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A Change in the Structure of V Chromatin Associated with TCR Allelic Exclusion¹

Department of Immunology, Duke University Medical Center, Durham, NC 27710

	Abstract

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To investigate chromatin control of TCR rearrangement and allelic exclusion, we analyzed TCR chromatin structure in double negative (DN) thymocytes, which are permissive for TCR recombination, and in double positive (DP) thymocytes, which are postallelic exclusion and nonpermissive for V to DJ recombination. Histone acetylation mapping and DNase I sensitivity studies indicate V and DJ segments to be hyperacetylated and accessible in DN thymocytes. However, they are separated from each other by hypoacetylated and inaccessible trypsinogen chromatin. The transition from DN to DP is accompanied by selective down-regulation of V acetylation and accessibility. The level of DP acetylation and accessibility is minimal for five of six V segments studied but remains substantial for one. Hence, the observed changes in V chromatin structure appear sufficient to account for allelic exclusion of many V segments. They may contribute to, but not by themselves fully account for, allelic exclusion of others.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
During T and B lymphocyte development, TCR and Ig gene variable (V), diversity (D), and joining (J) segments are assembled by the process of V(D)J recombination (1, 2). In both lineages, V(D)J recombination occurs in a highly ordered and tightly regulated fashion (3, 4, 5, 6). In the B lineage, IgH locus rearrangement initiates in pro-B cells and occurs in two steps, first D_H to J_H, and then V_H to DJ_H. Rearrangement of Ig L chain loci (Ig and Ig) initiates subsequently, at the pre-B cell stage, and occurs in a single step (V to J or V to J). In similar fashion, V(D)J recombination occurs at two distinct stages in the development of T lineage cells. TCR locus rearrangement initiates in CD4^-CD8^- double negative (DN)³ thymocytes and occurs in two steps, first D to J and then V to DJ. TCR locus rearrangement initiates subsequently in CD4⁺CD8⁺ double positive (DP) thymocytes and occurs in a single step (V to J).

A striking regulatory feature of V(D)J recombination is the phenomenon of allelic exclusion, which restricts precursor lymphocytes to the production of a single, functional Ag receptor gene at a given locus. Allelic exclusion functions in highly analogous fashion at the IgH and TCR loci. In each case, the presence of a functional VDJ rearrangement on one allele inhibits the V to DJ step of rearrangement on the second allele by a feedback mechanism (7, 8, 9). For TCR , the feedback signal depends on the assembly of a TCR polypeptide into a pre-TCR complex with pre-T and CD3, and on the activity of the nonreceptor protein tyrosine kinase lck (10, 11). Similarly, for IgH this signal depends on the assembly of a membrane Igµ polypeptide into a pre-B cell receptor (BCR) complex (12, 13). Pre-TCR and pre-BCR signaling also induces critical developmental transitions during T and B lymphocyte development (from DN to DP and pro-B to pre-B, respectively), as well as the proliferative expansion of developing lymphocytes (14, 15). How pre-TCR- and pre-BCR-derived signals impact the process of V(D)J recombination to inhibit V to DJ rearrangement at the TCR and IgH loci is not well understood.

V(D)J recombination is initiated by the recombinase-activating gene (RAG)-1 and RAG-2 proteins, which introduce double-strand breaks at recombination signal sequences (RSSs) flanking Ig and TCR gene segments (1, 2). One level at which V(D)J recombination can be regulated is by developmental activation and inactivation of RAG gene expression. For example, developmental inactivation of RAG gene expression can account for the termination of V to J rearrangement upon transition of DP thymocytes to the single positive stage, and for termination of Ig L chain rearrangement on transition of pre-B cells to the immature B cell stage (6, 16). However, other developmental changes in V(D)J recombination can occur in the face of ongoing RAG gene expression and recombinase activity. The inhibition of V to DJ and V_H to DJ_H rearrangement that characterizes allelic exclusion falls into this category. Although pre-TCR and pre-BCR signaling down-regulates RAG expression in late-stage DN thymocytes and pro-B cells, respectively, allelic exclusion is enforced despite the subsequent up-regulation of RAG expression that is associated with V to J rearrangement in DP thymocytes, and with V to J and V to J rearrangement in pre-B cells (6, 16). In general, developmental changes in recombinase targeting have been attributed to developmental changes in locus accessibility to the recombinase, with chromatin structure under the control of specific promoters and enhancers (3, 4, 5, 6, 17, 18, 19). However, data addressing the role of chromatin structure in the process of allelic exclusion have been limited (20).

The TCR locus spans roughly 700 kb (21). At the 3' end are two D-J-C clusters, as well as a single, inverted V gene segment. The majority of V segments are scattered across a region that extends from 250 to 500 kb upstream of the D-J-C clusters. This V domain is flanked on the 5' side and, remarkably, on the 3' side as well, by extended arrays of trypsinogen genes and gene fragments. Moreover, one V segment is located upstream of the 5' trypsinogen cluster, 650 kb away from the D-J-C clusters. To date, V(D)J recombination at the TCR locus has been shown to depend on two cis-elements. E, situated downstream of C2, is required for all D to J and V to DJ rearrangement events (22, 23), whereas a germline promoter (PD1), situated upstream of D1, is required specifically for rearrangement events involving the D1-J1 cluster (24, 25). Thus, E and PD1 cooperate to promote rearrangement of D1 and J1 segments, but the extent to which this regulation occurs at the level of accessibility, vs at a later step in the reaction, is not fully resolved (24, 25, 26, 27).

TCR allelic exclusion appears not to involve a developmental transition involving D-J-C chromatin and the activities of E and PD1. First, signal ends associated with D to J rearrangement are present even in DP thymocytes, suggesting that these segments maintain accessibility to the recombinase at this stage (24). Second, in RAG^-/- thymocytes induced to differentiate to the DP stage, D-J-C chromatin maintains the germline transcription and CpG hypomethylation that characterizes the DN compartment (28). These results leave open the possibility that V chromatin is regulated independently of E, and that allelic exclusion is regulated at the level of V chromatin. Indeed, recent data indicate that accessibility and transcription of upstream and downstream V segments are maintained in DN thymocytes of E-/- mice (27). However, only limited data speak to the status of V segments in DP thymocytes: levels of germline transcripts for several upstream V segments (but not the downstream V) were found to decline on transition from DN to DP (28, 29). Thus, the notion that TCR allelic exclusion is associated with a change in the structure of V chromatin remains largely untested. In this study we present a detailed comparison of TCR locus chromatin structure both before and subsequent to allelic exclusion signals that addresses this issue in some detail.

	Materials and Methods

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CHIP

Chromatin immunoprecipitation (CHIP) analysis was performed as described (30), using rabbit antisera to diacetylated histone H3 (AcH3), tetraacetylated histone H4 (Upstate Biotechnology, Lake Placid, NY), and control rabbit IgG (Sigma-Aldrich, St. Louis, MO). Serial 3-fold dilutions of nucleosomal DNA (20, 6.7, and 2.2 ng) isolated from Ab bound and unbound fractions were amplified by 25 cycles of PCR (45 s at 94°C, 1 min at 51°C, 2 min at 72°C) in a 25-µl reaction. PCR products were electrophoresed through 1.5% agarose gels, transferred to nylon, and detected by hybridization with DNA fragments labeled by random priming. PCR primers used to amplify mononucleosomal DNA or to produce probes for hybridization are listed in Table I. TCR locus primers were designed with reference to GenBank files MMAE000663, MMAE000664, and MMAE000665. Primers for analysis of Oct-2 were described previously (30). Blots were washed in 1x SSC and 0.5% SDS for 20 min at 23°C with one change. A single serial dilution series produced from each DNA fraction was used for all PCR included in a single experiment. Quantification was accomplished using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Raw acetylation values were derived by determining the displacement between titration curves for the bound fraction of the AcH3 immunoprecipitation and unbound fraction of the control immunoprecipitation. Several factors insured that PCR signals were specific. First, there is typically only 50–70% sequence homology among V coding segments and <50% sequence homology in promoter and 3' flank regions. Second, repetitive elements in noncoding regions were avoided. Third, our detection strategy involved multiple steps, each imparting a degree of specificity: an initial PCR step to generate products from mononucleosomes, a second PCR step using one primer from the first step and one new primer to generate an overlapping probe, and, finally, detection of PCR products by hybridization with radiolabeled PCR probe.

Thymocytes were permeabilized and treated with DNase I as described (31, 32). Purified DNA (4 µg) was restriction digested, electrophoresed through 0.7% agarose, transferred to nylon, and assayed by hybridization with ³²P-labeled probes generated by random priming. DNA fragments DJ (MMAE00665 153,405–153,996), T4T5 (MMAE00663 92,771–93,461), V11 (MMAE00664 23,621–24,290), V12 (MMAE00664 14,657–15,446), and V13 (MMAE00664 7,916–8,370) were produced by PCR. Hybridization was quantified using a PhosphorImager.

	Results

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Strategy

Acetylation is structural modification of the amino-terminal tails of core histones that is associated with an open, nuclease-sensitive chromatin configuration (33, 34, 35, 36, 37). A variety of approaches have recently established a very tight relationship between histone hyperacetylation and accessibility for V(D)J recombination (18, 19, 27, 30, 38, 39, 40, 41, 42). Based on these results, we used acetylation mapping in the current study to evaluate changes in the structure of TCR locus chromatin that might be associated with TCR allelic exclusion.

We used a CHIP assay to measure the acetylation status of TCR locus chromatin before and after allelic exclusion. As a source of nonallelically excluded chromatin we used thymocytes of RAG^-/- mice (43). These thymocytes are primarily DNIII, a stage in which V to DJ rearrangement is normally permitted. However, an allelic exclusion signal cannot be generated in these mice due to the lack of TCR rearrangement and pre-TCR expression on the RAG^-/- background. As a source of allelically excluded chromatin we used thymocytes of RAG^-/- mice complemented with a rearranged TCR transgene (44). These thymocytes are almost exclusively DP and would already have received an allelic exclusion signal from the TCR -containing pre-TCR at the DN stage. The lack of V(D)J recombination on the RAG^-/- and RAG^-/- x TCR backgrounds ensures that endogenous TCR gene segments will be uniformly in germline configuration and therefore directly comparable in both chromatin preparations. Mononucleosomes prepared from RAG^-/- and RAG^-/- x TCR chromatin were immunoprecipitated with anti-AcH3 or a control serum, and PCR was used to assess the representation of particular segments of TCR locus chromatin in equivalent quantities of DNA isolated from the Ab bound and unbound fractions.

Structure of TCR locus chromatin in DN thymocytes

Because the DNIII compartment is permissive for V to DJ rearrangement, both V chromatin and DJ chromatin were expected to be in an open or accessible configuration in this compartment. To assay V chromatin, we initially analyzed a series of sites spanning 40 kb of the V locus, including five functional V segments, that is situated 400 kb upstream of E (Fig. 1). Within this region, we assayed the conserved decamer sequence (45, 46) in several V promoters (V12P, V11P, V9P), the RSS elements flanking V segments (V13R, V12R, V11R, V9R, V6R), and several intergenic sites located between V segments. To assay DJ chromatin we analyzed a site in PD1 (47, 48), just upstream of the D1 gene segment. Acetylation at these sites was compared with that of Oct-2 as a negative control not expressed in thymocytes (30), and to CD3 as a positive control expressed at high levels in thymocytes. All TCR locus sites were chosen so as to exclude the TCR transgene from analysis.

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Schematic of the TCR locus. , Gene segments; , RSSs; •, E; arrows, promoters. Dots identify sites analyzed by CHIP.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. TCR locus H3 acetylation in DN and DP thymocytes. A, Serial 3-fold dilutions of bound (B) and unbound (U) fractions of anti-AcH3 and control immunoprecipitated DNA were analyzed by PCR at the indicated sites. VxP and VxR designate a promoter and an RSS site, respectively, associated with a particular V, and 12/11-1, 12/11-2, 11/9-1, and 11/9-2 designate intergenic sites. B, H3 acetylation is plotted as B/U, with B representing the bound fraction of the anti-AcH3 immunoprecipitate and U representing the unbound fraction of the control immunoprecipitate (taken as equivalent to input). Although results of a single experiment are plotted, almost all TCR locus sites analyzed here were analyzed in two or three independent CHIP experiments starting from independent mononucleosome preparations. Statistical analysis revealed only minor variability at all sites except those displaying the highest acetylation values (V11R in DN and PD in DP). Thus, site to site and DN to DP comparisons are highly reliable at low to moderate acetylation values. Experimental variation observed at V11R and PD does not impact conclusions about changes between DN and DP (compare B to Fig. 4A).

View larger version (13K): [in this window] [in a new window]   FIGURE 3. V9 and trypsinogen H3 acetylation in DN thymocytes. A, Acetylation was measured as in Fig. 2 at a series of sites across the V9 region. Sites are identified based on their distance upstream of V9P or downstream of V9R. The site designated -2500 is identical to 11/9-2 in Fig. 2 and is 8 kb downstream of V11. Site +1350 is 10 kb upstream of V23. B, Acetylation was measured at sites spanning the 5' and 3' trypsinogen clusters. T4/T5 and T14/T15 are intergenic sites.

V2 is isolated from all other V segments at the extreme 5' end of the TCR locus, upstream of the 5' trypsinogen cluster (Fig. 1). It was therefore of interest to characterize the structure of V2 chromatin. As for V segments in the main cluster, V2 was found to be hyperacetylated in DN thymocytes (Fig. 4A). We did not analyze V14, which is isolated at the 3' end of the locus, because we could not distinguish endogenous V14 from a copy contained in the TCR transgene used to induce the DN to DP transition.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. TCR locus H3 and H4 acetylation in DN thymocytes, DP thymocytes, and non-T cells. A, H3 acetylation is plotted as B/U with normalization to the acetylation level of -actin in the same sample. Non-T cells were splenocytes of C-/-C-/- mice. B, H4 acetylation is plotted as in A. Non-T cells were splenocytes of C-/- mice.

Comparison of TCR locus chromatin structure in DN and DP thymocytes

To determine whether a change in TCR locus chromatin structure accompanies allelic exclusion, we compared H3 acetylation along V and DJ chromatin in DN and DP thymocytes (Fig. 2). We found the control Oct-2 gene to be equivalently hypoacetylated in the DN and DP compartments, confirming the DN and DP samples to be comparable. CD3 acetylation was 3-fold higher in DP thymocytes than in DN thymocytes. With respect to the TCR locus, we found acetylation at PD1 to be nearly twice as high in the DP as the DN compartment. Thus DJ chromatin appears to be active in both thymocyte populations, consistent with inferences drawn from previous studies (24, 28). However, quite different results were obtained for V chromatin. At all points assayed, including both V segments and sites between V segments, H3 acetylation was lower in the DP compartment than in the DN compartment. This drop was in the 82–92% range for most V segments but was as little as 54% for V11. Importantly, these reductions occur despite elevated acetylation of PD1 in the same samples. Hence, there is a striking and highly selective change in the structure of V chromatin that accompanies allelic exclusion. Similar results were obtained when many of the same sites were analyzed in independent experiments (Fig. 4A and data not shown).

Comparison of TCR locus chromatin structure in thymocytes and non-T cells

Although acetylation at V11 was reduced in DP as compared with DN thymocytes, acetylation in the DP compartment was still substantial (Figs. 2 and 4A). To evaluate the significance of this observation, we compared acetylation in DN and DP cells to that in non-T cells at selected sites across the locus (Fig. 4). As a source of non-T cells, we examined splenocytes of either C^-/-C^-/- mice (Fig. 4A) or C^-/- mice (Fig. 4B), both of which lack T cells (50, 51). These splenocytes consist primarily of B cells and macrophages and should retain the entire V locus in germline configuration. To insure equivalence of the DN, DP, and non-T cell samples, we analyzed acetylation at a site in -actin (data not shown) and normalized the acetylation values for TCR locus chromatin on the basis of the -actin data. The validity of this approach was confirmed by the equivalent levels of histone H3 (Fig. 4A) and H4 (Fig. 4B) acetylation calculated for sites within trypsinogen chromatin, which appears to be inactive in all three cell populations.

Analysis of histone H3 (Fig. 4A) and H4 (Fig. 4B) acetylation in non-T cells revealed the TCR locus acetylation profiles to be largely T cell-specific. Strikingly, the residual H3 acetylation at V11 in DP cells was found to be still elevated with respect to non-T cells. A similar result was obtained for V9, although the level of residual DP acetylation was much lower in this case. Analysis of H4 acetylation yielded a similar picture. At two sites associated with V segments and two intergenic sites, H4 acetylation was substantially reduced in DP relative to DN thymocytes. However, the reductions in H4 acetylation were not as great as those for H3. Moreover, for V13 and V9, H4 acetylation in the DP compartment was somewhat elevated in DP cells as compared with non-T cells. We conclude that V11, in particular, appears to be in an intermediate rather than a fully repressed state in DP thymocytes. Several other V segments may display low-level activity as well.

One exception to the T cell specificity of the TCR locus acetylation patterns was V2. As for most other V segments, H3 acetylation at this site was dramatically down-regulated on transition from DN to DP. However, V2 acetylation was slightly higher in non-T cells than in DP thymocytes (Fig. 4A). Perhaps consistent with this, recent data indicated that V2 displays promiscuous activity in other lineages, as V2 germline transcripts were identified in NK cells and in a myeloid-enriched population of bone marrow cells (52).

DNase I sensitivity of TCR locus chromatin in DN and DP thymocytes

To understand whether the above documented structural transition in V chromatin reflects a functionally relevant difference in V segment accessibility, we directly probed the accessibility of TCR locus chromatin by measuring its general sensitivity to DNase I digestion (Fig. 5, A–F). Thymocytes were mildly permeabilized with detergent and incubated briefly with increasing concentrations of DNase I, following which genomic DNA was purified, digested with restriction enzymes, and analyzed by Southern blot. To minimize artifactual differences in sensitivity to DNase I digestion, we chose to compare fragments that avoided known promoters and thereby avoided potential hypersensitive sites, that were of similar size and therefore provided similar targets for DNase I digestion, and that were analyzed on a single blot. Digest and probe combinations were chosen so as to exclude the TCR transgene from analysis.

View larger version (32K): [in this window] [in a new window]   FIGURE 5. TCR locus sensitivity to DNase I digestion. Permeabilized thymocytes were incubated with increasing concentrations of DNase I. Genomic DNA was prepared and digested with PstI and PvuII for analysis of fragments V12 (2.66 kb), V13 (1.85 kb), T4/T5 (2.19 kb), and DJ (1.97 kb), or with PstI and HindIII for analysis of fragments V11 (2.29 kb), T4/T5 (2.76 kb), and DJ (1.97 kb). A, Sample analysis of V12, DJ, and T4/T5 in DP thymocytes. Shown are the results of simultaneous probing for all three fragments on a Southern blot (upper panel) and PhosphorImager quantification of residual signal for each fragment as a function of DNase I concentration (lower panel). B and C, PhosphorImager quantification of relative DNase I sensitivities of V12, V13, DJ, and T4/T5 in DN and DP thymocytes. Signals for T4/T5 are normalized to 100% at all DNase I concentrations, and residual signals for other fragments are expressed as a percentage of the residual T4/T5 signal at each concentration (i.e., percentage of V12 remaining/percentage of T4/T5 remaining x 100). Two independent blots prepared from independent restriction digests were each serially probed with the same sets of probes and analyzed by PhosphorImager several times, and mean and SE of five to seven total analyses of the two blots are provided. Similar conclusions were also drawn from an independent DNase I digestion series. D–F, PhosphorImager quantification of relative DNase I sensitivities of V11, DJ, and T4/T5 in DN thymocytes, DP thymocytes, and C-/- splenocytes. Analysis was as for B and C, except that only three replicates were conducted for F.

For additional comparative analyses that eliminated DNA loading as a variable, hybridization signals for trypsinogen chromatin were normalized to 100% at all DNase I concentrations, and residual hybridization signals for other fragments were expressed relative to the trypsinogen signal at each point (Fig. 5, B–F). In comparing DN to DP thymocytes, we found that DP thymocytes routinely require less DNase I than DN thymocytes to produce equivalent digests of bulk genomic DNA (data not shown). This may result from increased intrinsic fragility and permeability of DP thymocytes to DNase I or increased sensitivity to detergent permeabilization. Concentrations of DNase I that produced equivalent digests of bulk DNA revealed DJ chromatin to have roughly similar sensitivity to DNase I digestion, relative to trypsinogen chromatin, in the two compartments (Fig. 5, compare B to C and D to E). However, relative to trypsinogen and DJ chromatin, the DNase I sensitivity of sites within V chromatin was found to vary between DN and DP thymocytes. V12 and V13 display sensitivities in the DN compartment that are nearly equivalent to that of DJ chromatin, but they display sensitivities in the DP compartment that are much more like that of trypsinogen chromatin (Fig. 5, B and C). Thus, both V segments are highly accessible in DN thymocytes, with V12 accessibility slightly less than that of V13. V12 is converted to an inaccessible configuration in DP thymocytes, whereas V13 displays residual, albeit much diminished, accessibility.

V11 is notable for its unusually high level of acetylation in DP thymocytes (Figs. 2 and 4). DNase I analysis revealed V11 to be highly accessible in DN thymocytes and to display only a modest reduction in accessibility on transition to DP (Fig. 5, D and E). Strikingly, although V11 is significantly more accessible than trypsinogen chromatin in DP thymocytes, it is as inaccessible as trypsinogen chromatin in non T cells (Fig. 5, E and F). Overall, the conclusions drawn from DNase I sensitivity analysis of V11, V12, and V13 match well with the conclusions drawn from acetylation mapping.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
To gain a better understanding of the role of chromatin structure in the developmental regulation of TCR locus rearrangement and allelic exclusion, we have analyzed the structure of TCR locus chromatin in DN thymocytes, a stage that is permissive for TCR recombination, and in DP thymocytes, a stage which is postallelic exclusion and nonpermissive for V to DJ recombination. Our results are summarized schematically in Fig. 6. We find that both V segments and DJ segments reside in accessible chromatin in the DN compartment but that accessible chromatin domains are separated from each other by large stretches of inaccessible trypsinogen chromatin. The transition to DP occurs without any reduction in accessibility of DJ chromatin but is accompanied by conversion of V chromatin to a less-accessible configuration. Of note, this conversion is not uniform for all V segments. Thus, V12 and V13 undergo rather dramatic changes in accessibility as defined by DNase I sensitivity. Based on the observed parallels between the acetylation and accessibility transitions of these V segments, we predict that V2, V6, and V9, which were only studied at the level of acetylation, undergo dramatic changes in accessibility as well. In contrast, the accessibility change at V11 is rather mild, and significant V11 accessibility is maintained in DP thymocytes. Thus we document changes in V chromatin structure that appear sufficient to account for allelic exclusion of several V segments and that may contribute to, but may not by themselves fully account for, allelic exclusion of V11.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. Schematic representation of TCR locus chromatin structure in DN and DP thymocytes. A cartoon version of the H3 acetylation profile across prototypical regions of trypsinogen, V, and DJC chromatin is presented.

That locally elevated acetylation over individual V segments might reflect V promoter function begs the question of the relationship between acetylation levels and transcriptional activity. We note that although CD3 and V11 are both heavily acetylated in DN thymocytes, CD3 transcripts are detectable by Northern blot, whereas V11 transcripts are not (data not shown). Moreover, although we find substantial acetylation of V6 in DN thymocytes, V6 transcripts were not detected by RT-PCR (52). Although suggestive that acetylation and transcription do not correlate, an important caveat is that these approaches measure steady state levels of V transcripts rather than rates of V transcription. Nevertheless, discordance between acetylation and transcription is certainly possible, because recruitment of histone acetyltransferases to promoters can precede, and can be experimentally segregated from, transcriptional activity per se (53, 54). Additional work will be required to clarify this issue.

An important finding of ours is the down-regulated but nevertheless heterogeneous nature of V accessibility in the DP compartment. All V segments analyzed displayed reduced accessibility in the DP as compared with the DN compartment. Nevertheless, whereas most analyzed V segments (i.e., V12, V13, and, we predict, V2, V6, and V9 as well) become either inaccessible or nearly so, V11 clearly displays residual accessibility, at least as defined by sensitivity to DNase I digestion. Thus, whereas the changes in chromatin structure at V2, V6, V9, V12, and V13 have the potential to account for allelic exclusion of these gene segments, it is unclear whether the change at V11 is sufficient to account for its allelic exclusion. We considered the possibility that allelic exclusion might not be complete for V11, but V11 was shown to be efficiently allelically excluded in previous work (11).

An important issue with respect to V11 is how accurately measures of general accessibility within chromatin (i.e., acetylation, DNase I sensitivity) predict the frequency of RAG cleavage. Although acetylation seems to correlate well with accessibility for V(D)J recombination in numerous instances, another chromatin modification (55, 56) could be most relevant for accessibility to RAG. In addition, RSS positioning with respect to the underlying nucleosome can significantly impact RAG access and cleavage (41, 57). We note as well that accessibility is clearly not the only factor governing RAG cleavage in chromatin. Sequence variation among natural RSS heptamer and nonamers (58, 59), spacer regions (60), and coding flanks (61, 62, 63, 64) can significantly impact rearrangement frequencies as well. It is notable that V12, which is inaccessible in the DP compartment, carries a nonamer (GCAAAAACA) that diverges from the consensus (ACAAAAACC) at only the end positions. In contrast, V13, which displays low DP accessibility, and V11, which displays substantial DP accessibility, carry more divergent nonamers (GCACAAAGC and GCAAGAAAC, respectively), with substitutions at positions known to significantly inhibit recombination in test substrates (65, 66). Similarly, V12 has a permissive C nucleotide immediately 5' of the heptamer, whereas V13 has a T nucleotide that in some studies appears to inhibit recombination (61, 64). Thus, chromatin accessibility, as defined here, may integrate with factors like RSS positioning with respect to nucleosomes and with variation in RSS or flanking sequences to equalize V segments as recombination substrates in DP cells. In this manner, V11 could have a frequency of RAG cleavage in DP cells that is essentially equivalent to that of V12, despite the difference in general chromatin accessibility.

As a cautionary note, we point out that the fully germline TCR locus alleles analyzed in our study are not identical to the DJ rearranged alleles that are the physiological substrates for allelic exclusion. Fully down-regulated V accessibility could conceivably require prior D to J rearrangement, a possibility we cannot exclude at the present time.

The notion that TCR allelic exclusion is enforced primarily at the level of V segment accessibility would be consistent with prior analyses of the IgH locus, which demonstrated V_H RSSs to serve as substrates for exogenously introduced RAG in isolated nuclei of pro-B cells but not mature B cells (20), and demonstrated V_H segments to display greater sensitivity to DNase I digestion in pro-B cells as compared with pre-B or mature T cells (67). Taken together, these studies support the existence of a general mechanism for the allelic exclusion process. Nevertheless, given the ambiguous data for V11, we cannot exclude the possibility thatallelic exclusion is controlled, at least in part, by a non-chromatinbased mechanism that inhibits V to DJ rearrangement on an allele in which V and DJ segments retain some degree of accessibility to RAG. How this might occur is unclear, but one possibility is suggested by recent data demonstrating that V rearrangement can be mediated by the 12-bp RSS 5' of D1, but not those associated with J segments (68, 69). This raises the possibility of a coupling factor for V to DJ rearrangement that might itself be developmentally regulated. In summary, we have demonstrated clear structural and functional changes in V chromatin in thymocytes undergoing TCR allelic exclusion. Additional studies will be required to carefully evaluate the relative roles of chromatin-based and non-chromatin-based regulation in this process.

	Acknowledgments

 
We thank Dr. Barry Sleckman for helpful comments on the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants GM41052 and AI49934.

² Address correspondence and reprint requests to Dr. Michael S. Krangel, Department of Immunology, Duke University Medical Center, PO Box 3010, Durham, NC 27710. E-mail address: krang001{at}mc.duke.edu

³ Abbreviations used in this paper: DN, double negative; DP, double positive; AcH3, anti-diacetylated histone H3; BCR, B cell receptor; CHIP, chromatin immunoprecipitation; RAG, recombinase-activating gene; RSS, recombination signal sequence; B, bound; U, unbound.

Received for publication November 9, 2001. Accepted for publication December 21, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Fugmann, S. D., A. I. Lee, P. E. Shockett, I. J. Villey, D. G. Schatz. 2000. The RAG proteins and V(D)J recombination: complexes, ends and transposition. Annu. Rev. Immunol. 18:495.[Medline]
2. Grawunder, U., E. Harfst. 2001. How to make ends meet in V(D)J recombination. Curr. Opin. Immunol. 13:186.[Medline]
3. Sleckman, B. P., J. R. Gorman, F. W. Alt. 1996. Accessibility control of antigen receptor variable region gene assembly: role of cis-acting elements. Annu. Rev. Immunol. 14:459.[Medline]
4. Schlissel, M. S., P. Stanhope-Baker. 1997. Accessibility and the developmental regulation of V(D)J recombination. Semin. Immunol. 9:161.[Medline]
5. Hempel, W. M., I. Leduc, N. Mathieu, R. K. Tripathi, P. Ferrier. 1998. Accessibility control of V(D)J recombination: lessons from gene targeting. Adv. Immunol. 69:309.[Medline]
6. Hesslein, D. G. T., D. G. Schatz. 2001. Factors and forces controlling V(D)J recombination. Adv. Immunol. 78:169.[Medline]
7. Weaver, D., F. Costantini, T. Imanishi-Kari, D. Baltimore. 1985. A transgenic immunoglobulin µ gene prevents rearrangement of endogenous genes. Cell 42:117.[Medline]
8. Nussenzweig, M. C., A. C. Shaw, E. Sinn, D. B. Danner, K. L. Holmes, III H. C. Morse, P. Leder. 1987. Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin µ. Science 236:816.[Abstract/Free Full Text]
9. Uematsu, Y., S. Ryser, Z. Dembic, P. Borgulya, P. Krimpenfort, A. Berns, H. von Boehmer, M. Steinmetz. 1988. In transgenic mice the introduced functional T cell receptor gene prevents expression of endogenous genes. Cell 52:831.[Medline]
10. Aifantis, I., J. Buer, H. von Boehmer, O. Azogui. 1997. Essential role of the pre-T cell receptor in allelic exclusion of the T cell receptor locus. Immunity 7:601.[Medline]
11. Anderson, S. J., S. D. Levin, R. M. Perlmutter. 1993. Protein tyrosine kinase p56^lck controls allelic exclusion of T-cell receptor -chain genes. Nature 365:552.[Medline]
12. Kitamura, D., K. Rajewsky. 1992. Targeted disruption of µ chain membrane exon causes loss of heavy-chain allelic exclusion. Nature 356:154.[Medline]
13. Loffert, D., A. Ehlich, W. Muller, K. Rajewsky. 1996. Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development. Immunity 4:133.[Medline]
14. von Boehmer, H., H. J. Fehling. 1997. Structure and function of the pre-T cell receptor. Annu. Rev. Immunol. 15:433.[Medline]
15. Meffre, E., R. Casellas, M. C. Nussenzweig. 2000. Antibody regulation of B cell development. Nat. Immunol. 1:379.[Medline]
16. Nagaoka, H., W. Yu, M. C. Nussenzweig. 2000. Regulation of RAG expression in developing lymphocytes. Curr. Opin. Immunol. 12:187.[Medline]
17. Hernandez-Munain, C., M. McMurry, M. S. Krangel. 1999. Regulation of chromatin accessibility for V(D)J recombination. Cold Spring Harbor Symp. Quant. Biol. 64:183.[Medline]
18. Roth, D. B., Y. Roth. 2000. Unequal access: regulating V(D)J recombination through chromatin remodeling. Cell 103:699.[Medline]
19. Krangel, M. S.. 2001. V(D)J recombination becomes accessible. J. Exp. Med. 193:F27.[Free Full Text]
20. Stanhope-Baker, P., K. M. Hudson, A. L. Shaffer, A. Constantinescu, M. S. Schlissel. 1996. Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro. Cell 85:887.[Medline]
21. Rowen, L., B. F. Koop, L. Hood. 2000. The complete 685-kilobase DNA sequence of the human T cell receptor locus. Science 272:1755.[Abstract]
22. Bouvier, G., F. Watrin, M. Naspetti, C. Verthuy, P. Naquet, P. Ferrier. 1996. Deletion of the mouse T-cell receptor gene enhancer blocks T-cell development. Proc. Natl. Acad. Sci. USA 93:7877.[Abstract/Free Full Text]
23. Bories, J.-C., J. Demengeot, L. Davidson, F. W. Alt. 1996. Gene-targeted deletion and replacement of the T-cell receptor -chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility. Proc. Natl. Acad. Sci. USA 93:7871.[Abstract/Free Full Text]
24. Whitehurst, C. E., S. Chattopadhyay, J. Chen. 1999. Control of V(D)J recombinational accessibility of the D 1 gene segment at the TCR locus by a germline promoter. Immunity 10:313.[Medline]
25. Whitehurst, C. E., M. S. Schlissel, J. Chen. 2000. Deletion of germline promoter PD1 from the TCR locus causes hypermethylation that impairs D1 recombination by multiple mechanisms. Immunity 13:703.[Medline]
26. Hempel, W. M., P. Stanhope-Baker, N. Mathieu, F. Huang, M. S. Schlissel, P. Ferrier. 1998. Enhancer control of V(D)J recombination at the TCR locus: differential effects on DNA cleavage and joining. Genes Dev. 12:2305.[Abstract/Free Full Text]
27. Mathieu, N., W. M. Hempel, S. Spicuglia, C. Verthuy, P. Ferrier. 2000. Chromatin remodeling by the T cell receptor (TCR)- gene enhancer during early T cell development: implications for the control of TCR- locus recombination. J. Exp. Med. 192:625.[Abstract/Free Full Text]
28. Chattopadhyay, S., C. E. Whitehurst, F. Schwenk, J. Chen. 1998. Biochemical and functional analysis of chromatin changes at the TCR- gene locus during CD4^-CD8^- to CD4⁺CD8⁺ thymocyte differentiation. J. Immunol. 160:1256.[Abstract/Free Full Text]
29. Senoo, M., Y. Shinkai. 1998. Regulation of V germline transcription in RAG-deficient mice by the CD3-mediated signals: implications of V transcriptional regulation in TCR allelic exclusion. Int. Immunol. 10:553.[Abstract/Free Full Text]
30. McMurry, M. T., M. S. Krangel. 2000. A role for histone acetylation in the developmental regulation of V(D)J recombination. Science 287:495.[Abstract/Free Full Text]
31. Boyes, J., G. Felsenfeld. 1996. Tissue-specific factors additively increase the probability of the all-or-none formation of a hypersensitive site. EMBO J. 15:2496.[Medline]
32. Hernandez-Munain, C., B. P. Sleckman, M. S. Krangel. 1999. A developmental switch from TCR enhancer to TCR enhancer function during thymocyte maturation. Immunity 10:723.[Medline]
33. Lee, D., J. J. Hayes, D. Pruss, A. P. Wolffe. 1993. A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell 72:73.[Medline]
34. Hebbes, T. R., A. L. Clayton, A. W. Thorne, C. Crane-Robinson. 1994. Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken -globin chromosomal domain. EMBO J. 13:1823.[Medline]
35. Vettese-Dadey, M., P. A. Grant, T. R. Hebbes, C. Crane-Robinson, C. D. Allis, J. L. Workman. 1996. Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro. EMBO J. 15:2508.[Medline]
36. Krajewski, W. A., P. B. Becker. 1998. Reconstitution of hyperacetylated, DNase I-sensitive chromatin characterized by high conformational flexibility of nucleosomal DNA. Proc. Natl. Acad. Sci. USA 95:1545.
37. Schubeler, D., C. Francastel, D. M. Cimbora, A. Reik, D. I. K. Martin, M. Groudine. 2000. Nuclear localization and histone acetylation: a pathway for chromatin opening and transcriptional activation of the human -globin locus. Genes Dev. 14:940.[Abstract/Free Full Text]
38. Durum, S. K., S. Candeias, H. Nakajima, W. J. Leonard, A. M. Baird, L. J. Berg, K. Muegge. 1998. Interleukin 7 receptor control of T cell receptor gene rearrangement: role of receptor-associated chains and locus accessibility. J. Exp. Med. 188:2233.[Abstract/Free Full Text]
39. Cherry, S. R., D. Baltimore. 1999. Chromatin remodeling directly activates V(D)J recombination. Proc. Natl. Acad. Sci. USA 96:10788.[Abstract/Free Full Text]
40. McBlane, F., J. Boyes. 2000. Stimulation of V(D)J recombination by histone acetylation. Curr. Biol. 10:483.[Medline]
41. Kwon, J., K. B. Morshead, J. R. Guyon, R. E. Kingston, M. A. Oettinger. 2000. Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage of nucleosomal DNA. Mol. Cell 6:1037.[Medline]
42. Agata, Y., T. Katakai, S.-K. Ye, M. Sugai, H. Gonda, T. Honjo, K. Ikuta, A. Shimizu. 2001. Histone acetylation determines the developmentally regulated accessibility for T cell receptor- gene recombination. J. Exp. Med. 193:873.[Abstract/Free Full Text]
43. Shinkai, Y., G. Rathbun, K.-P. Lam, E. M. Oltz, V. E. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A. M. Stall, F. W. Alt. 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68:855.[Medline]
44. Shinkai, Y., S. Koyasu, K.-I. Nakayama, K. M. Murphy, D. Y. Loh, E. L. Reinherz, F. W. Alt. 1993. Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes. Science 259:822.[Abstract/Free Full Text]
45. Anderson, S. J., H. S. Chou, D. Y. Loh. 1988. A conserved sequence in the T-cell receptor -chain promoter region. Proc. Natl. Acad. Sci. USA 85:3551.[Abstract/Free Full Text]
46. Anderson, S. J., S. Miyake, D. Y. Loh. 1989. Transcription from a murine T-cell receptor V promoter depends on a conserved decamer motif similar to the cyclic AMP response element. Mol. Cell. Biol. 9:4835.[Abstract/Free Full Text]
47. Sikes, M. L., R. J. Gomez, J. Song, E. M. Oltz. 1998. A developmental stage-specific promoter directs germline transcription of DJ gene segments in precursor T lymphocytes. J. Immunol. 161:1399.[Abstract/Free Full Text]
48. Sikes, M. L., C. C. Suarez, E. M. Oltz. 1999. Regulation of V(D)J recombination by transcriptional promoters. Mol. Cell. Biol. 19:2773.[Abstract/Free Full Text]
49. Stevenson, B. J., O. Hagenbuchle, P. K. Wellauer. 1986. Sequence organisation and transcriptional regulation of the mouse elastase II and trypsin genes. Nucleic Acids Res. 14:8307.[Abstract/Free Full Text]
50. Mombaerts, P., A. R. Clarke, M. A. Rudnicki, J. Iacomini, S. Itohara, J. J. Lafaille, L. Wang, Y. Ichikawa, R. Jaenisch, M. L. Hooper, S. Tonegawa. 1992. Mutations in T-cell antigen receptor genes and block thymocyte development at different stages. Nature 360:225.[Medline]
51. Itohara, S., P. Mombaerts, J. Lafaille, J. Iacomini, A. Nelson, A. R. Clarke, M. L. Hooper, A. Farr, S. Tonegawa. 1993. T cell receptor gene mutant mice: independent generation of T cells and programmed rearrangements of TCR genes. Cell 72:337.[Medline]
52. Chen, F., L. Rowen, L. Hood, E. V. Rothenberg. 2001. Differential transcriptional regulation of individual TCR V segments before gene rearrangement. J. Immunol. 166:1771.[Abstract/Free Full Text]
53. Kuo, M.-H., E. vom Baur, K. Struhl, C. D. Allis. 2000. Gcn4 activator targets Gcn5 histone acetyltransferase to specific promoters independently of transcription. Mol. Cell 6:1309.[Medline]
54. Agalioti, T., S. Lomvardas, B. Parekh, J. Yie, T. Maniatis, D. Thanos. 2000. Ordered recruitment of chromatin modifying and general transcription factors to the IFN- promoter. Cell 103:667.[Medline]
55. Strahl, B. D., D. Allis. 2000. The language of covalent histone modifications. Nature 403:41.[Medline]
56. Jenuwein, T., C. D. Allis. 2001. Translating the histone code. Science 293:1074.[Abstract/Free Full Text]
57. Kwon, J., A. N. Imbalzano, A. Matthews, M. A. Oettinger. 1998. Accessibility of nucleosomal DNA to V(D)J cleavage is modulated by RSS positioning and HMG1. Mol. Cell 2:829.[Medline]
58. Ramsden, D. A., G. Wu. 1991. Mouse light-chain recombination signal sequences mediate recombination more frequently than those of light chain. Proc. Natl. Acad. Sci. USA 88:10721.[Abstract/Free Full Text]