The TCRβ-chain gene enhancer activates transcription and V(D)J recombination in immature thymocytes. In this paper we present a systematic analysis of the elements that contribute to the activity of the murine TCRβ enhancer in mature and immature T cell lines. We identified a region containing the βE4, βE5, and βE6 motifs as the essential core of the TCRβ enhancer in pro-T cells. In mature cells, the core enhancer had low activity and required, in addition, either 5′ or 3′ flanking sequences whose functions may be partially overlapping. Mutation of any of the six protein binding sites located within the βE4–βE6 elements essentially abolished enhancer activity, indicating that this core enhancer contained no redundant elements. The βE4 and βE6 elements contain binding sites for ETS-domain proteins and the core binding factor. The βE5 element bound two proteins that could be resolved chromatographically and that were both essential for enhancer activity.

Genes encoding αβ TCR heterodimers are assembled at discrete stages during T cell differentiation (1, 2). TCRβ-chain assembly takes place first and involves two rearrangement steps to produce V(D)J recombinants at one of two possible constant region genes. Production of TCRβ-chain protein is a critical check point during T cell differentiation, at which cells containing one functional V(D)J recombination are selected to progress further (2). This is most vividly illustrated by the analysis of mice deficient in the recombination activation genes 1 or 2 (RAG 1 or RAG 2)3 (3, 4) or the TCRβ gene (5, 6), in which thymocyte maturation is blocked at a very early CD4CD8 (double negative) stage of development. Provision of a transgenic recombined TCRβ-chain gene allows cells to progress into CD4+CD8+ (double positive) cells (7, 8). During normal development, sensing of TCRβ-chain protein is a signal for initiation of TCRα gene rearrangements, cell proliferation, and expression of the coreceptors CD4 and CD8. Thus, TCRβ-chain expression is a prerequisite for further differentiation.

Transcriptional regulation of the TCRβ-chain gene is complex. TCRβ transcripts can be detected in the most immature cells in the thymus, as well as in thymic NK1.1+ cells, both of which retain the ability to differentiate into multiple lineages (9, 10). In addition, TCRβ transcripts have also been detected in bone marrow cells (11, 12). These observations suggest that the gene may be activated in multipotential bone marrow precursors. Subsequently, TCRβ gene transcription must be maintained (or enhanced) in cells that commit to differentiate into T lymphocytes; conversely, TCRβ gene transcripion must be extinguished if the cells commit to differentiate into other lineages. A transcriptional enhancer located 3′ of the Cβ2 exons plays a major role in establishing the pattern of TCRβ gene expression, because in its absence the locus is not transcribed, nor does it undergo V(D)J recombination (13, 14).

The murine TCRβ enhancer is located ∼5 kb 3′ of the Cβ2 exons (Refs. 15, 16 and Fig. 1,A). Deletion analysis by Takeda et al. (17) defined 5′ and 3′ ends of the enhancer that were separated by ∼325 bp (Fig. 1 B). However, the indicated region was not tested for enhancer activity by itself. Seven protein binding motifs, termed βE1–βE7, were identified within this 325 bp using a combination of DNaseI footprinting and EMSA. Although no T cell-specific proteins were identified, competition assays suggested that some of the enhancer binding proteins were similar to factors that bound the IgH enhancer and the decamer element found in the Vβ8 promoter. In addition, several binding sites for the T cell-restricted GATA-3 protein have been identified within an 800-bp fragment of the murine TCRβ enhancer (18). One of these GATA sites falls within the βE1 motif, and mutation of this site has been shown to reduce enhancer activity by 20–50%. The role of GATA sites for TCRβ expression is further complicated by the demonstration that coexpression of a dominant-negative GATA-3 protein has no effect on β enhancer activity (19).

FIGURE 1.

Schematic representation of the murine TCR β locus (A) and enhancer (B). A 700-bp StuI-NcoI fragment containing the murine enhancer is indicated on the top line of B (151617 ). Arrows above this line denote the end points of 5′ and 3′ deletion mutants studied by Takeda et al. (17 ) and the activity of these mutants above that of an enhancerless reporter plasmid. Enhancer fragments examined extended from the indicated deletion end points either 3′ to the NcoI site or 5′ to the StuI site. βE1–βE7 indicate the sites that interact with DNA binding proteins as identified by DNase1 footprinting and EMSAs (17 ). Sites marked TE1, 2, and 4 are three of four binding sites for the T cell-restricted transcription factor GATA-3 (18 ). Geometric shapes indicate transcription factor binding sites in the minimal TCRβ enhancer identified and studied in this report. Ovals represent binding sites for ETS domain proteins; diamonds represent binding sites for the CBF, and the rectangles represent two binding sites identified in this report. Shown below are the several subfragments derived from the StuI-NcoI piece that were used in this study to identify the smallest enhancer fragment that activates transcription in T cells.

FIGURE 1.

Schematic representation of the murine TCR β locus (A) and enhancer (B). A 700-bp StuI-NcoI fragment containing the murine enhancer is indicated on the top line of B (151617 ). Arrows above this line denote the end points of 5′ and 3′ deletion mutants studied by Takeda et al. (17 ) and the activity of these mutants above that of an enhancerless reporter plasmid. Enhancer fragments examined extended from the indicated deletion end points either 3′ to the NcoI site or 5′ to the StuI site. βE1–βE7 indicate the sites that interact with DNA binding proteins as identified by DNase1 footprinting and EMSAs (17 ). Sites marked TE1, 2, and 4 are three of four binding sites for the T cell-restricted transcription factor GATA-3 (18 ). Geometric shapes indicate transcription factor binding sites in the minimal TCRβ enhancer identified and studied in this report. Ovals represent binding sites for ETS domain proteins; diamonds represent binding sites for the CBF, and the rectangles represent two binding sites identified in this report. Shown below are the several subfragments derived from the StuI-NcoI piece that were used in this study to identify the smallest enhancer fragment that activates transcription in T cells.

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The human TCRβ enhancer has also been identified and characterized (20, 21). Five regions (Tβ1-Tβ5) of a 480-bp fragment of the human β enhancer were shown to bind nuclear factors by DNaseI footprinting assays (20). In addition, Prosser et al. (22) previously identified closely juxtaposed binding sites for ETS domain proteins and core binding factor (CBF/AML1/PEBP2) in the Tβ3 and Tβ4 motifs of the human TCRβ enhancer. They showed that both motifs contributed to the inducible activation of the enhancer and that multimers of the Tβ3 element conferred phorbol ester (PMA) inducible transcription. These sites (Tβ3 and Tβ4) are well conserved in the murine enhancer and correspond to the βE4 and βE6 motifs, respectively. Alignment of the murine and human β enhancer sequences show that the murine βE1 and βE2 motifs (including a GATA site) fall within Tβ2, βE4 falls within Tβ3, βE5 and βE6 fall within Tβ4, and βE7 falls within Tβ5. Mutations or deletion of Tβ2 and Tβ3 reduced enhancer 30–50% (20). Deletions of Tβ4 gave more variable results, reducing activity to 10% (20) or 40% (21) in two different studies. Recently, Kim et al. (23) showed that mutation of individual ETS and CBF elements of the human TCRβ enhancer significantly decreased activity in BW5147 cells.

To understand the molecular basis of the TCRβ enhancer activity in T cells, in this paper we define a minimal domain of the murine TCRβ enhancer and characterize the contribution of individual elements to enhancer activity. We found that a fragment containing the βE1–βE6 elements was required for full enhancer activity in several phenotypically mature αβ T cell lines. Mutation of individual ETS or CBF binding sites in either βE4 or βE6 abolished enhancer activity. In addition, two protein binding sites were identified within βE5 that were also essential for enhancer activity. However, in the pro-T cell line 2017, only the βE4, βE5, and βE6 elements were sufficient for full enhancer activity, suggesting that enhancer requirements may be different at different stages of differentiation. These results suggest that at least six factors must come together to activate the murine TCRβ enhancer.

Jurkat, EL-4, and 2017 (Moloney virus-derived pro-T cell line (24)) cells were maintained in RPMI 1640 containing 10% heat-inactivated FCS, 105 M 2-ME, and 0.5% penicillin-streptomycin. D5h3 cells were grown in DMEM supplemented with 5% newborn calf serum + 5% inactivated FCS. All cell lines were maintained in an atmosphere of 5% C02 at 37°C.

Transient transfections of all cell lines were conducted by using the DEAE-dextran protocol (25). Typically, 1 × 107 (Jurkat) or 2 × 107 (2017) cells were transfected with 10 μg of supercoiled plasmid DNA. All transfections were done in duplicate and were repeated at least twice. Total cellular extracts were prepared 40–48 h after transfection, and CAT enzyme levels were assayed using a CAT ELISA kit following the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN).

A 4-kb HindIII DNA fragment cloned into the HindIII site of pBluescript was subjected to restriction enzyme digestion to obtain a 695-bp StuI-NcoI fragment containing the functional TCRβ enhancer previously identified (17). The 695-bp StuI-NcoI was digested to produce a 222-bp HinfI and a 295-bp HinfI-NcoI DNA fragment. The 222-bp fragment contains the βE1–βE3 binding protein motifs, and the 295-bp fragment contains the remaining βE4–βE7 (Fig. 1). Each DNA fragment (695 bp, 222 bp, and 295 bp) was treated with Klenow enzyme and cloned into the filled in SalI site of the Δ56CAT plasmid upstream of the c-fos promoter. The 695-bp fragment was also cloned into the EcoR V site of pBluescript (pB695).

β242, β191, and β158 fragments were generated by PCR using primers shown in Table I from the pB695 clone. The 242-bp fragment contains βE1–βE6, the 191-bp fragment contains βE3–βE6, and the 158-bp fragment contains βE4–βE6, respectively (Fig. 1). The PCR products were digested with XhoI and subcloned into the SalI site of Δ56CAT. The 242-bp fragment was also cloned into the XhoI site of pBluescript (pB242). Additionally, an 81-bp HinfI-DdeI fragment containing βE4 plus βE5 and a 61-bp DdeI fragment containing βE6 were obtained from pB242 by restriction enzyme digestion. Both fragments were treated with Klenow and separately subcloned into the SalI site of the Δ56CAT plasmid. Plasmids containing dimers and monomers of both motifs were used for transfection analyses. For the data presented, all reporter plasmids contained the β enhancer in the same 5′-3′ orientation as was present in the TCRβ locus. We also tested reporters containing enhancer fragments cloned in the opposite orientation with similar results (data not shown).

Table I.

Deletion oligonucleotide primers

NameaSequenceb
βE1.5′ 5′-CCG CTC GAG CGG AAG CAT CTC ACC CC-3′ (307–322) 
βE3.5′ 5′-CCG CTC GAG CAA GTA AGA ATG GCC-3′ (358–372) 
βE4.5′ 5′-CCG CTC GAG CAT CTC CAG GAG TCA C-3′ (387–402) 
βE6.3′ 5′-CCG CTC GAG CGC TGA GGT AGA AAG GGC-3′ (533–549) 
NameaSequenceb
βE1.5′ 5′-CCG CTC GAG CGG AAG CAT CTC ACC CC-3′ (307–322) 
βE3.5′ 5′-CCG CTC GAG CAA GTA AGA ATG GCC-3′ (358–372) 
βE4.5′ 5′-CCG CTC GAG CAT CTC CAG GAG TCA C-3′ (387–402) 
βE6.3′ 5′-CCG CTC GAG CGC TGA GGT AGA AAG GGC-3′ (533–549) 
a

5′ indicates that the primer sequence is complementary to the bottom strand of the indicated motif. 3′ indicates that the primer sequence is complementary to the top strand of the indicated motif.

b

Underlined is the region for the XhoI site introduced for direct cloning. Numbers in parentheses correspond to the nucleotide positions according to Takeda et al. (17).

PCR oligonucleotide mutagenic primers were designed to introduce nucleotide changes at the ETS and CBF binding sites of βE4 and βE6, as well as at the βE5 motif (Table II). The pB242 subclone was used as a wild-type template in the PCR reactions to introduce the site-specific mutations. The procedure requires a single mutant primer and two flanking primers (26, 27), which in this case annealed within the polylinker of pBluescript. The oligonucleotides used in these experiments introduced unique restriction enzyme sites. The mutagenic primer and the downstream flanking primer (pBluescript primer 1, T7) were used to generate a primary product. The PCR product from this first reaction was gel purified and used in a second PCR as a primer with pBluescript primer 2, T3, and the wild-type 242 fragment as the template. The second PCR product was then gel purified and subcloned into the Δ56CAT vector. Mutations were confirmed by restriction enzyme analysis and DNA sequencing. Conditions for the PCR reactions were as follows: 10 ng of wild-type template, 200 ng of each primer, 200 mM deoxynucleotide triphosphates, 10 mM Tris, 500 mM KCl, and 1.5 mM MgCl2. PCRs were performed for 33 cycles at 94°C for 1 min, 45°C for 1 min, and 72°C for 2 min.

Table II.

Site-directed mutagenesis: mutant oligonucleotide primer sequences

NameaSequenceb
βE4(E5′-CCA GGA GTC ACG AGC TCA TGT GGT TTG ACA-3′ (391–421) 
βE4(C5′-CAC AAC AGG ATG TTC TAG AAC ATT TAC CAG GTC C-3′ (399–433) 
βE6(E/C5′-GGG TTT GAA GAC AGG AGA ATT CAA GTG TGG TTC CCA-3′ (487–522)c 
βE6(C5′-GGA TGT GGC AAG AAG CTT TCC CAA AAT GCT CAG-3′ (487–509)c 
βE5M1 5′-GAC ATT TAC CAG GTA GAT CTC CTG GGT GCC TGT G-3′ (419–452) 
βE5M2 5′-GGT CCT ACA TCT CTG CAG CCT GTG-3′ (429–452) 
βE5M3 5′-TAC ATC TGG GGT GCC GAA TTC TGC TCC CCC ACT CAC-3′ (434–469) 
βE5M4 5′-GTG CCT GTG AAG TCG ACC CCA CTC-3′ (445–467) 
βE5M5 5′-GAA TGC TCC CCC ACG GAT CCA CAT TCT GAG-3′ (452–481) 
βE5M6 5′-CTC ACT CAC AAA GCT TGC ATT TTG GGA A-3′ (465–492) 
NameaSequenceb
βE4(E5′-CCA GGA GTC ACG AGC TCA TGT GGT TTG ACA-3′ (391–421) 
βE4(C5′-CAC AAC AGG ATG TTC TAG AAC ATT TAC CAG GTC C-3′ (399–433) 
βE6(E/C5′-GGG TTT GAA GAC AGG AGA ATT CAA GTG TGG TTC CCA-3′ (487–522)c 
βE6(C5′-GGA TGT GGC AAG AAG CTT TCC CAA AAT GCT CAG-3′ (487–509)c 
βE5M1 5′-GAC ATT TAC CAG GTA GAT CTC CTG GGT GCC TGT G-3′ (419–452) 
βE5M2 5′-GGT CCT ACA TCT CTG CAG CCT GTG-3′ (429–452) 
βE5M3 5′-TAC ATC TGG GGT GCC GAA TTC TGC TCC CCC ACT CAC-3′ (434–469) 
βE5M4 5′-GTG CCT GTG AAG TCG ACC CCA CTC-3′ (445–467) 
βE5M5 5′-GAA TGC TCC CCC ACG GAT CCA CAT TCT GAG-3′ (452–481) 
βE5M6 5′-CTC ACT CAC AAA GCT TGC ATT TTG GGA A-3′ (465–492) 
a

At the indicated motifs, an ETS site mutation is represented by E, a CBF site mutation is represented by C, and a double-mutant ETS/CBF is represented by E/C. M indicates a random mutation at the indicated motif.

b

Numbers in parentheses correspond to the nucleotide positions according to Takeda et al. (17). Nucleotide substitutions are shown in bold.

c

The nucleotides shown represent the complementary sequence for the top strand in that motif.

Nuclear extracts from all cell lines were prepared as described by Dignam et al. (28). EMSA reactions typically used 10 μg of nuclear extract in Buffer D (20 mM HEPES, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 20% glycerol), 3 μg of poly (dI-dC), and 20,000 cpm of a 32P-labeled DNA fragment. Reactions were conducted in a volume of 10 μl containing 2 μl of 10× lipage buffer (100 mM Tris (pH 7.5), 0.5 M NaCl, 100 mM 2-ME, 10 mM EDTA, and 40% glycerol). After a 10-min incubation on ice, the reaction products were electrophoresed through nondenaturing 4% polyacrylamide gels in 0.5× Tris-borate buffer (44 mM Tris borate, 44 mM boric acid, and 2 mM EDTA). The gels were dried and visualized on DuPont film with an intensifying screen.

Nuclear extracts were prepared from the pro-T cell line 2017 as described by Dignam et al. (28), with one modification. Nuclear proteins extracted in Buffer C (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 25% glycerol) where directly chromatographed on a 0.5-ml DEAE-Sephacel Pharmacia (Piscataway, NJ) column equilibrated with Buffer C. This step removes residual nucleic acids from the protein preparation. The flow-through peak was dialyzed against Buffer D (20 mM HEPES, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 20% glycerol) and concentrated by chromatography on heparin-Sepharose 4B. This column was equilibrated with Buffer D, and specific binding activity for βE5 was eluted at 400 mM KCl (HF400). The HF400 was dialyzed into Buffer D (50 mM KCl) and stored at −70°C.

The chromosomal context and the results of earlier studies of the murine TCRβ enhancer are summarized in Fig. 1. Takeda et al. (17) defined the ends of the murine TCRβ enhancer by a series of 5′ or 3′ deletions. Later, Henderson et al. (18) evaluated the role of GATA elements in the same enhancer by point mutational analysis. Nevertheless, the importance of most protein binding sites within the murine enhancer has not been systematically studied. Because many enhancers contain compensatory elements, the effects of point mutations in individual elements is often difficult to distinguish. To circumvent the problem of redundancy, we first identified the smallest enhancer domain that retained significant activity and then conducted further mutational studies.

Several fragments derived from the enhancer region (Fig. 1,B) were cloned into a CAT reporter vector and assayed by transient transfection into Jurkat cells. A 242-bp fragment containing the βE1–βE6 motifs (β242) provided maximal transcription enhancement, whereas the fragment β222 (containing βE1–βE3) was inactive (Fig. 2,A). Three other fragments, β191 (containing βE3–βE6), β295 (containing βE4–βE7) and β158 (containing βE4–βE6) were also partially active (Fig. 2,A). βE4–βE6 motifs were common to all active fragments, suggesting that they constituted an essential core of the TCRβ enhancer. The activity of the core was accentuated by additional flanking elements such as βE7 (in β295) and βE1/βE2 (in β242). Similar results were obtained when these fragments were tested by transfection into EL-4 cells or D5h3 T hybridoma cells (Fig. 2, B and C).

FIGURE 2.

Transfection analyses of TCRβ enhancer mutations. AC, Enhancer derivatives shown in Fig. 1 B were cloned 5′ of a basal c-fos gene promoter (indicated as Δ56) that directs transcription of a CAT reporter gene. Ten micrograms of plasmids, labeled according to the inserts they contain, were transiently transfected into Jurkat (A), D5h3 T hybridoma (B), or EL-4 (C) cells by the DEAE-dextran procedure. CAT enzyme levels in whole cell extracts were detected by colorimetric enzyme immunoassay (CAT ELISA) 40–48 h after transfection. The y-axis represents the amount of CAT protein in 100 μg of whole cell extracts. A CAT reporter plasmid containing the Moloney murine leukemia virus enhancer was used as a positive control (labeled J21). Results represent the average of three independent transfections conducted in duplicate. D, Mutations in the ETS (E) and CBF (C) sites of βE4 and βE6 were tested in the context of the β242 enhancer fragment that contains motifs βE1–βE6. In addition, dimers of fragments containing βE4 + βE5 (labeled βE4) or only βE6 were tested for enhancer activity in Δ56CAT reporter plasmid. Results shown are from three independent transfections conducted in duplicate in Jurkat cells.

FIGURE 2.

Transfection analyses of TCRβ enhancer mutations. AC, Enhancer derivatives shown in Fig. 1 B were cloned 5′ of a basal c-fos gene promoter (indicated as Δ56) that directs transcription of a CAT reporter gene. Ten micrograms of plasmids, labeled according to the inserts they contain, were transiently transfected into Jurkat (A), D5h3 T hybridoma (B), or EL-4 (C) cells by the DEAE-dextran procedure. CAT enzyme levels in whole cell extracts were detected by colorimetric enzyme immunoassay (CAT ELISA) 40–48 h after transfection. The y-axis represents the amount of CAT protein in 100 μg of whole cell extracts. A CAT reporter plasmid containing the Moloney murine leukemia virus enhancer was used as a positive control (labeled J21). Results represent the average of three independent transfections conducted in duplicate. D, Mutations in the ETS (E) and CBF (C) sites of βE4 and βE6 were tested in the context of the β242 enhancer fragment that contains motifs βE1–βE6. In addition, dimers of fragments containing βE4 + βE5 (labeled βE4) or only βE6 were tested for enhancer activity in Δ56CAT reporter plasmid. Results shown are from three independent transfections conducted in duplicate in Jurkat cells.

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To evaluate the role of the individual motifs within the core domain, we mutated either the ETS or the CBF motifs of βE4 and βE6 (referred to as E or C). The βE6 element contains an ETS site and two CBF binding sites, which we will refer to as the proximal and distal CBF sites relative to the ETS site. We mutated either the distal CBF alone (C) or a combination of the ETS and proximal CBF sites (E/C). EMSA using the ETS domain of Ets-1 and the DNA-binding Runt domain of CBFα confirmed that each mutation affected only the sites that had been targeted (data not shown). Enhancer mutants were assayed in the context of β242 by transient transfection into Jurkat cells, and each mutation was found to be significantly less active than the wild-type enhancer (Fig. 2,D). We conclude that each ETS and CBF protein binding site is essential for enhancer function. These results parallel those of Kim et al. (23), who analyzed similar mutations in the human TCRβ enhancer. To determine whether the ETS and CBF elements were sufficient for enhancer activity, we assayed dimers of either βE4 or βE6 elements. Neither dimer activated transcription (Fig. 2 D), showing that ETS proteins and CBF cooperate with other elements of the TCRβ enhancer to activate transcription.

The studies described above were conducted in cell lines that represent mature T cells. In such cells, the β enhancer activates transcription of the functionally rearranged TCRβ gene. However, the enhancer is normally activated at a much earlier stage of T cell development, when it is required to initiate V(D)J recombination of the TCRβ locus. To study β enhancer activity in early T cells, we transfected the pro-T cell line 2017. This line was derived by intrathymic injection of Moloney murine leukemia virus, and its very early immature status is defined by the phenotype Thy+LyCD4CD8Ly2; these cells express TCR γ and β, but not α, sterile transcripts (24).

The activity profiles of the various enhancer fragments in Jurkat and 2017 cells differed in one striking way. β158 activity was comparable to β242 in 2017 cells (Fig. 3,A), whereas it was significantly less active than β242 in the other cell lines (Fig. 2). We conclude that βE1–βE3 do not contribute significantly to enhancer activity in 2017 cells and that βE4–βE6 are sufficient for transcriptional enhancement. Comparable activity of β158 and β242 was also noted in a CD4CD8 cell line derived from RAG1/p53 double-deficient mice (data not shown). The reduced activity of β191 and β295, both of which contain motifs βE4–βE6, compared with β158 in these cells may be due to negative transcriptional elements interspersed within the additional sequences; we have not investigated this phenomenon further at present. A possible interpretation of these observations is that the relative levels of functional factors binding within the βE4–βE6 enhancer core may differ between mature and immature cell lines. These observations suggest that at an early stage of T cell development, the β enhancer is activated by fewer factors than that required at a later stage. In 2017 cells as well, each of the two ETS or CBF binding sites were necessary for function (Fig. 3,B). To determine whether the ETS/CBF motifs were sufficient for transcriptional enhancement, we assayed βE4 and βE6 dimers in 2017 cells. Neither reporter was detectably active in these cells (Fig. 3 B), suggesting that ETS and CBF proteins required the βE5 element present in β158 to activate the β enhancer in 2017 cells. Therefore, we examined the βE5 region more closely.

FIGURE 3.

Transfection analysis in 2017 pro-T cells. Enhancer derivatives described in Fig. 1,B or point mutations and dimers described in Fig. 2 D were transiently transfected into 2017 cells using DEAE-dextran. Whole cell extracts prepared after 40–48 h were assayed for CAT enzyme levels by immunoassays. CAT concentrations obtained from the various plasmids are shown on the y-axis. Results shown are the average of three independent transfections conducted in duplicate

FIGURE 3.

Transfection analysis in 2017 pro-T cells. Enhancer derivatives described in Fig. 1,B or point mutations and dimers described in Fig. 2 D were transiently transfected into 2017 cells using DEAE-dextran. Whole cell extracts prepared after 40–48 h were assayed for CAT enzyme levels by immunoassays. CAT concentrations obtained from the various plasmids are shown on the y-axis. Results shown are the average of three independent transfections conducted in duplicate

Close modal

The βE5 region was defined by an oligonucleotide used by Takeda et al. (17) to identify proteins that bind to the TCRβ enhancer. Using this oligonucleotide, they detected several nucleoprotein complexes with BW5147 cell extracts, and methylation interference assays were used to identify the contact residues shown in Fig. 4,A. Because the functional relevance of this sequence is not known, we generated six sets of point mutations within the βE5 sequence. One of these mutations affected residues identified by methylation interference (βE5 M5), and the other five were introduced to cover most of the remaining nucleotides in βE5 (Fig. 4 A). All mutants were analyzed in the context of β242 by transient transfection into Jurkat cells.

FIGURE 4.

Functional analysis of the βE5. A, Sequence of the wild-type βE5 region is shown in capital letters. The G residues complementary to the Cs indicated by arrows were previously identified by methylation interference assays to bind a nuclear factor (17 ). Six sets of clustered mutations were generated in βE5 marked M1 through M6; the altered nucleotides are indicated in lower case. One of these mutations (M5) alters residues previously identified by methylation interference. B, Transfection analysis of βE5 mutations. For functional analysis M1–M6 mutations were introduced into the β242 enhancer fragment and were assayed after cloning into Δ56CAT reporter vector as described in Materials and Methods. Jurkat cells were transiently transfected with reporter plasmids containing no enhancer (Δ56CAT), wild-type enhancer (β242), or mutated enhancers (M1–M6), and CAT level (y-axis) was determined by immunoassays as described. Results shown are averaged from three experiments conducted in duplicate.

FIGURE 4.

Functional analysis of the βE5. A, Sequence of the wild-type βE5 region is shown in capital letters. The G residues complementary to the Cs indicated by arrows were previously identified by methylation interference assays to bind a nuclear factor (17 ). Six sets of clustered mutations were generated in βE5 marked M1 through M6; the altered nucleotides are indicated in lower case. One of these mutations (M5) alters residues previously identified by methylation interference. B, Transfection analysis of βE5 mutations. For functional analysis M1–M6 mutations were introduced into the β242 enhancer fragment and were assayed after cloning into Δ56CAT reporter vector as described in Materials and Methods. Jurkat cells were transiently transfected with reporter plasmids containing no enhancer (Δ56CAT), wild-type enhancer (β242), or mutated enhancers (M1–M6), and CAT level (y-axis) was determined by immunoassays as described. Results shown are averaged from three experiments conducted in duplicate.

Close modal

Mutations βE5 M4 and βE5 M5 significantly decreased enhancer activity (Fig. 4 B), whereas mutations M1–M3 had no effect. M6 reduced enhancer activity to ∼30%, probably because it changed residues that flank the binding site of the protein affected more substantially by mutant M5. First, these results show that the βE5 region binds proteins (see below) that are essential for enhancer function and whose loss cannot be compensated by βE1–βE3. They also rule out the possibility that the region acts as stuffer DNA to maintain appropriate distance between βE4 and βE6. The region spanned by the inactive mutations M4 and M5 is 16 nt; if the partial effect of M6 is taken into consideration, the region increases to 24 nt. This stretch is unlikely to be the binding site for one factor. Therefore, activity of the βE5 sequence appears to be mediated by (at least) two factors.

Proteins that bind to the βE5 region were identified by EMSA using nuclear extracts from 2017 cells. A probe encompassing the βE5 region generated several nucleoprotein complexes, of which two appeared to be important based on competition assays with wild-type, or mutated, βE5 sequences (Fig. 5 A); interestingly, the corresponding region from the human TCRβ enhancer also produced a similar pattern of DNA/protein complexes (data not shown). Complex A was competed by all fragments except M4 and the unrelated AP-1 sequence. Conversely, complex B was competed by all fragments except M5 and AP-1. (Note that competition with M5 revealed a weak upper complex, which we do not define as complex A based on its slower mobility compared with that of A.) Because mutations M4 and M5 decreased enhancer activity, we concluded that the proteins that generate complexes A and B are good candidates to be the functionally relevant proteins. All other nucleoprotein complexes detected did not show a competition pattern consistent with the functional analysis of the mutations. These observations suggest that TCRβ enhancer activity requires two proteins that bind within βE5.

FIGURE 5.

Analysis of βE5 binding proteins. A, EMSA and competition analyses in 2017 nuclear extracts. Synthetic wild-type βE5 sequence (residues 436–488) (17 ) was cloned into the XhoI site of pBluescript and was excised as a DNA fragment for use as the radioactive probe. EMSA was conducted with 15 μg nuclear extracts from 2017 cells in the presence of no competitor DNA (lane 1), wild-type or mutated βE5 sequences (lanes 3–9), and an irrelevant DNA sequence containing an AP-1 binding site (lane 2). Competitors were used at 100 ng (200× molar excess). Sequence-specific complexes A and B are indicated. B, Chromatographic resolution of βE5 binding proteins. The 2017 nuclear extract depleted of nucleic acids was fractionated by adsorption to heparin agarose. Proteins were bound to the resin in 100 mM KCl-containing buffer and were eluted at increasing salt concentrations. Fractions were assayed by EMSA using a wild-type βE5 probe. The pattern with 2017 nuclear extracts is shown in lane 1 before the flow through (FT; lane 2) and salt concentrations, as indicated above the figure (lanes 3–13). Complexes A and B are discussed in the text and correspond to the same labeled complexes in A and in Fig. 6. FP, Free probe.

FIGURE 5.

Analysis of βE5 binding proteins. A, EMSA and competition analyses in 2017 nuclear extracts. Synthetic wild-type βE5 sequence (residues 436–488) (17 ) was cloned into the XhoI site of pBluescript and was excised as a DNA fragment for use as the radioactive probe. EMSA was conducted with 15 μg nuclear extracts from 2017 cells in the presence of no competitor DNA (lane 1), wild-type or mutated βE5 sequences (lanes 3–9), and an irrelevant DNA sequence containing an AP-1 binding site (lane 2). Competitors were used at 100 ng (200× molar excess). Sequence-specific complexes A and B are indicated. B, Chromatographic resolution of βE5 binding proteins. The 2017 nuclear extract depleted of nucleic acids was fractionated by adsorption to heparin agarose. Proteins were bound to the resin in 100 mM KCl-containing buffer and were eluted at increasing salt concentrations. Fractions were assayed by EMSA using a wild-type βE5 probe. The pattern with 2017 nuclear extracts is shown in lane 1 before the flow through (FT; lane 2) and salt concentrations, as indicated above the figure (lanes 3–13). Complexes A and B are discussed in the text and correspond to the same labeled complexes in A and in Fig. 6. FP, Free probe.

Close modal

To strengthen this conclusion, we attempted to separate the proteins that produced complexes A and B. Nucleic acid-depleted 2017 nuclear extracts were fractionated by adsorption to heparin agarose and eluates assayed by EMSA. The factor resulting in complex A (NF-βA) did not bind to heparin and was detected in the flow-through and wash fractions (Fig. 5,B, lanes 2-4). Note that these fractions also generated a faster migrating complex with the βE5 probe. The factor(s) resulting in complex B (NF-βB) bound to heparin and were eluted at higher salt concentration (Fig. 5 B, lanes 11 and 12). These observations confirm that two distinct proteins bind to βE5 sequences: NF-βA binds to the sequence mutated by M4, and NF-βB binds to the sequence mutated by M5. Use of mutant probes and competition assays with the 0.4 M salt fraction were entirely consistent with the results in total nuclear extracts (data not shown).

The βE5 probe was used to screen a library of nuclear extracts to determine the tissue distribution of NF-βA and NF-βB. The upper NF-βA complex was detected in most nuclear extracts examined (Fig. 6). NF-βB binding was much weaker but was detectable in most B and T lymphoid cell lines. Complex A in the various extracts was abolished when the βE5 M4 probe was used in binding assays, confirming that it was generated by a protein having the same sequence-specificity as that characterized in 2017 cell extracts (data not shown). A ubiquitous Igμ enhancer binding protein was used to normalize between the different cell extracts (Fig. 6, μE3). Thus, neither protein binding to βE5 is T cell specific.

FIGURE 6.

Tissue distribution of βE5 binding proteins. Nuclear extracts prepared from cell lines as indicated above the lanes were used in EMSA with a wild-type βE5 probe (top panel) or a probe containing the μE3 sequence (bottom panel) from the Igμ heavy chain gene enhancer that binds ubiquitously distributed leucine-zipper containing basic helix-loop-helix factors. Binding reactions were done with 12 μg extracts and autoradiographs exposed for 48h. Positions of functional complexes A and B are indicated. 2017 and 2052C, murine pre-T cell lines; Jurkat, CD4+ human T lymphoma; EL-4, murine thymoma; D5h3, T hybridoma; S194, murine plasmacytoma; BJAB, EW, Ramos, and Namalwa, human B lymphomas; 70Z, murine pre-B cells; RAW, murine macrophage cell; HeLa, human cervical carcinoma.

FIGURE 6.

Tissue distribution of βE5 binding proteins. Nuclear extracts prepared from cell lines as indicated above the lanes were used in EMSA with a wild-type βE5 probe (top panel) or a probe containing the μE3 sequence (bottom panel) from the Igμ heavy chain gene enhancer that binds ubiquitously distributed leucine-zipper containing basic helix-loop-helix factors. Binding reactions were done with 12 μg extracts and autoradiographs exposed for 48h. Positions of functional complexes A and B are indicated. 2017 and 2052C, murine pre-T cell lines; Jurkat, CD4+ human T lymphoma; EL-4, murine thymoma; D5h3, T hybridoma; S194, murine plasmacytoma; BJAB, EW, Ramos, and Namalwa, human B lymphomas; 70Z, murine pre-B cells; RAW, murine macrophage cell; HeLa, human cervical carcinoma.

Close modal

Our studies identify a region containing the βE4, βE5, and βE6 motifs as the essential core of the TCR β enhancer in pro-T cells. In more mature cells, the βE4–βE6 core required either 5′ or 3′ flanking sequences to activate transcription. Presumably, the flanking sequences are functionally redundant, so that either one can provide the requisite, albeit quantitatively different, activity. The importance of the βE4–βE6 core was emphasized by the observation that point mutations made within any of these elements abolished enhancer activity when assayed in the context of a fragment containing βE1–βE6. Two of the three motifs in the β enhancer core, βE4 and βE6, contain composite ETS/CBF elements that are a common feature of several Ag receptor gene enhancers (29, 30). Although these sites were identified earlier, a consensus opinion on their function in the TCRβ enhancer had proven hard to establish (20, 21) until the recent studies of the human TCRβ enhancer (23). Our observations conclusively demonstrate that individual elements of both composite motifs are essential for activity of the murine TCRβ enhancer. In addition, in this paper we show that the intervening βE5 element is also essential for enhancer activity.

Analyses of point mutations through the βE5 region showed that two protein binding sites within this region were required for TCRβ enhancer activity. Proteins that bind to these elements were identified by EMSA; neither protein appeared to be limited in expression to T cells. Neither the estimated m.w. of these proteins nor the nucleotide sequence of their binding sites offered clues to their identity. Although the NF-βB site (affected by mutation M5) bore some resemblence to the TGAXTCA motif that is bound by AP-1 and activating transcription factor (ATF) family members, an AP-1 binding site did not compete effectively for NF-βB binding. These observations suggest that NF-βB is not a basic leucine zipper protein. Identification of functional βE5 binding proteins completes the minimal cast of characters required to activate the TCRβ enhancer and provides the foundation for further mechanistic analysis of this enhancer.

Recently, Ferrier and colleagues (31) have defined a core domain of the TCRβ enhancer that activates V(D)J recombination in transgenic mice. The smallest fragment that scores positive in this assay is one that contains the βE3 and βE4 elements and 30 nt 3′ of βE4. The 3′ nucleotides are critical for recombination, even when the enhancer fragment extends further 5′ to include βE1 and βE2. Comparison of the recombination data with the transcriptional data presented in this manuscript suggests the following. First, the critical nucleotides 3′ of βE4 do not include the motifs we have defined within βE5. Our βE5 mutations M1–M3, which do not affect enhancer activity, fall within the 3′ sequence. Thus, the region between βE4 and βE6 contains nonoverlapping motifs that are required for transcription and recombination. Second, five protein binding sites that are essential for transcription (two within βE5, two CBF sites, and the ETS site in βE6) are not required to activate recombination. It is particularly curious that Jβ1 germline transcription in the recombination substrates is activated in the absence of the functional βE5 and βE6 motifs identified in this paper. The importance of the ETS/CBF sites in βE6 is indicated by in vivo footprints over both βE4 and βE6 motifs in CD4CD8 thymocytes (31).

Production of TCRβ protein signals further differentiation along the αβ T cell pathway, which includes activating TCRα-chain transcription and rearrangement (1, 2, 32). In transgenic assays the TCRβ and α enhancers closely recapitulate early and late activation of these genes, respectively (33, 34). Therefore, it is interesting to compare the organization of the core domains of the α and β enhancers (Fig. 7). There is a well-characterized ETS/CBF/CBF motif in the α enhancer (35, 36) that is very similar to the βE6 element. The extent of similarity includes the relative orientation of the ETS and CBF sites to each other, the overlap between the ETS and the first CBF sites, and the same distance between the first and second CBF sites. This particular combination of ETS/CBF/CBF sites has not been observed in other genes, and its striking conservation between the two TCR enhancers argues in favor of functional significance. A particularly appealing possibility is that it may, in part, determine T cell specificity of these enhancers. Other than this highly conserved element, the α and β enhancers contain two other elements that are unique to each enhancer. The β enhancer contains the βE5 element in the middle and a distal ETS/CBF element, whereas the α enhancer contains a LEF-1/T cell-specific factor-1 (TCF-1) binding site in the middle and a distal ATF/cAMP response element binding protein site in place of the ETS/CBF site of the β enhancer (Fig. 7). The spacing between elements in the α and β enhancers is not discernibly conserved; however, it is entirely possible that spacing of elements within the β enhancer is important as has been noted in the α enhancer (35) and the Igμ heavy chain gene enhancer (37).

FIGURE 7.

Comparison of the minimal domains of the TCR α and β gene enhancers. The minimal TCRα gene enhancer is contained in a 98-bp fragment that contains binding sites for ATF/cAMP response element binding protein (CREB), LEF-1/TCF-1, and ETS/CBF proteins as indicated. The nucleotide sequences of these elements are indicated above each motif. The minimal TCRβ enhancer active in pro-T cell lines defined in this study is shown on the lower line. The ETS/CBF binding sites and the βE5A and B elements are indicated. Sequences within these elements are indicated above each motif. The schematics are drawn approximately to scale to reflect the similarity in 1) the organization of the two enhancers and 2) the βE6 site and ETS/CBF/CBF element of the TCRα enhancer.

FIGURE 7.

Comparison of the minimal domains of the TCR α and β gene enhancers. The minimal TCRα gene enhancer is contained in a 98-bp fragment that contains binding sites for ATF/cAMP response element binding protein (CREB), LEF-1/TCF-1, and ETS/CBF proteins as indicated. The nucleotide sequences of these elements are indicated above each motif. The minimal TCRβ enhancer active in pro-T cell lines defined in this study is shown on the lower line. The ETS/CBF binding sites and the βE5A and B elements are indicated. Sequences within these elements are indicated above each motif. The schematics are drawn approximately to scale to reflect the similarity in 1) the organization of the two enhancers and 2) the βE6 site and ETS/CBF/CBF element of the TCRα enhancer.

Close modal

Several lines of evidence suggest that transcription of the TCRβ locus may be initiated before the commitment of a multipotent cell to the T cell lineage. In particular TCRβ transcripts have been detected in Lin bone marrow cells (11) and in NK1.1+ precursor cells in the thymus, which can differentiate into both NK and T cells (9). However, the TCRβ locus is not transcribed in B cells. These observations suggest that T cell specificity may not be determined by an early T cell-specific factor that activates the TCRβ enhancer, but rather by inactivating the enhancer in non-T cell lineages. This reasoning leads to two questions: what factors initiate the early activity of the TCRβ enhancer in multipotent cells, and how is its activity maintained in cells of the T lineage? One possibility that we favor is that ETS and CBF proteins activate the enhancer before T cell commitment. Consistent with this is the observation that CBFA2/AML1 (a CBFα gene) is expressed in hematopoietic precursors and that deletion of this gene affects several hematopoietic lineages (38, 39). Second, ETS/CBF composite elements are found in the regulatory regions of several B-, T-, and myeloid-specific genes, suggesting that this element does not confer T cell specificity.

Among the core enhancer binding factors, this leaves the βE5 element as the one most likely to maintain TCRβ enhancer activity in T cells. It is noteworthy that the location of βE5 relative to the other two elements of the TCRβ enhancer is the same as that of LEF/TCF-1 binding site in the TCRα gene enhancer, which is the element that probably determines the late activation of the TCRα. We are aware that a caveat to the suggestion that βE5 contributes to T cell specificity is that both βE5 binding proteins that we have identified are more broadly expressed. However, it is often difficult to conclusively establish lineage specificity of DNA binding proteins in the milieu of more ubiquitously expressed factors that recognize similar sequences. Indeed, LEF/TCF binding activity can be detected in both B and T cell extracts. Alternatively, it is possible that maintenance of TCRβ enhancer activity is mediated by factors that bind to elements that flank the core (βE4–βE6) enhancer. In our assays these sequences are necessary for enhancer function in mature cells.

1

This work was supported by National Institutes of Health Grant GM43874 (to R.S.). I.C. holds a graduate fellowship from Banco Interamericano de Desarrollo-Consejo Nacional de Investigaciones Científicas y Technologicas/Instituto Venezolano de Investigaciones Cientificas (Venezuela).

3

Abbreviations used in this paper: RAG, recombination activation gene; CBF, core binding factor; (also referred to as AML 1 (acute myeloid leukemia 1) or PEBP2, (polyoma enhancer binding protein 2)); CAT, chloramphenicol acetyltransferase; ATF, activating transcription factor; TCF-1, T cell-specific factor-1.

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