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Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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to DJ rearrangement step by the 5' D RS and is enforced at or before the DNA cleavage step of the V(D)J recombination reaction. In this study, the components of the 5' D RS required for enforcing the B12/23 rule are assessed on chromosomal substrates in vivo in the context of normal murine thymocyte development and on extrachromosomal substrates induced to undergo recombination in nonlymphoid cell lines. These analyses reveal that the integrity of the nonamer sequence as well as the highly conserved spacer nucleotides of the 5' D1 RS are critical for enforcing the B12/23 restriction. These findings have important implications for understanding the B12/23 restriction and the manner in which RS synaptic complexes are assembled in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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V, D, and J gene segments are flanked by recombination signal (RS)4 sequences composed of conserved heptamers and nonamers flanking relatively nonconserved 12- or 23-bp spacers (hereafter referred to as 12RSs and 23RSs, respectively) (3). Recombination occurs only between gene segments flanked by RSs of dissimilar spacer length, a restriction known as the 12/23 rule (3). The V(D)J recombination reaction can be generally divided into DNA cleavage and joining steps. The DNA cleavage step is initiated through the formation of a synaptic complex that includes an appropriate RS pair and the recombinase-activating gene (RAG) 1 and 2 proteins (4, 5). In the context of a synaptic complex, the RAG-1/2 proteins introduce DNA double-strand breaks at the RS coding segment border, resulting in the formation of blunt phosphorylated signal ends and hairpin-sealed coding ends. Processing and joining of these DNA ends is mediated by proteins of the nonhomologous end-joining pathway of DNA double-strand break repair (1).
RSs can impose significant constraints on variable region gene assembly beyond enforcing the 12/23 rule. This restriction, termed B12/23, has been defined in the TCR locus (6, 7). TCR variable region genes are assembled from V, D, and J gene segments. The murine TCR locus is composed of 
35 V gene segments and two D-J gene segment clusters, each with a single D gene segment (D1 and D2) and six functional J gene segments (6). V and J gene segments are flanked by 23RSs and 12RSs, respectively. D gene segments are flanked 5' by 12RSs and 3' by 23RSs. Although direct V to J rearrangement is permitted by the 12/23 rule, D gene segments are used in the assembly of essentially all TCR variable region genes (8). This is due to a requirement for the 5'D 12RS to efficiently target rearrangement of V 23RSs beyond simply enforcing the 12/23 rule (6, 7). At a minimum, the B12/23 restriction ensures D gene segment utilization which is important for generating a diverse repertoire of functional TCR chains (8).
The B12/23 restriction is imposed at or before the DNA cleavage step of the V(D)J recombination reaction and does not exhibit an absolute requirement for lymphoid-specific factors other than the RAG-1/2 proteins (9, 10, 11). Furthermore, the D1 12RS rearranges efficiently with V and J
23RSs, demonstrating that D 12RSs enforce the B12/23 restriction in a manner that does not require specific V/D RS synapsis (10). The 5' D 12RSs have heptamers and nonamers that approximate consensus heptamer and nonamer sequences. Notably, a cross-species comparison of D 12RSs spacer sequences reveals a relatively high level of sequence conservation (see below). Finally, the mouse 5' D1 12RS contains a consensus TATA box used by the PD promoter for the initiation of germline (GL) D1-J1 gene segment cluster transcripts (12, 13, 14). Although this consensus TATA box is not conserved in other D 12RSs, it is not known whether these RSs possess other transcript initiation sequences that could contribute to the B12/23 restriction.
In this study, we assess the requirements for different components of the 5' D RS in enforcing the B12/23 restriction. For this purpose, we generate mutant 5' D1 RSs and analyze their function in a TCR minilocus that undergoes D to J and V to DJ rearrangement in developing thymocytes and in extrachromosomal substrates that can recombine in nonlymphoid cell lines. Our findings have important implications for the manner in which rearrangement is regulated within the context of the B12/23 rule and for the assembly of RS synaptic complexes in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation of genomic DNA and Southern blot analysis were conducted as previously described using a 600-bp AccI fragment spanning the V14 gene segment (probe P) (7, 15). Band intensities were determined using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Band intensities were corrected for background and the ratio of VDJ rearrangement as a function of total transgene rearrangement was calculated as VDJ band intensity/(DJ band intensity + VDJ band intensity).
Cell culture
The M12 and ES cell lines were maintained and transfected with the TCRSPF minilocus as previously described (7, 12).
S1 nuclease protection assay
mRNA was isolated using TRIzol (Invitrogen, Carlsbad, CA). For the generation of S1 nuclease protection probes, 406-bp genomic DNA fragments spanning from immediately upstream of the 5' D1 RSs to the BglII site 3' of the D1 gene segment were subcloned into pBSSK. The 471-bp S1 nuclease probes were then generated by PCR using the T7 primer and the ClaIS1 primer: 5'-AGATCGATCTTTTAAAACAAAAC-3'. This results in S1 probes with 65 bp of nonhomologous polylinker DNA followed by 406 bp that are homologous to the 5' D1 RSs and downstream region. Initial hybridizations were conducted with 40 µg whole cell RNA and excess end-labeled probes in 15 µl of 80% Formamide (Fluka, Buchs, Switzerland), 40 mM PIPES (pH 6.4), 400 mM NaCl, and 1 mM EDTA at 51°C for 12 h after incubation for 5 min at 100°C. S1 nuclease digestion was conducted in 300 µl of 37°C for 30 min with 300–400 U of S1 nuclease (Promega, Madison, WI). Samples were size fractionated on 7 M urea/8% polyacrylamide gels.
Transient recombination assays
Transient recombination assays using the pC substrate were performed and analyzed as previously described (10).
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Results |
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SPF:D1 minilocus in thymocytes
The TCRPF minilocus is composed of a single V gene segment (V14), D gene segment (D1), and two J gene segments (J1.1 and J1.2) linked to the IgH intronic enhancer (Eµ) and constant region gene (Cµ; Fig. 1A) (7). Efficient DJ and VDJ rearrangement of this minilocus occurs during thymocyte development in chimeric mice generated by RAG-2-deficient blastocyst complementation (RDBC) using embryonic stem (ES) cells with integrated copies of the TCRPF minilocus (7). Furthermore, as is the case with the endogenous TCR locus, the 5' D1 12RS is required to efficiently target V rearrangement in the minilocus during thymocyte development due to constraints beyond simply enforcing the 12/23 rule (7).
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SPF:D1 minilocus was generated from the TCRPF minilocus by introducing restriction sites (HpaI and NheI) that flank the 5' D 12-RS, allowing for easy replacement of this RS with mutant RSs (Fig. 1A). Thus, the TCRPF and TCRSPF:D1 miniloci are identical except for the introduction of these restriction sites. Chimeric mice were generated by RDBC using four independently derived ES cell lines (TCRSPF:D1–163, 169, 184, and 189) containing two to four stably integrated copies of the TCRSPF:D1 minilocus (Fig. 1B) (7, 16). Rearrangement of the TCRSPF:D1 minilocus was assayed by Southern blotting of BamHI- and BglII-digested thymocyte genomic DNA isolated from chimeric mice with >5 x 107 thymocytes and normal fractions of thymocyte subsets as assessed by flow cytometry (Fig. 1B and data not shown). Robust levels of D to J and V to DJ rearrangement of the TCRSPF:D1 minilocus were observed in thymocytes from chimeric mice generated from all four TCRSPF:D1 minilocus containing ES cells (Fig. 1B).
Bands generated by probe hybridization to miniloci in the unrearranged (GL) DJ and VDJ configurations were quantitated by densitometry and the percentage of rearranged miniloci in the VDJ configuration was calculated as described in Materials and Methods (Table I). Since local integration effects would impact D to J and V to DJ rearrangement, the fraction of the minilocus in the VDJ configuration (VDJ/VDJ + DJ) was used to permit quantitative comparisons of the level of V to DJ rearrangement between thymocytes from chimeric mice generated from ES cell lines with distinct minilocus integrants. The fraction of rearranged TCRSPF:D1 miniloci in the VDJ configuration was similar (37–54%) when comparing seven chimeric mice derived from four independent-derived ES cells (Table I). Together these data demonstrate that during thymocyte development, the TCRSPF:D1 minilocus undergoes consistently efficient V to DJ rearrangement.
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1 12RS nonamer impacts targeting of V rearrangement
In the endogenous TCR locus, replacing the 5' D1 12RS with the J1.2 12RS results in a block in V to D rearrangement (6). The TCRSPF:JH/N minilocus was generated by replacing 5' D1 12RS of the TCRSPF:D1 minilocus with a chimeric RS (JH/N) composed of the J1.2 heptamer/nonamer sequences and the 5' D1 12RS spacer sequence (Figs. 2 and 3A). Thus, the TCRSPF:JH/N and TCRSPF:D1 miniloci are identical except for the altered 5' D1 12RS heptamer/nonamer sequences. Chimeric mice were generated by RDBC from two independently derived ES lines harboring the TCRSPF:JH/N minilocus (TCRSPF:JH/N-26 and -33; Fig. 3A). Southern blot analysis of thymocytes isolated from these mice revealed that the TCRSPF:JH/N minilocus undergoes efficient D to J rearrangement but has a severe block in V to DJ rearrangement (Fig. 3A and Table I).
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1.2 heptamer and 5' D1 spacer and nonamer (JH) or the J1.2 nonamer with the 5' D1 heptamer and spacer (JN) were generated (Figs. 2 and 3, B and C). Whereas the TCRSPF:JH minilocus exhibits a modest reduction (2-fold) in VDJ rearrangement, the TCRSPF:JN minilocus exhibits profound reduction in VDJ rearrangement (Fig. 3C and Table I). Together these findings demonstrate that the 5' D1 12RS nonamer sequence is critical for enforcing the B12/23 restriction.
The B12/23 restriction is dependent on the 5' D1 RS spacer
The JSP 12RS is composed of the 5' D 12RS heptamer and nonamer flanking the J1.2 spacer (Fig. 2). Strikingly, the TCRSPF:JSP minilocus exhibits a reduction in V to DJ rearrangement of similar magnitude to that observed for the TCRSPF:JN minilocus (Fig. 3A and Table I). Unlike the J 12RS spacers, the 5' D 12RS spacers exhibit a high degree of sequence homology across different mammalian species (Fig. 4). Five of the 12 nt are absolutely conserved and 2 additional nucleotides are conserved in all but single RSs (Fig. 4). To test whether these conserved nucleotides are important for enforcing the B12/23 restriction, the CSM 12RS was generated by replacing the seven conserved nucleotides in the 5' D 12-RS with the corresponding bases in the J1.2 spacer resulting in 4 bp changes (Fig. 2). Similar to what was observed for the TCRSPF:JSP minilocus, a severe reduction in V to DJ rearrangement was observed for the TCRSPF:CSM minilocus, highlighting the importance of these conserved spacer nucleotides in enforcing the B12/23 restriction (Table I).
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1 RS TATA box is not required to target V rearrangement
As demonstrated above, targeting of V rearrangement by the 5' D1 12 RS relies on features of the nonamer and spacer sequences. A consensus TATA box, utilized by the PD1 promoter, spans these sequences and is not present in the mutant 12RSs (JSP, CSM, JH/N, and JN) that fail to efficiently target V rearrangement (Fig. 2). Unlike the 5' D1 12RS, the 5' D2 12RS does not contain a consensus TATA box sequence (Fig. 2). To determine whether this RS is capable of efficiently targeting the V rearrangement, the TCRSPF:D2 minilocus was generated in which the 5' D1 12RS was replaced with the 5' D2 12RS (Fig. 2). This minilocus undergoes efficient V to DJ rearrangement, demonstrating that the 5' D2 12RS is capable of efficiently targeting V rearrangement (Fig. 5 and Table I).
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2 12RS may possess cryptic transcript initiation sequences. To investigate this possibility, the TCRSPF:D1 and TCRSPF:D2 miniloci were stably introduced into the M12 cell line and transcript initiation from the PD1 promoter was assayed by S1 nuclease protection (Fig. 6). These analyses revealed a predominant transcript initiating from the TCRSPF:D1 minilocus 20 bp downstream of the 5' D1 RS TATA (+20, Fig. 6). These transcripts are similar to PD1 promoter-specific transcripts previously observed in the endogenous locus during thymocyte development and in the TCR minilocus in the M12 cell line (12, 14). Transcripts that initiate at this position or others downstream of the 5' D2 RS were notably absent in M12 cells harboring the TCRSPF:D2 minilocus (Fig. 6). Together, these findings demonstrate that, unlike the 5' D1 12RS, the 5' D2 12RS contains neither a consensus TATA box nor sequences that mediate transcript initiation immediately downstream of the RS, yet the 5' D2 12RS is capable of efficiently targeting the V rearrangement.
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1 12RS to TGTAAA (Fig. 2). Analysis of transcripts initiating from the TCRSPF:TGT minilocus in M12 cells revealed a complete loss of the TATA-specific +20 transcripts observed in the TCRSPF:D1 minilocus (Fig. 6). However, the TCRSPF:TGT minilocus exhibited only a mild reduction (2-fold) in V to DJ rearrangement in developing thymocytes (Table I). Together, these findings demonstrate that a functional TATA box in, or transcript initiation from, the 5' D 12RSs is not required for enforcement of the B12/23 restriction. The B12/23 nonamer and spacer sequences requirements are enforced on extrachromosomal substrates in nonlymphoid cell lines
The pC competitive extrachromosomal recombination substrate has three positions (P1, P2, and P3) for RSs cloning (Fig. 7) (10). The pC substrate containing appropriate RS combinations can undergo rearrangement in nonlymphoid cell lines that transiently express the RAG-1/2 proteins (10, 17). Rearrangement of the RSs cloned at P1 to RSs cloned at P2 or P3 deletes the transcriptional terminator, permitting expression of the chloramphenicol acetyltranferase (CAT) gene which allows quantitation of rearrangement efficiency after bacterial transformation as previously described (10, 17). Rearrangement of the RS at P1 to the RS at P2 or P3 is assayed by PCR.
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14 23RS at P1 and the 5' D1 12RSS at P2 and P3 (10). As previously demonstrated, an intrinsic bias for rearrangement of the V14 23RS to the 5' D1 12RSs at P2 or P3 is not observed in Chinese hamster ovary cells expressing the RAG-1/2 proteins (Table II) (10). The pC:V14DJH/N substrate was generated from the pC:V14DD substrate by replacing the 5' D1 12RS at P3 with the J H/N 12RSS. This substrate exhibits a profound bias for rearrangement of the V14 23RS to the 5' D1 12RS at P2 over the J H/N 12RS at P3 similar in magnitude to that observed for the pC:V14DJ1.2 substrate which has the J1.2 12RS at P3 (Table II) (10). Analysis of extrachromosomal recombination substrates with the J H (pC:V14DJH) and J N (pC:V14DJN) RSs reveals that replacing the 5' D RS nonamer (J N) has a more profound effect on V RS targeting than replacing the heptamer (J H) which had only a modest effect (Table II). Finally, the pC:V14DDC substrate exhibits severely diminished levels of rearrangement of the V 23RS to the CSM 12RS, as compared with the 5' D 12RS (Table II). Together these findings demonstrate that, similar to what was observed for rearrangement of the minilocus in vivo during thymocyte development, enforcing the B12/23 restriction on extrachromosomal substrates in nonlymphoid cells appears to be critically dependent on the 5' D1 12RS nonamer and spacer.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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variable region genes during lymphocyte development is ordered with D to J rearrangement preceding V to DJ rearrangement. J 12RSs readily rearrange with 3' D 23RSs yet fail to rearrange efficiently with V 23RSs that rearrange efficiently with 5' D 12RSs due to B12/23 constraints imposed on V rearrangement by the 5' D 12RSs (6, 7). In this study, we show that the B12/23 restriction is critically dependent on the nonamer and spacer sequences of the 5' D 12RS. This nonamer and spacer sequence requirement was observed for rearrangement both on chromosomal substrates in the setting of thymocyte development and on extrachromosomal substrates in nonlymphoid cell lines.
The CAC trinucleotide of the consensus heptamer sequence (CACAGTG) is essential for RS function (18, 19). As expected, this CAC trinucleotide is absolutely conserved in the 5' D and J 12RS heptamer sequences. With respect to the four remaining nucleotides, the D 12RS heptamers (CACAATG) differ from consensus at one position whereas the J 12RSs, except for J1.1, differ from consensus at two or three of these positions (20). Deviation of these four nucleotides from consensus can affect the efficiency of recombination of extrachromosomal substrates (18, 19). However, the heptamer sequence differences between the 5' D and J 12RS do not appear to be the critical factor for enforcing the B12/23 rule as evidenced by the modest reduction (2-fold) in V 23RSS rearrangement to the JH 12RSS on chromosomal and extrachromosomal substrates. Thus, features of the 5' D 12RSS heptamer, other than the essential CAC trinucleotide, do not significantly impact the B12/23 restriction imposed on V to D rearrangement.
In striking contrast to the heptamer, replacing the nonamer of the 5' D 12RS with the J1.2 nonamer (JN RS) results in a profound block in V to D rearrangement of the TCR minilocus and on extrachromosomal substrates. Nonamer sequences vary widely with some deviating considerably from consensus (ACAAAAACC). Although there are no strict nonamer sequence requirements, alterations in one or both bases of the nonamer AA dinucleotide (ACAAAAACC) have led to reduced recombination efficiency on extrachromosomal substrates (18). This AA dinucleotide is intact in the 5' D, but not the J1.2, 12RS nonamer. Although this difference may contribute to the B12/23 restriction, it is not likely to be the sole determinant of the nonamer requirement as many other J 12RS nonamers have the AA dinucleotide (20).
The strict requirement for RS spacer nucleotide length is well established (18, 19). Single nucleotide deviations in spacer nucleotide length result in a reduction in rearrangement efficiency, and a gain or loss of two or more nucleotides essentially abolishes RS function (18, 19). Although the importance of spacer nucleotide sequence is less well established, a low level of spacer sequence conservation has been noted upon analysis of a large number of RSs (20). Furthermore, spacer sequence variations have been implicated in affecting the efficiency of RS utilization (21, 22, 23, 24). Perhaps most notably, V RS spacer sequences affect rearrangement efficiency on extrachromosomal substrates with these effects likely impacting differential V gene segment utilization in vivo (22). Analysis of the 5' D 12RS spacer sequences across species reveals a remarkable level of conservation with 5 of the 12 nt absolutely conserved and 7 conserved in all but 2 of the D 12RSs. This high level of conservation is not observed for J 12RS spacer sequences. Strikingly, replacing four of the conserved 5' D 12RS spacer nucleotides with the corresponding J1.2 12RS spacer nucleotides (CSM 12RS) results in a profound reduction in V to D rearrangement both in the minilocus and on extrachromosomal substrates. Thus, the 5' D 12RS spacer nucleotides are critical for enforcing the B12/23 restriction.
It is conceivable that independent mechanistic constraints rely on features of the nonamer and spacer sequences to enforce the B12/23 restriction. It is unlikely, though, that this reflects a requirement for the binding of lymphoid-specific factors to nonamer and/or spacer sequences since these sequences are required to enforce the B12/23 restriction on extrachromosomal substrates in a nonlymphoid cell line. Alternatively, and perhaps more plausibly, a common B12/23 mechanistic constraint relies on features of both the nonamer and spacer sequences. Notably, this is not due to a requirement for transcription initiation sequences spanning the nonamer and spacer such as the TATA box present in the 5' D1 RS. The 5' D2 12RS efficiently targets V rearrangement yet lacks a TATA box or other sequences that serve to initiate transcription. Furthermore, mutation of the TATA box in the 5' D1 RS has only a modest effect on targeting the V rearrangement.
The B12/23 restriction is enforced at or before the DNA cleavage step of the V(D)J recombination reaction and does not absolutely require lymphoid-specific factors (9, 10, 11). Furthermore, specific synapsis between the 5' D 12RS and a V 23RS is also not required (9). Within these contexts how might the 5' D 12RS nonamer and spacer sequences enforce the B12/23 restriction? Synaptic complex formation is initiated by RAG-1/2 binding to an RS. Purified RAG-1 can bind a single RSS in vitro (25, 26, 27). Although RAG-1 makes contacts with RS nonamer and spacer nucleotides, binding appears to rely primarily on the contacts made with the nonamer (25, 26, 28, 29, 30). In the case of 12RSs, the addition of RAG-2 to this complex promotes more extensive contacts with the spacer nucleotides, including those proximal to the heptamer that are highly conserved in the 5' D 12RS spacer (28, 29, 30, 31). The notion that these contacts may be important for RAG-1/2 binding to 12RSs is supported by the partial disruption of this binding upon chemical modification of the heptamer proximal spacer nucleotides (29). Thus, it is possible that the nonamer and spacer sequence requirements for the B12/23 restriction reflect, in part, a requirement for efficient binding of RAG-1/2 to the 5' D 12RS before functional synaptic complex formation with a V 23RS.
Functional synaptic complex formation in vitro occurs most efficiently between a RS bound by RAG-1/2 and an unbound RSS (32, 33). Thus, the 5' D 12RS may serve to "nucleate" the formation of a functional V 23RS/D 12RS synaptic complex in vivo by efficiently binding RAG-1/2 followed by complex formation with an unbound V 23RS. In this regard, the J 12RSs may bind RAG-1/2 much less efficiently and as such be unable to readily nucleate functional synapsis with V 23RSs. The notion that the J 12RSs are generally less efficient at mediating recombination than the D 12RSs is consistent with recent analyses of the functional effect of RS sequence variations (34, 35). However, rearrangement between the 3' D 23RS and J 12RSs occurs efficiently in the endogenous TCR locus and on extrachromosomal recombination substrates (11). In this regard, it is conceivable that the 3' D 23RS functions to nucleate functional synapsis for D to J rearrangement. The notion that the 3' D 23RSs are generally more efficient at mediating recombination than the V 23RSs is consistent with the observation that replacing the V14 23RS with the 3' D 23RS in the endogenous TCR locus results in a dramatic increase in V14 gene segment utilization (36). Thus, it is plausible that the B12/23 restriction may reflect a requirement that, at a minimum, one RS of a recombining pair has the capacity to nucleate functional synaptic complex formation through efficient and independent binding of the RAG-1/2 proteins. In this regard, specific nonamer and spacer sequence combinations may serve as optimal substrates for RAG-1/2 binding. Importantly, this would not preclude additional roles for these sequences in promoting synaptic complex stability once the complex forms (37). The general assembly of functional synaptic complexes in this manner in vivo has potentially important evolutionary implications because it would require that the sequence features permitting efficient nucleation of RAG-1/2 binding be strictly conserved at a single RS of a functional RS pair.
Note added in proof. Since the acceptance of this manuscript, three papers (38, 39, 40) with findings relevant to spacer and nonamer sequence function have been published.
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Footnotes |
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2 M.M.H. and R.E.T. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Barry P. Sleckman, Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8118, St. Louis, MO 63110-1093. E-mail address: Sleckman{at}Immunology.WUSTL.edu
4 Abbreviations used in this paper: RS, recombination signal; RAG, recombinase-activating gene; RDBC, RAG-deficient blastocyst complementation; ES, embryonic cell.
Received for publication August 13, 2003. Accepted for publication October 15, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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locus V, D, and J gene segments. Proc. Natl. Acad. Sci. USA 97:7975. to D rearrangement by RSSs can be mediated by the V(D)J recombinase in the absence of additional lymphoid-specific factors. J. Immunol. 170:5.J gene segments in precursor T lymphocytes. J. Immunol. 161:1399.1 gene segment at the TCR locus by a germline promoter. Immunity 10:313.[Medline]
-chain gene in pro-T cells. Blood 93:3017.
enhancer in and 
T cells. Immunity 7:505.[Medline]
usage in vivo. J. Exp. Med. 187:1495.3) expression correlates with allelic polymorphism in the spacer region of the recombination signal sequence. J. Exp. Med. 179:1707.14 driven by the 3'D1 recombination signal sequence. Immunity 18:75.[Medline]
locus. J. Immunol. 171:3605.Related articles in The JI:
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), the genes encoding resistance to ampicillin (Amp) and chloramphenicol (Cam) and the 1233 and CAT3 primers used for PCR analyses.