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The Journal of Immunology, 2003, 171: 3605-3611.
Copyright © 2003 by The American Association of Immunologists

DNA-Rag Protein Interactions in the Control of Selective D Gene Utilization in the TCR{beta} Locus1

Alexandru Olaru{dagger}, Dimeka N. Patterson{dagger}, Isabelle Villey{dagger} and Ferenc Livák2,*

* Department of Microbiology and Immunology, Graduate Program in Molecular and Cellular Biology, University of Maryland School of Medicine, Baltimore, MD 21201; and {dagger} Developpement Normal et Pathologique du Systeme Immunitaire, Institut National de la Santé et de la Recherche Médicale Unité 429, Hopital Necker Enfants Malades, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ordered assembly of Ag receptor genes by VDJ recombination is a key determinant of successful lymphocyte differentiation and function. Control of gene rearrangement has been traditionally viewed as a result of complex reorganization of the nucleochromatin mediated by several nuclear factors. Selective recombination of the variable (V) genes to the diversity (D), but not joining (J), gene segments within the TCR{beta} locus has been shown to be controlled by recombination signal (RS) sequences that flank the gene segments. Through ex vivo and in vitro recombination assays, we demonstrate that the Rag proteins can discriminate between the RS of the D and J genes and enforce selective D gene incorporation into the TCR{beta} variable domain in the absence of other nuclear factors or chromatin structure. DNA binding studies indicate that discrimination is not simply caused by higher affinity binding of the Rag proteins to the isolated 12RS of the D as opposed to the J genes. Furthermore, we also demonstrate that the 12RS within the TCR{beta} locus is functionally inferior to the consensus 12RS. We propose that selective gene segment usage is controlled at the level of differential assembly and/or stability of synaptic RS complexes, and that evolutionary "deterioration" of the RS motifs may have been important to allow the VDJ recombinase to exert autonomous control over gene segment use during gene rearrangement.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Somatic rearrangement of Ag receptor genes is essential for the development of mature lymphocytes and for the generation of a functional Ag recognition repertoire (1, 2). The assembly of Ag receptor variable (V), diversity (D), and joining (J) genes through VDJ recombination is catalyzed by a relatively simple enzymatic machinery encoded in the two recombination activating genes (RAG-1 and RAG-2)3 (for reviews see Refs. 3 and 4). In contrast, physiological control of VDJ recombination has been proposed to be achieved by a multitude of regulatory mechanisms (for reviews see Refs. 5, 6, 7, 8). However, accumulating data suggest that even complex regulation, such as, for example, the nonrandom use of gene segments during Ag receptor gene rearrangement, could be determined by the Rag proteins (9, 10, 11, 12, 13).

During VDJ recombination the Rag proteins are targeted to the Ag receptor genes through binding to the recombination signal (RS) sequences that flank each gene segment. The RS is composed of highly conserved heptamer and nonamer motifs separated by less conserved spacers of 12 or 23 bp in length (14). According to the 12/23 rule, only gene segments flanked by an RS of different length rearrange efficiently (15). The molecular basis of this restriction has been explained by the identification of a stable precleavage paired synaptic complex that in vitro contains the Rag proteins, a 12RS and a 23RS (16). DNA cleavage by the Rag proteins in vitro or in vivo will occur only if a 12/23RS synaptic complex can be assembled (17, 18). Joining of the gene segments introduces junctional variations through deletion and insertion of nucleotides and is mediated by components of the ubiquitous DNA double-strand break repair apparatus (Ref. 4 and references therein).

Recombination exhibits cell type, locus, and gene segment-specific regulation (reviewed in Refs. 5, 6, 7, 8 and 19). Rearrangement of the TCR{beta} locus, for example, occurs before that of the TCR{alpha} (20) and proceeds in an ordered fashion with D to J gene recombination followed by V to DJ rearrangement (21). Rearrangement of V{beta} genes to either the D or J segments would be permitted by the 12/23 rule; however, in vivo only complete V-D-J{beta} rearrangements have been identified (21). A variety of transcriptional regulatory elements, chromatin reorganization, and nuclear trans-acting factors have been invoked to explain such highly specific control (5, 6, 7, 8). However, recent elegant experiments with transgenic minilocus and knock-in mutants have successfully identified the flanking RS as the major determinants of restricted V-D rearrangement in the murine TCR{beta} locus (11, 22). In addition, while this manuscript was in preparation molecular studies have demonstrated that the Rag proteins are sufficient to reproduce these restrictions in ex vivo transfection and in vitro DNA cleavage assays (23, 24).

In this report we confirm that interactions between the Rag proteins and the RS are sufficient to restrict V to D{beta} recombination. DNA binding assays demonstrate differences in simple Rag protein-single 12RS interactions are not sufficient to explain restricted recombination, indicating that additional steps of the reaction control selective D{beta} gene use. We propose that evolution of the RS has endowed the VDJ recombinase with a previously unsuspected level of sophistication in the regulation of Ag receptor gene segment use.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombination substrates

The basic structure of the competitive recombination substrates has been described (9) and is shown on Fig. 1. Additional substrates were created by replacing the RS of existing constructs with new synthetic oligonucleotides, all containing the RS and 6 nt of the endogenous coding flank. The sequence of the RS and coding flanks used in this study are shown in Table I. All constructs were sequence verified before use. Several constructs were subcloned both into pBKS-based and pCMV-based backbones. The latter, but not the former, plasmids are able to replicate in HEK293T cells because of the expression of the SV40 T Ag. For the in vitro cleavage assays, PvuI-AflIII (from pBKS constructs) or AseI-DraIII (from pCMV constructs) fragments were purified and used.



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  		FIGURE 1. Schematic representation of the recombination substrates. Open triangles indicate 23RS; closed/shaded triangles indicate 12RS. Boxes represent the six base endogenous coding flanks. Possible rearranged coding joint products of the ex vivo transfection assays (shown on Fig. 2) and cleavage products of the in vitro cleavage assays (shown on Fig. 3) are indicated. Arrows and numbers show the position of the PCR primers used in the transfection assays and the expected sizes of the PCR products, respectively. A thick line above the cleavage products marks the position of the hybridization probe. Note that the in vitro cleavage assays do not involve PCR amplification.

 

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  		Table I. DNA sequence of the coding flank and RS of gene segments used in this study

 
Ex vivo VDJ recombination assay

Transient transfections were conducted with 1 µg of substrate and 5 µg of each RAG expression plasmid (25) using the Ca phosphate precipitation method. The transfected DNA was recovered using the alkaline lysis protocol of a commercial plasmid mini prep kit (Invitrogen, San Diego, CA). PCR amplification was performed on one-fiftieth of the transfections for 30 cycles using the same specific primer pair for all reactions. Cycle numbers 27, 30, and 35 were tested and gave identical results on selected samples. PCR were separated on agarose gel electrophoresis, hybridized to a specific radiolabeled probe, and analyzed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Western blot analyses were routinely performed on 5 x 104 cell-equivalent lysates that were separated through 8% SDS-PAGE, blotted onto polyvinylidene difluoride membrane (Millipore, Bedford, MA), and incubated with rabbit anti-RAG-2 (a gift from D. Schatz, Yale University, New Haven, CT) or monoclonal anti-myc-epitope tag (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) Abs.

In vitro VDJ cleavage assay

Maltose binding protein-fused murine, core Rag-1 protein from BL21 bacteria, and GST-fused murine core Rag-2 protein from HEK293T cells were purified as described previously (25). A total of 3–5 ng of purified substrate DNA, 10 nM RAG-1, and 10 nM Rag-2 proteins were incubated in a 10-µl cleavage assay in the presence of Mg2+ and bacterially expressed, purified, His6-tagged high mobility group (HMG)-2 protein (26) at 37°C for 90 min. We did not observe further cleavage beyond 90 min. Deproteinized reactions were separated on agarose gel electrophoresis, hybridized to a specific radiolabeled probe, and analyzed on a PhosphorImager.

DNA binding studies

Synthetic oligonucleotides corresponding to the entire RS, six base coding flank and 10-nt additional sequence on both ends were purified through native PAGE. The top strand was radiolabeled and annealed to the bottom strand. The 2 nM double-strand radiolabeled oligonucleotides (consensus or endogenous) were incubated with approximately 30 nM purified, core Rag-1, 20 nM purified Rag-2 and HMG-2 proteins in the presence of Ca2+ at 30°C for 20 min as described (26), (note that Mg2+ has been replaced with Ca2+). An identical amount of radioactive reactions were separated on native 6% PAGE and analyzed on a PhosphorImager. Rag protein-bound complexes were visualized as shifted bands with distinctly slower mobility than the free probe and not apparent in lanes without the addition of Rag-1 and Rag-2 proteins.

Competitive DNA binding studies

The relative binding affinity of the purified Rag proteins to the isolated RS was estimated from competitive DNA binding assays, similar to published studies (26). Competitive assays were performed by adding 0.5 nM labeled consensus 12RS oligonucleotides mixed with 10- to 5000-fold excess of unlabeled 12RS of consensus or endogenous origin. Binding curves of the ratio of bound fractions in the presence vs absence of competitor DNA were plotted against the concentration of the competitor DNA. The relative binding affinities (Kd dissociation constants) were calculated, and the graphs were created with SigmaPlot 4.14 (Jandel, San Rafael, CA) following the formula: PD+/PD- = (K + Pt)/(K + Pt + Ct), where PD+ and PD- are the fraction of protein bound to DNA with or without competitor, respectively; Pt is the total effective Rag protein concentration; Ct is the concentration of the competitor DNA; and K is the dissociation constant. Once the K value for the consensus 12RS has been determined, it is used to calculate the Kx value of the endogenous 12RS by using the formula: PD+/PD- = Kx(K + Pt)/[(Kx(K + Pt) + Kx(Ct)] (26). It must be noted that the effective protein concentration was not determined experimentally. We conservatively estimated ~10%, 2.5 nM concentration of the added Rag proteins to be active in the aforementioned equations. For this reason, not the absolute, but the relative affinity values are shown in Table II, assuming that the differences in affinity to the various RS are independent of the active protein concentration.


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  		Table II. Comparison of the affinity of the consensus (Con) and endogenous TCR{beta} 12RS to core Rag proteins

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experimental approach

To gain insight into the molecular mechanism of restricted TCR{beta} locus recombination, we have designed ex vivo cell transfection experiments and in vitro DNA cleavage and DNA-binding assays using purified Rag proteins. With the combination of these approaches, we expected to determine the importance of Rag protein-RS interactions, and to define the possible molecular mechanism of these interactions in controlling restricted gene segment usage.

To determine whether lymphoid-specific factors and/or endogenous TCR{beta} locus sequences, other than the RS and the coding flanks, were required, we have adopted an ex vivo recombination assay (14). The nonlymphoid HEK293T cell line was transiently transfected with extrachromosomal recombination substrates that contain only the RS and six base coding flank motifs of the murine TCR{beta} locus (Fig. 1) along with expression constructs of the murine RAG genes. Recombination is detected by PCR amplification with one pair of primers followed by hybridization to a specific probe. Competitive substrates were built such that one 23RS can rearrange to either of two 12RS resulting in two distinct PCR products upon rearrangement. Permutations in the position of the 12RS serve as an internal control for the potential influence of the location of the RS and of the different efficiency of PCR amplification (see Fig. 1). We have used either full-length, wild-type, or truncated core RAG-expression constructs (27, 28). The latter ones are also used to purify Rag proteins (see below). Western blot assays were performed to document consistent transfection efficiency (data not shown).

To study the molecular mechanism of restricted recombination, we have established an entirely in vitro system by using purified core Rag proteins and naked DNA substrates or synthetic oligonucleotides. To validate the use of the in vitro approach, we performed DNA cleavage assays on competitive substrates identical with those used in the transfection experiments. To investigate the molecular interactions between the Rag proteins and the RS, we performed gel EMSAs using synthetic 12RS oligonucleotides.

Restricted V-D{beta} rearrangement can be reproduced in nonlymphoid cells and with purified Rag proteins in vitro

We have been able to reproduce the preferential rearrangement of the V{beta} 23RS to the 12RS of the D instead of the J{beta} gene using either full length or truncated RAG expression constructs (data not shown, and Refs. 23 and 24). Very low-level rearrangement occurs between the V{beta} 23 and the J{beta} 12RS, even if no other 12RS are present in the construct (data not shown). Because preferential recombination of a V{beta} 23RS can be mediated by either the full-length or truncated Rag proteins on extrachromosomal substrates, all subsequent experiments were performed with truncated constructs to allow better comparison to the in vitro assays.

We have analyzed several 23RS (V{beta}8, 11, and 14) in combination with the 12RS of the D1 vs J1.1, J2.5, and J2.7 genes. The analyses with the 12RS of the J{beta}2.5 and 2.7 genes extend the results of the published in vivo (11, 22) and in vitro (23, 24) studies, because those studies tested only the RS of the first J{beta} cluster. J{beta}2.5 and 2.7 are the most frequently used genes in the second cluster (9), and the flanking RS are also among the most efficient J{beta} RS in cell transfection assays (A. Olaru, D. Patterson, H. Cai, and F. Livak, manuscript in preparation). The results demonstrate that the 23RS of all three V{beta} genes preferentially recombine with the 12RS of the D1 instead of the J{beta} genes (Fig. 2, lanes 1–6 and 9–14). Importantly, recombination of the D1 and J1.1 12RS with the 23RS 3' of D1 is comparable (Fig. 2, lanes 7 and 8) in accordance with the in vivo observations of efficient D to J{beta} rearrangements (9) and the documented occurrence of D1 to D2 rearrangements in murine thymocytes (29).



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  		FIGURE 2. Comparison of TCR{beta} RS in the ex vivo transfection assay. PhosphorImager analysis of PCR amplification of recombination between the 12RS of the D1 vs J1.1, J2.5, and J2.7 genes and the 23RS of V8, 11, and 14 genes (lanes 1–6 and 9–12). Lane 7 shows a representative control of similar recombination pattern using two identical 12RS, and lane 8 shows efficient recombination between the 23RS 3' of D1 and the 12RS 5' of either the D1 or J1.1 genes. The expected position for the substrate (Subst) and for the 12B and 12A coding joints are indicated by arrows. Numbers on the right denote m.w. marker in base pairs. All transfections are done with truncated, core RAG-expression constructs. One of two (lanes 9–12) to three (lanes 1–8) representative experiments is shown.

 
The data are shown with substrates that are expected to replicate in HEK293T cells. However, we obtained similar results with some substrates that could not replicate in these cells (see Materials and Methods, data not shown). Therefore, nucleosomal organization, which could form on replicating extrachromosomal plasmids, is not likely to influence the interpretation. The results also indicate that the less conserved portions of the Rag proteins are not required for preferential V-D{beta} recombination (30, 31) and validate the use of purified core Rag proteins in the biochemical experiments described below.

In vitro DNA cleavage experiments were performed with purified, core, murine Rag proteins, HMG protein, and linearized DNA substrates derived from the extrachromosomal plasmids in the presence of Mg2+. The amount of double-strand coordinated cleavage introduced by the Rag proteins is monitored by direct visualization of the cleaved DNA molecules (32). The intensity of the two distinct bands corresponding to the two possible cleaved products is proportional to the extent to which the two 12RS participated in the reaction. In addition, a varying level of single-site cleavage can also be observed (32). Analysis of the V{beta}8, 11, and 14 23RS in combination with the 12RS of the D1 and J1.1 genes shows preferential cleavage at the D instead of the J1.1 RS for all combinations (Fig. 3, A and B). Positional permutations of the 12RS demonstrate that location does not significantly influence the efficiency of cleavage. Cleavage at two identical D1 12RS with the 23RS is similar in efficiency, whereas cleavage at the J1.1 12RS is inefficient under all circumstances (Fig. 3A). In contrast, the 12RS of the J genes can be efficiently cleaved with the 23RS of the D gene (9). Interestingly, a consensus 23RS (33) exhibits the same bias of cleavage at the 5'D1, as opposed to J1.1, 12RS (Fig. 3A, lanes 6–9), in agreement with published results (23) and our own transfection experiments (data not shown). We have found varying levels of single-site cleavage, i.e., double-strand cleavage at one of the 12 (or the 23) RS. These products are not detectable in vivo (34), although they have been observed in cell transfection assays (17). We have only observed single cleavage at the D1 12RS, which normally also supports coordinated cleavage and recombination in these assays (Fig. 3B).



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  		FIGURE 3. Comparison of TCR{beta} RS in the in vitro VDJ cleavage assay. A, Top: schematic representation of the cleavage substrate. Middle: PhosphorImager analysis of in vitro cleavage assays to compare the relative functional capacity of the 12RS of the D1 and J1.1 genes with the 23RS of the V{beta}14 gene (lanes 2–5) or a consensus (Con, lanes 6–9) sequence (33 ). MW denotes a m.w. marker generated by restriction enzyme digestion of a substrate. The positions of the signal end (SE) products of the two 12/23 coordinated (A, B) and the single 23RS cleavage and the substrate are indicated on the left with symbols and on the right with text. The vertical bar on the left shows the area traced for quantitative analysis. Bottom: area quantitation of the cleavage reactions. One of three representative experiments is shown. B, Additional in vitro cleavage assays using the 23RS of the V8, 11, or 14 genes. Symbols are as in A. Note that these substrates and the MW marker on the right were derived from a different plasmid construct. Therefore, the position of the bands is slightly different from the ones shown in A. The positions of the signal ends (SE) of the single 12RS cleavage are also shown. One of two representative experiments is shown.

 
We conclude that neither lymphoid-specific trans-acting factors nor endogenous chromosomal sequences, besides the RS and short coding flanks, are required to reproduce preferential V-D{beta} recombination. In addition, the purified core Rag proteins are sufficient to enforce restricted preferential V to D{beta} recombination in vitro, justifying the following biochemical experiments. These data are also in accordance with recent reports that were published while this manuscript was in preparation (23, 24).

Rag protein-12RS DNA interactions in the TCR{beta} locus

A plausible mechanism of selective V to D{beta} recombination would be the preferential binding of the Rag proteins to the 12RS of the D{beta}, as opposed to the J{beta}, genes. To determine whether this was the case, we performed EMSA analyses with radiolabeled 12RS oligonucleotides, purified Rag, and HMG proteins in the presence of Ca2+. The oligonucleotides are incubated with the proteins and separated by using native PAGE. Bound oligonucleotides appear as slower migrating, shifted bands, specifically only in the presence of both Rag-1 and -2 proteins. As a positive control, a consensus 12RS with consensus heptamer, nonamer, and optimized spacer sequences was used (33).

Binding of the Rag proteins to the isolated 12RS of the D1 and various J{beta} genes appears to be substantially weaker than to the consensus 12RS (Fig. 4). Although binding was reproducibly detected in four to six independent experiments, with at least two different Rag-1 and four different Rag-2 protein preparations, the weak interactions between the endogenous TCR{beta} 12RS and the Rag proteins prevented us from accurate, quantitative comparison of binding affinities. Instead, we estimated the relative binding affinities of these RS in competitive EMSA analyses using the radiolabeled consensus 12RS mixed with unlabeled 12RS of the D and J{beta} genes (Fig. 5A). The ratio of the protein-bound fraction of the labeled consensus 12RS in the presence vs absence of competitor was plotted against the concentration of the competitor DNA. From this analysis it is apparent that the consensus 12RS binds substantially better to the Rag proteins (i.e., its binding curve is shifted to the left) than any of the endogenous TCR{beta} 12RS (Fig. 5B). Importantly, when the estimated Kd values of the endogenous 12RS are compared with the consensus, the best binding affinity was observed for the 12RS of the J1.1 and 2.5 genes, followed by D1, and J.2.2 and 2.7 genes (Table II). Nonetheless, all of these Kd values were 4- to 20-fold lower than that of the consensus 12RS (Table II).



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  		FIGURE 4. Binding of purified Rag proteins to isolated 12RS in vitro. PhosphorImager analysis of EMSAs with radiolabeled oligonucleotides corresponding to the consensus (Con) 12RS and the 5'D1, J1.1, J2.2 and J2.5 12 RS. Purified core (c) Rag and HMG-2 proteins were incubated with labeled oligonucleotides. Unlabeled consensus oligonucleotides were added in 10-fold molar excess as competitor (Compet). Arrows indicate the position of the free probe and the specific shifted single-end complex (SC) between the RS and the Rag proteins. Other visible, but unmarked, bands depend on the presence of the Rag proteins and may represent partial complexes (26 ), but were not confirmed specifically.

 


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  		FIGURE 5. Competitive binding assay of endogenous 12RS. A, PhosphorImager analysis of EMSA with radiolabeled consensus 12RS oligonucleotides in competition (Compet) with unlabeled consensus (Con) 5'D1, J1.1, J2.2, J2.5, and J2.7 12RS. The molar excess of competitor oligonucleotides was 10-, 20-, 200-, 1000-, and 5000-fold (5000 only with endogenous RS). Arrows indicate the position of the free probe and the specific shifted single-end complex (SC) between the RS and the Rag proteins. Lane 1 indicates the maximum shift in the absence of competitor. B, Quantitative analysis of the SC bands shown in A. The relative amount of Rag protein-bound labeled consensus oligonucleotide in the presence vs absence of competitor DNA is plotted against the concentration of the competitor DNA (expressed in nanomoles). The average values of two to three experiments with the SDs are shown. The curves were fitted by using SigmaPlot 4.14. The relative Kd values were calculated as described (see Materials and Methods and Ref. 26 ) and are shown in Table II.

 
We conclude that binding of the Rag proteins to isolated 12RS does not correlate with the preferential use of the D{beta}1 gene during TCR{beta} V to D/J recombination. At least two J{beta} 12RS (J1.1 and 2.5) bind as well or better than the D1 RS to the Rag proteins. These results are inconsistent with the simple explanation that preferential V to D{beta} recombination is the result of significantly superior binding of the Rag protein to the isolated 12RS of the D1 gene. In addition, we also found that all analyzed TCR{beta} 12RS bind substantially less efficiently to the purified Rag proteins than a consensus 12RS, regardless of the origin (D or J gene) of the sequence.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Developmental control of VDJ recombination has long been interpreted as the result of complex interactions between the nuclear chromatin of the endogenous Ag receptor loci and trans-acting, perhaps lymphoid-specific, factors (5, 6). Therefore, it was surprising to learn that simple replacement of RS motifs within the D-J{beta} cluster could determine preferential rearrangement of the V{beta} genes to the D, but not the J, genes (11). In this study we demonstrated that this control can be sufficiently explained by interactions between the VDJ recombinase and the RS/coding flank sequences. We cannot rule out that in vivo, additional factors participate in enforcing selective D gene usage, but our results suggest that the role of these factors, if any, is relatively minor compared with the effect of the Rag proteins. While this manuscript was in preparation, two groups have reported data that support the aforementioned conclusion (23, 24). However, we have provided a series of additional data that go beyond the recently published reports. We have performed most of our transfection experiments using core Rag-expression constructs and formally demonstrate that the less conserved, noncore domains of the Rag proteins (30, 31) are not required for selective V to D{beta} recombination. We have also performed experiments using 12RS from the second J{beta} cluster. This cluster could not be tested in vivo because of the structure of the knock-in model (22), but would be a natural target for V to J recombination in the endogenous locus. Our results show that selective V to D rearrangement also operates within the second J{beta} cluster. Most importantly, we have provided the first direct analysis of binding of the Rag proteins to the 12RS of the endogenous D and J{beta} genes, which are also the first EMSA analyses performed with endogenous RS from any of the TCR loci.

In contrast to the less well-defined mechanism of chromosomal accessibility, biochemical tools have been developed to study how RAG-RS/coding flank interactions may influence gene segment usage during VDJ recombination. Specific changes in four steps of the cleavage reaction may, alone or in combination, explain the preferential recombination between the V{beta} and D{beta} genes. It has been proposed that the Rag proteins bind first to an isolated RS, followed by formation of a stable paired complex between the 12 and 23RS (16, 35, 36). After nicking the top strand of the DNA between the RS and the coding end, the nicked DNA is converted into a double-strand cleavage that generates the hairpin-terminated coding end (32). Although the joining reaction may also be sensitive to particular gene segment combinations (37), this is difficult to assess currently biochemically because of the very low efficiency of the joining step in vitro (38, 39). The simplest explanation for the selective use of the D gene during V{beta} recombination would be to postulate that the Rag proteins bind to the 12RS of the D{beta} genes far more efficiently than to that of the J{beta} genes. This would be consistent with the remarkable evolutionary conservation of the D{beta} 12RS (22). Although it would also imply that the 12RS of the J{beta} genes are inherently weaker, those RS could still effectively mediate D to J{beta} rearrangement, because D to J{beta} and V to DJ{beta} recombination are temporally separated during T cell development (21, 40).

However, our experiments do not support this scenario. Competitive EMSA analyses indicate that the 12RS of the J1.1 and 2.5 genes bind the Rag proteins more efficiently than that of D1. Although in vitro binding assays could be misleading, because of the use of core Rag proteins, lack of influence from the chromatin, etc., the same J1.1 and 2.5 RS could not dictate recombination or cleavage in the context with the V{beta} 23RS in other assays (see Figs. 2 and 3). Therefore, we conclude that differential binding of the Rag proteins to isolated 12RS does not explain selective V to D{beta} recombination.

It will be interesting to determine the efficiency of the additional steps of the cleavage reaction with the natural endogenous 12RS. Because of the weak binding of the Rag proteins to the 12 and 23RS (Fig. 4 and data not shown), we have not been able to demonstrate synaptic complex formation between the V{beta} and D/J{beta} RS, although we could detect a synaptic complex between a consensus 23RS and the D and some of the J{beta} 12RS (data not shown). Nonetheless, selective formation of a synaptic complex between the V and D, but not the V and J RS, is a possible mechanistic explanation for "beyond the 12/23 regulation" (24) of TCR{beta} locus recombination. Efficiency of VDJ recombination is also sensitive to coding flank variations because of an effect on the nicking and double-strand cleavage steps (12, 41, 42). The single-site cleavage seen occasionally at the 12RS of the D, but not J, gene (see Fig. 3B and data not shown) may reflect the negative effect of certain J{beta} coding ends on recombination (24). Both the 12 and 23RS of the D{beta} genes are flanked by identical, highly efficient (43) coding ends in contrast to the more divergent coding ends of the V and J genes. These variations could also play an important role in skewing V gene rearrangement to the D instead of the J genes. In vitro analysis of nicking and paired complex formation, coupled with site directed mutagenesis, should resolve these questions.

In contrast to the recently proposed evolutionary conservation of the Ig VH RS (44), several of the TCR{beta} RS appear to have undergone significant functional "deterioration" as measured in DNA binding (Fig. 4), cleavage (Fig. 3A), transfection (23), and most recently in vivo knock-in experiments (45). Because many of the variations are conserved between mouse and man (46), it is likely that such sequence variations play an important role in establishing the primary Ag receptor repertoire. One such function may be to ensure incorporation of the D gene segment during VDJ recombination. In the IgH locus, this has been achieved by the unique reorganization of the 12 and 23RS, such that the V and J genes can never recombine according to the 12/23 rule (15). In the TCR{beta} locus this control appears to operate beyond the 12/23 rule (22) because of unique sequence variations in the RS. However, it is interesting that the RS 5' of the DH genes have also undergone functional deterioration to prevent 12/23-compatible inversional rearrangements (47, 48). It could be hypothesized that the 23RS of the VH genes has a unique capacity to form synaptic complex with the 5' 12RS of the DH genes, similar to the preferential interactions between the V{beta} 23RS and 5' D{beta} 12RS. What specific features of the 12 and 23RS determine preferential synapsis in either the IgH or TCR{beta} loci remains to be determined. It is interesting that a minimum of 20 different functional V{beta} 23RS, as well as an unrelated "consensus" 23RS, derived from the Ig{kappa} locus, show the same bias in preferential D{beta} use (see Fig. 3A and Ref. 23). It is tempting to speculate that unique characteristics of the evolutionarily highly conserved 3'D1 23RS play a crucial role in enforcing restricted TCR{beta} locus recombination, although this RS clearly does not discriminate between the 5'D1 and the J{beta} 12RS (see Fig. 2 and Ref. 45).

Accumulating evidence suggests that specific RAG-RS/coding flank interactions may explain other examples of nonrandom gene segment usage (9, 10, 12). It appears that sequence variations within the RS establish an additional code beyond simply marking the rearranging gene segments. The evolution of this code is not well understood yet. Because chromosomal accessibility also plays an important role in gene rearrangement (5, 6, 8), it is not surprising that minor nucleotide sequence variations in the promoter elements may have also evolved in a similar fashion to regulate gene segment usage (49). We propose that evolutionary changes, not only in the transcriptional regulatory sequences but also in the RS/coding flank, played a major role in the establishment of the tight and specific control of VDJ recombination.


   Acknowledgments
 
We thank H. Cai for technical assistance, D. G. Schatz for reagents and advice, M. F. Flajnik for comments on the manuscript, D. G. Schatz and B. P. Sleckman for discussions, and especially Karla Rodgers for advice and assistance on the competitive EMSA analyses and Kd value calculations.


   Footnotes
 
1 This work was supported in part by funds from the University of Maryland Bressler Foundation. Back

2 Address correspondence and reprint requests to Dr. Ferenc Livák, Department of Microbiology and Immunology, School of Medicine, University of Maryland, 655 West Baltimore Street, BRB 13-017, Baltimore, MD 21201. E-mail address: Almassy{at}rocketmail.com Back

3 Abbreviations used in this paper: RAG, recombination activating gene; HMG, high mobility group; RS, recombination signal. Back

Received for publication February 5, 2003. Accepted for publication July 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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