SJL Tcrb locus lacks an 80-kb region within the Vβ gene cluster encompassing Vβ5.2 to Vβ9 (Fig. 1). Isolation of LN T cells expressing Vβ9 or Vβ11 (each missing on the SJL allele) provided a T cell population in which the functional rearrangement must have occurred on the B6 Tcrb allele. By studying the chromatin structure and activity of Vβ gene segments upstream of Vβ11, but absent from SJL, we could restrict our analysis to those Vβ gene segments situated upstream of a functional VDJβ rearrangement.

To examine the chromatin structure of Vβ gene segments on an allele that had not undergone Vβ to DJβ rearrangement, we analyzed M4/SJL F1 hybrid mice. M4 mice carry a strain 129 Tcrb locus that lacks the Dβ2-Jβ2 gene cluster and includes a mutation in the 5′Dβ1 RSS that renders it nonfunctional (25) (Fig. 1). Therefore, the M4 Tcrb locus is incapable of Vβ to DJβ rearrangement. By studying the chromatin structure and activity of Vβ gene segments that are missing on the SJL allele, we could restrict our analysis to those Vβ gene segments contained on the nonrearranged M4 allele.

To determine the chromatin structure of Vβ gene segments directly upstream of a functional VDJβ rearrangement, we prepared chromatin from B6/SJL LN T cells expressing Vβ9 or Vβ11 and performed ChIP using Abs specific for diacetylated histone H3 and dimethylated histone H3 K4. As a comparison, we performed the same ChIPs on both CD3ε+ LN T cells and total DP thymocytes from M4/SJL mice to determine chromatin structure of the corresponding Vβ gene segments on a nonrearranged allele. ChIP was also performed on a non-T cell sample to serve as a negative control. We could not perform an analysis of the rearranged allele in DP thymocytes because the relevant Vβ9low or Vβ11low cells could not be isolated at a sufficient purity. Substantial contamination with other Vβlow cells could lead to artifactual ChIP signals, reflecting the state of these Vβ segments when included in functional VDJβ rearrangements.

Using real-time PCR to evaluate enrichment of Vβ promoter and RSS sequences in immunoprecipitated chromatin, we detected significant differences in H3 acetylation and H3 K4 dimethylation on the rearranged and nonrearranged alleles (Fig. 2, left panels). Vβ12, Vβ13, and Vβ8.1 lie 10.5, 16.5, and 20 kb upstream of Vβ11, respectively. On the nonrearranged allele, all three Vβ gene segments displayed H3 acetylation and H3 K4 dimethylation at levels that were similar to the non-T cell control. However, on the rearranged allele, Vβ12 displayed high levels of H3 acetylation and H3 K4 dimethylation, whereas Vβ13 displayed intermediate levels. Vβ8.1 displayed levels of H3 acetylation and H3 K4 dimethylation that were not distinguishable from those on the nonrearranged allele. Control analysis revealed elevated H3 acetylation and H3 K4 dimethylation at Eβ and low levels at inactive trypsinogen genes in all samples (Fig. 2, right panels). We interpret the pattern of histone modifications on the rearranged allele to indicate that Eβ exerts control over the chromatin structure of Vβ gene segments situated immediately upstream of a functional VDJβ rearrangement, and that its influence diminishes with distance.

FIGURE 2.

Histone H3 diacetylation and H3 K4 dimethylation of Vβ gene segments on rearranged and nonrearranged alleles. Chromatin was prepared from Vβ11+Vβ9+ LN T cells isolated from B6/SJL mice (purity 80–88%) for analysis of the rearranged (R) allele, from CD3ε+ LN T cells isolated from M4/SJL mice (purity 85–89%) and DP thymocytes isolated from M4/SJL mice (purity 89–99%) for analysis of the nonrearranged (NR) allele, and from unfractionated splenocytes isolated from Tcrb−/−Tcrd−/− mice (non-T cells). Immunoprecipitations were analyzed at both Vβ promoter (P) and RSS sites. The strategy allowed analysis of Vβ segments on a single allele, except in the case of non-T cell chromatin, in which both alleles were assayed simultaneously. Similarly, in all chromatin preparations, both alleles were assayed simultaneously at Eβ and at T4/T5 (a site within the inactive trypsinogen gene segments upstream of the Vβ cluster). Bound and input fractions were quantified using real-time PCR, and ratios of bound/input were expressed relative to the values for Cad in each sample. The data shown are the mean ± SEM for three to four independent ChIP experiments.

FIGURE 2.

Histone H3 diacetylation and H3 K4 dimethylation of Vβ gene segments on rearranged and nonrearranged alleles. Chromatin was prepared from Vβ11+Vβ9+ LN T cells isolated from B6/SJL mice (purity 80–88%) for analysis of the rearranged (R) allele, from CD3ε+ LN T cells isolated from M4/SJL mice (purity 85–89%) and DP thymocytes isolated from M4/SJL mice (purity 89–99%) for analysis of the nonrearranged (NR) allele, and from unfractionated splenocytes isolated from Tcrb−/−Tcrd−/− mice (non-T cells). Immunoprecipitations were analyzed at both Vβ promoter (P) and RSS sites. The strategy allowed analysis of Vβ segments on a single allele, except in the case of non-T cell chromatin, in which both alleles were assayed simultaneously. Similarly, in all chromatin preparations, both alleles were assayed simultaneously at Eβ and at T4/T5 (a site within the inactive trypsinogen gene segments upstream of the Vβ cluster). Bound and input fractions were quantified using real-time PCR, and ratios of bound/input were expressed relative to the values for Cad in each sample. The data shown are the mean ± SEM for three to four independent ChIP experiments.

Close modal

To obtain an independent assessment of chromatin structure and function, we used RT-PCR to compare germline transcription of Vβ gene segments that were located upstream of a functional VDJβ rearrangement or within a nonrearranged Vβ array. For these experiments, we used flow cytometry to isolate two independent preparations of B6/SJL LN T cells expressing Vβ11 or Vβ9 for analysis of the rearranged allele, as well as CD3ε+ LN T cells from M4/SJL mice for analysis of the nonrearranged allele. We also analyzed DN thymocytes of Rag2−/− mice and DP thymocytes of R × β mice as positive and negative controls, respectively. Analysis of the rearranged allele revealed germline transcription at Vβ12 that was equal to that seen in DN thymocytes (Fig. 3). Germline transcripts were very low at Vβ13, but could be readily detected further upstream at Vβ8.1. In contrast, germline transcription of the same Vβ segments was nearly undetectable on the nonrearranged allele.

FIGURE 3.

Germline transcription of Vβ gene segments on rearranged and nonrearranged alleles. PCR was performed on 3-fold serial dilutions of cDNA (indicated by wedges) prepared from Rag2−/− (DN thymocytes), from R × β (DP thymocytes), from two independent preparations of Vβ11+Vβ9+ LN T cells from B6/SJL mice (rearranged allele; purity 97%), and from CD3ε+ LN T cells from M4/SJL mice (nonrearranged (NR) allele, purity 89%). The wild-type allele was of C57BL/6 origin. PCR was also performed from control samples prepared without reverse transcriptase (−RT). RT-PCR of Actb was used to assess cDNA loading.

FIGURE 3.

Germline transcription of Vβ gene segments on rearranged and nonrearranged alleles. PCR was performed on 3-fold serial dilutions of cDNA (indicated by wedges) prepared from Rag2−/− (DN thymocytes), from R × β (DP thymocytes), from two independent preparations of Vβ11+Vβ9+ LN T cells from B6/SJL mice (rearranged allele; purity 97%), and from CD3ε+ LN T cells from M4/SJL mice (nonrearranged (NR) allele, purity 89%). The wild-type allele was of C57BL/6 origin. PCR was also performed from control samples prepared without reverse transcriptase (−RT). RT-PCR of Actb was used to assess cDNA loading.

Close modal

We have shown that Vβ gene segments directly upstream of a functional VDJβ rearrangement possess several hallmarks of rearrangement-permissive chromatin. We expect that many Tcrb alleles will contain VDJβ1 rearrangements, thereby leaving the DJβ2 gene cluster as a potential substrate for secondary Vβ to DJβ2 recombination (30). If accessibility were the sole constraint on recombination, we would predict that Vβ gene segments situated upstream of a functional VDJβ1 rearrangement could undergo secondary rearrangement to the DJβ2 gene cluster when recombinase is re-expressed in DP thymocytes.

To test for secondary recombination events, we assayed for the presence of recombination intermediates within the DP compartment. We used flow cytometry to isolate DN3 and DP thymocytes from 129/M4 F1 hybrid mice. We also isolated DP thymocytes enriched for low level Vβ11 expression (Fig. 4 A) so we could measure recombination events at V segments upstream of functional Vβ11 rearrangements. Although Vβ11low cells could not be isolated to homogeneity, the enriched population should allow an assessment of whether these recombination events occur at higher frequency than in unfractionated DP thymocytes. We then prepared genomic DNA and performed ligation-mediated-PCR to detect signal ends at Vβ12 and Vβ13. To control for effective linker ligation, we also assayed for signal ends at Jα42 in the Tcra locus. Jα42 signal ends should not be detected in DN3 thymocytes, but should be easily detected in association with Tcra recombination in DP thymocytes.

FIGURE 4.

Feedback inhibition of Vβ segments upstream of a functional VDJβ rearrangement. A, Flow cytometric analysis of DP thymocytes from 129/M4 mice before (thin line) and after (bold line) enrichment for Vβ11low thymocytes. The Ig isotype control (dashed line) was used to set the Vβ11low sorting gate. B, Detection of signal end recombination intermediates in thymocyte subpopulations of 129/M4 mice. The DN3 and DP no. 1 samples were obtained at 96% purity from the same pool of unfractionated thymocytes. DP no. 2 (96% pure) and Vβ11low (enriched) DP no. 2 samples were similarly obtained from the same population of unfractionated thymocytes. Vβ11+ thymocytes were enriched at least 8-fold in Vβ11low DP no. 2 as compared with unfractionated DP thymocytes. Three-fold serially diluted samples of linker-ligated genomic DNA (wedges) were analyzed by PCR and Southern blot using 32P-labeled oligonucleotide probes. Linker-ligated splenocyte DNA, nonlinker-ligated thymocyte DNA, and a sample without DNA served as controls. Cd14 amplification was used to assess DNA loading. The data are representative of two independent experiments.

FIGURE 4.

Feedback inhibition of Vβ segments upstream of a functional VDJβ rearrangement. A, Flow cytometric analysis of DP thymocytes from 129/M4 mice before (thin line) and after (bold line) enrichment for Vβ11low thymocytes. The Ig isotype control (dashed line) was used to set the Vβ11low sorting gate. B, Detection of signal end recombination intermediates in thymocyte subpopulations of 129/M4 mice. The DN3 and DP no. 1 samples were obtained at 96% purity from the same pool of unfractionated thymocytes. DP no. 2 (96% pure) and Vβ11low (enriched) DP no. 2 samples were similarly obtained from the same population of unfractionated thymocytes. Vβ11+ thymocytes were enriched at least 8-fold in Vβ11low DP no. 2 as compared with unfractionated DP thymocytes. Three-fold serially diluted samples of linker-ligated genomic DNA (wedges) were analyzed by PCR and Southern blot using 32P-labeled oligonucleotide probes. Linker-ligated splenocyte DNA, nonlinker-ligated thymocyte DNA, and a sample without DNA served as controls. Cd14 amplification was used to assess DNA loading. The data are representative of two independent experiments.

Close modal

Signal end intermediates at Vβ12 and Vβ13 were readily detected in control DN3 thymocytes, but were reduced by at least 90% in DP thymocytes (Fig. 4,B). Importantly, these signal end intermediates were similarly reduced in Vβ11-enriched DP thymocytes. These reductions were apparent despite readily detectable levels of available DJβ2 recombination substrates in the various DP preparations (data not shown). Moreover, the reductions were apparent despite substantial up-regulation of Jα42 signal ends in all DP samples (Fig. 4 B). We conclude that although Vβ gene segments directly upstream of a functional rearrangement reside in accessible chromatin, their rearrangement is suppressed in the DP compartment when recombinase is re-expressed.

We have used the deletion in the SJL Tcrb locus as a window to examine Vβ chromatin structure in an allele-specific fashion during normal T cell development. Previous studies of Vβ chromatin structure in thymocytes were performed on nonrearranged alleles in mice that lack recombinase activity (20, 22). These studies showed that the chromatin encompassing the main Vβ cluster transitions to a less active state as thymocytes differentiate to the DP compartment. Consistent with this, our allele-specific analysis of physiological T cell populations revealed Vβ gene segments on the nonrearranged allele to reside in inactive chromatin in both DP thymocytes and peripheral T cells. In contrast, analysis of Vβ gene segments on the rearranged allele showed that those Vβ segments located directly upstream of the functional VDJβ rearrangement displayed hallmarks of accessible and active chromatin. Because the rearranged allele displayed a gradient of Vβ segment activation that was inversely related to the distance from Eβ, we attribute this activation to an interaction with Eβ as opposed to an intrinsic feature of the Vβ segments.

Of note, Vβ12, the first Vβ gene segment upstream of a functional Vβ11 rearrangement, showed levels of H3 diacetylation, H3 K4 dimethylation, and germline transcription that were similar to those seen in the DN compartment (23). Despite these findings, Vβ12 signal ends remained low in DP thymocytes enriched for Vβ11 rearrangements. Because we do detect low levels of signal ends in DP thymocytes, we cannot formally exclude that Vβ to DJβ recombination can still proceed at low levels in this compartment. However, our data clearly indicate that recombination is substantially suppressed in DP thymocytes and imply that additional developmental constraints restrict Vβ to DJβ rearrangements to the DN compartment. One potential caveat is that we did not assess Vβ12 chromatin structure on the rearranged allele specifically in DP thymocytes. Due to low TCR surface expression, we were unable to isolate Vβ11low DP thymocytes to the purity necessary for ChIP and RT-PCR studies. Rather, our conclusions rely on the analysis of the rearranged allele in mature T cells. However, we have no reason to believe that Vβ segments should be less accessible in DP thymocytes than in mature T cells. In fact, multiple studies have shown that the DJCβ region, to which the Vβ segments are approximated, displays increased accessibility in DP as compared with DN thymocytes (17, 21, 22).

Although on the rearranged allele we detected coordinate increases in histone modifications and germline transcription at Vβ12, at Vβ13 we detected elevated histone modifications with minimal increase in germline transcription, and at Vβ8.1 we detected the reciprocal pattern. These results suggest that histone modifications and germline transcription may be distinct consequences of Eβ proximity. Under conditions in which the Vβ13 and Vβ8.1 promoters may be competing with more proximal promoters for the influence of Eβ, differences in promoter structure could direct distinct outcomes of enhancer-promoter interactions at the two gene segments. As compared with Vβ12 accessibility, Vβ13 and Vβ8.1 accessibility may be only partial or may occur at reduced frequency on alleles with Vβ9 or Vβ11 rearrangements.

Our germline transcription data differ from that reported in a previous publication (31). That study analyzed T cell hybridomas and simultaneously addressed germline transcription upstream of rearranged Vβ gene segments on a pair of rearranged alleles. The authors failed to detect germline transcription of Vβ gene segments upstream of the rearranged Vβ segments. However, analysis was by Northern blot, and the authors did not demonstrate the sensitivity to detect germline transcripts even in DN thymocytes. Another study addressed germline transcription upstream of a Vβ10 gene segment brought into proximity of Eβ by a large deletion (32). However, in this case, Northern blots did identify germline transcripts for Vβ segments upstream of Vβ10, and showed that they were not down-regulated between the DN and DP stages. Although these data were obtained by simultaneous analysis of both alleles in genetically manipulated mice, the results are consistent with our own data.

This study as well as other published work suggest that feedback inhibition of Vβ gene segments operates at a level beyond accessibility. Unlike other Vβ segments, Vβ14 displays increased germline transcription, increased histone H3 acetylation, and increased accessibility to digestion with restriction enzymes in DP thymocytes as compared with DN thymocytes (17, 20, 21). Nevertheless, allelic exclusion is still enforced. Vβ14 rearranges by inversion and must form chromosomal coding joints and signal joints, and it has been suggested that feedback inhibition may be enforced by slow signal joint formation (17). However additional mechanisms, similar to those constraining Vβ to DJβ on a rearranged allele, could also be at play. Allelic exclusion was also maintained for the Vβ10 gene segment when it was repositioned to the Dβ1 region through a large scale deletion (32). Despite an increase in rearrangement frequency in DN thymocytes and enhanced accessibility in DP thymocytes as a result of its proximity to Eβ, functional Vβ10 rearrangements were restricted to a single allele and feedback inhibition was apparently enforced. Finally, work from our laboratory has shown that the 5′ Vβ gene cluster could be forced to adopt an accessible configuration in DP thymocytes by ectopic placement of the TCRα enhancer, but Vβ recombination in the DP compartment remained suppressed (23). Together, the data argue for a unique feedback mechanism imposed on Vβ segments that may act independently of accessibility.

It remains possible that accessibility as defined by increased germline transcription, H3 diacetylation, H3 K4 dimethylation, and increased sensitivity to nuclease digestion may not be sufficient to allow RAG access to Vβ RSSs. Because the nonamer portion of the RSS has been shown to act as a nucleosome positioning sequence, V(D)J recombination may require additional chromatin modifications and substantial nucleosome disruption specifically at the RSS (33). An undetected histone modification, or recruitment of a critical chromatin remodeler such as Brahma-related gene 1, could possibly distinguish Tcrb alleles in DN and DP (16). We cannot formally exclude this possibility. However, while TCRα enhancer has the ability to recruit all components necessary for Tcra recombination in DP thymocytes (34, 35), it cannot initiate Vβ to DJβ recombination at the same developmental stage (23). This implies that additional locus-specific and developmental stage-specific factors or events may be required for efficient Vβ to DJβ recombination.

Most recently, elegant studies using fluorescence in situ hybridization have revealed a change in the subnuclear positioning of Ig alleles before and after V(D)J recombination (36, 37, 38, 39). Before recombination, the Ig alleles are repositioned away from the nuclear periphery, where they undergo a large scale contraction event that is thought to bring distal V segments into proximity of D and J segments (38, 40). After successful recombination, the loci decontract, and one allele becomes associated with centromeric heterochromatin (37, 39). Thus, locus decontraction and movement of an allele to a silent region in the nucleus have been implicated in the feedback mechanism. However, neither of these mechanisms can account for the feedback imposed on a functionally rearranged allele in which the recombination process itself provides a means for both gene segment approximation and positioning in an active nuclear environment.

In summary, our data suggest that Vβ gene segments upstream of a functional rearrangement are maintained in an active chromatin environment due to the influence of Eβ. Nevertheless, these Vβ segments are restricted from further recombination in DP thymocytes despite proximity to Dβ2 and expression of recombinase. The data imply a requirement for additional developmental stage-specific factor(s) that regulates the Vβ to DJβ recombination step in either the DN or DP compartment. It remains possible that this factor(s) could determine additional chromatin modifications needed for RSS accessibility. Alternatively, the factor(s) could regulate the proximity of TCRβ locus RSSs to recombinase activity in thymocyte nuclei, or could otherwise impact the formation or stabilization of synaptic complexes. Regardless, the data suggest an additional control point for Tcrb alleles that seem otherwise poised for further recombination. This raises the question of whether such regulation could be reversed under any circumstances. In this regard, we wonder whether regulation at this level might play a role in the occasional reports of receptor revision in peripheral T cells (41, 42, 43, 44, 45).

We thank L. Martinek of the Duke University Cancer Center Flow Cytometry Facility for help in cell sorting and analysis, H. Boutrid for technical assistance, and Y. Zhuang, J. Jia, and B. Sleckman for critical review of the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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This work was supported by National Institutes of Health Grant AI35748 (to M.S.K.). A.M.J. was supported by a National Science Foundation Graduate Research Fellowship.

3

Abbreviations used in this paper: RSS, recombination signal sequence; Cad, carbamoyl transferase dihydrorotase; ChIP, chromatin immunoprecipitation; DN, double negative; DP, double positive; Eβ, Tcrb enhancer; LN, lymph node.

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