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The Journal of Immunology, 2000, 164: 6332-6339.
Copyright © 2000 by The American Association of Immunologists

Functional Analysis of the Murine TCRß-Chain Gene Enhancer1

Irvith M. Carvajal and Ranjan Sen2

Rosenstiel Research Center and Department of Biology, Brandeis University, Waltham, MA 02254


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TCRß-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 TCRß enhancer in mature and immature T cell lines. We identified a region containing the ßE4, ßE5, and ßE6 motifs as the essential core of the TCRß 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 ßE4–ßE6 elements essentially abolished enhancer activity, indicating that this core enhancer contained no redundant elements. The ßE4 and ßE6 elements contain binding sites for ETS-domain proteins and the core binding factor. The ßE5 element bound two proteins that could be resolved chromatographically and that were both essential for enhancer activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Genes encoding {alpha}ß TCR heterodimers are assembled at discrete stages during T cell differentiation (1, 2). TCRß-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 TCRß-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 TCRß gene (5, 6), in which thymocyte maturation is blocked at a very early CD4-CD8- (double negative) stage of development. Provision of a transgenic recombined TCRß-chain gene allows cells to progress into CD4+CD8+ (double positive) cells (7, 8). During normal development, sensing of TCRß-chain protein is a signal for initiation of TCR{alpha} gene rearrangements, cell proliferation, and expression of the coreceptors CD4 and CD8. Thus, TCRß-chain expression is a prerequisite for further differentiation.

Transcriptional regulation of the TCRß-chain gene is complex. TCRß 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, TCRß 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, TCRß gene transcription must be maintained (or enhanced) in cells that commit to differentiate into T lymphocytes; conversely, TCRß gene transcripion must be extinguished if the cells commit to differentiate into other lineages. A transcriptional enhancer located 3' of the Cß2 exons plays a major role in establishing the pattern of TCRß gene expression, because in its absence the locus is not transcribed, nor does it undergo V(D)J recombination (13, 14).

The murine TCRß enhancer is located ~5 kb 3' of the Cß2 exons (Refs. 15, 16 and Fig. 1GoA). Deletion analysis by Takeda et al. (17) defined 5' and 3' ends of the enhancer that were separated by ~325 bp (Fig. 1GoB). However, the indicated region was not tested for enhancer activity by itself. Seven protein binding motifs, termed ßE1–ßE7, 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 Vß8 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 TCRß enhancer (18). One of these GATA sites falls within the ßE1 motif, and mutation of this site has been shown to reduce enhancer activity by 20–50%. The role of GATA sites for TCRß expression is further complicated by the demonstration that coexpression of a dominant-negative GATA-3 protein has no effect on ß enhancer activity (19).



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  		FIGURE 1. Schematic representation of the murine TCR ß locus (A) and enhancer (B). A 700-bp StuI-NcoI fragment containing the murine enhancer is indicated on the top line of B (15 16 17 ). 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. ßE1–ßE7 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 TCRß 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.

 
The human TCRß enhancer has also been identified and characterized (20, 21). Five regions (Tß1-Tß5) of a 480-bp fragment of the human ß 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 Tß3 and Tß4 motifs of the human TCRß enhancer. They showed that both motifs contributed to the inducible activation of the enhancer and that multimers of the Tß3 element conferred phorbol ester (PMA) inducible transcription. These sites (Tß3 and Tß4) are well conserved in the murine enhancer and correspond to the ßE4 and ßE6 motifs, respectively. Alignment of the murine and human ß enhancer sequences show that the murine ßE1 and ßE2 motifs (including a GATA site) fall within Tß2, ßE4 falls within Tß3, ßE5 and ßE6 fall within Tß4, and ßE7 falls within Tß5. Mutations or deletion of Tß2 and Tß3 reduced enhancer 30–50% (20). Deletions of Tß4 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 TCRß enhancer significantly decreased activity in BW5147 cells.

To understand the molecular basis of the TCRß enhancer activity in T cells, in this paper we define a minimal domain of the murine TCRß enhancer and characterize the contribution of individual elements to enhancer activity. We found that a fragment containing the ßE1–ßE6 elements was required for full enhancer activity in several phenotypically mature {alpha}ß T cell lines. Mutation of individual ETS or CBF binding sites in either ßE4 or ßE6 abolished enhancer activity. In addition, two protein binding sites were identified within ßE5 that were also essential for enhancer activity. However, in the pro-T cell line 2017, only the ßE4, ßE5, and ßE6 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 TCRß enhancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

Jurkat, EL-4, and 2017 (Moloney virus-derived pro-T cell line (24)) cells were maintained in RPMI 1640 containing 10% heat-inactivated FCS, 10-5 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.

Transfections and chloramphenicol acetyltransferase (CAT) enzyme analysis

Transient transfections of all cell lines were conducted by using the DEAE-dextran protocol (25). Typically, 1 x 107 (Jurkat) or 2 x 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).

Plasmids construction

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 TCRß 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 ßE1–ßE3 binding protein motifs, and the 295-bp fragment contains the remaining ßE4–ßE7 (Fig. 1Go). 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 {Delta}56CAT plasmid upstream of the c-fos promoter. The 695-bp fragment was also cloned into the EcoR V site of pBluescript (pB695).

ß242, ß191, and ß158 fragments were generated by PCR using primers shown in Table IGo from the pB695 clone. The 242-bp fragment contains ßE1–ßE6, the 191-bp fragment contains ßE3–ßE6, and the 158-bp fragment contains ßE4–ßE6, respectively (Fig. 1Go). The PCR products were digested with XhoI and subcloned into the SalI site of {Delta}56CAT. The 242-bp fragment was also cloned into the XhoI site of pBluescript (pB242). Additionally, an 81-bp HinfI-DdeI fragment containing ßE4 plus ßE5 and a 61-bp DdeI fragment containing ßE6 were obtained from pB242 by restriction enzyme digestion. Both fragments were treated with Klenow and separately subcloned into the SalI site of the {Delta}56CAT plasmid. Plasmids containing dimers and monomers of both motifs were used for transfection analyses. For the data presented, all reporter plasmids contained the ß enhancer in the same 5'-3' orientation as was present in the TCRß locus. We also tested reporters containing enhancer fragments cloned in the opposite orientation with similar results (data not shown).


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  		Table I. Deletion oligonucleotide primers

 
Site-directed mutagenesis

PCR oligonucleotide mutagenic primers were designed to introduce nucleotide changes at the ETS and CBF binding sites of ßE4 and ßE6, as well as at the ßE5 motif (Table IIGo). 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 {Delta}56CAT 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.


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  		Table II. Site-directed mutagenesis: mutant oligonucleotide primer sequences

 
EMSA

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 10x 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 0.5x 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.

Fractionation of nuclear extracts

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 ßE5 was eluted at 400 mM KCl (HF400). The HF400 was dialyzed into Buffer D (50 mM KCl) and stored at -70°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The chromosomal context and the results of earlier studies of the murine TCRß enhancer are summarized in Fig. 1Go. Takeda et al. (17) defined the ends of the murine TCRß 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.

TCRß enhancer activity in T cell lines

Several fragments derived from the enhancer region (Fig. 1GoB) were cloned into a CAT reporter vector and assayed by transient transfection into Jurkat cells. A 242-bp fragment containing the ßE1–ßE6 motifs (ß242) provided maximal transcription enhancement, whereas the fragment ß222 (containing ßE1–ßE3) was inactive (Fig. 2GoA). Three other fragments, ß191 (containing ßE3–ßE6), ß295 (containing ßE4–ßE7) and ß158 (containing ßE4–ßE6) were also partially active (Fig. 2GoA). ßE4–ßE6 motifs were common to all active fragments, suggesting that they constituted an essential core of the TCRß enhancer. The activity of the core was accentuated by additional flanking elements such as ßE7 (in ß295) and ßE1/ßE2 (in ß242). Similar results were obtained when these fragments were tested by transfection into EL-4 cells or D5h3 T hybridoma cells (Fig. 2Go, B and C).



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  		FIGURE 2. Transfection analyses of TCRß enhancer mutations. A–C, Enhancer derivatives shown in Fig. 1GoB were cloned 5' of a basal c-fos gene promoter (indicated as {Delta}56) 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 ßE4 and ßE6 were tested in the context of the ß242 enhancer fragment that contains motifs ßE1–ßE6. In addition, dimers of fragments containing ßE4 + ßE5 (labeled ßE4) or only ßE6 were tested for enhancer activity in {Delta}56CAT reporter plasmid. Results shown are from three independent transfections conducted in duplicate in Jurkat cells.

 
To evaluate the role of the individual motifs within the core domain, we mutated either the ETS or the CBF motifs of ßE4 and ßE6 (referred to as E- or C-). The ßE6 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 CBF{alpha} confirmed that each mutation affected only the sites that had been targeted (data not shown). Enhancer mutants were assayed in the context of ß242 by transient transfection into Jurkat cells, and each mutation was found to be significantly less active than the wild-type enhancer (Fig. 2GoD). 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 TCRß enhancer. To determine whether the ETS and CBF elements were sufficient for enhancer activity, we assayed dimers of either ßE4 or ßE6 elements. Neither dimer activated transcription (Fig. 2GoD), showing that ETS proteins and CBF cooperate with other elements of the TCRß enhancer to activate transcription.

The studies described above were conducted in cell lines that represent mature T cells. In such cells, the ß enhancer activates transcription of the functionally rearranged TCRß 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 TCRß locus. To study ß 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+Ly-CD4-CD8-Ly2-; these cells express TCR {gamma} and ß, but not {alpha}, sterile transcripts (24).

The activity profiles of the various enhancer fragments in Jurkat and 2017 cells differed in one striking way. ß158 activity was comparable to ß242 in 2017 cells (Fig. 3GoA), whereas it was significantly less active than ß242 in the other cell lines (Fig. 2Go). We conclude that ßE1–ßE3 do not contribute significantly to enhancer activity in 2017 cells and that ßE4–ßE6 are sufficient for transcriptional enhancement. Comparable activity of ß158 and ß242 was also noted in a CD4-CD8- cell line derived from RAG1/p53 double-deficient mice (data not shown). The reduced activity of ß191 and ß295, both of which contain motifs ßE4–ßE6, compared with ß158 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 ßE4–ßE6 enhancer core may differ between mature and immature cell lines. These observations suggest that at an early stage of T cell development, the ß 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. 3GoB). To determine whether the ETS/CBF motifs were sufficient for transcriptional enhancement, we assayed ßE4 and ßE6 dimers in 2017 cells. Neither reporter was detectably active in these cells (Fig. 3GoB), suggesting that ETS and CBF proteins required the ßE5 element present in ß158 to activate the ß enhancer in 2017 cells. Therefore, we examined the ßE5 region more closely.



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  		FIGURE 3. Transfection analysis in 2017 pro-T cells. Enhancer derivatives described in Fig. 1GoB or point mutations and dimers described in Fig. 2GoD 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

 
Mutational analysis of ßE5

The ßE5 region was defined by an oligonucleotide used by Takeda et al. (17) to identify proteins that bind to the TCRß 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. 4GoA. Because the functional relevance of this sequence is not known, we generated six sets of point mutations within the ßE5 sequence. One of these mutations affected residues identified by methylation interference (ßE5 M5), and the other five were introduced to cover most of the remaining nucleotides in ßE5 (Fig. 4GoA). All mutants were analyzed in the context of ß242 by transient transfection into Jurkat cells.



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  		FIGURE 4. Functional analysis of the ßE5. A, Sequence of the wild-type ßE5 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 ßE5 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 ßE5 mutations. For functional analysis M1–M6 mutations were introduced into the ß242 enhancer fragment and were assayed after cloning into {Delta}56CAT reporter vector as described in Materials and Methods. Jurkat cells were transiently transfected with reporter plasmids containing no enhancer ({Delta}56CAT), wild-type enhancer (ß242), 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.

 
Mutations ßE5 M4 and ßE5 M5 significantly decreased enhancer activity (Fig. 4GoB), 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 ßE5 region binds proteins (see below) that are essential for enhancer function and whose loss cannot be compensated by ßE1–ßE3. They also rule out the possibility that the region acts as stuffer DNA to maintain appropriate distance between ßE4 and ßE6. 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 ßE5 sequence appears to be mediated by (at least) two factors.

Nuclear factors that interact with ßE5

Proteins that bind to the ßE5 region were identified by EMSA using nuclear extracts from 2017 cells. A probe encompassing the ßE5 region generated several nucleoprotein complexes, of which two appeared to be important based on competition assays with wild-type, or mutated, ßE5 sequences (Fig. 5GoA); interestingly, the corresponding region from the human TCRß 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 TCRß enhancer activity requires two proteins that bind within ßE5.



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  		FIGURE 5. Analysis of ßE5 binding proteins. A, EMSA and competition analyses in 2017 nuclear extracts. Synthetic wild-type ßE5 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 ßE5 sequences (lanes 3–9), and an irrelevant DNA sequence containing an AP-1 binding site (lane 2). Competitors were used at 100 ng (200x molar excess). Sequence-specific complexes A and B are indicated. B, Chromatographic resolution of ßE5 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 ßE5 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. 6Go. FP, Free probe.

 
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-ßA) did not bind to heparin and was detected in the flow-through and wash fractions (Fig. 5GoB, lanes 2-4). Note that these fractions also generated a faster migrating complex with the ßE5 probe. The factor(s) resulting in complex B (NF-ßB) bound to heparin and were eluted at higher salt concentration (Fig. 5GoB, lanes 11 and 12). These observations confirm that two distinct proteins bind to ßE5 sequences: NF-ßA binds to the sequence mutated by M4, and NF-ßB 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 ßE5 probe was used to screen a library of nuclear extracts to determine the tissue distribution of NF-ßA and NF-ßB. The upper NF-ßA complex was detected in most nuclear extracts examined (Fig. 6Go). NF-ßB 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 ßE5 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. 6Go, µE3). Thus, neither protein binding to ßE5 is T cell specific.



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  		FIGURE 6. Tissue distribution of ßE5 binding proteins. Nuclear extracts prepared from cell lines as indicated above the lanes were used in EMSA with a wild-type ßE5 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies identify a region containing the ßE4, ßE5, and ßE6 motifs as the essential core of the TCR ß enhancer in pro-T cells. In more mature cells, the ßE4–ßE6 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 ßE4–ßE6 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 ßE1–ßE6. Two of the three motifs in the ß enhancer core, ßE4 and ßE6, 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 TCRß enhancer had proven hard to establish (20, 21) until the recent studies of the human TCRß enhancer (23). Our observations conclusively demonstrate that individual elements of both composite motifs are essential for activity of the murine TCRß enhancer. In addition, in this paper we show that the intervening ßE5 element is also essential for enhancer activity.

Analyses of point mutations through the ßE5 region showed that two protein binding sites within this region were required for TCRß 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-ßB 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-ßB binding. These observations suggest that NF-ßB is not a basic leucine zipper protein. Identification of functional ßE5 binding proteins completes the minimal cast of characters required to activate the TCRß enhancer and provides the foundation for further mechanistic analysis of this enhancer.

Recently, Ferrier and colleagues (31) have defined a core domain of the TCRß enhancer that activates V(D)J recombination in transgenic mice. The smallest fragment that scores positive in this assay is one that contains the ßE3 and ßE4 elements and 30 nt 3' of ßE4. The 3' nucleotides are critical for recombination, even when the enhancer fragment extends further 5' to include ßE1 and ßE2. Comparison of the recombination data with the transcriptional data presented in this manuscript suggests the following. First, the critical nucleotides 3' of ßE4 do not include the motifs we have defined within ßE5. Our ßE5 mutations M1–M3, which do not affect enhancer activity, fall within the 3' sequence. Thus, the region between ßE4 and ßE6 contains nonoverlapping motifs that are required for transcription and recombination. Second, five protein binding sites that are essential for transcription (two within ßE5, two CBF sites, and the ETS site in ßE6) are not required to activate recombination. It is particularly curious that Jß1 germline transcription in the recombination substrates is activated in the absence of the functional ßE5 and ßE6 motifs identified in this paper. The importance of the ETS/CBF sites in ßE6 is indicated by in vivo footprints over both ßE4 and ßE6 motifs in CD4-CD8- thymocytes (31).

Production of TCRß protein signals further differentiation along the {alpha}ß T cell pathway, which includes activating TCR{alpha}-chain transcription and rearrangement (1, 2, 32). In transgenic assays the TCRß and {alpha} 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 {alpha} and ß enhancers (Fig. 7Go). There is a well-characterized ETS/CBF/CBF motif in the {alpha} enhancer (35, 36) that is very similar to the ßE6 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 {alpha} and ß enhancers contain two other elements that are unique to each enhancer. The ß enhancer contains the ßE5 element in the middle and a distal ETS/CBF element, whereas the {alpha} 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 ß enhancer (Fig. 7Go). The spacing between elements in the {alpha} and ß enhancers is not discernibly conserved; however, it is entirely possible that spacing of elements within the ß enhancer is important as has been noted in the {alpha} enhancer (35) and the Igµ heavy chain gene enhancer (37).



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  		FIGURE 7. Comparison of the minimal domains of the TCR {alpha} and ß gene enhancers. The minimal TCR{alpha} 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 TCRß enhancer active in pro-T cell lines defined in this study is shown on the lower line. The ETS/CBF binding sites and the ßE5A 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 ßE6 site and ETS/CBF/CBF element of the TCR{alpha} enhancer.

 
Several lines of evidence suggest that transcription of the TCRß locus may be initiated before the commitment of a multipotent cell to the T cell lineage. In particular TCRß 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 TCRß 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 TCRß 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 TCRß 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 CBF{alpha} 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 ßE5 element as the one most likely to maintain TCRß enhancer activity in T cells. It is noteworthy that the location of ßE5 relative to the other two elements of the TCRß enhancer is the same as that of LEF/TCF-1 binding site in the TCR{alpha} gene enhancer, which is the element that probably determines the late activation of the TCR{alpha}. We are aware that a caveat to the suggestion that ßE5 contributes to T cell specificity is that both ßE5 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 TCRß enhancer activity is mediated by factors that bind to elements that flank the core (ßE4–ßE6) enhancer. In our assays these sequences are necessary for enhancer function in mature cells.


   Footnotes
 
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). Back

2 Address correspondence and reprint requests to Dr. Ranjan Sen, Rosenstiel Research Center (MS-029), Brandeis University, 415 South Street, Waltham, MA 02454-9110. Back

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. Back

Received for publication August 3, 1999. Accepted for publication March 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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