From the Centre d'Immunologie de Marseille-Luminy, INSERM, CNRS, Université de la Méditerranée, 13288 Marseille, France
Received for publication, December 11, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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To assess the role of the T cell receptor
(TCR) V(D)J recombination, one example of developmentally
regulated DNA rearrangement known to occur in higher eukaryotes, is
required for T cell receptor
(TCR)1 and Ig gene assembly
and for T and B lymphocyte differentiation. This process is mediated by
an enzymatic complex (the VDJ recombinase) whose targets (the
recombination signal sequences or RSSs) flank dispersed V, D, and J
gene segments and consist of conserved seven- and
nine-nucleotide sequences (the heptamer and nonamer) separated by a non-conserved 12- or 23-nucleotide spacer (1). The
recombination-activating-gene (RAG)-1 and -2 products constitute the
core components of the recombinase (2). The RAG genes
(possibly together with the original RSSs) are thought to have been
transferred, in the form of a composite transposon, from the
prokaryotic world to the germline of a common ancestor of the jawed
vertebrates (3).
V(D)J recombination has been divided into two phases, based on in
vitro recombination studies and the biochemical characterization of rearrangement products (4). In the first phase, the RAG factors
(assisted by architectural factors; i.e. the high mobility group-1 and -2 proteins) initiate recombination by binding to, and introducing DNA cleavage at, two RSSs with spacers of dissimilar lengths. Ensuing DNA double strand breaks (DSBs) yield two pairs of
products that consist of 5'-phosphorylated, blunt-ended RSSs (called
signal ends, SEs), and the hairpinned, adjacent coding sequences
(called coding ends, CEs). In a second phase, which depends on the
coordinated action of the RAG and DNA repair non-homologous end-joining
(NHEJ) factors (including Ku70/86, DNA-PK/Artemis, XRCC4, and
DNA ligase IV), the two CEs are rapidly processed (this involves
opening of the hairpins and, often, deletion and/or addition of
nucleotides) and ligated to form a coding joint (CJ). With slower
kinetics, the SEs are precisely joined to form a signal joint (SJ). As
in the case of various recombination systems in prokaryotes (5),
synaptic complexes of V(D)J recombination have been characterized that
contain all or part of the aforementioned nucleotide sequences and
catalytic factors (reviewed in Ref. 6).
V(D)J recombination is confined to immature lymphocytes because of the
restricted expression of the RAG genes. In addition, it is tightly
controlled with respect to lymphoid cell lineage and within a given
lineage to the developmental stage and possibly also the TCR/Ig allele
used. For example, the TCR In the mouse germline, the ~500-kb TCR Mice--
Single knockout, RAG-1-deficient (Rag), E Thymocyte Preparation and Cell Culture Conditions--
Thymocyte
preparation and cell culture have been described previously (15). 150 µg of anti-CD3- Flow Cytofluorometry Analyses and Cell
Sorting--
Cell-staining conditions, flow cytometric analyses, and
cell purification by cell sorting were carried out as described by Leduc et al. (14).
Molecular Analyses of V(D)J Recombination Products and Chromatin
Structure at the TCR Production of the
We started our evaluation of TCR
The small accumulation of D
Sequence analysis of D Analysis of
Although SEs 3' of D
HJs are non-standard V(D)J rearranged products that result from the
attack of a hairpinned CE by the SE liberated from the opposite gene
segment participating to the recombination complex (schematized in Fig.
3). The reaction, which is mechanistically similar to a transposition,
was partially reproduced in vitro using purified, truncated
forms of RAG proteins (catalytically active core RAGs), in the absence
of the NHEJ factors (23). In vivo, HJs are found at a low
level in wt lymphocytes. In line with the in vitro results,
it is widely accepted that HJs predominate in developing lymphocytes
with NHEJ deficiencies. However, a recent study (24) suggests a more
complex situation, as the non-core regions in full-length RAGs seem to
down-modulate HJ levels in the absence of NHEJ. Intriguingly, HJs
involving CEs 3' of D TCR Developmentally Regulated Activity of the VDJ Recombinase May Be
Compromised at E E
We have tested this model and analyzed chromatin structure at discrete
TCR
Histone acetylation has emerged as an important regulator of chromatin
structure (29) and of locus accessibility for V(D)J recombination
in vivo (30). We used ChIP-PCR to compare histone H3
acetylation at D E
As expected, we detected SEs at V
Processing of V A Critical Function for E A Mechanism of Enhancement of D
We stress that the effect of E Implications for T Cell Differentiation and the Control of Allelic
Exclusion at the TCR
In DP thymocytes, the 3' end of the TCR
Based on in vitro studies, cleaved SEs are thought to form
excellent substrates for RAG-mediated transposition, with the risk to
compromise genomic stability in lymphocytes leading possibly to
translocation and leukemia (26). One would predict that
Our findings indicate that E gene enhancer (E
) in regulating the processing of VDJ
recombinase-generated coding ends, we assayed TCR
rearrangement of
E
-deleted (
E
) thymocytes in which cell death is inhibited via
expression of a Bcl-2 transgene. Compared with
E
,
E
Bcl-2
thymocytes show a small accumulation of TCR
standard recombination
products, including coding ends, that involves the proximal D
-J
and V
14 loci but not the distal 5' V
genes. These effects are
detectable in double negative pro-T cells, predominate in double
positive pre-T cells, and correlate with regional changes in
chromosomal structure during double negative-to-double positive
differentiation. We propose that E
, by driving long range
nucleoprotein interactions and the control of locus expression and
chromatin structure, indirectly contributes to the stabilization of
coding ends within the recombination processing complexes. The results
also illustrate E
-dependent and -independent changes in
chromosomal structure, suggesting distinct modes of regulation of
TCR
allelic exclusion depending on the position within the locus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and TCR
genes (assembled from V
,
D
, and J
and from V
and J
gene segments, respectively) are,
with a few exceptions, rearranged exclusively in the T cell lineage,
with TCR
gene rearrangement in double negative (DN) pro-T cells
preceding that of TCR
in double positive (DP) pre-T cells. Moreover,
at the TCR
locus, D
-to-J
rearrangement occurs first in
CD44+CD25+ DN thymocytes and, presumably,
simultaneously on both alleles, followed by complete V
-to-DJ
assembly in more mature CD44
/loCD25+ cells,
possibly with no allele synchronicity. Formation of a productive
V
-to-DJ
joint (i.e. that maintains an open reading frame within the TCR
gene) and expression of a TCR
chain
(conditional for
lineage normal development past the
CD44
/loCD25+ DN stage) result in the arrest
of further V
rearrangement to mediate TCR
allelic exclusion (7).
To a large extent, these controls are thought to involve the regulated
modulation of RSS accessibility to the recombinase (8). The findings,
using transgenic and knock-out mice, that transcriptional regulatory
elements (enhancers/promoters) modulate cis-rearrangement
and chromatin structure at TCR/Ig gene segments and/or loci gave a
first hint toward an understanding of how recombinational accessibility
is achieved (4, 9).
locus consists of ~35
distinct V
genes that, for the most part, are spread over a large
DNA region extending from 200-450 kb upstream of the duplicated D
1-J
1-C
1/D
2-J
2-C
2 clusters, except for one (V
14),
which lies, in opposite orientation, ~10 kb downstream (10). A single TCR
gene enhancer (E
) has been described that is located within the C
2-V
14 intervening region (11). Targeted deletion of E
has
revealed a striking phenotype. In the T cell lineage, D
-to-J
CJs
are drastically reduced at the targeted TCR
allele(s)
(>50-100-fold compared with the wild-type (wt)), with an even more
severe defect in V
-to-DJ
CJs. In homozygously deleted
(E
/
, hereafter
E
) mice, no TCR
chains are
made, and no
T cells can develop (12-14). Moreover, comparative
analysis of molecular markers for chromatin structure in
developmentally arrested DN, CD25+ pro-T cells from either
Rag
/
(hereafter Rag) or combinatorial
(Rag
/
×
E
; Rag
E
) mice provided compelling
evidence for a primary function of E
in regulating chromatin opening
within a limited (~25 kb) upstream domain comprised of the
D
-J
-C
clusters, with a minor effect on the 5' distal V
genes or 3' proximal V
14 (15). However, RAG-mediated SEs at D
and
J
gene segments (as well as the corresponding SJs) can readily be
detected at E
-deleted alleles, although at a level 10-30-fold lower
compared with the wt (16). The facts that TCR
rearrangement was
initiated in
E
thymocytes, but that formation of CJs may be more
severely impaired, suggested an additional function for E
in CE
processing. Here, using
E
mice expressing an anti-apoptotic
Bcl-2 transgene, we attempt to better delineate the actual
impact of E
in enhancing DNA repair/CJ formation during TCR
locus
recombination, relative to its effects on chromosomal accessibility.
Our findings are consistent with a model in which E
impinges on the
stabilization of CEs within the post-cleavage synaptic complex, in
addition to its primary functions in regulating chromosomal access and locus expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-deleted
(
E
) mice, and double knockout (Rag
E
) mice, as well as
mouse housing and analyzing conditions, were as described previously
(16). wt C57BL/6J and CB17 SCID (Scid) mice, transgenic
Eµ-Bcl-2 (B2) and P14 TCR
/V
8.1-DJ
2.4 (p14) mice (17, 18),
and combinatorial knock-out and transgenic (
E
B2) mice were
handled similarly.
(2C11; Pharmingen) monoclonal antibody were
utilized for intraperitoneal injection of 4-week-old animals.
Locus--
Nucleic acid extraction, assays for
SE, SJ, CJ, and hybrid joints (HJ) products, sequencing, and RT-PCR
analyses, as well as ligation-mediated (LM)-PCR analysis of restriction
enzyme accessibility and chromatin immunoprecipitation (ChIP)-PCR
analysis of histone H3 acetylation were performed as described
previously (13, 15, 16). Assays for CE products were performed
according to Zhu et al. (19), with PCR products for CEs/SEs
being separated through polyacrylamide gels (instead of agarose gels,
as for the analysis of the amplified products in all other PCR assays).
All PCR experiments were performed at least twice with consistent
results. A list of oligonucleotide primers used in these experiments is
available upon request.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E
B2 Mice and Characterization of Their
TCR
Gene Recombination Profile--
A defect in resolving
RAG-mediated DSBs that form at E
-deleted (E
)
alleles must result in cell death of the particular thymocytes. This
could mask the actual levels of TCR
recombination products (e.g. SEs) in
E
thymi and the role played by E
in
promoting RSS accessibility versus post-cleavage assembly
in vivo. To overcome this problem, we analyzed TCR
gene
recombination at E
alleles in the situation where cell
death is inhibited;
E
mice were bred with mice that express an
anti-apoptotic human Bcl-2 transgene (Tg Bcl-2)
in T lineage cells (B2 mice) (17). Cell counting and flow cytometric
analysis indicated that constitutive Bcl-2 expression results in a
slightly reduced proportion of DN cells in
E
B2 versus
E
thymi (from ~33 to 25.6%) and an increase in that of DP
cells (from ~58 to 69%) although TCR
+ and genuine
single positive cells are still missing (Table
I). Moreover, Bcl-2 expression was found
to prolong cell survival of E
-deleted DN and DP thymocytes without
rescuing the CD44
/loCD25+ DN developmental
block and accompanying cell proliferation defect (data not shown).
These findings are in agreement with those from earlier studies
demonstrating that Tg Bcl-2 expression inhibits cell death
without substituting for major selection processes in the developing T
cells such as pre-TCR-based
-selection or TCR
-based positive
selection (17, 20-22).
Absolute numbers and percentages of thymocytes in wt, E
, and
E
B2 mice
E
and
E
B2 single positive (SP)
subsets were mostly of low to intermediate intensities, as opposed to
those within the wt SP subsets that demonstrated mostly CD4high
and CD8high cells. Three color flow cytometric analysis further
demonstrated that CD4+ CD8
and CD4
CD8+ cells within the
E
B2 thymus do not express
cell-surface TCR
indicating that genuine SP thymocytes are still
missing in this strain.
gene rearrangement in Tg
Bcl-2-expressing thymocytes using well defined,
semi-quantitative PCR assays to test for SE, SJ, and CJ products at the
D
2-J
2 cluster (Fig. 1A).
In agreement with earlier findings (16), both 3' D
2 SEs and
D
2-to-J
2.6 SJs were detected in
E
thymocytes, at decreased
levels compared with the wt (Fig. 1, B (lanes
1-5) and C, lanes 1-12), whereas
D
2-to-J
2.1/J
2.6 CJs appeared to be more severely reduced (Fig.
1D, lanes 1-5). According to PhosphorImager scanning and densitometric analysis (Fig. 1E), SEs and SJs
without E
were reduced to ~16-20% of those in wt thymi whereas
CJs were reduced to ~7%, arguing that E
deletion indeed impacts
on D
/J
RSS cleavage with an additional, weaker effect on CE
resolution. Significantly, all the three types of recombination
products were amplified at increased levels in
E
B2 compared with
E
thymocytes, including D
-to-J
CJs (Fig. 1, B,
C, and D, lanes 5-7,
9-20, and 5-7,
respectively). By densitometric comparison of
E
and
E
B2
thymocytes (Fig. 1E), we estimated that SEs and SJs
increased from, respectively, ~16 to 29% (~1.8×) and ~20 to
26% (~1.3×) relative to those in wt cells, whereas CJs showed a
greater rescue, from 7 to 30% (~4.3×). Overall, the effect of Bcl-2
in rescuing D
-to-J
CJs may thus result from a small accumulation
of intermediate SE/CE products combined to a specific, additional
enhancement of CE processing (formally, because the CJ effect should be
the product of SE/CE formation (or abundance) and CE resolution, the apparent effect on CE resolution in this case could be ~2.4×
(4.3/1.8) only). Consistent with this, the germline fragment containing D
2/J
2 gene segments was detected at high levels in both
E
and
E
B2 but not wt thymocytes (Fig. 1D). Also, as
judged from similar SE and CJ assays, recombinase activity in
E
B2 cells generally appears less marked within the D
1-J
1 cluster
compared with D
2-J
2 (data not shown).
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Fig. 1.
D 2-to-J
2 SE,
SJ, and CJ products in
E
B2 thymocytes. A, schematic representation of the
PCR assays used to analyze SE, SJ, and CJ products within the
D
2-J
2 gene cluster. RSSs of 23- or 12-nucleotide spacer are
figured by shaded and open triangles,
respectively. Oligonucleotide primers and the linker used in LM-PCR are
schematized by horizontal arrows and asymmetric pairs
of bold lines. B, genomic DNA from thymocytes of the
indicated mice (including two
E
B2 individuals) was analyzed by
LM-PCR for SEs 3' of D
2 (D
SE). Lanes 1-4,
serial dilution analysis using linker-ligated DNA from a wt thymus
and/or kidney (lane 1, undiluted thymus; lanes 2 and 3, thymus/kidney, 1/5 and 1/25 dilutions; lane
4, undiluted kidney). PCR amplifications for a C
2-containing
DNA fragment (C
) were carried out in parallel to control
for sample loading. C, DNA samples were analyzed by PCR for
D
2-to-J
2.1 SJs (DJ
SJ). Digestion of amplified DNAs
by the restriction enzyme ApaLI (arrows) was
performed to check the accuracy of RSS ligation to form the SJs.
D, PCR analysis of D
2-to-J
2.1/2.6 CJs (DJ
CJ; top panels) and control C
2 amplifications
(C
; bottom panels). Gl,
D
2/J
2-containing germline fragment. The asterisk
indicates a nonspecific band that was also amplified from kidney DNA.
Serial dilution was as in B except that genomic
(non-ligated) DNAs were used; dilution in lanes 2 and
3 was 1/4 and 1/8, respectively. E, quantitation
of SE, SJ, and CJ products. PhosphorImager signals for the
recombination products were quantified by densitometric scanning and
corrected according to the signal from the C
control. Graphic
representation for each product is shown, relative to a 100% value as
defined from the corresponding signals in wt thymocytes.
2-to-J
2 recombination products in
E
B2 thymocytes prompted us to also check for the presence of
V
-to-DJ
CJs. Surprisingly, V
14-to-DJ
2 CJs were found in thymocytes from the
B2 mice (at levels varying from ~12 to 48% of those in the wt) whereas these products were routinely not
detected in cells from
E
littermates (e.g. Fig.
2). Analysis of SEs at V
14 once again
argued for a specific effect of Bcl-2 expression on the rescue of CEs
compared with SEs (see below). However, V
-to-DJ
2 assembly in
E
B2 thymocytes seems to be restricted to V
14 only
(i.e. the single V
gene located on the 3' end of the
TCR
locus), as no accumulation of CJs was detected for several 5'
V
s, including members located in the proximal (V
18), median
(V
20, V
11, and V
5), or distal (V
4) parts of the 5' V
gene cluster (Fig. 2) (data not shown).
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Fig. 2.
V 14-to-DJ
2 CJ
products in
E
B2
thymocytes. Long range PCR analyses of V
14- and
V
5-to-(D)J
2.1/2.6 CJs (V
14 CJ and V
5
CJ, respectively) in the indicated thymocytes are shown as
outlined in the legend to Fig. 1. Quantitation of V
14 CJs in
E
B2 thymocytes was performed by densitometric scanning of
PhosphorImager signals from each amplified fragment, yielding 48%
(lane 7) and 17% (lane 8) of those in wt
thymocytes. The preferential amplifications of low sized
V
14DJ
2.5/2.6-containing fragments in
E
B2 compared with
control thymocytes underlies the lower level of V
14 CJs in the
former cells.
2-to-J
2.5/2.6 CJs from
E
B2
thymocytes generally showed the hallmarks of normal CE processing prior to joining, including occasional P and/or N nucleotide additions and
short deletions; one D
2-to-J
2.5 CJ showed an unusually long (11-bp) N region (data not shown) (also, in Fig. 1C, SE
resolution in
E
B2 T cells appears to mostly result in standard
SJs as they were digested by restriction enzyme ApaLI that
cleaves the perfect fusion of two heptamers). Parallel analysis of
V
14-to-DJ
2 rearrangement similarly revealed canonical CJ/SJ
features and, as expected, joining by DNA inversion.
E
B2 Thymocytes for CE and HJ Products--
The
above data indicate that constitutive Bcl-2 expression effects a small
accumulation of TCR
SE, SJ, and CJ products in E
-deleted
thymocytes that is confined to gene segments located proximal to the
E
deletion, including the D
-J
upstream segments and downstream
V
14 gene. We next investigated whether this effect also impacts on
the accumulation of other forms of V(D)J recombination products within
this domain, namely CEs and HJ products.
gene segments can be found in thymocytes from
the
E
mice (e.g. Fig. 1), the corresponding CEs could not be detected by LM-PCR of genomic DNA treated with mung bean nuclease (to open the hairpin structures) and T4 DNA polymerase (to
blunt occasional DNA overhang
extremities)2 (for details on
this strategy, see Ref. 19). However, CEs are difficult to detect, even
in wt thymocytes, because of their rapid processing and resolution into
CJs. Conversely, CEs readily accumulate in developmentally arrested
lymphocytes from NHEJ-deficient mice such as the Scid
(DNA-PK-deficient) mice (19). Using the aforementioned strategy, CEs 3'
of D
2 were indeed observed in thymocytes from a Scid mouse (together
with SEs 5' of D
2), but not in those from a
E
B2 mouse (Fig.
3, upper panels; in
E
B2
DNA, the faint signal at a size close to that of CEs was not
reproducibly observed), arguing that there is no accumulation of CEs at
E
alleles, even under the condition of constitutive
Bcl-2 expression.
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Fig. 3.
Analysis of CE and HJ products in
E
B2 thymocytes.
Thymocyte DNA was analyzed by LM-PCR for the presence of 3' D
2 CEs
(upper panels: M, 190-bp size marker) and by PCR
for the presence of D
2-to-J
2.6 HJs (bottom panels);
DNA from Rag
/
(Rag) and Scid or wt mice were
used as negative and positive controls, respectively. The diagram
schematizes the two assays and the region of the TCR
locus that was
analyzed (outlined as in Fig. 1A), emphasizing the
D
2/J
2 cleavage/recombination sites that were investigated. The
vertical arrow indicates the position of 5' D
2 SEs that,
in the LM-PCR assay, can be amplified simultaneously with 3' D
2 CEs.
Horizontal arrows indicate the relative location of
oligonucleotide primers used for PCR amplifications. The size of the
amplified fragments is indicated.
2 and J
2 SEs could not be detected in
E
or
E
B2 thymocytes (Fig. 3, bottom panels).
Thus, Tg Bcl-2 expression in E
thymocytes
results in an accumulation of SE/SJ/CJ products of D
-to-J
rearrangement, but it has a negligible impact on parallel accumulation
of CEs and HJs. Overall, these data do not support a role for E
in
the recruitment of NHEJ factors to the recombination complex (see
"Discussion"). However, they do not exclude an indirect effect of
E
on CJ formation; e.g. the stabilization of CE
intermediates within the PCS complex.
Gene Expression in E
-Deleted Thymocytes--
At
TCR/Ig loci, activation of regional transcription and V(D)J
recombination frequently (but not always) correlate (4). Earlier
studies have shown that transcription of the unrearranged (germline) D
-J
loci is strongly inhibited at E
compared with E
+ alleles in early developing T cells,
whereas that of V
genes is not significantly altered (15). We have
analyzed TCR
gene expression in
E
B2 versus
E
thymocytes, using RT-PCR assays to study transcription through either
germline J
or V
gene segments (J
Gl or V
Gl) or through
partially (DJ
) or completely (V
DJ
) rearranged products
(DJ
Rg or V
Rg). We found no J
Gl transcription in
E
B2 thymocytes at the D
1-J
1 or D
2-J
2 loci; also,
DJ
Rg transcription was negative in these cells (Fig.
4A, upper two panels) (data not shown). Therefore, despite the evidence of
D
-to-J
recombination at E
alleles, there is
no evidence of transcription through these loci (including in the Tg
Bcl-2 expressing cells). This is yet another example of
differential activation of the two processes. In addition, we found
V
Gl transcription for V
5, V
11, and V
14 at
E
alleles, but no V
Rg transcription, including for
V
14 and the
E
B2 thymocytes (Fig. 4A) (data not
shown), in agreement with the lack of TCR
+ cells (and
-selection) in the
E
/
E
B2 mice, as evidenced by flow
cytometry. Whereas V
Gl transcription of V
5 (and V
11) appeared
to be reduced in
E
B2 compared with
E
thymocytes, that of
V
14 was unchanged (or increased slightly; e.g. see Fig. 4A). These differential profiles likely result from a
reduced DN/DP cell ratio in
E
B2 versus
E
thymi,
coupled with E
-independent developmental changes in V
Gl
transcription (rather than from intrinsic differences between the two
types of
E
B2 and
E
cells). Indeed, we found a dramatic
down-regulation of V
5 and V
11 Gl transcripts in purified DP
versus DN
E
B2 thymocytes and steady-state (or
slightly increased) levels of V
14 Gl transcripts (Fig.
4B) (data not shown). Therefore, V
Gl transcription
profiles in DP
E
B2 cells (5' V
Gl
/V
14
Gl+) correlate with those of V
-to-DJ
CJs in
E
B2 thymi. This lead us to investigate whether, in this situation,
TCR
recombination also depends on DN-to-DP development and,
potentially, E
-independent changes in chromosomal access.
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Fig. 4.
TCR gene expression
in E
-deleted thymocytes. A,
transcripts initiating upstream of the unrearranged J
2 gene segment
(J
Gl), rearranged D
2-J
2 CJs (DJ
Rg),
and unrearranged or V
-to-(D)J
rearranged V
5 and V
14 genes
(V
Gl or V
Rg) in thymocytes from the
indicated mice were analyzed by RT-PCR. Thymocyte (T) and
kidney (K) RNA from a Rag mouse and thymocyte RNA from a wt
mouse were used as controls. RT-PCR products for
-actin are shown.
Note the differential J
Gl versus DJ
Rg expression
profiles in Rag but not wt or B2 thymocytes, confirming the nature of
the analyzed transcripts. B, same as in A,
exemplifying V
5 Gl (but not V
14 Gl) drop in expression in DP
versus DN
E
B2 thymocytes.
-Deleted Alleles--
To investigate whether the
predominance of TCR
SE, SJ, and CJ products in
E
B2
versus
E
thymi also correlates with their differences
in cell distribution, we purified DN and DP thymocytes from both types
of mice and analyzed the rearrangement of their TCR
locus, as
described above. We first focused on D
2-to-J
2 rearrangement.
Remarkably, we found SEs 3' of D
2 and D
2-to-J
2-6 SJs
predominantly in DP thymocytes from both
E
and
E
B2 mice and at higher levels in Tg Bcl-2 expressing cells; however,
the effect of Bcl-2 on the accumulation of rearrangement products was
also visible in DN pro-T cells (Fig. 5,
A (lanes 3-6) and B (lanes
5-12)). Likewise, in E
-deleted animals, we detected D
2-to-J
2 CJs predominantly in
E
B2 DP thymocytes; Bcl-2
also had an effect on CJ accumulation in DN cells (Fig. 5C).
Notably, CJs in
E
B2 DN cells were detected at a slightly higher
level (~1.2×) compared with those in
E
DP cells despite a bias
for SEs (~2×) in favor of the latter (Fig. 5, C and
A, lanes 4 and 5, respectively) (data
not shown). Further quantitation analysis (Fig. 5D)
indicated that SEs without E
are reduced to ~7% (DN) and ~26%
(DP) of those in the corresponding wt cells, whereas CJs are reduced to
~4% (DN) and ~10% (DP). By comparison, in
E
B2 thymocytes,
SEs increased slightly to, respectively, ~12% (DN; ~1.7×) and
~42% (DP; ~1.6×) whereas CJs increased to 24% (DN; ~6×) and
~36% (DP; ~3.6×). These data confirm our previous results of a
preferential, although limited effect of Bcl-2 on D
/J
CE resolution at E
alleles, which is apparent in both DN
(~3.5× (6/1.7)) and DP (~2.2× (3.6/1.6)) cells. They further
suggest that, in most E
-deleted thymocytes, recombinase activity at
the D
/J
RSSs is extended/delayed to cells that have developed to
the DP stage. Significantly, we also found high levels of SEs 3' of
D
2 and D
2-to-J
2-6 SJs in both DN and DP wt thymocytes (Fig.
5, A (lanes 1 and 2) and B (lanes 1-4)), in agreement with similar findings at the
D
1-J
1 gene cluster (25). Because unresolved DSBs generated in DN
cells would arrest cell proliferation during DN-to-DP cell
differentiation, the detection of SEs 3' of D
in DP cells from wt
mice must reflect cell autonomous activity of the RAG factors at
unrearranged D
/J
loci (25) and/or at D
-to-J
SJs within
extrachromosomal circles (26).
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Fig. 5.
D 2-to-J
2 SE,
SJ, and CJ products in E
-deleted DN and DP
thymocytes. A-C, genomic DNA was prepared from
sorted CD4
CD8
(DN) and
CD4+CD8+ (DP) thymocytes in the
indicated mouse lines. PCR assays for the presence of SEs 3' of D
2
(A), D
2-J
2.6 SJs (B), or D
2-J
2.1/6
CJs (C), as well as quantitations of SE and CJ products
(D) are shown as outlined in the legend to Fig. 1. Sample
loadings for experiments in A and B were
identical.
-Independent Changes in Chromatin Structure at the TCR
Locus
during T Cell Development--
The extended lifespan conferred by
a Bcl-2 transgene is likely to provide an extended time
window per cell for V(D)J recombination (27), thus accounting for one
aspect of our results (i.e. the rescue of CJ formation; see
below). However, other mechanisms must account for the accumulation of
SEs on a large scale in DP cells (including DP cells that develop in
the absence of
-selection) (14) and for the profile of V
gene
recombination in
E
B2 thymocytes. One possibility would be that,
upon DP development, accessibility to the RAG factors is established
along the D
/J
loci in an E
-independent manner, whereas a
repressive structure invades the 5' V
genes but not V
14. This
would impact on the controls of both TCR
gene recombination and
allelic exclusion during T cell differentiation.
regions using nuclei from Rag and Rag
E
thymocytes (E
+ and E
DN cells, respectively) and
enzyme restriction/LM-PCR chromosomal accessibility assays, as
described previously (15). Thymocytes from Rag and Rag
E
mice
treated by intraperitoneal injection of anti-CD3-
monoclonal
antibody (to mimic pre-TCR signaling) (28) were used as a source of
DP-enriched nuclei. Fibroblasts were used as a non-lymphoid control. As
predicted, we found that the J
1 region is more likely to be cleaved
in E
DP (Rag
E
CD3) and in E
DN
(Rag) or DP (Rag CD3) thymic nuclei, compared with E
DN (Rag
E
) thymic or to fibroblastic nuclei (Fig.
6A, top panels; consistent results were also obtained at the J
2 locus). Furthermore, we found V
5 to be more resistant to cleavage in both
E
+ and E
DP (Rag CD3 and Rag
E
CD3) nuclei relative to E
+ or E
DN (Rag
and Rag
E
) nuclei (Fig. 6A, middle upper
panels; similar results were found at V
11) (data not shown).
Finally, we found V
14 to be cleaved in T cell nuclei, independent of
the developmental (DN or DP) stage and the presence of E
(Fig.
6A, middle lower panels).
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Fig. 6.
E -independent
changes in TCR
chromatin structure in DP
versus DN thymocytes. A, thymocyte nuclei from
Rag
/
(Rag) and Rag
/
E
/
(Rag
E
) mice and from
littermates that have been injected with anti-CD3-
monoclonal
antibody (Rag CD3 and Rag
E
CD3) or nuclei
from 3T3 fibroblasts (fibro.) were treated with increasing
amounts of the restriction enzyme RsaI and analyzed by
LM-PCR for enzyme cleavage in various parts of the TCR
locus, as
described previously (15). The bottom panels show C
2 PCR
amplifications (C
) using RsaI-restricted,
linker-ligated DNA samples. The J
1 cleavage profile, with three
visible bands (instead of the two in Ref. 15), is because of a longer
gel migration and better separation of the RsaI restricted
fragments. The results shown are representative of three independent
experiments. B, histone H3 acetylation at J
1-, D
1-,
D
2-, V
5-, and V
14-associated sequences was investigated in Rag
E
and Rag
E
CD3 thymocytes by ChIP, in parallel with that
at the hyperacetylated TCR
gene enhancer (E
) and the
hypoacetylated Oct-2 (Oct) gene. ChIP was performed using
either an anti-AcH3 anti-serum (
AcH3) or no antibody
(control). Two concentrations (dilution 1/0 and 1/3) are
shown for amplification of the
AcH3-precipitated fractions and input
materials. The graphics represent the
AcH3-precipitated/input
material ratios, determined after PhosphorImager scanning of the
hybridizing images. The 100% value was attributed to the acetylated
E
control.
-J
and V
loci in Rag
E
CD3 (DP)
versus Rag
E
(DN) thymocytes. We found that, during
the course of anti-CD3-
-induced DN-to-DP differentiation, H3
acetylation of E
thymocytes (i) increases slightly at
J
1 and D
1 and, more readily, at D
2; (ii) decreases (by
~2-fold) at V
5; and (iii) is maintained at a steady state level at
V
14 (Fig. 6B). Overall, these data support our model of
E
-independent, DN-to-DP regulated changes in chromosomal
organization (including histone H3 acetylation) at distinct regions
throughout the TCR
locus.
-Independent DSB Cleavage at V
14 in DP Thymocytes--
The
drop in accessibility of 5' V
genes in CD3-
triggered Rag
thymocytes likely mimics a physiological mechanism involved in the
feedback inhibition of V
gene rearrangement in response to
pre-TCR-induced signaling (i.e. allelic exclusion). In this context, persistent accessibility of the D
-J
and V
14 locus regions potentially threatens allelic exclusion so that this process must be regulated differently at the 3' end of the TCR
locus. The
finding of V
14-to-DJ
CJs in
E
B2 thymocytes also raises the
question as to whether E
, in conjunction with cell death control,
could participate in this regulation. To address these issues, we
tested E
+ and E
thymocytes for the
presence of SEs at both V
5 and V
14 and of V
5-/V
14-to-DJ
2
CJs. As a source of E
+ or E
thymocytes,
we used wt mice and TCR
transgenic mice (p14, a model for TCR
allelic exclusion (18)) or the
E
and
E
B2 mice, respectively.
5 predominantly in DN thymocytes
from the E
+ animals and at a reduced level in p14
compared with wt cells (a >6-fold decrease as judged from
densitometric analysis of PhosphorImager signals) whereas, in agreement
with previous findings, these products were hardly visible in
E
(
E
and
E
B2) thymocytes (Fig.
7A, top panel). In
contrast, we found V
14 SEs to predominate in DP cells from the p14,
E
, and
E
B2 mice (Fig. 7A, middle
panel; V
14 SEs were occasionally detected, at a lower level, in
DN and/or DP cells from wt mice) (data not shown). Yet both V
5 and
V
14 CJs were normally found in wt thymocytes and were strongly
reduced in p14 cells (although, possibly, to a lesser extend for the
V
14 CJs); as expected, CJs were not detected at E
alleles except for V
14 and the
E
B2 cells (Fig.
7B). The latter findings, coupled to those of V
14 SEs in
E
DP thymocytes (Fig. 7A, lane 7), are
consistent with a specific effect of Bcl-2 on CE processing also at
V
14. Two other elements should also be considered. First, a 6-fold
decrease of V
5 SEs between wt and p14 DN cells (LM-PCR assays of
Fig. 7A, lanes 2 and 4) can account for the drop of the corresponding CJs (PCR assays of Fig.
7B, lanes 4 and 6; also see
lanes 2 and 3), implying that exclusion of V
5
rearrangement is likely to be regulated primarily at the level of
chromosomal access. In p14 thymocytes, V
5 SEs may correspond to
normally rearranging alleles; e.g. in a few DN cells that do not express the
transgene. We cannot exclude, however, that inhibition of V
5 rearrangement is regulated beyond the step of DSB cleavage in a small population of TCR
rearranging cells. Second,
V
5 and V
14 CJs look similar in wt thymocytes (Fig. 7B, lanes 4 and 5). It is thus reasonable to assume
that, similar to V
5, most of the V
14 CJs detected in wt DP cells
are indeed generated in DN cells and then expanded by
-selection.
The failure to detect V
14 SEs in wt DN thymocytes may be because of
a specific feature(s) in the processing of these products, linked to
the mode of V
14 rearrangement by DNA inversion and the constraint to
preserve chromosome integrity at the site of SJ formation (31). Conversely, increased accumulation of V
14 SEs in DP thymocytes from
p14 and E
-deleted mice may reveal a disorder of the latter control
in these cells and, indeed, a unique mode of allelic exclusion at the
3' end of the TCR
locus, evidenced here by the high frequency of
attempted V
14 rearrangement. Given the accessibility of the V
14/DJ
loci, the level at which V
14 23-nucleotide RSS cleavage can be observed in total DP cells is predicted to depend on the proportion of complementary 5' D
12-necleotide RSS left available for synapsis (4, 32, 33).
View larger version (42K):
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Fig. 7.
Analysis of V SE and
CJ products in DN and DP thymocytes from
E
+ and
E
mice. A and
C, lower panels, genomic DNA from total and/or
DN- and DP-sorted thymocytes in the indicated mouse lines was analyzed
by LM-PCR for the presence of V
5 or V
14 SEs (V
5 SE
and V
14 SE, respectively) and by long range PCR for the
presence of V
5-(D)J
2 or V
14-(D)J
2 CJs (V
5 CJ
and V
14 CJ, respectively; B and C,
upper panels). In A, thymus DNA from a Rag mouse
was used as a negative control. Serial dilution analyses of wt thymus
DNA shown in B and C were as in Fig.
1D. C, PhosphorImager scanning analysis of
recombination products in p14 B2 versus p14 thymocytes gave
the following results (after normalization to C
controls): CJs,
2.53/0.88; SEs, 2.2/1.8, respectively. Because control C
2
amplifications (C
) used oligonucleotide primers located,
respectively, upstream of (5' primer) and within (3' primer) exon I of
C
2, the p14 transgene was not detected in this assay.
14 SEs into CJs at E
alleles can be
rescued, to some extent, by constitutive Bcl-2 expression. To check
whether this also occurs in the presence of E
, we analyzed
thymocytes from p14 B2 double transgenic mice. Indeed, we found
increased levels (~2.9×) of V
14 CJs in p14 B2 compared with p14
thymocytes, whereas V
14 SEs were roughly equivalent in DP cells from
both types of mice (Fig. 7C). Altogether, the above data
strongly suggest that exclusion of V
14 rearrangement proceeds
through a unique mechanism, one aspect of which could be a specific
defect in the resolution of discrete DNA DSBs and, most likely, the
induction of cell death. E
does not appear to interfere with these
processes, including the initial steps of the recombination reaction
(synapsis and RAG-mediated DNA cleavage) during attempted
V
14-to-DJ
rearrangement.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in Promoting Access to the D
-J
Domains in pro-T Cells--
We have analyzed TCR
gene rearrangement
in
E
B2 thymocytes to better assess E
function in the
regulation of CJ formation during V(D)J recombination. Compared with
E
,
E
B2 thymocytes show a small accumulation of TCR
standard recombination products, most notably CJs, that is confined to
proximal D
-J
and V
14 loci. Although detectable in DN
CD25+ pro-T cells, these effects predominate in cells that
have differentiated to the DP stage. Developmental cell selection is
unlikely to account for these findings, as no TCR
rearranged
products could be detected at either the RNA or protein levels.
Instead, evidence for VDJ recombinase activity at these loci also in
E
+ DP thymocytes, and the fact that the rearrangement
profiles at distinct TCR
loci in DN and DP
E
thymocytes
correlate with their level of chromosomal accessibility in the
particular cell subset, argue for delayed access of the recombinase to
E
alleles. Besides pointing to a possible, likely
indirect effect of E
in assisting CJ formation, these data most
notably emphasize this critical function of the enhancer for chromatin
opening within the D
-J
domains, which, unexpectedly, appears also
limited to an early window of T cell development only (i.e.
anterior to the CD44
/loCD25+ DN cell stage).
-to-J
CJ Formation by
E
?--
Resolution of Rag-mediated DNA breaks that can
happen in E
-deleted thymocytes is improved by Tg Bcl-2
expression, apparently with a slightly greater impact on CJ compared
with SJ formation (e.g. Fig. 2). May this tell us something
about a putative role of E
in enhancing CE processing? First, the
effects conferred by Bcl-2 transgenes on developmental
processes in lymphoid cells (including V(D)J recombination) have
generally been attributed to cell extended lifespan (27, 34), although
a non-conventional role of Bcl2 and incidental effect(s) on the
processing of injured DNA cannot be formally ruled out (35). Second,
the resolution of CE and SE products is thought to proceed along two
different pathways, involving distinct requirements and kinetics (4). Thus, although a deficiency in any of the factors of the NHEJ apparatus
results in a CJ defect, some proceed with unaltered SJ formation
(e.g. the SCID defect resulting from DNA-PK deficiency). Also, whereas SEs accumulate in lymphoid cells undergoing V(D)J recombination (and are eventually resolved after RAG expression is
down-regulated), CEs are difficult to detect in the wild-type situation
(they accumulate in DNA-PK-deficient lymphocytes), indicating that CJ
formation must be tightly linked to RSS cleavage. In possible relation
to these distinct behaviors, in vitro studies have suggested diametrically opposed stability of V(D)J recombination post-cleavage complexes depending on product content; complexes consisting of the two
CEs and two SEs appear to be highly unstable whereas those consisting
of the two SEs bound by the RAG factors are resistant to dissociation
challenges (reviewed in Ref. 6). We believe that a direct effect of
E
in mediating the recruitment of DNA-PK (or a DNA-PK/Artemis
complex (36)) is unlikely, based on the CJ sequences from
E
and
E
B2 thymocytes that do not show the typical abnormalities
associated with the SCID defect (i.e. extensive deletions,
frequent usage of short sequence homologies, long palindromic junctional inserts), along with the seeming lack of CEs and HJs at
E
loci in Tg Bcl-2 expressing T cells (16)
(this study). However, an effect of E
on the stabilization of
post-cleavage synaptic complexes would be compatible with the small
accumulation of CJs observed in
E
B2 thymocytes; unstable CEs
(especially at E
alleles) would have more chances of
being resolved when cell survival is extended. As for HJ formation (not
found in
E
or
E
B2 thymocytes), it could be especially
sensitive to suboptimal conformation and/or stability of the
post-cleavage synaptic complexes (24).
on chromatin structure at the
D
-J
region and an incidental effect on CJ formation may not be
mutually exclusive. First, the maintenance of an open structure may be
required for NHEJ repair. Second, E
modulation of chromosomal accessibility almost certainly involves nucleo-protein interactions including additional cis-regulatory element(s)
(e.g. the D
1 upstream promoter (25)). Within such
structures, recombination synaptic complexes (D
-to-J
and,
subsequently, V
-to-DJ
) could be optimally organized for RAG
cleavage and/or the processing of the cleaved extremities. As an
integral component, E
(and bound factors) may thereby contribute to
optimizing V(D)J repair processes, for example through the
anchorage/tethering of loose extremities within interacting distances
or, in a more sophisticated way, by favoring catalytic transitions
within the synaptic complexes. As the efficiency of CJ formation is
likely influenced by the efficiency with which loose CEs are recaptured
by the post-cleavage complex (37), DNA tethering should improve this
process. A model of post-synaptic conformational isomerization of a
recombination complex has also been described during phage Mu DNA
transposition (38). Importantly, enhancers at other TCR/Ig loci may
share the dual functions of E
on coupled regulation of chromatin
modulation and CJ formation, which could be revealed once the two
effects can be distinguished.
Locus--
Allelic exclusion at the TCR
locus is regulated at the level of V
-to-DJ
joining. In this
context, evidence of chromatin compaction at 5' V
genes during the
DN-to-DP cell transition, including reduced histone acetylation, has
accumulated (39-41) (this study). This change in organization likely
contributes to lock in allelic exclusion, at least within alleles
carrying a germline 5' V
cluster (because we used Rag mice in our
analyses, it is still unclear whether unrearranged V
genes upstream
of a V
DJ
CJ unit behave similarly). We now demonstrate that
developmentally regulated re-organization of the 5' V
genes does not
require E
, in line with their previously reported E
-independent
transcription in earlier DN thymocytes. Although long range
accessibility within antigen receptor loci has been inferred to depend
on matrix attachment regions (42), targeted deletion of matrix
attachment region
(located ~400 bp upstream of E
) does not
change TCR
transcription and V(D)J recombination in developing T
cells (43). Likewise, targeted deletion of J
2-C
2 intronic
cis-elements does not alter these processes (44). Therefore,
the re-organization at 5' V
genes could rather involve as yet
undefined regulatory sequences within the upstream part of the TCR
locus. Assuming that the promoters of 5' V
genes and of V
14
behave similarly (45), our finding that V
14 is spared by the
repressive effect makes the individual 5' V
promoters unlikely
candidates. Indeed, a 5' V
gene was no longer under allelic
exclusion control when inserted, together with associated promoter
sequences, in the region upstream of D
1 (46).
locus containing the
D
-J
-C
and V
14 domains maintains (or gains in the case of E
alleles and the D
-J
-C
clusters) chromosomal
accessibility. This might involve the regulated activation of
additional cis elements that normally act redundantly with
E
within these loci (40). At this stage of development and in the
wild-type situation, fully derepressed chromatin may be required to
ensure high levels of expression of a rearranged V
DJ
-C
unit.
The ensuing drawback is cleavage by the RAG machinery past the
-selection checkpoint, with consequences such as the specific
accumulation of D
and V
14 SEs and DJ
CJs in DP thymocytes (see
Fig. 5, A and C and see Fig. 7A) (25).
Nevertheless, the fact that levels of V
14 CJs remain extremely low
in p14 DP cells (Fig. 7B) indicates that these consequences
on TCR
allelic exclusion are minimized, possibly involving regulated
cell death (Fig. 7C). Recent results (47) suggest that
programmed cell death may be a parameter that also limits ongoing
rearrangements along the TCRJ
locus in DP thymocytes. In this
context, the particular situation of V
14 and inversional mode of
rearrangement could concur to the surprisingly opposite outcomes of
V
14-to-DJ
versus D
-to-J
attempted recombination in DP cells. Notably, intrachromosomal V
14-to-D
SJs within this accessible part of the locus could be ideal targets for DSB formation by a RAG-mediated nick-nick mechanism (26). It is, however, important
to stress that the actual levels of V(D)J recombination at D
-J
and V
14 loci in DP thymocytes is unclear and could be quite low
given the large amounts of germline-sized D
-J
containing fragments detected by PCR and Southern assays of DNA from
E
(
E
B2) and E
+ (p14)
thymocytes.3 The reasons for
these paradoxical effects of combining chromosomal accessibility and
low levels of recombination within RAG expressing cells (48) warrant
further investigating efforts.
E
B2 mice
may be subject to these effects. However, neoplasia does not
significantly affect these animals (but translocation into an
enhancerless TCR
locus may not result in oncogene-deregulated expression in the developing lymphocytes), and we did not find evidence
of TCR
genomic instability using FISH analysis and PCR assays
(including assays to search for TCR
-to-TCR
/
interlocus recombination). Specific pathways might exist to counteract such events
in DP thymocytes involving, for example, a role of the recombination
machinery in the prevention of transposition (49, 50) and/or sensors of
injured DNA (e.g. p53, ATM) acting to uncover and eliminate
such damages and/or the damaged cells (51). The latter possibility is
supported by the finding that introduction of an E
mutation onto a
p53-deficient background accelerates tumor development in T
cells.4
is not involved in the sophisticated
controls that, following
-selection, secure allelic exclusion at the
TCR
locus. However, allele asynchronicity of V
gene assembly in
earlier DN pro-T cells is a prerequisite for allelic exclusion, as this
should leave cells enough time to test for V
-to-DJ
productivity before V
gene rearrangement proceeds on the other allele (7). The
molecular basis for this phenomenon are still unclear. It may involve a
recombination machinery that incidentally operates at suboptimal
efficiency or structural features to ensure that a single allele
rearrange at a time, including specific properties of D
-flanking RSS
(4) and/or an epigenetic mark(s) established at antigen receptor genes
early in development (52). We stress that E
could be involved in any
of these controls (except may be for the latter), as evidenced by the
differential effect exerted on activation of the D
-J
-
versus the V
-containing chromosomal domains and
associated regulatory elements (15). Our current results of a rigorous
control by E
in regulating chromosomal access in early pro-T cells
further sustain this hypothesis. This may explain the evolutionary
constrain to maintain a function of E
that is limited both spatially
(the D
-J
domains) and temporally (prior to the
CD44
/loCD25+ DN cell stage).
![]() |
ACKNOWLEDGEMENT |
---|
This manuscript was improved by the helpful criticisms of an anonymous reviewer.
![]() |
FOOTNOTES |
---|
* This work was funded by institutional grants from INSERM and the CNRS and by specific grants from the Association pour la Recherche sur le Cancer, the Commission of the European Communities, and the Fondation Princesse Grace de Monaco.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: CEA, Centre d'Etudes Nucléaires, 60/68
Avenue du Général Leclerc, BP6, 92 265 Fontenay-aux-Roses
Cedex, France.
§ Present address: Immunologie Rétrovirale et Moléculaire, IRD CNRS/CTS, 240 Avenue Pr. Emile Jeanbraud, 34090 Montpellier, France.
¶ Present address: Département de Biologie Cellulaire et Moléculaire, Pfizer Global Research & Development/Fresnes Laboratories, 3-9 rue de la Loge, 94265 Fresnes Cedex, France.
Present address: Institut de Signalisation, Biologie du
Développement et Cancer, CNRS-UMR 6543, Centre Antoine
Lacassagne, 33, Avenue Valombrose, 06189 Nice, France.
** To whom correspondence should be addressed: CIML, Case 906, 13288 Marseille Cedex 9, France. Tel.: 33-491-269435; Fax: 33-491-269430; E-mail: ferrier@ciml.univ-mrs.fr.
Published, JBC Papers in Press, March 14, 2003, DOI 10.1074/jbc.M212647200
2 W. M. Hempel and N. Mathieu, unpublished results.
3 N. Mathieu, unpublished results.
4 J. Chen, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TCR, T cell receptor; DNA-PK, DNA-dependent protein kinase; DN, double negative; DP, double positive; wt, wild-type; LM, ligation-mediated; RT, reverse-transcribed; CE, coding end; SE, signal end; CJ, coding joint; SJ, signal joint; HJ, hybrid joint; DSB, double strand break; RSS, recombination signal sequence; RAG, recombination-activating-gene; NHEJ, non-homologous end-joining; ChIP, chromatin immunoprecipitation; Gl, germ line.
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REFERENCES |
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