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Cutting Edge |
to D Rearrangement by RSSs Can Be Mediated by the V(D)J Recombinase in the Absence of Additional Lymphoid-Specific Factors1
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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variable region genes is ordered during
thymocyte development with D to J rearrangement preceding V to
DJ rearrangement. The 5'D 12-RSS is required to precisely and
efficiently target V rearrangement beyond simply enforcing the 12/23
rule. By prohibiting direct V to J rearrangement, this
restriction ensures D gene segment use in the assembly of
essentially all TCR variable region genes. In this study, we show
that rearrangement of V 23-RSSs is significantly biased to the D
12-RSS over J 12-RSSs on extrachromosomal recombination substrates
in nonlymphoid cells that express the recombinase-activating gene-1/2
proteins. These findings demonstrate that targeting of V to D
rearrangement can be enforced by the V(D)J recombinase in the absence
of lymphoid-specific factors other than the recombinase-activating
gene-1/2 proteins. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Assembly of variable region genes exhibits lineage and developmental stage specificity and, in some loci, is regulated in the context of allelic exclusion (5, 9). In loci containing D segments, gene assembly can be ordered with D to J preceding V to DJ rearrangement. Regulation in these various contexts can be achieved through modulation of V, D, and J gene segment accessibility to the V(D)J recombinase by alterations in transcription, methylation, and chromatin structure of recombining gene segments (5, 9). In addition, regulation can occur at the level of the recombination reaction, for example, through differences in the ability of specific RSSs to serve as substrates for the V(D)J recombinase affected by RSS and/or adjacent coding sequence variations (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21).
TCR variable region genes are assembled during thymocyte development
from V, D, and J gene segments (22). This process
is ordered with D to J rearrangement preceding V to DJ
rearrangement and allelic exclusion enforced at the V to DJ
rearrangement step (22). The V and J gene segments
are flanked by 23- and 12-RSSs, respectively, whereas D gene
segments are flanked 5' and 3' by 12- and 23-RSSs, respectively. Direct
V to J rearrangement, which would satisfy the 12/23 rule, occurs
rarely, if at all, due to a requirement for the 5'D 12-RSS to target
V rearrangement (23, 24). This restriction, which is
enforced at the DNA cleavage step, promotes rearrangement of V gene
segments from up to a megabase away precisely to the 5'D 12-RSS and
not the J 12-RSSs that are <1 kb downstream (25).
Furthermore, appending the 5'D 12-RSS to a J gene segment
efficiently targets V rearrangements specifically to this J
gene segment, suggesting that it can function in a position-independent
fashion (23).
The mechanism by which the 5'D RSS targets V rearrangement is
unknown, but may depend on common features of the V(D)J recombination
reaction. Alternatively, it may rely on the expression of specific
factors that promote use of the 5'D RSS or V/5'D RSS pair by
the V(D)J recombinase during thymocyte development. In this study, we
have assayed recombination between V, D, and J RSSs on
extrachromosomal substrates in nonlymphoid cells. These
extrachromosomal substrates were designed to allow for direct
comparison of the efficiency of recombination between V 23-RSSs and
the 5'D and J 12-RSSs. Our findings suggest that targeting of
V to D rearrangement is due, at least in part, to constraints
imposed by the V(D)J recombinase without a requirement for
lymphoid-specific factors other than the RAG-1/2 proteins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pJH290 was modified by introducing restriction sites that
allowed for cloning of RSSs at three positions, P1
(NcoI/XhoI), P2
(HpaI/NheI), and P3
(XmaI/SacII) (Fig. 1
A) (26). A
300-bp spacer fragment of nonspecific DNA was introduced between the
RSS in P1 and the OOP sequence to increase the distance between
the recombining RSSs. RSSs with four nucleotide-coding flanks were
cloned into the different positions to generate the recombination
substrates described in the text. All substrates used the V14
23-RSS-coding flank (heptamer-TCTG) at P1 and the 5'D1 12-RSS-coding
flank (heptamer-GGGA) at P2. The 5'D1 12-RSS coding flank was also
used at P3 in
pC:J
VV.
In all other substrates the J1.1/J1.2 12-RSS-coding flank
(heptamer-CAAA) was used at P3.
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Transient recombination assays and analysis were performed in the Chinese hamster ovary (CHO) cell line as previously described using 3 µg each of RAG-1 and RAG-2 expression vectors (pJH548 and pJH549, respectively) and 2 µg of the recombination substrate with 6 µl of SuperFect (Qiagen, Valencia, CA) per microgram of DNA (27). Recovered plasmids were analyzed for rearrangement by PCR using the oligonucleotide primers 1233, 5'-AGCGGATAACAATTTCACACAGGA-3' and CAT3, 5'-GGTGGTATATCCAGTGATTTT-3'. PCRs were conducted on colony stabs in 100-µl reactions containing 20 pmols of each primer, 50 mM KCl, 10 mM Tris (pH 8.0), 2 mM MgCl2, dNTPs, and Taq. PCR conditions were 3 min, 93°C for 1 cycle and 30 cycles of 1 min, 93°C; 1 min, 55°C and 1 min, 72°C. PCR products from rearrangements to P2 and P3 were resolved on 1.2% agarose gels.
Ligation-mediated PCR (LMPCR)
Isolated recombination substrates were ligated to the BW linker using previously described conditions (25). Heminested PCRs were performed using the BW-1H, 5'-CCGGGAGATCTGAATTCCAC-3' and 3P1, 5'-GATGAGAGGATCGACGAC-3' in the primary LMPCR and the BW-1H and 3P2, 5'-GACGACATGGCTCGATTG-3' primers in the secondary PCR. Primary and secondary LMPCR conditions were as previously described. LMPCR products were size-fractionated on 1% agarose gels and Southern blotting was performed with the 5P1 oligonucleotide probe, 5'-CTGCAGTCGACTCTCAT-3', as previously described (25).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmid-based extrachromosomal recombination substrates
containing appropriate RSS pairs recombine in nonlymphoid cell lines
upon expression of the RAG-1 and RAG-2 proteins. The prototypical
extrachromosomal recombination substrate, pJH290, contains the J1
23-RSS and the V8 12-RSS pair flanking a prokaryotic transcription
terminator and imparts bacterial resistance to ampicillin
(26). Recombination between the two RSSs results in
deletion of the transcription terminator and expression of the
chloramphenicol acetyl transferase gene allowing for isolation of
recombined plasmids based on their ability to impart bacterial
resistance to ampicillin and chloramphenicol. To generate the
competitive recombination substrate, pC, we modified pJH290 as
described in Materials and Methods such that a single RSS
could be introduced 3' of the transcription terminator (position 1, P1)
and two RSSs 5' of the transcription terminator (positions 2 and 3, P2
and P3) (Fig. 1
A). Thus, RSSs introduced at P1 can rearrange
to RSSs introduced at P2 and P3.
To determine whether rearrangement to P2 and P3 occurred in a
position-independent manner, the
pC:JVV
substrate was generated by introducing the J1 23-RSS at P1 and the
V8 12-RSS at P2 and P3 of pC (Fig. 1, A and
B). The coding nucleotide flanks at P2 and P3 of the
pC:JVV
were held constant as described in Materials and Methods.
The
pC:JVV
substrate was introduced into the nonlymphoid CHO cell line with and
without RAG-1 and RAG-2 expression vectors followed by recovery of
the substrate after 
60 h. Substrates recovered from cells that did
not express RAG-1/2 had not recombined; however, 2% of the
pC:JVV
substrate recovered from the RAG-1/2-expressing cells had recombined as
evidenced by the fraction of ampicillin-resistant colonies that were
also resistant to chloramphenicol (Table I). Analysis of individual
ampicillin/chloramphenicol-resistant colonies by PCR revealed that
essentially equivalent numbers had undergone J1 23-RSS
rearrangements to the V8 12-RSS at either P2 or P3 (Table I and Fig. 1C). Sequence analyses revealed that most isolated
rearrangements were unique (data not shown). Together these data
demonstrate that the pC substrate rearranges efficiently in CHO cells
and without inherent biases for rearrangement to P2 or P3.
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to D rearrangement in nonlymphoid cells
The pC:V14DD substrate was generated by
introducing the V14 23-RSS at P1 and the 5'D1 12-RSS at P2 and P3
of pC. The flanking coding nucleotides at P2 and P3 of
pC:V14DD, and all subsequent substrates,
corresponded to those at 5' D RSS and J1.1 RSS, respectively, as
described in Materials and Methods. Approximately 0.05% of
pC:V14DD recovered from CHO cells expressing
RAG-1 and RAG-2 had recombined with near equivalent use of the 5'D
12-RSSs at P2 and P3 (Table II). The
5'D1 12-RSS at P3 of pC:V14DD was replaced
with the J1.1 12-RSS to generate
pC:V14DJ1.1. The efficiency
of recombination of
pC:V14DJ1.1 in CHO cells
was similar to that of pC:V14DD; however, all
V rearrangements were to the 5'D 12-RSSs at P2 with no detectable
rearrangement to the J1.1 12-RSS at P3 (Table II). Thus, the bias
for V to D rearrangement observed in the endogenous TCR locus
in thymocytes appears to be recapitulated on extrachromosmal
recombination substrates in the CHO cell line.
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23-RSSs and the 5'D 12-RSS
was also observed when the J1.1 12-RSS of
pC:V14DJ1.1 was replaced
with the J1.2 12-RSS
(pC:V14DJ1.2) or when the
V14 23-RSSs was replaced with either the V 2 or 15 23-RSS
(pC:V2DJ1.1 or
pC:V15DJ1.1, respectively)
(Table II). Thus, the bias for V to D rearrangement on
extrachromosomal substrates is not dependent on specific V or J
RSSs. Finally, exchanging the position of the 5'D and J1.1
12-RSSs (pC:V14J1.1D and
pC:V2J1.1D) did not alter
the bias for V 23-RSS to 5'D 12-RSS rearrangement (Table II).
Targeting of V to D rearrangement by the 5'D RSS in the
endogenous TCR locus occurs at the DNA cleavage step of the V(D)J
recombination reaction (25). DNA cleavage intermediates
from recombination on extrachromosomal substrates were assayed by
LMPCR. These analyses revealed similar levels of signal end
intermediates from cleavage at the V RSSs at P2 and P3 of the
pC:JVV
substrate consistent with the equal level of recombination to these two
positions in this substrate (Table I and Fig. 2). As expected, detection of these
signal ends was dependent on the expression of RAG-1/2 and the addition
of ligase (Fig. 2). Analysis of signal end cleavage intermediates from
the RSSs at P2 and P3 of the
pC:V2DJ1.1 and
pC:V2J1.1D substrates
revealed detectable signal ends only from cleavage at the 5'D1
12-RSS and not the J1.1 12-RSS. These findings demonstrate that, as
in the endogenous TCR locus, targeting of V rearrangement by the
5'D RSS on extrachromosomal substrates occurs at the DNA cleavage
step of the V(D)J recombination reaction.
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to D RSS rearrangement in
nonlymphoid cells
Previous analyses have demonstrated that RSSs with consensus
heptamer/nonamer sequences generally function better than those that
deviate from consensus (20). In this regard, the
heptamer/nonamer sequences of the 5'D1 12-RSS are closer to
consensus than those of the J 12-RSSs. The bias for V to D
rearrangement could be due to features of the 5'D1 12-RSS that make
it a better substrate than the J 12-RSSs regardless of its 23-RSS
partner, or to specific constraints imposed by the V/D RSS pair.
To distinguish between these possibilities, we initially generated the
pC:V14DV substrate by
replacing the J1.1 12-RSS of
pC:V14DJ1.1 with the Vk8
12-RSS (Table II). The heptamer/nonamer sequence of the V8 12-RSS is
closer to the consensus than that of the 5'D 12-RSS (Fig. 1B). In CHO cells,
pC:V14DV and
pC:V14DJ1.1 rearrange with
equal efficiency (Table II). However, in striking contrast to
pC:V14DJ1.1, rearrangement
of the V14 23-RSS is no longer specifically targeted to the 5'D
12-RSS on pC:V14DV as
83% of recombined plasmids exhibit V14 23-RSS rearrangement to the
V8 12-RSS (Table II). Similar results were obtained when positions
of the V8 and 5'D 12-RSSs were exchanged
(pC:V14VD) (Table II).
Furthermore, the
pC:JDJ1.1 and
pC:JJ1.1D substrates, in
which the V14 23-RSS was replaced with the J1 23-RSS, exhibit a
bias for rearrangement of the J1 23-RSS to the 5'D over J 1.1
12-RSS (Table II). Together, these findings suggest that the
preferential targeting of V rearrangement to the 5'D over J
RSSs is due to intrinsic properties of these RSSs rather than a
specific requirement for V/D RSS synapsis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene segment rearrangement is
targeted by the 5'D 12-RSS (23, 24). In this study, we
show that on extrachromosomal substrates in the nonlymphoid CHO cell
line, rearrangement of V 23-RSSs is also preferentially targeted to
the 5'D 12-RSS over J 12-RSSs. Similar to what was observed in
the endogenous TCR locus, this constraint is mediated at the DNA
cleavage step of the V(D)J recombination reaction on extrachromosomal
substrates (25). These findings demonstrate that targeting
of V rearrangement by the 5'D RSS may be enforced in part by the
features of the V(D)J recombination reaction that do not require
substrate chromosomal context or lymphoid-specific factors other than
the RAG-1/2 proteins.
The generation of DNA DSBs during V(D)J recombination occurs through
the initial binding of the RAG-1/2 proteins to RSSs followed by
synaptic complex formation and DNA cleavage. The bias for use of the
5'D 12-RSS over J 12-RSSs could be mediated at any, or all, of
these steps. Importantly, our findings demonstrate that this bias is
due to independent features of the 5'D 12-RSS and not to a specific
requirement for synapsis with V 23-RSSs. This is evidenced by the
bias for rearrangement of the J 23-RSS to the 5'D
12-RSS over the J 1.1 12-RSS and the bias for rearrangement of V
23-RSSs to the near consensus V 12-RSS over the 5'D 12-RSS.
The ordered assembly of TCR variable region genes (D to J
preceding V to DJ) may be determined, in part, by processes that
promote accessibility of DJ gene segments before V gene segments
during thymocyte development. Targeting of V gene segment
rearrangement to the D gene segment, instead of otherwise accessible
downstream J gene segments, could then be determined by the more
efficient use of the 5'D 12-RSS, as compared with the J 12-RSSs.
Notably, recent in vitro analyses have demonstrated that synapsis and
cleavage occurs most efficiently through complexing of a RAG-1/2-bound
RSS with an unbound complementary RSS (28, 29).
Furthermore, these studies demonstrated that faithful preservation of
the 12/23 restriction is more readily observed when the RAG-RSS complex
forms initially on a 12-RSS (28). Thus, targeting of V
rearrangement by the 5'D RSS in vivo could occur through the
preferential binding of the RAG-1/2 proteins to the 5'D 12-RSS, over
the J 12-RSSs, followed by synaptic complex formation with a V
23-RSS. However, our findings are not inconsistent with the possibility
that additional factors promote V to D rearrangement.
Specifically, the V/5'D RSS pair catalyzes recombination on
extrachromosomal substrates much less efficiently than the V/J
RSS pair. However, a large fraction of mature T cells have two complete
VDJ rearrangements, suggesting that V to D rearrangement
proceeds efficiently during thymocyte development (B. Khor and B.
P. Sleckman, unpublished observations) (23, 30). Thus,
additional factors may directly or indirectly promote V to D
rearrangement in developing thymocytes.
RSSs can affect the relative use of specific gene segments in the
formation of variable region genes. Strikingly, the differential
ability of the 5'D vs J 12-RSSs to be used by the V(D)J
recombinase would affect the assembly of all TCR variable region
genes. This constraint ensures D gene segment use which may be
important for the generation of a TCR chain repertoire with a normal
CDR3 loop length distribution. In addition, it ensures that
assembly occurs through two rearrangement steps, D to J followed
by V to DJ, thus increasing the potential for variable region
gene diversification due to the imprecise joining process. As such,
this restriction is likely important for the generation of a diverse
repertoire of functional 
TCRs.
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Acknowledgments |
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Footnotes |
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2 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
3 Abbreviations used in this paper: RAG, recombinase-activating gene; DSB, DNA double-strand break; CHO, Chinese hamster ovary; LMPCR, ligation-mediated PCR.
Received for publication September 20, 2002. Accepted for publication October 10, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene A2b, which is associated with increased susceptibility of Navajos to Haemophilus influenzae type b disease. J. Immunol. 161:6068. usage in vivo. J. Exp. Med. 187:1495. light-chain recombination signal sequences mediate recombination more frequently than do those of 
light chain. Proc. Natl. Acad. Sci. USA 88:10721.3) expression correlates with allelic polymorphism in the spacer region of the recombination signal sequence. J. Exp. Med. 179:1707. locus. Curr. Opin. Immunol. 14:230.[Medline]
locus V, D, and J gene segments. Proc. Natl. Acad. Sci. USA 97:7975. locus. Immunity 7:601.[Medline]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. Also shown are
schematics for pC substrates that have undergone P1 to P2 and P1 to P3
rearrangements. The expected size products for PCR of recombined
substrates with the 1223 and CAT3 primers are indicated. The schematics
are not drawn to scale. B, Sequences of RSSs. The
consensus heptamer and nonamer sequences are shown in bold.
C, PCR analysis of seven individual ampicillin- and
chloramphenicol-resistant bacterial colonies generated from
recombination of the
pC:J