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The four T cell receptor genes (Tcra, Tcrb, Tcrg, Tcrd) are assembled by V(D)J recombination according to distinct programs during intrathymic T cell development. These programs depend on genetic factors, including gene segment order and recombination signal sequences. They also depend on epigenetic factors. Regulated changes in chromatin structure, directed by enhancers and promoter, can modify the availability of recombination signal sequences to the RAG recombinase. Regulated changes in locus conformation may control the synapsis of distant recombination signal sequences, and regulated changes in subnuclear positioning may influence locus recombination events by unknown mechanisms. Together these influences may explain the ordered activation and inactivation of T cell receptor locus recombination events, and the phenomenon of Tcrb allelic exclusion.
The somatic assembly of T cell receptor (TCR) genes (Tcra, Tcrb, Tcrg, Tcrd) generates a diverse T cell repertoire and is an essential component of thymocyte development that instructs numerous cell lineage and cell fate decisions. TCR genes are assembled through V(D)J recombination, a site specific recombination process directed by the lymphoid-specific recombinase (RAG, composed of RAG1 and RAG2) and ubiquitously expressed DNA repair proteins . RAG proteins create double-strand breaks at recombination signal sequences (RSSs) that flank TCR variable (V), diversity (D) and joining (J) gene segments, and these breaks are subsequently resolved by nonhomologous end joining. The four TCR genes are assembled according to distinct developmental programs. Recombination of Tcrd, Tcrg and Tcrb all occur during the CD4−CD8−double-negative (DN) 2 (CD44+CD25−) and DN3 (CD44−CD25+) stages of thymocyte development, during an initial period of recombinase expression. Thymocytes commit to the αβ or γδ lineages concurrent with, and likely as an outcome of, these TCR gene recombination events . Successful recombination of Tcrd and Tcrg promotes assembly of a γδ TCR, whereas successful recombination of Tcrb promotes assembly of TCRβ with pre-Tα to form a pre-TCR. Pre-TCR signals then provoke downregulation of recombinase expression, several rounds of proliferation, and differentiation of thymocytes to the CD4+CD8+ double positive (DP) compartment, within which Rag genes are re-expressed and Tcra recombination initiated. The specificity of assembled αβ TCR for intrathymic ligands then dictates cell survival and differentiation into the CD4, CD8 or other αβ T cell lineages.
TCR gene assembly is regulated through controls exerted at the level of chromatin structure, at the level of locus conformation, and at the level of locus positioning in thymocyte nuclei [3;4]. Recombination biases are also dictated by information encrypted in RSSs themselves. This review outlines current thinking on how these regulatory influences are integrated to orchestrate the unique developmental programs at murine TCR loci.
The Tcrg locus has two properties that have provoked substantial interest. First, certain Vγ-to-Jγ recombination events are developmentally programmed, with Vγ usage defining γδ T cell subsets that populate distinct anatomical locations . Second, Vγ-to-Jγ recombination events are directed by γc family cytokines. The murine Tcrg locus is composed of three functional Vγ-Jγ-Cγ clusters, Cγ1, Cγ2 and Cγ4 (Figure 1). The Cγ1 cluster is the best studied, because its four Vγ gene segments are used in a highly regulated way: the Jγ1-proximal Vγ3 and Vγ4 gene segments undergo recombination in early fetal thymocytes and encode invariant γδ TCRs of intraepithelial lymphocytes in the epidermis and in vaginal and tongue epithelia, respectively. The more distal Vγ2 and Vγ5 gene segments rearrange subsequently and contribute, along with Vγ1.1 (Cγ4 cluster) and Vγ1.2 (Cγ2 cluster), to the diverse γδ repertoires in secondary lymphoid organs. Vγ5 usage predominates in the intestinal epithelium.
RSS accessibility at antigen receptor loci is mediated by regulatory elements such as enhancers and promoters that disrupt nucleosome structure through effects on transcription, on histone modifications, and by recruitment of chromatin remodeling complexes [3;4]. Two elements in the Cγ1 cluster, transcriptional enhancer 3′ECγ1 and DNAse hypersensitive site HsA, function together to regulate transcription of rearranged Tcrg genes, but have only a modest impact on Tcrg recombination . In contrast, Vγ promoters have been implicated as critical developmental regulators of Vγ to Jγ1 recombination. As measured by germline transcription and histone acetylation, the fetal Vγ3 gene segment is active in fetal but not adult thymocytes, whereas the adult Vγ2 gene segment is active at both stages . Exchange of the Vγ3 and Vγ2 promoters in a transgenic recombination substrate reprogrammed Vγ3 and Vγ2 rearrangement in adults, favoring Vγ3 rearrangement over Vγ2 . However, promoter exchange did not impact the bias towards Vγ3 rearrangement in fetal thymocytes. Rather, the fetal bias depends on gene order, because exchange of Vγ3 and Vγ2 in a transgenic recombination substrate caused Vγ2 rather than Vγ3 rearrangement to predominate in fetal thymocytes, even though both displayed accessible chromatin . Moreover, excision of Vγ3 and Vγ4 from the endogenous Tcrg locus increased Vγ2 rearrangement in fetal thymocytes without modifying Vγ2 chromatin structure . Thus, all Vγ gene segments in the Cγ1 cluster are accessible in fetal thymocytes, with proximity to Jγ1 biasing rearrangement to Vγ3 and Vγ4; this bias is then overcome in adults by specific repression enforced by the Vγ3 and Vγ4 promoters. This suppression depends on transcription factor E2A . Interestingly, an analogous proximal to distal switch occurs for VH segments between fetal and adult B cell development. The suppression of proximal VH segments in the adult occurs, in part, through Ezh2-dependent trimethylation of histone H3K27 [11;12]. Thus in both loci, proximal V segments outcompete distal V segments unless the proximal V segments are specifically suppressed. However, in comparison to Igh, the Cγ1 cluster is extremely compact with Vγ segments tightly clustered. Therefore the bias towards proximal Vγ usage may depend on a specific locus conformation rather than overall distance, or perhaps by recombinase tracking along the DNA .
Il7−/− and Il7ra−/− mice lack γδ T cells because IL-7/IL-7R signaling in DN thymocytes is essential for accessibility and recombination of Tcrg [14–16]. IL-7R signaling activates STAT5, and indeed there are STAT5 binding sites in Jγ promoters and 3′Eγ. Moreover, an activated STAT5 could induce γδ T cell development by activating Jγ chomatin and Vγ-to-Jγ recombination [17;18]. A role for STAT5 in Tcrg recombination was questioned based on the analysis of Stat5a−/− Stat5b−/− mice with hypomorphic Stat5 alleles , but mice carrying null alleles indeed mimic the dramatic phenotype of Il7−/− and Il7ra−/− mice . A distinct γc cytokine, IL-15, specifically induces Vγ5-to-Jγ1 recombination through effects on Vγ5 and Jγ1 chromatin . Like IL-7, IL-15 activates STAT5, and the selective activation of Vγ5 is STAT5-dependent. The basis for distinct responses to IL-7 and IL-15 remains an open question.
A hallmark of Tcrb is developmentally ordered recombination, with Dβ-to-Jβ preceding Vβ-to-DβJβ recombination. All Tcrb recombination events depend strictly on Eβ, which is essential for chromatin modifications throughout a 25 kb domain encompassing the two Dβ-Jβ-Cβ clusters  (Figure 1). Eβ cooperates with the promoter PDβ1, which is tightly associated with Dβ1, to stimulate Dβ1-to-Jβ1 recombination  and likely cooperates with a second promoter, PDβ2 , to stimulate Dβ2-to-Jβ2 recombination. Dβ1 appears to be the critical control point for Dβ1-to-Jβ1 recombination, because although Eβ can independently influence chromatin structure across a broad region, a specific interaction between Eβ and PDβ1 is required to make the Dβ1 gene segment accessible to the recombinase [25;26]. This interaction recruits the SWI-SNF chromatin remodeling complex to Dβ1; recruitment of SWI/SNF is necessary for Dβ-to-Jβ recombination and, in the context of a minilocus V(D)J recombination reporter substrate, can replace PDβ1 function .
These experiments provide insight into accessibility control of Dβ-to-Jβ recombination, but do not directly address the basis for two-step, developmentally ordered rearrangement. What prevents direct Vβ-to-Jβ recombination? Control is exerted by RSSs themselves. RSSs have either 12- or 23-bp spacers separating conserved heptamer and nonamer elements, and recombination is restricted to gene segments with dissimilar RSSs (the 12/23 rule). Dβ-to-Jβ recombination is directed by a 3′ Dβ 23 RSS and a Jβ 12 RSS; the Vβ-to-Dβ step is directed by a Vβ 23 RSS and a 5′ Dβ 12 RSS. Although direct Vβ 23 RSS-to-Jβ 12 RSS recombination is permissible according to the 12/23 rule, it does not occur (a restriction known as “beyond 12/23” or B12/23) . However, direct Vβ-to-Jβ recombination can occur when a Jβ RSS is replaced by the 5′ Dβ1 RSS or when a Vβ RSS is replaced by the 3′ Dβ1 RSS [28;29]. These restrictions are apparent even on nonchromatinized substrates and appear to depend on both RSS and flanking sequences . In fact, B12/23 restrictions can be replicated using RAG and high mobility group proteins in vitro, with the reaction blocked at early steps of RSS nicking and synaptic complex formation .
Although B12/23 restrictions enforce a two step recombination process, they do not impose order. Why Dβ-to-Jβ recombination invariably precedes Vβ-to-Dβ recombination has been uncertain. Notably, the 3′ Dβ1 and 3′ Dβ2 23 RSSs were shown to include a highly conserved binding site for the transcription factor AP-1, and the AP-1 component c-Fos was found to recruit RAG to the 3′ Dβ1 23 RSS . Although stimulatory for plasmid Dβ1-to-Jβ1 recombination, this recruitment inhibited Vβ-to-Dβ1 recombination, presumably because it sterically interferes with RAG access to the nearby 5′ Dβ1 12 RSS. Notably, deletion of the Dβ1 23 RSS  or gene-targeted loss of c-Fos  made Tcrb permissive for Vβ-to-Dβ1 recombination. This suggests that during T cell development, removal of the 3′ Dβ1 23 RSS through Dβ1-to-Jβ1 recombination may signal to initiate Vβ-to-DβJβ recombination.
The Vβ-to-DβJβ step of Tcrb recombination is subject to allelic exclusion, thus insuring that developing T lymphocytes assemble a functional Tcrb gene on only a single allele. Allelic exclusion can be thought of as occurring in two phases [34;35]. First, in the initiation phase, it must be highly unlikely that both alleles attempt Vβ-to-DβJβ recombination within the same time frame. Second, in the maintenance phase, in-frame recombination on one allele must provoke a feedback signal that suppresses further Vβ-to-DβJβ recombination on the other allele.
From a mechanistic perspective, we have a much better understanding of the maintenance phase of allelic exclusion. Feedback inhibition is established by pre-TCR signaling, which provokes downregulation of Rag-1 and Rag-2 expression, entry of DN3 thymocytes into the cell cycle, and differentiation to the DP stage . Vβ-to-DβJβ recombination is then suppressed in DP thymocytes despite re-expression of Rag-1 and Rag-2. Almost all Vβ gene segments are several hundred kb distant from Dβ-Jβ-Cβ and are regulated independently; these Vβ segments transition to a less accessible chromatin structure in DP thymocytes . However, chromatin accessibility cannot fully account for feedback inhibition because a genetic manipulation that maintained accessible Vβ chromatin in DP thymocytes failed to override feedback inhibition . Locus conformation is thought to provide an additional level of regulation. Use of three dimensional fluorescence in-situ hybridization (3D-FISH) and the chromosome conformation capture (3C) crosslinking technique have shown that the two ends of the Tcrb locus are farther apart in DP than in DN thymocytes . This “decontraction” may limit synapsis of Vβ and DβJβ segments in DP thymocytes. Similar decontraction of the Igh locus has been linked to Igh allelic exclusion . The molecular mechanisms mediating large-scale locus conformational changes are unknown. However an intriguing candidate would be the transcription factor CTCF, which functions to recruit cohesins [41;42]and can tether distal portions of the β-globin locus .
Notably, it was recently shown that overexpression of transcription factor E2A, whose activity is normally downregulated in response to pre-TCR signals, can override feedback inhibition in DP thymocytes . E2A was shown to be a dosage-dependent regulator of Vβ accessibility [44;45], but one might predict that it must also regulate locus conformation. It should also be noted that there are examples in which Vβ segments are close to Dβ and apparently accessible in DP thymocytes, yet are still subject to feedback inhibition: (1) Vβ14, which is situated downstream of Cβ2 in an inverted orientation ; (2) unrearranged Vβ gene segments upstream of a Vβ-Dβ -Jβ1 rearrangement [47;48]; (3) Vβ10 on an allele with a 475 kb deletion . These examples suggest that feedback inhibition could involve constraints beyond reduced accessibility and locus decontraction.
Effective feedback inhibition requires that in the initiation phase of allelic exclusion in DN thymocytes, the two Tcrb alleles must attempt Vβ-to-DβJβ recombination one allele at a time. Asynchronous recombination could occur through two types of mechanisms: (1) a deterministic mechanism, in which each DN thymocyte carries one Tcrb allele that provides an initial substrate for Vβ-to-DβJβ recombination; or (2) a stochastic mechanism, in which inefficient recombination on both alleles makes simultaneous Vβ-to-DβJβ recombination highly unlikely. Recent data appears consistent with the latter. Tcrb alleles in DN thymocytes were shown by 3D Immuno-FISH to have the unusual property of associating at high frequency with the nuclear lamina and with foci of pericentromeric heterochromatin, two nuclear compartments often thought to be suppressive for gene expression [39;50]. Notably, associated alleles were less likely to have undergone Vβ-to-DβJβ recombination in DN thymocytes ; however, these associations were not strictly monoallelic. Rather, associated and free alleles distributed stochastically in DN thymocyte nuclei, with most nuclei having either one or two associated alleles. Thus, it was proposed that frequent and stochastically distributed associations with inhibitory subnuclear compartments reduce the likelihood that two Tcrb alleles attempt Vβ-to-DβJβ recombination simultaneously . Nevertheless, formal proof that these interactions inhibit Vβ-to-DβJβ recombination, and the mechanism by which they do so, are lacking. In this regard, single cell analysis showed Vβ8.2 transcription to be biallelic in all DN3 thymocytes . Thus, inhibition may not involve modulation of Vβ accessibility.
The Tcra/Tcrd locus is characterized by an even more complex developmental progression of recombination events . Approximately 100 V gene segments are arrayed across 1.5 mb of the locus, with Dδ, Jδ and Cδ gene segments, 61 Jα gene segments and Cα, spanning 100 kb at the 3′ end of the locus (Figure 1). Vδ-to-Dδ-to-Jδ recombination occurs in DN thymocytes, whereas Vα-to-Jα recombination (which deletes partially or fully rearranged Tcrd genes [52–54]) occurs in DP thymocytes. This developmental switch in recombinase targeting depends on the differential activation of developmental stage-specific enhancers, Eδand Eα [55–57]. The locus displays regulated V segment usage, as only a small fraction of the V gene segments contribute substantially to the Tcrd repertoire (reviewed in ). Moreover, there are multiple rounds of Vα-to-Jα recombination in DP thymocytes, with primary rearrangements using 3′ Vα and 5′ Jα segments, and secondary rearrangements using more 5′Vα and more 3′ Jα gene segments .
Only a handful of V gene segments dominate the adult Vδ repertoire (reviewed in ). Some (TRDV2-2, TRDV5) are situated proximal to Dδ gene segments, whereas others (TRAV15 family) are hundreds of kb distant. All members of this select group are characterized by active transcription and an accessible chromatin configuration in DN thymocytes , suggesting that promoter activity in DN thymocytes may define these as Vδ segments. Upon transition to DP, many additional V gene segments become active, particularly within the proximal 500 kb of the V segment array; this activation depends on the long-distance influence of Eα . A concurrent locus contraction event brings proximal V gene segments much closer to Jα and Cα segments . This conformational change may facilitate, or may be facilitated by, enhancer-promoter interactions. Regardless, both contraction and proximal V promoter activation may bias towards usage of 3′ Vα segments in primary Vα-to-Jα recombination. Notably, TRDV4, the dominant fetal Vδ, appears inactive in adult DN and DP thymocytes ; like Vγ3 and Vγ4, it may be subject to specific suppression in adults .
The targeting of primary Vα-to-Jα recombination events depends on the activity of two Eα-dependent promoters, T-early α (TEA) and Jα49, associated with 5′ Jα gene segments [59;60]. Transcriptional elongation from TEA was shown to be critical for chromatin remodeling and accessibility to the recombinase at Jα segments extending 12 kb downstream from the promoter [61;62]. Transcription-coupled trimethylation of histone H3K4, which is high across this region, may promote RAG protein binding and activity [63;64]. Nevertheless, transcription appears to suppress cryptic promoter activity and chromatin accessibility at Jα segments further downstream [60–62]. Primary Vα-to-Jα recombination deletes the TEA (and often the Jα 49) promoter, but places the promoter of the introduced Vα gene segment at the 5′ end of the residual Jα array. Secondary Vα-to-Jα recombination is then targeted to Jα segments immediately downstream of the primary Vα-Jα, presumably a consequence of chromatin remodeling events directed by transcription from the introduced Vα promoter . These regulatory influences continually retarget recombination events to the most 5′ of the available Jα gene segments, thereby promoting efficient use of the Jα array. Estimates suggest, on average, five rounds of Vα-to-Jα recombination on each allele , with the progression of recombination events terminated by either positive selection [67;68], which downregulates recombinase expression, or cell death . Multiple rounds of Vα-to-Jα recombination on both alleles, in the absence of allelic exclusion, provides thymocytes multiple opportunities to generate a TCRα chain that can support positive selection [70–72].
Although we are developing a reasonable understanding of the factors that dictate programs of V(D)J recombination at the different TCR loci, in many cases we have only scratched the surface when it comes to underlying mechanisms. Accessibility aside, it is still unclear how RAG proteins find RSSs and assemble synaptic complexes in vivo. How and to what sites is RAG recruited, or are RSSs recruited to RAG? We can only describe locus conformation in crude terms, and we can only guess at the molecular events directing conformational changes. We also lack mechanistic insight regarding changes in subnuclear positioning and their influence on recombination. Finally, it would seem that precise temporal regulation of recombination events should be critical for thymocyte development, since this would allow time to test TCR proteins for functionality. Without appropriate pacing, a productive Vβ-Dβ 1-Jβ 1 rearrangement could be excised by Vβ-to-Dβ 2-Jβ 2 rearrangement before it could induce β-selection, and a productive Vα-to-Jα rearrangement could be excised by further Vα-to-Jα rearrangement before it could induce positive selection. Might chromatin structure provide the “pacemaker”? Only time will tell.
I would like to thank Eugene Oltz, Hrisavgi Kondilis and Hanyu Shih for critical reading of the manuscript. Work in the author’s laboratory was supported by the National Institutes of Health (R37 GM41052 and RO1 AI49934).
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