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Variable Gene Segment Chromatin Structure1
Department of Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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rearrangement. Allelic exclusion is achieved through asynchronous V to DJ recombination as well as feedback inhibition that terminates recombination once a functional rearrangement has occurred. Because the accessibility of V gene segment chromatin is diminished as thymocytes undergo allelic exclusion at the CD4–CD8– (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 V to DJ recombination on one or both alleles before the enforcement of feedback. On rearranged alleles, V 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 V segments on a nonrearranged allele, those situated upstream of a functionally rearranged V segment are contained in active chromatin as judged by histone H3 acetylation, histone H3 lysine 4 (K4) methylation, and germline transcription. Nevertheless, these V 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 V to DJ recombination after the double-negative stage of thymocyte development. |
Introduction |
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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 DJC gene clusters whose accessibility and rearrangement are regulated by Tcrb enhancer (E) (13). The V gene segments are located further 5' and are separated from the DJC segments by a 250-kb region containing trypsinogen genes that are inactive in T cells. The exception is V14, which is situated on the 3' edge of the locus. The V gene segments are associated with their own promoters and are regulated independently of E before rearrangement (13, 17). However, VDJ recombination places the promoter of the rearranged V within the regulatory influence of E, where together they coordinate transcription of the mature Ag receptor gene (18).
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to J rearrangement on both alleles. V to DJ rearrangement then follows on a single allele. Asynchronous V to DJ rearrangement allows the cell to test for a functional rearrangement on the first allele before initiating V to DJ rearrangement on the second allele. The production of a TCR 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 V to DJ 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 V to DJ rearrangement and feedback inhibition once a functional VDJ rearrangement has been formed (4, 5).
Although progress has been made in understanding the requirements for activating VDJ rearrangement, the mechanisms underlying feedback inhibition of the Tcrb locus are not well understood. The observed loss of V chromatin accessibility within the 5' V gene cluster during DN to DP differentiation suggested that decreased V accessibility may prevent further rearrangements in the DP compartment (17, 20, 21, 22). However, we recently showed that forced activation of the distal V 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 V 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 V segments have addressed the status of V segments in circumstances in which they remain in a germline configuration and far from E (17, 20, 21, 22). However, the status of V segments upstream of a rearranged V in physiological T cell populations has not previously been addressed. These V segments are much closer to E and may as a consequence be accessible for secondary V to DJ2 rearrangements when RAG is re-expressed in DP thymocytes. This study addresses the accessibility status of these V segments and the need for additional constraints on V rearrangement to enforce allelic exclusion.
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Materials and Methods |
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Rag2–/– mice, Rag2–/– x Tcrb transgenic mice (R x ) (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.
Antibodies
Anti-CD16 and CD32 (clone 2.4G2), anti-V11 (clone RR3-15), anti-V9 (clone MR10-2), anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), anti-CD3
(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.
Isolation of lymph node (LN) T cell populations
To isolate peripheral T cells for analysis of the nonrearranged allele, 100 x 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 x 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-CD3.
To isolate peripheral T cells expressing TCRs using V9 or V11, 200–300 x 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 x 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 V11 and V9, washed twice, and incubated with anti-biotin magnetic beads (Miltenyi Biotec). Approximately 10 x 106 V9+V11+ 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-CD3, FITC-conjugated anti-V11 and anti-V9, 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– CD3+ V11+V9+ gate and sorted a second time using the same parameters.
Isolation of thymocyte subpopulations
To isolate DP thymocytes for ChIP analysis of the nonrearranged allele, 30 x 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 V11 surface expression, 100 x 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-V11. Cell sorting and analysis were conducted using a FACVantage SE and CellQuest software. CD4+CD8+ thymocytes were collected with and without a V11low gate.
ChIP
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. V8.1 RSS primers were: 5'-GCTTCCCTTTCTCAGACAGCTG-3' and 5'-CTTCCTGGGGTACACAGAGAGC-3'. V11P and T4-T5 primers (22); V12, V13, and Cad primers (23); and E primers (16) were previously described.
Germline transcription
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 V primers were positioned in the leader sequence and downstream of the RSS, respectively. V8.1 primers were 5'-GCGAACCTGCCTTAGTTCTG-3' and 5'-CTTCCTGGGGTACACAGAGAGC-3'. V12, V13, and Actb primers were previously described (23). The V8.1 probe was 5'-GCTTCCCTTTCTCAGACAGCTG-3', and the Actb probe was 5'-GTCATCACTATTGGCAACGAG-3'.
Analysis of V recombination intermediates
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 J
42 were 5'-GAGGATGCTCTAAGCCTTCCC-3' and 5'-GGGAAGATGATGTCGCTTTTC-3', respectively. Primers and probes for V12, V13, and Cd14 (23) and the linker and linker primer (29) were described previously.
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Results |
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To examine the chromatin structure of V gene segments on alleles that had previously undergone a V to DJ rearrangement, we analyzed B6/SJL F1 hybrid mice. The SJL Tcrb locus lacks an 80-kb region within the V gene cluster encompassing V5.2 to V9 (Fig. 1). Isolation of LN T cells expressing V9 or V11 (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 V gene segments upstream of V11, but absent from SJL, we could restrict our analysis to those V gene segments situated upstream of a functional VDJ rearrangement.
To examine the chromatin structure of V gene segments on an allele that had not undergone V to DJ rearrangement, we analyzed M4/SJL F1 hybrid mice. M4 mice carry a strain 129 Tcrb locus that lacks the D2-J2 gene cluster and includes a mutation in the 5'D1 RSS that renders it nonfunctional (25) (Fig. 1). Therefore, the M4 Tcrb locus is incapable of V to DJ rearrangement. By studying the chromatin structure and activity of V gene segments that are missing on the SJL allele, we could restrict our analysis to those V gene segments contained on the nonrearranged M4 allele.
Differential regulation of V gene segments on rearranged and nonrearranged alleles
To determine the chromatin structure of V gene segments directly upstream of a functional VDJ rearrangement, we prepared chromatin from B6/SJL LN T cells expressing V9 or V11 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 CD3+ LN T cells and total DP thymocytes from M4/SJL mice to determine chromatin structure of the corresponding V 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 V9low or V11low cells could not be isolated at a sufficient purity. Substantial contamination with other Vlow cells could lead to artifactual ChIP signals, reflecting the state of these V segments when included in functional VDJ rearrangements.
Using real-time PCR to evaluate enrichment of V 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). V12, V13, and V8.1 lie 10.5, 16.5, and 20 kb upstream of V11, respectively. On the nonrearranged allele, all three V 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, V12 displayed high levels of H3 acetylation and H3 K4 dimethylation, whereas V13 displayed intermediate levels. V8.1 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 E 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 E exerts control over the chromatin structure of V gene segments situated immediately upstream of a functional VDJ rearrangement, and that its influence diminishes with distance.
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gene segments that were located upstream of a functional VDJ rearrangement or within a nonrearranged V array. For these experiments, we used flow cytometry to isolate two independent preparations of B6/SJL LN T cells expressing V11 or V9 for analysis of the rearranged allele, as well as CD3+ 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 x mice as positive and negative controls, respectively. Analysis of the rearranged allele revealed germline transcription at V12 that was equal to that seen in DN thymocytes (Fig. 3). Germline transcripts were very low at V13, but could be readily detected further upstream at V8.1. In contrast, germline transcription of the same V segments was nearly undetectable on the nonrearranged allele.
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We have shown that V gene segments directly upstream of a functional VDJ rearrangement possess several hallmarks of rearrangement-permissive chromatin. We expect that many Tcrb alleles will contain VDJ1 rearrangements, thereby leaving the DJ2 gene cluster as a potential substrate for secondary V to DJ2 recombination (30). If accessibility were the sole constraint on recombination, we would predict that V gene segments situated upstream of a functional VDJ1 rearrangement could undergo secondary rearrangement to the DJ2 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 V11 expression (Fig. 4A) so we could measure recombination events at V segments upstream of functional V11 rearrangements. Although V11low 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 V12 and V13. To control for effective linker ligation, we also assayed for signal ends at J42 in the Tcra locus. J42 signal ends should not be detected in DN3 thymocytes, but should be easily detected in association with Tcra recombination in DP thymocytes.
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12 and V13 were readily detected in control DN3 thymocytes, but were reduced by at least 90% in DP thymocytes (Fig. 4B). Importantly, these signal end intermediates were similarly reduced in V11-enriched DP thymocytes. These reductions were apparent despite readily detectable levels of available DJ2 recombination substrates in the various DP preparations (data not shown). Moreover, the reductions were apparent despite substantial up-regulation of J42 signal ends in all DP samples (Fig. 4B). We conclude that although V 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. |
Discussion |
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chromatin structure in an allele-specific fashion during normal T cell development. Previous studies of V 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 V 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 V gene segments on the nonrearranged allele to reside in inactive chromatin in both DP thymocytes and peripheral T cells. In contrast, analysis of V gene segments on the rearranged allele showed that those V segments located directly upstream of the functional VDJ rearrangement displayed hallmarks of accessible and active chromatin. Because the rearranged allele displayed a gradient of V segment activation that was inversely related to the distance from E, we attribute this activation to an interaction with E as opposed to an intrinsic feature of the V segments.
Of note, V12, the first V gene segment upstream of a functional V11 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, V12 signal ends remained low in DP thymocytes enriched for V11 rearrangements. Because we do detect low levels of signal ends in DP thymocytes, we cannot formally exclude that V to DJ 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 V to DJ rearrangements to the DN compartment. One potential caveat is that we did not assess V12 chromatin structure on the rearranged allele specifically in DP thymocytes. Due to low TCR surface expression, we were unable to isolate V11low 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 V segments should be less accessible in DP thymocytes than in mature T cells. In fact, multiple studies have shown that the DJC region, to which the V 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 V12, at V13 we detected elevated histone modifications with minimal increase in germline transcription, and at V8.1 we detected the reciprocal pattern. These results suggest that histone modifications and germline transcription may be distinct consequences of E proximity. Under conditions in which the V13 and V8.1 promoters may be competing with more proximal promoters for the influence of E, differences in promoter structure could direct distinct outcomes of enhancer-promoter interactions at the two gene segments. As compared with V12 accessibility, V13 and V8.1 accessibility may be only partial or may occur at reduced frequency on alleles with V9 or V11 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 V gene segments on a pair of rearranged alleles. The authors failed to detect germline transcription of V gene segments upstream of the rearranged V 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 V10 gene segment brought into proximity of E by a large deletion (32). However, in this case, Northern blots did identify germline transcripts for V segments upstream of V10, 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 V gene segments operates at a level beyond accessibility. Unlike other V segments, V14 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. V14 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 V to DJ on a rearranged allele, could also be at play. Allelic exclusion was also maintained for the V10 gene segment when it was repositioned to the D1 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 E, functional V10 rearrangements were restricted to a single allele and feedback inhibition was apparently enforced. Finally, work from our laboratory has shown that the 5' V gene cluster could be forced to adopt an accessible configuration in DP thymocytes by ectopic placement of the TCR enhancer, but V recombination in the DP compartment remained suppressed (23). Together, the data argue for a unique feedback mechanism imposed on V 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 V 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 TCR enhancer has the ability to recruit all components necessary for Tcra recombination in DP thymocytes (34, 35), it cannot initiate V to DJ 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 V to DJ 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 V gene segments upstream of a functional rearrangement are maintained in an active chromatin environment due to the influence of E. Nevertheless, these V segments are restricted from further recombination in DP thymocytes despite proximity to D2 and expression of recombinase. The data imply a requirement for additional developmental stage-specific factor(s) that regulates the V to DJ 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 TCR 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).
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 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. 
2 Address correspondence and reprint requests to Dr. Michael S. Krangel, Department of Immunology, P.O. Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address: krang001{at}mc.duke.edu
3 Abbreviations used in this paper: RSS, recombination signal sequence; Cad, carbamoyl transferase dihydrorotase; ChIP, chromatin immunoprecipitation; DN, double negative; DP, double positive; E, Tcrb enhancer; LN, lymph node.
Received for publication July 6, 2005. Accepted for publication August 18, 2005.
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), promoters (bent arrows), and E
