INTRODUCTION

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Precursor lymphocytes diversify immunoglobulin (Ig) and T-cell receptor (TCR) variable-region genes via a program of DNA recombination involving large arrays of variable (V), diversity (D), and joining (J) gene segments. All rearrangement events are mediated by a common V(D)J recombinase activity that targets conserved recognition sequences flanking each gene segment (32, 39). Despite these shared features, the rearrangement of antigen receptor loci proceeds in a tissue-, stage-, and allele-specific manner (39). For example, thymocytes specifically target TCR D and J gene segments for recombination upon commitment to the T-cell lineage. In turn, DJ joins rearrange with one of 30 upstream V elements to complete assembly of a variable-region coding exon. The resultant expression of TCR protein signals for a cessation of TCR recombination and the initiation of TCR gene assembly (42). Likewise, precursor B cells execute an ordered program of rearrangements at the Ig heavy-chain (IgH) and light-chain loci (8, 22). These observations indicate that precursor lymphocytes must direct and then redirect V(D)J recombinase activity to specific regions within antigen receptor loci at distinct stages of their development.

Recent studies have shown that the tissue- and stage-specific aspects of V(D)J rearrangement are governed by changes in the accessibility of gene segment clusters to recombinase proteins RAG-1 and RAG-2 (25, 41). An important role for enhancer elements in regulating the recombinational accessibility of linked gene segments has been deduced from numerous experimental approaches (reviewed in reference 39). For example, targeted deletion of Ig or TCR enhancers severely impairs recombination of gene segments specifically at the mutated alleles (1, 3, 6, 35, 40, 47). In addition, transgenic TCR miniloci undergo rearrangement in precursor lymphocytes only upon inclusion of Ig or TCR enhancers (4, 11, 12, 29). In studies using a recombinase-inducible cell line, the latter results have been extended to show that any active enhancer, including those derived from a viral genome, directs efficient DJ recombination within chromosomal miniloci (30). Despite these findings, the precise function of enhancer elements in regulating the rearrangement of associated gene segments remains unclear (39).

Prevailing models for enhancer-mediated control of V(D)J recombination invoke at least one of three effects exerted by these regulatory elements on neighboring gene segments. First, Ig and TCR enhancers activate transcription of germ line gene segments at developmental time points that coincide with their rearrangement (21, 31, 48). Second, the IgH enhancer (Eµ) has been shown to promote regional access to DNA-binding proteins, presumably via directed alterations in local chromatin configurations (17). Third, transcriptional enhancers protect chromosomal gene segments from active methylation and target hypermethylated sequences for demethylation (9, 19, 23). Each of these enhancer-dependent effects has been correlated with active recombination of linked gene segments (7, 24, 30, 34); however, their independent contributions to rearrangement efficiencies have not been established (39). Thus, it remains possible that enhancers directly regulate V(D)J recombination through their intrinsic abilities to potentiate the accessibility of neighboring chromatin. In this case, transcription and demethylation of gene segments would be simply by-products of enhancer function. Alternatively, enhancer-dependent activation of germ line promoters may be the critical parameter for directing efficient assembly of antigen receptor loci.

To dissect the role of transcriptional control elements in targeting V(D)J recombination, we have used a recombinase-inducible cell system to assess the rearrangement efficiency of chromosomal TCR miniloci. Here, we show that enhancer activity is insufficient to target these substrates for DJ recombination. Instead, a recently identified promoter situated 5' to the D1 gene segment (PD [37]) is absolutely required for enhancer-dependent rearrangement of chromosomal gene segments. Importantly, substitution of PD with a synthetic promoter completely restores recombination of substrates lacking a distal enhancer element, despite their retention of a hypermethylated DNA status. Together, these findings suggest that activation of germ line promoter elements is the primary mechanism by which enhancers initiate assembly of variable-region gene segments.

	MATERIALS AND METHODS

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Generation of stable TDR19 transfectants. The recombinase-inducible cell line TDR19 was generated by cotransfection of linearized pTET-R1, pTET-R2, pTET-tTAk (36), and pSV-HIS vectors (15) into the recombinase-null B-cell line M12. Transfected cells were selected in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.01% penicillin-streptomycin, 50 µM -mercaptoethanol, tetracycline (0.5 µg/ml), and histidinol (3 mM). Southern blot analyses demonstrated that the TDR19 clone contained more than 10 copies each of the pTET-R1 and pTET-R2 vectors (38).

Construction of TCR recombination substrates. To dissect the components of recombinational accessibility, we generated a TCR parental vector (D/E) that contained unique cloning sites at locations 5' (NotI) and 3' (XhoI) to the J1 and J2 gene segments. For this purpose, the 4.8-kb HindIII fragment spanning murine V14 and the 600-bp BglII/BamHI fragment containing murine J1 and J2 segments were sequentially inserted into the HindIII and BamHI sites, respectively, of pGEM 11Z creating the D/E/Cµ construct. The Sµ/Cµ region of the D/E construct was prepared from a 7.1-kb XbaI/EcoRI genomic fragment that was modified to destroy the internal XhoI site and linker ligated to replace the 5' XbaI site with a unique XhoI site. The modified Sµ/Cµ fragment was cloned into the corresponding XhoI/EcoRI sites in D/E/Cµ polylinker sequences to yield the D/E vector. Finally, a blunt-ended 475-bp AluI/AluI fragment spanning iE was cloned into the blunted XhoI site of D/E to produce the D/E⁺ construct. To generate the /iE minilocus, the AccI/BglII fragment spanning D1 (430 bp), which lacks PD, was ligated to NotI linkers and inserted into the unique NotI site present in D/E⁺. Likewise, other promoter-D1 combinations were inserted as either blunt-ended or NotI-linkered fragments into the unique NotI site located between the V14 and J gene segments.

PCR analyses of coding and signal joins. For coding join analyses, genomic DNA was harvested from transfectants that were propagated in the presence or absence of tetracycline for 5 days. To minimize amplification of unrearranged TCR miniloci, each DNA sample was digested with XbaI and ApaI, which both cleave at sites situated between the D1 and J1 gene segments. Extrachromosomal DNAs (30 µl) were prepared for signal join assays from 5 × 10⁶ cells that were cultured in the presence or absence of tetracycline for 48 h (28).

Reverse transcription-PCR (RT-PCR) assay for germ line DJ transcription. Total cellular mRNA was harvested by the LiCl method from clonal transfectants that were propagated for 72 h subsequent to tetracycline withdrawal. Reaction mixtures (20 µl) consisting of mRNA (3 µg), deoxynucleoside triphosphates (250 µM), random hexanucleotides (5 pmol), dithiothreitol (8.75 mM), and RNAsin (20 U; Promega) were preincubated at 65°C for 10 min to denature the RNAs and cooled rapidly to 42°C. Reverse transcription was initiated by immediate addition of MuLV reverse transcriptase (100 U; Perkin-Elmer) to each sample. The reaction mixtures were incubated at 42°C for 60 min, heat inactivated (75°C for 15 min), and stored at 20°C. To examine germ line expression, the resultant cDNAs (3 µl) were amplified with oligonucleotide primers specific for either DJCµ (27 cycles) or -actin (25 cycles) transcripts, using the conditions described for DJ coding join assays. Amplification products were separated on a 1% agarose gel, blotted to ZetaProbe membranes, and hybridized to the appropriate radiolabeled probes (Table 1).

Primers and probes. The sequences of oligonucleotide primers used for PCR amplification reactions and probes used for blot hybridizations are shown in Table 1.

DNA methylation analysis. The methylation status of chromosomal Ptet (see below) substrates was evaluated in transfectants maintained in the absence of tetracycline (in the presence of tTAk [see below]) for 5 days. Genomic DNAs (10 µg) were digested with appropriate combinations of restriction enzymes HindIII, HpaII, and MspI, separated on a 1% agarose gel, and transferred to ZetaProbe membranes. Southern blots were probed with a radiolabeled XbaI/BamHI fragment spanning the J1 and J2 gene segments as specified by the manufacturer (Bio-Rad).

	RESULTS

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Enhancer-dependent regulation of DJ rearrangement in recombinase-inducible cells. To expedite analyses of the molecular mechanisms that govern V(D)J recombination, we developed a B-cell system (TDR19) in which recombinase activity is expressed in an inducible manner. This strategy circumvents rearrangement of transfected substrates prior to their stable integration and allows us to specifically monitor the recombination potential of chromosomal gene segments. Prior studies have shown that coexpression of the recombination-activating genes RAG-1 and RAG-2 is sufficient to generate V(D)J recombinase activity in most mammalian cell lines (28, 32). Therefore, we prepared the TDR19 cell system by stable transfection of a recombinase-null B cell (M12) with RAG-1/2 expression vectors that were placed under the transcriptional control of a tetracycline-inducible promoter (Ptet [36]). In addition, the RAG cDNAs were cotransfected with an autoregulated expression vector encoding the chimeric Ptet-activating protein (tTAk [14, 36]), which binds to Ptet upon removal of tetracycline from the culture medium. The resultant TDR19 clone expressed undetectable levels of RAG transcripts in the presence of tetracycline. In contrast, 24 h after tetracycline withdrawal, levels of RAG gene expression in TDR19 were similar to those observed in primary thymocytes (38).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Schematic depiction of modified TCR miniloci. Unique restriction sites used for cloning promoter (NotI [N]) and enhancer (XhoI [X]) sequences are indicated. Arrows represent the relative positions of primers used for PCR amplification of DJ coding joins (A and B) or DJCµ cDNAs (C and D). Transcriptional regulatory elements: 5'D, 2.3-kb fragment of endogenous D1 sequences; PD, minimal 377-bp D1 germ line promoter (37); PV, the murine V21C promoter (13); PGK, the rat PGK promoter (44); teto, a heptamer of the bacterial tetracycline operon (14); iE, the murine Ig intronic enhancer (13).

View larger version (43K): [in this window] [in a new window]   FIG. 2.   Enhancer-dependent recombination of TCR miniloci requires 5' D1 sequences. Levels of DJ rearrangements in chromosomal TCR miniloci were analyzed by a semiquantitative PCR assay using primers A and B (Fig. 1). Letters above the lanes identify independent TDR19 clones harboring the substrates 5'D/iE (lanes 1 to 4), 5'D/ (lanes 5 and 6), and /iE (lanes 7 to 11). Transfectants were incubated in the presence (without RAG) or absence (with RAG) of tetracycline for 5 days. The relative positions of amplification products corresponding to germ line miniloci (gl), as well as DJ1 (DJ1) and DJ2 (DJ2) rearrangements, are shown at the left. Control assays for recombinase activity (VJ rearrangement) and total DNA content (C) in each sample are shown in the middle and bottom panels, respectively. The linearity of the DJ coding join assay was confirmed by serial dilutions (lanes 12 to 17) of the 5'D/iE sample shown in lane 2 with DNA harvested from the 5'D/ sample shown in lane 6.

View this table: [in this window] [in a new window]   TABLE 2.   Summary of germ line expression and D-J rearrangement in TCR minilocus transfectantsa

Distal enhancer activity is insufficient to target rearrangement of TCR miniloci. In recent studies, we have shown that a promoter located directly 5' to the D1 gene segment (PD) regulates germ line transcription of D1/J gene segments in an enhancer-dependent manner (37). As an initial attempt to dissect the individual roles of promoter and enhancer elements in targeting recombination, we deleted 2 kb of 5' D1 sequences from a TCR minilocus that contained iE. The resulting construct (/iE [Fig. 1]), which lacks PD, was stably transfected into TDR19 cells and assayed for DJ rearrangement subsequent to induction of recombinase activity. Deletion of the 5'D1 sequences severely impaired recombination of the TCR minilocus in all clones examined (Fig. 2, lanes 7 to 11; Table 2). In addition to PD, the deleted 5'D sequences span two regions that display DNase hypersensitivity in developing thymocytes (5). To test whether these regions were required for rearrangement of TCR miniloci, we inserted the 377-bp minimal PD element (37) at its native position in /iE to generate the PD/iE substrate. Importantly, restoration of PD was sufficient to support normal levels of DJ joining following recombinase induction (Fig. 3A, lanes 5 to 8). Together, these data clearly demonstrate that enhancer activity per se is insufficient to direct recombination of linked gene segments. Instead, positive regulation of TCR accessibility requires enhancer-dependent activation of the D1 germ line promoter.

View larger version (62K): [in this window] [in a new window]   FIG. 3.   Promoter activation mediates DJ rearrangement in chromosomal TCR substrates. (A) Levels of DJ rearrangements within TCR/iE substrates containing the minimal PD (lanes 5 to 8), the V21C (lanes 9 and 10), or the PGK (lanes 11 and 12) promoter element. Control PCRs with TDR19 transfectants containing the 5'D/iE (lanes 1 and 2) or the promoterless (lanes 3 and 4) substrates are included for comparison. (B) Rearrangement levels in TDR19 transfectants harboring the Ptet/iE (lanes 5 and 6), Ptet/ (lanes 7 to 12), or iE/PD (lanes 13 to 18) minilocus. Other notation is as for Fig. 2.

Promoter activation targets DJ rearrangement. The results presented in Fig. 2 and 3 favor a model in which promoter activation is the critical parameter for mediating efficient DJ rearrangement. Alternatively, physical interactions between promoter- and enhancer-bound proteins might act to loop out intervening gene segments from the chromosome and thereby facilitate their recognition by recombinase. Indeed, prior studies have shown that promoter-enhancer interactions can significantly alter the structure of intervening chromatin (10). Further support for this loop model is provided by the regulatory architecture of all antigen receptor loci, in which germ line gene segments are flanked by 5' promoter and 3' enhancer elements.

Germ line DJ transcription correlates precisely with substrate recombination potential. The activation of enhancer elements within antigen receptor loci has been linked temporally with the onset of germ line transcription and V(D)J recombination at participating gene segments (4, 33). To assess whether DJ joining in modified TCR miniloci correlated with their germ line transcription, we analyzed TDR19 transfectants in an RT-PCR assay that specifically detected hybrid DJCµ transcripts derived from unrearranged substrates (Fig. 1, primers C and D). In multiple independent transfectants, removal of either iE or 5'D sequences spanning the PD element abolished not only substrate rearrangement but germ line DJ transcription as well (Fig. 4, lanes 1 to 7). In contrast, DJCµ transcripts were readily detected in recombinationally active substrates containing iE linked to either the minimal PD element or heterologous promoters (lanes 8 to 16). These findings are fully consistent with prior studies, which have shown that PD is functional in B cells when linked to active enhancer elements (30, 37). Importantly, germ line transcription and rearrangement were restored in miniloci lacking a distal enhancer by positioning tet_o upstream from the consensus TATA element within the D1 recognition sequence (lanes 17 to 20). These functional correlations held true even for rare clones in which promoter/iE substrates lacked both DJ rearrangement and germ line transcripts (Table 2). As shown previously, these transfectants likely harbor substrate integrations into regions of heterochromatin (18). Thus, the results presented in Fig. 4 provide a direct correlation between the transcriptional activity of DJ gene segments and their rearrangement potential.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Germ line expression of TCR miniloci correlates with DJ recombination. Total cellular RNAs were harvested from individual transfectants maintained in the presence (without tTAk) or absence (with tTAk) of tetracycline (TET) for 3 days. The resultant RNAs were subjected to RT-PCR amplification with primers C and D (Fig. 1), and the reaction products were analyzed by Southern blotting using an oligonucleotide probe derived from J1 coding sequences (top panel). The relative positions of amplification products corresponding to germ line transcripts that were processed at either J1 (DJ1Cµ) or J2 (DJ2Cµ) 3' splice sites are shown at the left. Total cDNA levels were controlled in each sample by using a PCR assay specific for -actin transcripts (bottom panel). The linearity of each assay was confirmed with serial dilutions of cDNA (lanes 21 to 26) derived from the 5'D/iE transfectant shown in lane 1.

Transcription and rearrangement of TCR miniloci are independent of methylation status. In addition to transcription and recombination, enhancers direct the active demethylation of linked antigen receptor gene segments (7, 23). Several groups have proposed that demethylation leads to alterations in regional chromatin structure that promote accessibility to nuclear proteins, including RNA polymerase and V(D)J recombinase (26, 27). However, the lack of tractable experimental model systems has hampered previous efforts to establish causal relationships between enhancer activity, demethylation, transcription, and recombination of gene segments.

View larger version (28K): [in this window] [in a new window]   FIG. 5.   Ptet activation targets DJ rearrangement independent of substrate demethylation. (A) Schematic depiction of the DJ regions within the Ptet/iE and Ptet/ substrates. The relative positions of HpaII/MspI (H/M) restriction sites within the parental HindIII fragments are highlighted. The sizes of predicted restriction fragments are shown below each diagram. (B) Methylation status of independent transfectants harboring Ptet/iE (lanes 1 to 5) or Ptet/ (lanes 6 to 10) substrates. Genomic DNA from each clone was digested with HindIII alone (; lanes 1 and 6) or in combination with either HpaII (H; lanes 2, 4, 5, 7, 9, and 10) or MspI (M; lanes 3 and 8). Digested DNAs were analyzed by Southern blotting procedures using a radiolabeled probe spanning the J1/J2 gene segments (Fig. 6A). The relative positions (arrows) and sizes (left) of restriction fragments resulting from digestion at the HindIII sites (H3) or from further digestion by HpaII and MspI (H3 + H/M) are indicated.

Germ line promoter activation is required for generation of DJ signal joins. Emerging studies suggest that in addition to controlling the initial access of DJ gene segments to recombinase, the TCR enhancer (E) may affect the efficiency of coding join formation (16). A potential mechanistic explanation for these findings invokes transcriptional regulatory elements in the recruitment of DNA repair complexes to chromosomal breaks generated by recombinase cleavage. In contrast to coding join formation, DJ signal ends were resolved with similar efficiencies in mice harboring wild-type or E / loci (16). As such, we reasoned that the levels of signal joins generated from each TCR substrate would provide a more direct readout for gene segment accessibility. Moreover, since the DJ intervening sequences are identical in all modified TCR miniloci, the kinetics of signal join formation should be similar regardless of their substrate origin.

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Promoterless and enhancerless substrates do not generate DJ signal joins. (A) Diagram of the PCR assay used for signal join detection. The locations of amplification primers (C and E) as well as the predicted sizes of PCR products from DJ1 and DJ2 rearrangements are indicated. The relative positions of flanking promoter and enhancer elements are shown in the top diagram. (B) Levels of signal joins in TDR19 transfectants harboring the indicated miniloci 48 h after tetracycline withdrawal. The relative positions of amplification products corresponding to DJ1 and DJ2 signal joins are shown at the left. Control assays for recombinase activity (VJ signal joins) are presented in the bottom panel. The linearity of each assay was confirmed by serial dilutions (lanes 15 to 20) of the 5'D/iE sample shown in lane 2.

	DISCUSSION

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Promoter activation targets V(D)J recombination. The tissue, stage, and allele specificity of antigen receptor gene assembly is achieved through programmed alterations in the efficiency of V(D)J recombination at individual gene segment clusters (39). The results presented in this report provide novel insights into the critical role played by transcriptional control elements in targeting recombinase to chromosomal gene segments. Specifically, we show that positive regulation of DJ rearrangement within TCR miniloci can be dissociated from intrinsic properties of enhancer elements (Fig. 2). Instead, efficient recombination of these gene segments requires the presence of a germ line promoter located directly upstream from the DJ cluster (Fig. 3A). In light of these findings, we propose that the observed inhibition of endogenous TCR rearrangement by targeted deletion of E (1, 3) may indirectly result from the strict enhancer dependence of PD activity (37). Indeed, deletion of sequences spanning PD1 specifically impairs D1 rearrangement at the endogenous TCR locus without altering levels of D2J recombination (46). Similarly, removal of a regulatory region located 5' to the J cluster that includes a functional germ line promoter has been shown to preferentially impair rearrangement of proximal J gene segments (45). Taken together, these studies suggest that the primary function of Ig and TCR enhancers for targeting V(D)J recombination is to provide cell type and stage specificity to the activity of individual germ line promoters.

Uncoupling V(D)J recombination from substrate demethylation. Prior studies have established a correlation between the demethylation of antigen receptor loci and their recombination (7, 9, 19, 23). In turn, demethylation of gene segments, as well as their transcription and recombination, have been firmly linked to the activation of enhancer elements in cis. For example, Eµ and iE protect TCR transgenes from de novo methylation in murine lymphocytes (11) and promote demethylation of substrates in cell models (7, 23). Despite extensive correlations, it has been difficult to dissect the individual contributions of these distinct processes to recombinational accessibility. We now demonstrate that a synthetic promoter (Ptet) can efficiently direct germ line transcription and DJ rearrangement but not regional demethylation of TCR miniloci (Fig. 5B). These results indicate that Ptet lacks a hallmark feature associated with enhancers that drive Ig and TCR gene expressionthe ability to direct demethylation of neighboring sequences in a chromosomal context. More importantly, these data indicate that hypomethylation of TCR miniloci is not essential for conferring recombinational accessibility to its composite gene segments. In this regard, prior studies have shown that demethylated IgH gene segments may be refractory to recombinase activity in the absence of germ line transcription (6). Overall, these findings strongly suggest that demethylation is a functional consequence of enhancer activation within a given locus, rather than a prerequisite for antigen receptor gene assembly.

Regulatory studies in recombinase-inducible cell models. The physiological relevance of the TDR19-TCR model system is supported by its ability to recapitulate enhancer- and promoter-dependent recombination of the endogenous TCR locus (1, 3, 46). Although targeted deletion of endogenous regulatory elements can be used to judge their relative contributions to locus rearrangement, the TDR19-TCR system provides several distinct advantages for dissecting the molecular determinants of recombinational accessibility. For example, the framework TCR minilocus, which lacks PD and iE, is devoid of elements that direct either germ line transcription or rearrangement, but the substrate can be manipulated to include a broad panel of regulatory sequences. In contrast, endogenous loci harbor numerous promoter and enhancer elements that may partially overlap in their regulatory functions (1, 16, 35, 46). Furthermore, the TDR19 system permits transcriptional analysis of gene segments at the precise time point that rearrangement occurs (i.e., upon induction of recombinase activity). Analogous studies in murine models are complicated by fluctuations in promoter-enhancer activities that accompany the developmental progression of lymphocyte populations (29). Thus, our ability to directly compare transcription and rearrangement of substrates in TDR19 will be extremely valuable for future studies designed to dissect the molecular mechanisms by which promoter activation targets V(D)J recombination.