T cell receptor (TCR) α/δ locus enhancer identity and position are critical for the assembly of TCR δ and α variable region genes

Abstract

T cell receptor (TCR) δ and α variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCRδ segments are situated between TCRα segments. The TCRα enhancer (Eα) located at the 3′ end of the TCRα/δ locus functions over a long chromosomal distance to promote TCRα rearrangement and maximal TCRδ expression; whereas the TCRδ enhancer (Eδ) is located among the TCRδ segments and functions with additional element(s) to mediate TCRδ rearrangement. We used gene-targeted mutation to evaluate whether the identity of Eα and the position of Eδ are critical for the developmental stage-specific assembly of TCR δ and α variable region genes. Specific replacement of Eα with Eδ, the core Eα element (EαC), or the Ig heavy chain intronic enhancer (iEμ), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCRα rearrangement beyond that observed in the absence of Eα. Therefore, the identity and full complement of Eα-binding sites are critical for promoting accessibility within the TCRα locus. In the absence of the endogenous Eδ element, specific replacement of Eα with Eδ also did not promote TCRδ rearrangement. However, deletion of intervening TCRα/δ locus sequences to restore the inserted Eδ to its normal chromosomal position relative to 5′ sequences rescued TCRδ rearrangement. Therefore, unlike Eα, Eδ lacks ability to function over the large intervening TCRα locus and/or Eδ function requires proximity to additional upstream element(s) to promote TCRδ accessibility.

The exons that encode T cell receptor (TCR) and Ig variable regions are assembled during lymphocyte development from component variable (V), diversity (D), and joining (J) gene segments (1, 2). Both αβ and γδ T cells develop from CD4⁻/CD8⁻ (double negative, DN) thymocytes in which TCRβ, -γ, and -δ genes are all assembled (3). Productive VDJβ rearrangements generate TCRβ chains that associate with surrogate TCRα chains to form preTCRs that signal expansion and differentiation to the CD4⁺/CD8⁺ (double positive, DP) thymocyte stage (4, 5). Functional VJα rearrangements in DP cells generate TCRα chains that, when expressed as surface αβ TCR, direct cellular selection and development to the CD4⁺ or CD8⁺ (single positive, SP) thymocytes that exit the thymus as mature αβ T cells (6). Alternatively, productive VDJδ and VJγ rearrangements in DN progenitor (pro-) T cells generate TCRδ and -γ chains that form cell surface γδ TCR and direct γδ T cell development (4, 5).

The TCRα/δ locus spans 1 Mb and contains variable region gene segments for two distinct antigen receptors (7). The 5′ end of the locus consists of a cluster of ≈90 Vα/Vδ gene segments with Vδs concentrated near the 3′ end of the cluster, and the 3′ end of the locus consists of a cluster of 50 Jα segments that spans ≈60 kb followed by the TCRα constant region (Cα). The Dδ (Dδ1 and Dδ2) and Jδ (Jδ1 and Jδ2) segments, the TCRδ constant region (Cδ) and Vδ5 lie between the Vα/Vδ and Jα clusters. In developing αβ T cells, Vα-to-Jα rearrangement normally occurs on both alleles and deletes TCRδ segments from both chromosomes (7, 8). Consequently, the assembly of TCRδ and -α genes is strictly regulated within the context of lineage-specificity (e.g., TCRα gene assembly in αβ, but not γδ, T cells) and developmental stage specificity (e.g., TCRα gene assembly in DP, but not DN, thymocytes).

Transcriptional enhancers and germ-line promoters cooperate to mediate tissue-, lineage-, and developmental-stage-specific V(D)J recombination (1, 2, 7). The TCRα/δ locus contains two known enhancers, the TCRδ enhancer located between Jδ2 and Cδ and the TCRα enhancer (Eα) located just downstream of Cα, identified via their ability to enhance transcription of reporter genes (9–13). Germ-line promoters are associated with each Vα/Vδ segment (7), the T early α (TEA) promoter and a second Jα germ-line promoter reside upstream of the most 5′ Jαs (14, 15), and a transcriptional promoter may reside between Dδ2 and Jδ1 (16). Eδ functions with additional element(s) to mediate TCRδ germ-line transcription and TCRδ accessibility in DN pro-T cells (17, 18), but only partially contributes to the transcription of assembled VDJδ genes in mature γδ T cells (17). Eα is required for Jα germ-line transcription and TCRα rearrangements involving all Jα segments in DP thymocytes, as well as transcription of assembled VJα genes in mature αβ T cells and assembled VDJδ genes in mature γδ T cells (19). The TEA promoter is only required for TCRα rearrangement to the most 5′ Jαs (20).

The molecular mechanisms through which transcriptional enhancers promote the rearrangement of particular gene segments over large distances remain to be established (1, 2). In the endogenous TCRα/δ locus, Eα clearly mediates recombinational accessibility at a distance of at least 70 kb to the most 5′ Jαs as well as transcription at distances of ≈100 kb to the promoters of assembled VDJδ segments (19). Eδ is known to stimulate local recombinational accessibility in a transgenic TCRδ minilocus (21) and, in the murine TCRα/δ locus, can at least mediate transcription and accessibility at a distance of 18 kb to Dδ1 (17). However, despite a similar distance between Eδ and the Jα cluster, Eδ does not direct Vδ or Vα-to-Jα rearrangement. The ability of Eδ and Eα to promote the assembly of, respectively, TCRδ and TCRα variable region genes may be mediated by intrinsic properties of these enhancers and/or directed by additional cis-acting elements within the endogenous locus. Therefore, we used gene-targeted mutation to evaluate whether the identity of Eα and the position of Eδ within the locus are critical for the developmental stage-specific assembly of TCR α and δ variable region genes.

Materials and Methods

Targeting Constructs and Plasmids.

All targeting constructs contain the same 5′ homology arm and vector backbone as the EαKO construct (19). The 3′ homology arm is a 4.1-kb KpnI/SacI fragment comprised of a 3-kb BamHI genomic fragment 3′ of Eα and either a 1.1-kb ClaI/NdeI Eδ fragment (EδRKI), the 116-bp mouse Eα core fragment (EαCRKI), or a 700-bp XbaI/EcoRI iEμ fragment (EμRKI) subcloned into pBSK. The pMC–CreN plasmid contains the Cre recombinase gene with a nuclear localization signal expressed via the herpes simplex virus/thymidine kinase promoter.

Generation of Targeted Embryonic Stem (ES) Cells.

ES cells were transfected as described (19). Targeted clones were identified by Southern blot analysis with the 5′EαKO probe on EcoRV-digested DNA and confirmed with the 3′EαKO probe on BglII-digested DNA. The identity of the targeted allele (CBA versus B6) was established via Southern blot analysis with the Eα probe on StuI-digested DNA (19). For removal of phosphoglycerate kinase-neo^r, one million cells were transiently transfected with 30 μg of pMC–CreN. Cre-deleted clones were identified via Southern blot analysis with the 5′ probe on EcoRV-digested DNA. The F₁ EδΔEαΔ and F₁ EδΔ ES cells have been described (17). F₁ ES clones with Cre-mediated deletion of TCRα/δ locus sequences between the loxP sites inserted in place of the endogenous Eδ element and just 5′ of EδR and EαΔ were identified via Southern blot analysis with the 5′Eδ knockout (KO) probe on XbaI-digested DNA. Successive Cre–loxP-mediated gene targeting in TC1 ES cells (22) was used to generate EδR/EδR ES cells. Generation of ES cells harboring the different miniloci was carried out as described (23).

Generation of Mice.

Mice and lymphocytes were generated via recombinase-activated gene (RAG) 2-deficient blastocyst complementation (RBDC) with the indicated ES cells as described (24). For most genotypes, multiple mice were generated from at least two independently targeted ES clones. Germ-line EαCR/EαCR mice were analyzed.

Southern Blotting and PCR.

Southern blot analysis was conducted as described (19). The 5′ and 3′EαKO probes (19), 5′EδKO probe (17), probe B (23), TCRα/δ probes 1, 3, 4, 8, and Cδ (25–29), and the Neo probe (30) have all been described. PCR conditions were 10 pmol of each primer/50 mM KCl/10 mM Tris, pH 8.0/1.5 mM MgCl₂/200 μM dNTPs, and Taq in 100-μl reaction volumes. Cycling conditions were 3 min; 93°C for 1 cycle and 1 min; 93°C, 1 min; 60°C and 1 min; 72°C for 30 cycles. Primers used were V, 5′-GGCAAGCAAGCTGGTGTGT-3′; C, 5′-TCATGAACAAAGTCACTG-3′ and P, 5′-TATATTAGAAATTAAAGACAC-3′.

Flow Cytometry.

Single-cell suspensions were prepared as described (19). Cells were stained with FITC-conjugated anti-CD8, anti-β TCR chain, and anti-CD4 as well as phycoerythrin-conjugated anti-CD4 and anti-Vα2 antibodies (PharMingen) and analyzed via a FACScan (Becton Dickinson).

Generation and Analysis of T Cell Hybridomas.

The isolation of purified γδ and αβ T cells and the generation of γδ and αβ T cell hybridomas were conducted as described (19). TCRα and -δ gene rearrangements were analyzed as described (17, 19, 28).

Results

Eδ Does Not Promote Efficient Jα Accessibility When Located 3′ of Cα.

Eδ does not mediate Jα accessibility from its normal chromosomal location; however, the position of enhancers relative to target promoters within multigenic loci can be important (31). Therefore, to evaluate whether Eδ can replace Eα to promote endogenous Vα-to-Jα rearrangement, we generated ES cells and normal lymphocytes in mice in which Eα is replaced with Eδ. The murine TCRδ enhancer was defined as a 766-bp HaeII fragment based upon its ability to activate transcription from a heterologous promoter in γδ T cell hybridomas (10). To ensure this element is competent to promote accessibility, we first evaluated whether the 1.1-kb ClaI/NdeI genomic fragment containing this HaeII fragment that was deleted in EδΔ mice (17) could direct the enhancer-dependent rearrangement of a transgenic TCRβ minilocus (TCRβ^PF) (Fig. 1A) (23, 32). Southern blot and PCR analysis of thymocytes demonstrated this genomic fragment can promote both Dβ-to-Jβ and Vβ-to-DJβ rearrangement of TCRβ^PF and, thus, this Eδ-containing DNA segment comprises a functional recombinational enhancer (Fig. 1 B and C).

Figure 1.

Eδ promotes rearrangement of a TCRβ minilocus. (A) Schematic representation of TCRβ^PF, Eδ, and its position in Eδ⁺TCRβ^PF. The Vβ14, Dβ1, Jβ1.1, and Jβ1.2 segments and the locations of the BglII (Bg) and BamHI (B) sites and the V, P, and C oligonucleotides used for analysis are shown. (B) Southern blot analysis of thymocyte DNA from Eδ⁺ (Eδ⁺14.2 and 19.1) and Eδ⁻ (Eδ⁻69.1 and 9.2) TCRβ^PF and nontransfected (E) and transfected (ET) ES cells using probe B. The positions of molecular weight markers and bands generated by DJ and VDJ rearrangements are indicated. (C) Southern blotting of VDJ rearrangements in thymocyte DNA from mice containing Eδ⁻ (Eδ⁻ 9.2), Eδ⁺ (Eδ⁺19.1), or iEμ⁺ (iEμ⁺1.163) amplified by PCR with V and C primers and probed with the P oligonucleotide. Threefold serial dilutions of template DNA are shown. RAG2 control PCRs are also shown.

We used Cre–loxP-mediated gene-targeting to replace the 1 kb PvuII/BamHI genomic fragment containing Eα that was deleted in EαΔ mice (19) with a 1.1-kb genomic Eδ-containing segment on the CBA allele of F₁ ES cells (33), creating the EδR allele (Fig. 4, which is published as supporting information on the PNAS web site, www.pnas.org). To characterize the influence of this enhancer replacement on T cell development and TCRα rearrangement, we used F₁ Eα/EδR ES cells (Fig. 2A) to generate chimeric mice via RBDC. Flow cytometric (FACS) analysis of thymocytes isolated from F₁ Eα/EδR chimeras, as well as all other F₁ chimeras described below, revealed relatively normal populations of DN, DP, and SP thymocytes as compared with wild-type controls (Fig. 2B); although the relative levels of SP cells compared with DP cells appeared slightly reduced. In addition, FACS analysis of cells isolated from the spleen and lymph nodes of F₁ Eα/EδR mice revealed normal populations of CD4⁺ and CD8⁺ peripheral αβ T cells as well as peripheral γδ T cells (data not shown). Therefore, replacement of Eα with Eδ on a single allele did not substantially perturb αβ and/or γδ T cell development.

Figure 2.

Schematic of F₁ Eα/EδR ES cells and characterization of F₁ Eα/EδR thymocytes. (A) Schematic representation of the TCRα/δ loci in F₁ Eα/EδR cells. (B) FACS analysis of thymocytes isolated from F₁ Eα/EδR and wild-type mice with anti-CD4 and anti-CD8 antibodies. The numbers of thymocytes from representative mice are indicated.

Wild-type peripheral αβ T lymphocytes have Vα-to-Jα rearrangements on both alleles, resulting in the placement of TCRδ genes on extrachromosomal circles that are lost upon cell division (34, 35). In contrast, T cells heterozygous for deletion of Eα exhibit Vα-to-Jα rearrangement on only the Eα⁺ allele (19). To investigate whether EδR promotes Vα-to-Jα rearrangement, we assayed for the presence of TCRδ genes and germ-line Jα segments on the EδR CBA alleles in a panel of 135 F₁ Eα/EδR αβ T cell hybridomas. For this purpose, we took advantage of the fact that CBA and B6 alleles exhibit restriction fragment length polymorphisms associated with Dδ, Jδ, Cδ, and Jα gene segments that permit identification of allelic TCR δ and α rearrangements (17, 19, 28, 33). Southern blot analysis of MspI digested DNA with a Cδ probe and Eco1091 digested DNA with a probe that hybridizes 5′ of the Jαs (probe 8) demonstrated all 135 hybridomas retained chromosomal TCRδ genes and germ-line Jα segments on the EδR CBA allele (Table 1). Therefore, Eδ does not replace Eα to mediate efficient TCRα rearrangement in the endogenous TCRα/δ locus.

Table 1.

Characterization of TCRα rearrangement in hybridomas generated from Eα replaced mice

Genotype	Type	n	Allele	Germ line Jα
F1Eα/EδR	αβ	135	CBA	135
			B6	0
F1Eα/EμR	αβ	83	CBA	83
			B6	0
F1Eα/EαCR	αβ	60	CBA	60
			B6	0

3′ Enhancer Identity Is Critical for Efficient Jα Accessibility.

Transcriptional enhancers function through the hierarchical assembly of specific multiprotein complexes on a number of closely linked trans-factor binding sites (36). Thus, efficient Vα-to-Jα rearrangement may require the entire or a specific complement of Eα binding sites. In this regard, a 116-bp core fragment of human Eα that lacks several trans-factor binding sites has been shown to promote the complete assembly of a transgenic TCRδ minilocus (37). Furthermore, heterologous enhancer elements can promote accessibility in many contexts (38), including within the endogenous TCRβ locus where Eα and iEμ can replace Eβ function (39, 40). To further evaluate the extent to which TCRα/δ locus 3′ enhancer identity may be critical for chromosomal Vα-to-Jα rearrangement, we investigated whether EαC or iEμ could substitute for Eα function.

We used Cre–loxP-mediated gene-targeting to replace Eα on the CBA allele of F₁ ES cells with either the 116-bp murine core Eα element (EαC) or the 700-bp iEμ fragment used to replace the endogenous Eβ element (39), generating F₁ Eα/EαCR and F₁ Eα/EμR ES cells (Fig. 5, which is published as supporting information on the PNAS web site). To investigate whether EαC or iEμ mediates Jα accessibility in DP thymocytes, we analyzed Jα rearrangements in 60 F₁ Eα/EαCR and 88 F₁ Eα/EμR αβ T cell hybridomas generated via the RDBC approach. Southern blot analysis of Eco1091 digested DNA with probe 8 demonstrated all 60 F₁ Eα/EαcR hybridomas and the 83 F₁ Eα/EμR hybridomas that were fully characterized retained a germ-line Jα band on the CBA allele (Table 1). Therefore, although both EαC and iEμ, like Eδ, can promote V(D)J recombination in transgenic miniloci, neither EαC nor iEμ can also replace Eα to mediate efficient Vα-to-Jα rearrangement in the endogenous TCRα/δ locus.

3′ Enhancer Identity Is Critical for αβ T Cell Development and Diversification of the Vα Repertoire.

Mice with homozygous deletion of Eα (EαΔ/EαΔ) exhibit a block in αβ T cell development at the DP thymocyte stage with the accumulation of low numbers of peripheral αβ T cells that express only Vα2 gene segments (19). To more rigorously evaluate whether 3′ enhancer identity and the full complement of Eα binding sites are critical for Eα function, we analyzed T cell development in EδR/EδR and EαCR/EαCR mice. FACS analysis of thymocytes demonstrated that, identical to EαΔ/EαΔ mice, development in both is blocked at the DP stage (Fig. 3A). FACS analysis of peripheral lymphocytes demonstrated that, like EαΔ/EαΔ mice, EδR/EδR mice exhibit peripheral αβ T cells expressing only Vα2 (Fig. 3B). However, although EαCR/EαCR mice similarly contain a low number of peripheral αβ T cells expressing only Vα2, they also contain a lower number of peripheral αβ T cells expressing other Vα's as well (Fig. 3B). The development of these αβ T cells could result from the ability of EαC to promote slightly more efficient Vα-to-Jα rearrangement and/or increased cellular selection due to more efficient expression of these rearrangements compared with the EαΔ allele (19). Therefore, in the endogenous TCRα/δ locus, 3′ enhancer identity and the full complement of Eα binding sites are critical for DP to SP thymocyte development, generation of a normal number of peripheral αβ T cells, and diversification of the Vα repertoire.

Figure 3.

Defective αβ T cell development in EδR/EδR and EαCR/EαCR mice. (A) FACS analysis of thymocytes isolated from EδR/EδR, EαCR/EαCR, EαΔ/EαΔ, and wild-type mice with anti-CD4 and anti-CD8 antibodies. The numbers of thymocytes from representative mice are indicated. (B) FACS analysis of peripheral lymphocytes isolated from the lymph nodes of the same mice with anti-Vα2 and anti-TCRβ antibodies. The percentage of the overall number of cells expressing Vα2⁺ αβ TCR and αβ TCR of other Vαs are indicated.

Eδ Position Within the TCRα/δ Locus Is Critical for Its Function.

Eα clearly mediates accessibility over a distance of at least 70 kb to the most 5′ Jαs and transcription over a distance of at least 120 kb to assembled VDJδ segments (19). To evaluate whether Eδ can promote accessibility over a comparable distance when located in the normal chromosomal location of Eα, we used gene-targeting to generate F₁ EδΔEδR ES cells containing both the EδR mutation and deletion of the endogenous Eδ element on the CBA allele (Fig. 6A, which is published as supporting information on the PNAS web site). To determine whether EδR directs TCRδ accessibility in pro-T cells, we analyzed TCRδ gene rearrangement in a panel of 34 F₁ EδΔEδR γδ T cell hybridomas generated via the RDBC approach. Southern blotting of MspI- and BglII-digested DNA with a series of TCRδ probes demonstrated an incomplete assembly of endogenous TCRδ genes on the EδΔEδR CBA allele (Table 2), similar to that observed on the CBA allele of F₁ EδΔ γδ T cell hybridomas (17). Because EδR does not promote efficient Vα-to-Jα rearrangement in αβ T cells, we also analyzed TCRδ gene rearrangement in a panel of 35 F₁ EδΔEδR αβ T cell hybridomas to determine whether, when located in the normal chromosomal location of Eα, Eδ function is restricted to DP thymocytes. Southern blot analysis demonstrated a block in the assembly of TCRδ genes on the EδΔEδR CBA allele similar to that observed in F₁ EδΔEδR γδ T cell hybridomas (Table 2). Therefore, when situated in the normal chromosomal location of Eα, Eδ does not promote TCRδ accessibility in either developing γδ or αβ T cells.

Table 2.

Characterization of TCRδ rearrangement in hybridomas from EδR mice

Genotype	Type	n	Allele	Germ line	VD(D)Jδ	V(D)Dδ	D(D)Jδ	DDδ
F1EδΔEδR	γδ	34	CBA	0	8	8	11	7
			B6	0	34	0	0	0
F1EδΔEδR	αβ	35	CBA	0	13	11	7	4
			B6	NA	NA	NA	NA	NA
F1EδΔΔEδR	γδ	26	CBA	0	16	0	10	0
			B6	0	26	0	0	0
F1EδΔΔEαΔ	γδ	24	CBA	0	5	10	4	5
			B6	0	24	0	0	0

NA, not applicable. 

The lack of TCRδ gene rearrangement on the EδΔEδR allele could reflect the intrinsic ability of Eδ to function over only a limited distance, the requirement of additional sequences that were not included in our replacement, or the inhibition of Eδ function by cis-acting elements within the TCRα/δ locus. To evaluate whether we could rescue Eδ-mediated TCRδ accessibility through the deletion of sequences between EδR and the normal position of Eδ, we expressed the Cre recombinase in F₁ EδΔEδR ES cells to delete sequences between the EδΔ and EδR loxP sites, creating F₁ EδΔΔEδR ES cells (Fig. 6A) This manipulation results in the deletion of Cα, the entire Jα cluster, Vδ5, and Cδ and, thus, places Eδ back into its normal chromosomal location relative to the 5′ TCRδ segments and any uncharacterized 5′ cis elements. Southern blot analysis of a panel of 26 F₁ EδΔΔEδR γδ T cell hybridomas generated via the RDBC approach demonstrated the majority of CBA alleles contained completely assembled TCRδ genes (Table 2). Furthermore, the overall TCRδ rearrangement pattern on the EδΔΔEδR CBA alleles [only complete VD(D)Jδ and incomplete D(D)Jδ rearrangements] is similar to that observed on CBA alleles in wild-type F₁ γδ T cell hybridomas (Table 2) (17). As a control for the presence of Eδ, we also used F₁ EδΔEαΔ ES cells to delete sequences between the EδΔ and EαΔ loxP sites, creating F₁ EδΔΔEαΔ ES cells (Fig. 6B). Southern blot analysis of a panel of 24 F₁ EδΔΔEαΔ γδ T cell hybridomas generated via the RDBC approach demonstrated a level and pattern of incompletely assembled TCRδ genes on the EδΔΔEαΔ CBA alleles similar to that observed on the CBA alleles in F₁ EδΔEδR γδ T cell hybridomas (Table 2). Consequently, the rescue of TCRδ gene rearrangement in F₁ EδΔΔEδR γδ T cell hybridomas depends on the presence of the Eδ element.

Discussion

The Identity of Eα and Position of Eδ Are Critical.

We used gene-targeted mutation to evaluate whether the identity of Eα and the position of Eδ are critical for the developmental stage-specific assembly of TCR δ and α variable region genes. This approach permits the analysis of enhancer function on the complete repertoire of endogenous TCR δ and α gene segments in the context of other chromosomal cis elements. Specific replacement of Eα with Eδ and EαC did not promote TCRα rearrangement significantly beyond the level observed in the absence of Eα, although EαC did mediate slightly more efficient Vα-to-Jα rearrangement and/or increased selection of cells expressing diverse Vα's compared with the EαΔ allele. Furthermore, and in contrast to Eβ replacement in the TCRβ locus (39), replacement of Eα with iEμ did not rescue levels of rearrangement beyond that of the enhancer deleted allele. Therefore, the identity and full complement of Eα binding sites are critical for efficient Vα-to-Jα rearrangement within the TCRα/δ locus.

Gene-targeted mutation has demonstrated transcriptional enhancers promote recombinational accessibility over chromosomal distances at least to (D)J segments and possibly as far as V segments, whereas promoters mediate local accessibility of adjacent gene segments (1). The molecular mechanisms through which enhancers function with promoters to mediate recombinational accessibility remain to be established but may involve active V segment transcription, localized enhancer-dependent 5′ (D)J promoter-based chromatin remodeling, and more global enhancer-based chromatin reorganization (41, 42). In this context, Eα functions over distances of between 70 and 100 kb to stimulate transcription from TEA and the promoter of assembled VDJδ segments and mediate histone acetylation (19, 21).

Eδ, EαC, and iEμ all promote recombinational accessibility and, where assayed, germ-line transcription from heterologous promoters at distances of ≈2–4 kb in TCRβ- or TCRδ-based transgenic V(D)J recombination substrates (18, 32, 37, 43, 44). Eδ also mediates accessibility at a distance of at least 18 kb to Dδ1 in the endogenous locus (17) and histone acetylation over a distance that extends ≈4 kb to Dδ3, but not 12 kb to Vδ1 in a human TCRδ minilocus (21). Endogenous iEμ promotes accessibility at a distance of at least 4 kb to assembled DJ_H1 complexes and, after V_H-to-DJ_H rearrangement, appears capable of stimulating transcription of an 5′ germ-line V_H promoter at a distance of 17.5 kb (45). Furthermore, iEμ can also replace Eβ function within the endogenous TCRβ locus to promote Dβ1 accessibility (39, 40), a process dependent on activation of the Dβ1 germ-line promoter (46, 47), at a distance of 21 kb.

Although Eδ, EαC, and iEμ can promote recombinational accessibility, these enhancer elements may intrinsically function over more limited chromatin distances than Eα and, thus, simply lack the ability to activate TEA transcription and/or promote histone acetylation at a distance of 70 kb. Because of the unique organization of the TCRα/δ locus, Eα may have evolved to function over a much larger chromosomal distance than other antigen receptor transcriptional enhancers. In this context, the complete, long-range TCRα recombinational enhancer may consist of the classical transcriptional enhancer (Eα) and cis elements of the TCRα/δ locus control region (48), some of which could be deleted elements outside of the core Eα sequence (49). Another related possibility is that the binding factors of Eδ, EαC, and iEμ may also lack the intrinsic ability to efficiently form functional interactions with the binding proteins of TEA and other Jα germ-line promoters. In this context, interactions between TEA and Eα may have evolved under more stringent selective pressure than other antigen receptor promoter–enhancer interactions to ensure efficient transcriptional activation at such a long chromatin distance.

Factors That May Restrict Eδ Function in a Position-Dependent Manner.

We also demonstrated that Eδ cannot promote TCRδ gene rearrangement from the normal chromosomal location of Eα; however, deletion of intervening sequences to place Eδ back into its normal chromosomal location relative to 5′ TCRδ elements restored Eδ-mediated TCRδ accessibility. Therefore, although important intrinsic differences exist between Eδ and Eα, the function of Eδ may also be influenced by the chromosomal location of this element. As discussed above, Eδ may only function over a limited chromosomal distance and, thus, simply lack the ability to promote TCRδ gene rearrangement at a distance of 100 kb when located 3′ of Cα. In this context, Eδ-mediated TCRδ accessibility may depend on the proximity of Eδ to germ-line Dδ/Jδ promoters and other chromosomal elements, possibly including Dδ/Jδ matrix attachment regions (50), that direct TCRδ gene rearrangement in the absence of Eδ (17). Another related possibility is that, although the Eδ fragment used in the Eα replacement is sufficient to promote accessibility of a TCRβ minilocus, the functional endogenous TCRδ recombinational enhancer may consist of Eδ and additional elements within the TCRα/δ locus not included in the Eα replacement, but that were restored with Eδ on Cre-mediated deletion. In this context, similar to the cooperation between the TCRγ 3′ enhancer and an upstream transcriptional element (51), Eδ and upstream element(s) may function together as a locus control region to establish recombinational accessibility of only intervening gene segments. Finally, our findings are also consistent with the possibility that the ability of Eδ to promote TCRδ accessibility on the EδΔEδR allele could be blocked by inhibitory elements located between the normal chromosomal positions of Eδ and Eα, perhaps the inactive factor-bound TEA promoter (51).

Despite the close proximity between TCRδ gene segments and the Jα cluster, Eδ does not direct Vδ- or Vα-to-Jα rearrangement; therefore, molecular mechanisms operate to prevent Eδ-mediated Jα accessibility in normal DN thymocytes. Initially, a cis element with enhancer blocking activity (BEAD-1) located between the human Vδ5 and Jα segments was proposed to prevent Eδ-mediated Jα accessibility (52); however, gene-targeted deletion of the corresponding sequences did not permit Jα rearrangement in normal pro-T cells. Consequently, various other mechanisms, such as promoter preference/competition, directionality of Eδ function, and reciprocal developmental specificity of Eδ and TEA function, have been proposed to account for the lack of Eδ-mediated Jα accessibility (53, 54). Our analysis of EδR and EδΔEδR alleles provides further insight into the molecular mechanisms that may prevent Jα accessibility in normal DN thymocytes. The absence of TEA transcription and Jα rearrangement on EδR alleles containing two Eδ elements demonstrates the lack of Eδ-mediated Jα accessibility is not simply the result of a preferential interaction between Eδ and the Vδ5 promoter versus the TEA promoter (53, 54). Furthermore, the absence of Jα rearrangement on the EδR allele indicates the lack of Eδ-mediated Jα accessibility also is not simply caused by a 3′ restriction of Eδ activity by the Vδ5 promoter or potential insulators located between Eδ and Vδ5 (53, 54). Together, our findings indicate the lack of Jα accessibility in normal DN thymocytes may reflect the absence of an intrinsic functional interaction between Eδ and Jα germ-line promoters, the function of a boundary element in addition to BEAD-1, and/or the establishment of Eδ-mediated accessibility only between Eδ and an upstream cis element. Further gene-targeted mutation in EδR, EδΔEδR, and other tailored TCRα/δ loci should facilitate a complete elucidation of the factors that restrict Eδ function in a position dependent manner and prevent Eδ-mediated Jα accessibility in DN thymocytes.

Abbreviations

TCR

T cell receptor

RAG

recombinase-activating gene

D

diversity

RBDC

RAG-2-deficient blastocyst complementation

ES

embryonic stem

FACS

fluorescence-activated cell sorting