By
From the * Department of Biology, University of California San Diego, La Jolla, California 92093;
and the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
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Abstract |
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A key feature of B and T lymphocyte development is the generation of antigen receptors
through the rearrangement and assembly of the germline variable (V), diversity (D), and joining (J) gene segments. However, the mechanisms responsible for regulating developmentally ordered gene rearrangements are largely unknown. Here we show that the E2A gene products
are essential for the proper coordinated temporal regulation of V(D)J rearrangements within
the T cell receptor (TCR) and
loci. Specifically, we show that E2A is required during adult
thymocyte development to inhibit rearrangements to the
and
V regions that normally recombine almost exclusively during fetal thymocyte development. The continued rearrangement of the fetal V
3 gene segment in E2A-deficient adult thymocytes correlates with increased levels of V
3 germline transcripts and increased levels of double-stranded DNA breaks
at the recombination signal sequence bordering V
3. Additionally, rearrangements to a number of V
and V
gene segments used predominately during adult development are significantly reduced in E2A-deficient thymocytes. Interestingly, at distinct stages of T lineage development, both the increased and decreased rearrangement of particular V
gene segments is
highly sensitive to the dosage of the E2A gene products, suggesting that the concentration of
the E2A proteins is rate limiting for the recombination reaction involving these V
regions.
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Introduction |
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The ability of lymphocytes to respond to a vast array of
antigens is dependent on the generation of unique surface receptors with diverse binding specificities. The antigen receptors are assembled during lymphocyte development from germline variable (V), diversity (D), and joining
(J) gene segments by the process of V(D)J recombination
(1). Products of recombination activating gene 1 (Rag-1)1
and Rag-2 recognize and cleave DNA at recombination
signal sequences (RSS) that flank all rearranging gene segments (2). The restricted expression of Rag-1 and Rag-2
to B and T cells accounts for the lymphoid specificity of
the recombinase (3, 5). However, within the lymphoid
lineages, the process of V(D)J recombination is ordered and
developmentally regulated at a number of different levels.
For example, Ig V region genes are fully rearranged only in
B lymphocytes and TCR V region genes are rearranged
only in T lymphocytes. In addition, assembly of the V region genes is regulated in a developmentally stage-specific
manner within developing B and T lineage cells. That is,
most differentiating B cells assemble Ig heavy chain V regions before light chain V regions, and developing T cells
rearrange chain V regions before
chain V regions (8- 10). During the assembly of the Ig heavy chain and the
TCR
chain, the D to J rearrangement step normally occurs before the assembly of the V regions (9, 11). These
types of regulations are proposed to be affected by altering
the accessibility of the substrate gene segments to the recombinase (12, 13).
Control of V(D)J recombination can also be regulated at
the level of V gene usage. During murine B cell development, the 3'-most V gene segments are preferentially used
in pre-B cells in fetal liver and adult bone marrow, whereas
mature B cell populations use a wide range of VH segments
(14). A similar level of regulation exists in the rearrangement of the TCR and
genes. During thymic ontogeny, the
and
V gene rearrangements occur in waves.
V
3 and V
4, which are most proximal to the J
gene segments, are the most frequently rearranged V
gene segments during early fetal thymic development (18). Rearrangements to V
3 and V
4 peak at approximately
embryonic day 15 and decline thereafter. In the adult thymus, V
3 is rarely rearranged. In contrast, rearrangements to the more 5' V regions, such as V
2, begin late in fetal
development and predominate in the adult (22). Within
the
locus, V
1 rearrangements predominate during early
fetal thymic development, but are rare in the adult,
whereas V
5 usage begins later and predominates in the
adult (23, 24). Thus, within the
and
loci there is a regulated switch in the use of V gene segments between fetal and adult thymocyte populations. It is likely that the regulation of this developmental switch is controlled, in part, by
changes in gene segment accessibility and/or selective recruitment of the recombination enzymes.
Several studies have suggested that transcriptional promoters and enhancers play important roles in the regulation
of VDJ recombination by modulating the accessibility of
the gene segments to the recombination machinery (13,
25). However, evidence for the involvement of specific
transcription factors in this type of regulation is lacking.
The basic helix-loop-helix (bHLH) family of transcriptional regulatory proteins has been implicated in the regulation of Ig and TCR gene rearrangement based on the ability of these proteins to bind to and activate transcription from the Ig and TCR gene enhancers (26). The E2A
gene products, E12 and E47, are broadly expressed members of the HLH family of transcription factors and play important functional roles in the early stages of both B and /
T lymphocyte development. In the absence of E2A activity, B lymphocytes are blocked at a stage before the initiation of Ig gene rearrangements (29). Ectopic expression
of either E12 or E47 in the E2A-deficient background allows the activation of several B lineage-restricted genes
(32). Interestingly, E2A-deficient mice display an incomplete block at a developmentally similar stage of
/
T cell
development (33). A specific role for E2A in B lineage development is also inferred from experiments demonstrating
the induction of B cell-specific traits upon overexpression
of E47 in cell lines. Ectopic expression of E47 in non-B
cell lines results in the activation of a number of B lineage-
specific genes, including Rag-1, TdT, and
5 (34). Overexpression of E47 in a pre-T cell line leads to the induction of IgH DJ rearrangements as well (36).
Here we describe a functional role for the E2A gene
products during the development of the /
T lymphocytes. The absence of E2A results in the impaired development of the
/
T cells found in the secondary lymphoid
organs and the intraepithelial layers of the intestine. Rearrangements to the V regions used most predominately by
these
/
T cells are significantly reduced in E2A-deficient
thymocytes. In contrast, skin intraepithelial
/
T cells develop normally in E2A-deficient mice, and rearrangements to V
3 and V
1, the V regions used exclusively by the skin
/
T cells, are present at wild-type levels in the E2A-deficient fetal thymus. Remarkably, both V
3 and V
1 continue to rearrange coordinately in the E2A-deficient adult
thymus to an adult configuration of D and J segments,
whereas developing thymocytes in wild-type adult thymocytes rarely use these V regions. Interestingly, the data
indicate that the regulation of rearrangement to specific V
gene segments is dosage sensitive, as mice heterozygous for E2A show significant alterations in rearrangement levels.
We propose that the concentration of the E2A proteins is a
key factor in regulating, both positively and negatively, the
rearrangement of several V
and V
gene segments.
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Materials and Methods |
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Gene Targeting.
Targeting of the E2A gene has been described previously (29).Flow Cytometric Analysis.
Isolation ofRearrangement Southern Blot.
Genomic DNA was prepared from thymocytes by DNAzol. 10 µg DNA was digested with EcoRI, electrophoresed on a 0.8% agarose gel, and transferred to Nytran (Schleicher & Schuell). The blot was hybridized with probe 4, then stripped and reprobed sequentially with the VRearrangement PCR.
500 ng of adult or E18-19 fetal thymus genomic DNA (isolated using DNAzol; GIBCO BRL) was analyzed by PCR in a 25 µl reaction volume containing 100 ng of each primer in 10 mM Tris, pH 8.3, 50 mM KCl, and 2 mM MgCl2. All VLigation-mediated PCR.
Adult thymus genomic DNA was prepared by incubation of thymocytes overnight at 55°C with 50 µg/ml proteinase K in 50 mM Tris, pH 8.0, 100 mM EDTA, 100 mM NaCl, and 1% SDS. The DNA was phenol/chloroform extracted, chloroform extracted, and precipitated with 2 vol of ethanol. The DNA was washed in 70% ethanol and dissolved in ddH2O. 3 µg DNA was ligated to annealed BW-1/BW-2 linkers for 12-14 h before inactivation of the ligation reaction as described previously (43). 12 rounds of PCR were performed on 1/20 of the ligation reaction using 100 ng of the BW-1H primer and 100 ng of the following locus-specific primer: VReverse-transcription PCR.
Total RNA was prepared from thymocytes isolated from 4-6-wk-old E2A-deficient mice and heterozygous littermates by TriZOL (GIBCO BRL). 10 µg of total thymocyte RNA was DNase treated, and 3 µg of the DNase-treated RNA was reverse transcribed as described previously (29). The PCR reaction was run on a 2.5% Nusieve gel, transferred to Nytran, and hybridized with gene-specific probes. The actin and V ![]() |
Results |
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The E2A proteins are expressed at high levels
in prothymocytes and are important in the development of
committed /
T cells from uncommitted progenitors (33,
44). Since both
/
and
/
T lymphocytes develop from
the same progenitor thymocytes, we analyzed E2A-deficient mice for the presence of
/
T cells by flow cytometry (45). E2A-deficient mice display significantly reduced numbers of
/
T lymphocytes in the thymus,
spleen, and lymph node compared with their heterozygous
littermates (Fig. 1 A, and data not shown). In the thymus
and lymph nodes,
/
T cell numbers are reduced from 8- to 40-fold (Fig. 1 A). Similarly, the number of
/
T cells
is decreased ~20-fold in the intestinal epithelium of E2A-deficient mice (Fig. 1 B). Surprisingly, mice that lacked
E2A showed almost normal numbers of skin intraepithelial lymphocytes, suggesting that the E2A gene products differentially control
/
T cell development (Fig. 1 C).
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/
T lymphocytes can be divided into two broad
subtypes which differ in the type of V region used, their
junctional diversity, and their ability to home to specific
sites (48; Fig. 2 A). Skin intraepithelial lymphocytes represent one subtype, while
/
T cells in the secondary lymphoid organs and the intestinal epithelium are members of
the second subtype (48). The first
/
T cells to develop in
the thymus express an invariant receptor composed of
and
chains that have used exclusively V
3 and V
1 (19, 20, 40, 49). These
/
cells migrate to the skin (50).
/
T cells that populate the secondary lymphoid organs express variable receptors using predominately the
2,
1,
and
5 V regions, and intestinal
/
cells express variable
receptors often using V
5 and V
4 (23, 41, 52, 53; Fig. 2
A). Rearrangements to these V regions begin late in fetal
development and predominate in the adult (48). Since the
development of each subtype of
/
cells is associated with
specific
and
rearrangements, the development of only a
subset of
/
T cells in the E2A-deficient mice suggests that
and/or
rearrangements may be affected. Committed
/
T lymphocytes retain previously rearranged
genes in both chromosomal and extrachromosomal DNA
(39, 47, 54). Thus, the frequency of specific V
rearrangements can be analyzed in total thymus DNA despite the
fact that 95% of the cells are committed to the
/
T cell
lineage. To determine the relative frequency of V
gene
usage, we analyzed adult thymus DNA from E2A-deficient
mice and heterozygous littermates by Southern blotting. As
a probe, we used a genomic DNA fragment, termed probe
4(J
1-J
2), which is located between the J
1 and J
2 gene
segments and allows for the detection of TCR
genomic
rearrangements (39; Fig. 2 A). Southern blot analysis with
radiolabeled probe 4(J
1-J
2) identified a series of bands
present in both heterozygous controls (Fig. 2 B). Several of
these bands have been previously identified and are indicated by arrows (23, 39). Interestingly, thymus DNA from
the E2A-deficient mice gave a distinctly different pattern of
DNA fragments hybridizing to probe 4 (Fig. 2 B). Many of
the bands that are clearly visible in the wild-type DNA
samples are nearly undetectable in the E2A-deficient DNA
samples. However, there are two strongly hybridizing fragments in the E2A-deficient DNAs, one of which is undetectable in wild-type DNAs (Fig. 2 B, fragment indicated
by a bent arrow). To determine the identity of the bands,
the blot was rehybridized with V
5-, V
4-, and V
1-specific probes (Fig. 2, C and D, and data not shown). Bands
corresponding to V
5DJ
and V
4DJ
rearrangements
were detectable in both heterozygous DNAs but were virtually absent in the E2A-deficient DNA samples (Fig. 2 C,
and data not shown). These data demonstrate that the predominate
rearrangements in a wild-type adult thymus,
which involve the
5 and
4 V regions, are significantly
underrepresented in thymocyte DNA derived from E2A-deficient mice.
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The flow cytometric data showed that the /
T cells in
the skin, which express a TCR composed exclusively of
V
3 and V
1, are readily detectable at close to wild-type
levels in E2A-null mutant mice (Fig. 1 C). V
1 rearrangements occur predominately during fetal development, and
a significant proportion of V
1 rearrangements use the J
2
region, which results in the deletion of the DNA hybridizing to probe 4(J
1-J
2) (24, 41, 48; Fig. 2 A). Thus, V
1
rearrangements are not normally detectable in wild-type adult thymus DNA (23). Consistent with these data, only
the germline band is visible in the heterozygous DNA samples when hybridized with a V
1-specific probe (Fig. 2 D).
Surprisingly, the V
1 probe hybridized to two DNA fragments in the E2A-deficient DNA samples (Fig. 2 D). These
V
1-specific rearrangements comigrate with the two predominate fragments recognized by probe 4(J
1-J
2) (Fig.
2, B and D). Based on their sizes, the bands likely represent V
1DJ
1 rearrangements and V
1D
2 intermediate rearrangements (23). These data indicate that rearrangements
involving V
1, which are normally not detectable in wild-type mice, make up the majority of the rearrangements in
adult E2A-deficient thymocytes. The appearance of V
1
rearrangements and the lack of V
5 and V
4 rearrangements indicate that a deficiency in E2A leads to a deregulation of V region usage at the TCR
locus in adult thymocytes.
To confirm the rearrangement data
described above and to examine the level of
gene rearrangements, we analyzed total thymus DNA for V
and V
rearrangements by PCR using forward primers specific for
the V regions and reverse primers recognizing the J gene
segments. Only upon rearrangement are the primers
brought into sufficient proximity to allow PCR amplification. The data shown are derived from three independent
sets of littermates, designated as a, b, and c (Fig. 3, A and B).
As expected, all E2A-deficient mice analyzed displayed reduced levels of V
5DJ rearrangements, although the decreases varied from 3- to 50-fold (Fig. 3 B). As predicted
from the Southern blot analysis, V
1 rearrangements were
increased ~10-50-fold in thymus DNA derived from E2A-deficient mice compared with control littermates (Fig. 3 B).
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The differential V gene usage observed in the locus in
E2A-deficient mice was evident in the
locus as well.
PCR analysis using specific V
forward primers in conjunction with a J
reverse primer demonstrated significant
decreases in the levels of V
2 rearrangements in E2A-deficient adult thymus DNAs (Fig. 3 A). The decreases in the
level of V
2 rearrangements from 10 independent E2A-
deficient mice ranged from 2- to 10-fold. Within any one E2A-deficient thymus, rearrangements to V
5 were generally more significantly decreased than rearrangements to
V
2. Analysis of E2A-deficient thymus DNA for recombination to the V
5 gene segment demonstrated that V
5 rearrangements are not significantly affected by the absence
of E2A (Fig. 3 A). These data demonstrate that the proper
V(D)J rearrangement of particular V gene segments in both
the
and
locus requires the E2A gene products.
The TCR V gene segment, V
3, recombines coordinately with V
1 during early embryogenesis. To determine
whether V
3 rearrangements, like those of V
1, are increased in adult thymocytes derived from E2A-deficient
mice, genomic DNA was analyzed by PCR using a primer specific for V
3 (Fig. 3 A). Remarkably, rearrangements to
V
3 are also significantly increased in DNA isolated from
E2A-deficient thymocytes (Fig. 3 A). Thus, the absence of
E2A leads to a coordinate increase in the rearrangement of
the
and
V gene segments normally used predominately
during early fetal development to an adult configuration of
D and J segments. These data suggest that during adult thymocyte development, the E2A gene products act as both positive and negative regulators of
and
V gene usage,
and are essential for the temporally ordered recombination
of the TCR
and
loci.
Because the fetal and adult thymus
differ with respect to the V regions predominately rearranged and expressed, we analyzed DNA from E19 fetal
thymocytes for the presence of and
rearrangements. As
in the adult, V
5 rearrangements were present at relatively normal levels in DNA derived from E2A-deficient fetal
thymi, whereas V
2 rearrangements were decreased an average of three- to fourfold (Fig. 4 A). Additionally, we observed a striking decrease in the level of V
5 rearrangements in the E2A-deficient fetal thymus DNAs compared
with the wild-type littermates (~ 50-fold; Fig. 4 B). Surprisingly, we consistently detected three- to fourfold decreases in the level of V
5 rearrangements in fetal thymus
DNAs derived from mice heterozygous for E2A compared
with wild-type littermates (Fig. 4, B and C). Thus, the absence of the E2A gene products during fetal thymocyte development appears to have a more significant effect on rearrangement to the
5 V region than to the
2 V region. In
addition, the frequency of rearrangement to the
5 V gene
segment during fetal thymocyte development is sensitive to
the dosage of the E2A proteins.
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V3 and V
1 rearrangements, which are virtually undetectable in the adult thymus, predominate during fetal thymic development. As analyzed by PCR, the frequency of
usage of these V regions was similar in fetal thymus DNA
derived from E2A-deficient and control mice (Fig. 4, A
and B). These data suggest that the E2A gene products play
multiple and distinct roles in regulating
and
V(D)J recombination. The E2A gene products are nonessential in
the initiation of V
1 and V
3 rearrangements early in fetal thymic development. However, the presence of the E2A
gene products is required at the later stages of thymocyte
differentiation to inhibit the usage of V
3 and V
1, which
are normally dormant in wild-type mice. Additionally, activity of the E2A gene products is important for initiating
the proper rearrangement of the
5 and
2 V regions.
The observation that the frequency of V5 usage in fetal
thymus development is dependent on the dosage of the
E2A proteins led us to examine more carefully whether V
region usage during adult thymocyte development is correspondingly dosage sensitive. Thus, we analyzed DNAs
from wild-type and heterozygous littermates by PCR to
determine the relative levels of V gene rearrangements. Unlike in the fetal thymus DNAs, V
5 rearrangements
were comparable in adult thymocyte DNA derived from
E2A heterozygous and wild-type mice (Fig. 4 C). Similarly, rearrangements involving all other V regions were
unaffected in E2A heterozygous mice compared with wild-type littermates, with the exception of V
1 (Fig. 4 C, and data not shown). Unexpectedly, we found that the frequency of V
1 gene usage is increased dramatically in E2A
heterozygous mice compared with wild-type littermates
(Fig. 4 C). Therefore, the proper inhibition of V
1 rearrangement during adult thymus development is additionally dosage dependent. In summary, the data indicate that
the activity and concentration of the E2A gene products in thymocytes undergoing site-specific recombination are key
factors in inducing proper V
5 rearrangement during fetal
thymus development and for inhibiting rearrangements to
V
1 during adult thymocyte development.
The process of V(D)J recombination is tightly regulated and proceeds in well-defined stages. During the assembly of the Ig heavy chain and the TCR chain, the D
to J rearrangement step normally precedes the assembly of
the V regions (9, 11). However, recombination at the
locus is unique in that V to D rearrangement normally represents the initial recombination event and precedes rearrangement to the downstream J regions (24). As described
above, the end products of the recombination reaction involving V
5 are significantly decreased in adult thymus
DNA from E2A-deficient mice, whereas those involving
V
1 are strikingly increased. To further define the stage at
which the recombination events involving these V regions
are deregulated, we analyzed total thymus DNA from
E2A-deficient mice and wild-type controls for the presence
of rearrangement intermediates. V to D rearrangement intermediates can be detected using a V region-specific forward primer and a reverse primer located 3' of the D
2
coding sequence (Fig. 5 A). Since fully rearranged VDJ
products result in the deletion of the DNA hybridizing to
the reverse 3' D
2 primer, only intermediate V-D rearrangements can be PCR amplified (Fig. 5 A). Like the fully
rearranged products, V
5-D
2 intermediate rearrangement
products are present at significantly reduced levels in E2A-deficient adult thymus DNA (Fig. 5 C). Likewise, the V
1-D
2 intermediate rearrangements are increased to a similar
extent as the fully rearranged V
1DJ products in thymus
DNA lacking E2A (Fig. 5 C). Similar results were obtained
using a reverse primer located 3' of D
1 (data not shown).
Interestingly, the presence of V
1D
1 rearrangements in
the E2A-deficient thymus demonstrates that these V
1 rearrangements are not from residual fetal cells, as fetally derived V
1 rearrangements use exclusively the D
2 gene
segment. These data suggest that the deregulation of TCR
V to D rearrangement is not D region specific.
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Although recombination at the locus normally involves an initial V to D rearrangement step, the D
2 and
J
1 elements have been shown previously to be involved in
D
2-J
1 and D
1-D
2-J
1 intermediate rearrangements
which do not involve V gene rearrangement (54, 55). To
examine the efficiency of D to J joining, we analyzed total
thymus DNA by PCR using a forward primer located 5' of
the D
2 RSS and a reverse primer specific for J
1 (Fig. 5
B). Since rearrangement of any gene segment to D
2 results in the deletion of the DNA hybridizing to the forward
primer, only D
2-J
1 intermediates can be PCR amplified
(Fig. 5 B). As shown, D
2-J
1 and D
1-J
1 intermediate
rearrangements are present at normal levels in adult thymus
DNA derived from E2A-deficient mice (Fig. 5 D, and data
not shown). Thus, E2A plays an important role in regulating the rearrangement of the V gene segments within the
TCR
locus, but is dispensable for normal D
-J
rearrangement.
The recombination process that assembles the functional and
TCR genes involves cleavage at the RSS that border the
coding segments and then joining of the coding ends (56,
57). The RAG-1 and RAG-2 proteins were previously
shown to be sufficient for cleavage of the V(D)J recombination signal on extrachromosomal and in vitro substrates
(3, 4, 58). As described above, rearrangement to several V
and V
gene segments is deregulated in the absence of
E2A. In adult E2A-deficient mice, the most dramatic phenotype observed is the increased rearrangement to V
3 and
V
1. To determine whether the increased usage of V
3 reflected an increase in the cleavage of the V
3 RSS, we assayed for the presence of double-stranded breaks at the
RSS 3' of V
3 using LM-PCR (Fig. 6 A). Double-stranded breaks at V
3 were absent in E2A heterozygous and wild-type adult thymus DNA, but could be detected in
the E2A-deficient thymus DNA (Fig. 6 B). In contrast,
both the E2A-deficient DNA and the control DNA displayed similar levels of broken ends at the RSS 5' of JH2
(Fig. 6 B). These data suggest that the E2A gene products
inhibit rearrangement of the
3 V region by modulating the accessibility of
3 gene segments to the recombination
machinery.
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Several studies have demonstrated that gene rearrangement is correlated with prior transcription of the
unrearranged genes. Indeed, there is a striking correlation
between the level of gene rearrangement and the level of
expression of the unrearranged genes (59). In fact, production of sterile transcripts has been postulated to direct
recombination through the alteration of gene segment accessibility. In the locus, decreases in V
3 rearrangements correlate with decreases in the level of V
3 germline transcripts (22). To determine whether the increased cleavage
of the
3 V region is paralleled by increases in V
3 germline transcripts, we analyzed total thymus RNA from E2A-deficient mice and wild-type littermates by reverse-transcription (RT)-PCR. Consistent with the presence of
3
rearrangements, V
3 sterile transcripts were readily detectable in the thymus of the E2A-deficient mice, but were absent from the thymus of control littermates (Fig. 6 C). Thus, the absence of the E2A proteins during adult thymocyte development allows for the continued transcription
of the unrearranged
3 V gene segment, likely rendering
this V region accessible to the recombination machinery.
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Discussion |
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The E2A gene products have the ability to bind to the
enhancer elements within the Ig heavy and light chain
genes as well as the TCR and
genes (66). The ability of these enhancer elements to promote lineage-specific
activation of recombination suggests that the E2A gene
products might be involved in regulating Ig and TCR gene
rearrangements (70). However, whether the E2A proteins are important for recombination of the Ig or TCR
and
loci is still unclear. Here we have examined the rearrangement efficiency of the two other TCR genes, the
and
genes, within E2A-deficient
/
T cells. Committed
/
T cells, which represent >90% of total thymocytes,
retain previously rearranged
and
genes in both chromosomal and extrachromosomal DNA (39, 47, 54). Importantly, committed
/
T lymphocytes are selected and expanded on the basis of
chain rearrangements, and not
on the basis of
and
rearrangements (74). Thus, the
relative level of V
and V
gene rearrangements in total
thymus DNA reflects the frequency of V
and V
gene usage during double-negative thymocyte development. The
data presented here demonstrate that the E2A gene products are critical regulators of
and
V(D)J rearrangement during fetal and adult thymocyte development. First, they
are required during fetal development to allow efficient rearrangement of at least one TCR V
gene segment and to a
lesser extent a TCR V
gene segment. Second, the presence of the E2A proteins during adult thymocyte development is required to prevent the inappropriate rearrangement of V regions that primarily recombine in fetal
thymocytes. The decreased usage of particular V gene segments correlates well with the deficiency of the
/
subpopulations expressing TCRs that use those V regions.
Thus, the E2A gene products, which are required at two
distinct stages of
/
T cell development, regulate the development of the
/
subpopulations through the ability to
influence V gene usage during the recombination process.
E2A-deficient mice have significantly reduced levels of rearrangements to particular V regions, including V5 and V
2. There are various ways in which the
bHLH proteins might positively regulate VDJ rearrangement. Several studies have suggested that transcriptional enhancers play important roles in modulating the accessibility of the gene segments to the recombination machinery, and E2A protein binding sites have been identified in
the enhancer element of the
locus (13, 25, 77, 78). However, others have shown that the TCR
enhancer is important for J segment accessibility (79). In transgenic mice
lacking the
enhancer, V to D rearrangement is intact, but
V-D to J rearrangement is inhibited (79). Since this phenotype is clearly distinct from that observed in the E2A-deficient mice, it is unlikely that bHLH proteins are functioning through the TCR
enhancer.
The data described here raise the possibility that bHLH
proteins play a role in the initiation of germline transcription. Indeed, there are several E box elements in the V2
upstream sequences but not in the V
3 upstream sequences
(Raulet, D., personal communication). However, we have
examined for the presence of V
2 germline transcripts in
E2A-deficient and wild-type thymocytes and have not
found a tight correlation between the level of V
2 rearrangements and the level of V
2 germline transcripts (Bain,
G., unpublished observations). An alternative mechanism is
that bHLH proteins directly target the recombinase to the
RSS. We note the presence of consensus E box binding
sites in the linker sequences separating the heptamer and
nonamer of almost all V
and V
gene segments. E2A and
HEB proteins bind to these sites with relatively high affinity using nuclear extracts derived from thymocytes (Bain,
G., and C. Murre, unpublished results). To determine how
the E2A proteins positively regulate V(D)J recombination,
it will be important to examine the functional significance
of the E box binding sites in the promoter and spacer regions by mutational analysis using knock-in strategies, and
to determine the transcriptional activity of particular V region promoters in the absence of all E box binding activity.
Normally, V3 and V
1 rearrange coordinately
during early thymic development and give rise to
/
T
cells that migrate to the epithelium of the skin (20, 48). In
thymocytes that develop in the adult mouse, rearrangements to these V regions occur only at a very low frequency (23). Interestingly, we show here that both V
3
and V
1 gene rearrangements are significantly overrepresented in DNA derived from E2A-deficient adult thymi.
Thus, the presence of E2A is essential in order to properly
regulate the ordered rearrangement of these TCR loci in
adult thymocytes.
The question then becomes, how does E2A restrict the
usage of the 1 and
3 V regions in wild-type thymocytes?
As discussed above, positive regulation of recombination to
the
5 and
2 V gene segments is impaired in E2A-deficient mice. It is conceivable that the differences in V region
usage observed in the E2A-deficient thymus are simply the
result of competition between V gene segments. That is, if
V
5 and V
2 are prevented from rearranging efficiently in
the absence of E2A, then V
3 and V
1 rearrangements,
which don't require E2A, might continue to rearrange and
be overrepresented. However, we consider it unlikely that
a competition model could explain the data observed, particularly within the
locus, for the following reasons. First,
thymus DNAs isolated from adult E2A heterozygous mice
show normal levels of V
5 rearrangements, but at the same
time display significantly increased levels of V
1 rearrangements compared with wild-type littermates (Fig. 4 C).
Thus, we observe increased recombination to the
1 V region in the absence of a decrease in rearrangement to the
5 V region. In addition, V
5 rearrangements are significantly decreased in fetal thymus DNAs derived from E2A-deficient and E2A heterozygous mice despite the fact that
the V
1 rearrangements in these mice occur at a similar
frequency as in the wild-type fetal thymus (Fig. 4 B).
Taken together, we favor a model in which the E2A gene
products normally function in restricting the accessibility of
V
1 and V
3 to the recombinase.
Previous studies have identified a repressor element located in the V3-V
4 intergenic region that functions on
heterologous promoters in transient transfection assays (80).
It has been proposed that the activation of this repressor element during adult thymocyte development renders the
3
gene segment inaccessible to the recombinase (80). Several
consensus protein binding sites have been identified in the
repressor region; however, no E2A protein binding sites
are present. Nevertheless, it will be important to examine
whether E2A activity is required, indirectly, for the V
3-V
4 repressor activity. Additionally, it will be interesting to
determine whether a similar repressor element exists in the
locus. Taken together, we favor a model in which the
absence of the E2A proteins during adult thymocyte development allows for the continued transcription of unrearranged fetal V gene segments, rendering continued access
to the recombination machinery.
An intriguing result of the data presented
here is that the regulation of 5 rearrangement during fetal
thymic development appears to be highly sensitive to the
dosage of E2A. We would like to consider the possibility
that the concentration of E2A molecules in a double-negative T cell actively undergoing V
5 rearrangement is limiting to the recombination process. Site-specific recombination in B lymphocytes is controlled in such a way that if the
initial VH to DJH rearrangement results in the production of
a functional heavy chain, a signal is sent to shut down the heavy chain rearrangement process. As a result, an individual B cell expresses only one functional heavy chain. However, if the initial rearrangement is nonproductive, an additional VH to DJH rearrangement can be initiated on the
other allele (9). This model predicts that VH gene rearrangement occurs only on one allele at a time. These data
raise the question whether a limiting concentration of some
factor is important in regulating this aspect of V(D)J recombination. The results described here indicate that the
concentration of E2A proteins present in fetal thymocytes
undergoing TCR
rearrangements is rate limiting. Thus, it is conceivable that, within any one T lymphocyte, the
level of E2A is such that only one allele of the TCR
locus is undergoing rearrangement. In addition, these data
suggest that the level of E2A activity is significantly lower
in fetal thymocytes and relatively higher in adult thymocytes. Although E2A transcript levels are comparable
between fetal and adult thymus RNAs, Id-2 transcript levels are threefold higher in fetal thymocytes compared with
adult thymocytes (Bain, G., and C. Murre, unpublished
data). Since the Id-2 protein can function as a negative regulator of E2A activity, it is likely that E2A activity is significantly lower during fetal thymocyte development.
Like the TCR and
loci, the murine Ig heavy chain
locus displays preferential rearrangement of particular V
gene segments. For example, B cell precursors characteristically use a restricted set of VH segments, with the 3' V gene
segments being preferentially rearranged, whereas mature B
cell populations use a wide range of VH segments (14).
There is a dosage-dependent effect of E2A on differentiation through the B cell lineage as well, since mice heterozygous for E2A have ~50% of the wild-type numbers
of progenitor B cells (31, 81). These data raise the question
of whether the E2A gene products control Ig gene rearrangements in a similar dosage-dependent fashion as described here for the TCR
locus.
![]() |
Footnotes |
---|
Address correspondence to Cornelis Murre, Department of Biology/MC 0366, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0366. Phone: 619-534-8796; Fax: 619-534-7550; E-mail: murre{at}biomail.ucsd.edu
Received for publication 24 August 1998 and in revised form 6 November 1998.
We thank Drs. F. Livak and D. Schatz (Yale University, New Haven, CT) for providing the Southern
probes, and Dr. D. Raulet (University of California, Berkeley, CA) for providing V
2 and V
3 plasmids.
This work was supported by grants from the National Institutes of Health (to C. Murre and W.L. Havran), the Council for Tobacco Research, and the Malinkrodt Foundation (to C. Murre).
Abbreviations used in this paper bHLH, basic helix-loop-helix; LM, ligation-mediated; RAG, recombination activating gene; RSS, recombination signal sequence(s); RT, reverse transcription.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tonegawa, S.. 1983. Somatic generation of antibody diversity. Nature. 302: 575-581 [Medline]. |
2. | Schatz, D., M. Oettinger, and M. Schlissel. 1992. V(D)J recombination: molecular biology and regulation. Annu. Rev. Immunol. 10: 359-383 [Medline]. |
3. | Oettinger, M., D. Schatz, C. Gorka, and D. Baltimore. 1990. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science. 248: 1517-1523 [Medline]. |
4. | McBlane, J., D. van Gent, D. Ramsden, C. Romeo, C. Cuomo, M. Gellert, and M. Oettinger. 1995. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell. 83: 387-395 [Medline]. |
5. | Mombaerts, P., J. Iacomini, R.S. Johnson, K. Herrup, S. Tonegawa, and V.E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lymphocytes. Cell. 68: 869-877 [Medline]. |
6. | Shinkai, Y., G. Rathbun, K.-P. Lam, E.M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A.M. Stall, and F.W. Alt. 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell. 68: 855-867 [Medline]. |
7. | Schatz, D., M. Oettinger, and D. Baltimore. 1989. The V(D)J recombination activating gene, Rag-1. Cell. 59: 1035-1048 [Medline]. |
8. | Chen, J., and F.W. Alt. 1993. Gene rearrangement and B cell development. Curr. Opin. Immunol. 5: 194-200 [Medline]. |
9. | Alt, F., G. Yancopoulos, T. Blackwell, C. Wood, E. Thomas, M. Boss, R. Coffman, N. Rosenberg, S. Tonegawa, and D. Baltimore. 1984. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO (Eur. Mol. Biol. Organ.) J. 3: 1209-1219 [Abstract]. |
10. | Kisielow, P., and H. von Boehmer. 1995. Development and selection of T cells: facts and puzzles. Adv. Immunol. 58: 87-209 [Medline]. |
11. | Strominger, J.. 1989. Developmental biology of T cell receptors. Science. 244: 943-950 [Medline]. |
12. | Stanhope-Baker, P., K. Hudson, A. Shaffer, A. Constantinescu, and M. Schlissel. 1996. Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro. Cell. 85: 887-897 [Medline]. |
13. | Sleckman, B., J. Gorman, and F. Alt. 1996. Accessibility control of antigen-receptor variable-region gene assembly: role of cis-acting elements. Annu. Rev. Immunol. 14: 459-481 [Medline]. |
14. | Yancopoulos, G., B. Malynn, and F. Alt. 1988. Developmentally regulated and strain-specific expression of murine VH gene families. J. Exp. Med. 168: 417-435 [Abstract]. |
15. | Wu, G., and C. Paige. 1986. VH gene family utilization in colonies derived from B and pre-B cells detected by the RNA colony blot assay. EMBO (Eur. Mol. Biol. Organ.) J. 5: 3475-3481 [Abstract]. |
16. | Marshall, A., G. Wu, and C. Paige. 1996. Frequency of VH81X usage during B cell development. Initial decline in usage is independent of Ig heavy chain cell surface expression. J. Immunol. 156: 2077-2084 [Abstract]. |
17. | Dildrop, R., U. Krawinkel, E. Winter, and K. Rajewsky. 1985. VH-gene expression in murine lipopolysaccharide blasts distributes over the nine known VH-gene groups and may be random. Eur. J. Immunol. 15: 1154-1156 [Medline]. |
18. |
Raulet, D..
1989.
The structure, function, and molecular
genetics of the ![]() ![]() |
19. |
Ito, K.,
M. Bonneville,
Y. Takagaki,
N. Nakanishi,
O. Kanagawa,
E. Krecko, and
S. Tonegawa.
1989.
Different ![]() ![]() |
20. | Havran, W., and J. Allison. 1988. Developmentally ordered appearance of thymocytes expressing different T cell antigen receptors. Nature. 335: 443-445 [Medline]. |
21. | Garman, R., P. Doherty, and D. Raulet. 1986. Diversity, rearrangement and expression of murine T cell gamma genes. Cell. 45: 733-742 [Medline]. |
22. |
Goldman, J.,
D. Spencer, and
D. Raulet.
1993.
Ordered rearrangement of variable region genes of the T cell receptor ![]() |
23. |
Iwashima, M.,
A. Green,
M. Davis, and
Y. Chien.
1988.
Variable region (V![]() ![]() |
24. |
Chien, Y.,
M. Iwashima,
D. Wettstein,
K. Kaplan,
J. Elliott,
W. Born, and
M. Davis.
1987.
T cell receptor ![]() |
25. | Schlissel, M., and P. Stanhope-Baker. 1997. Accessibility and the developmental regulation of V(D)J recombination. Semin. Immunol. 9: 161-170 [Medline]. |
26. | Nelsen, B., and R. Sen. 1992. Regulation of immunoglobulin gene transcription. Int. Rev. Cytol. 133: 121-148 [Medline]. |
27. | Murre, C., G. Bain, M.A. van Dijk, I. Engel, B.A. Furnari, M.E. Massari, J.R. Matthews, M.W. Quong, R.R. Rivera, and M.H. Stuiver. 1994. Structure and function of helix-loop-helix proteins. Biochim. Biophys. Acta. 1218: 129-135 [Medline]. |
28. | Kadesch, T.. 1992. Helix loop helix proteins in the regulation of immunoglobulin gene transcription. Immunol. Today. 13: 31-36 [Medline]. |
29. | Bain, G., E. Maandag, D. Izon, D. Amsen, A. Kruisbeek, B. Weintraub, I. Krop, M. Schlissel, A. Feeney, M. van Roon, et al . 1994. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell. 79: 885-892 [Medline]. |
30. | Sun, X.-H.. 1994. Constitutive expression of the Id1 gene impairs mouse B cell development. Cell. 79: 893-900 [Medline]. |
31. | Zhuang, Y., P. Soriano, and H. Weintraub. 1994. The helix-loop-helix gene E2A is required for B cell formation. Cell. 79: 875-884 [Medline]. |
32. | Bain, G., E.C. Robanus, Maandag, H.P. te Riele, A. Feeney, A. Sheehy, M. Schlissel, S. Shinton, R. Hardy, and C. Murre. 1997. Both E12 and E47 allow commitment to the B cell lineage. Immunity. 6: 145-154 [Medline]. |
33. |
Bain, G.,
I. Engel,
E.C. Robanus,
Maandag,
H.P. te Riele,
J. Voland,
L. Sharp,
J. Chun,
B. Huey,
D. Pinkel, and
C. Murre.
1997.
E2A deficiency leads to abnormalities in ![]() ![]() |
34. |
Kee, B., and
C. Murre.
1998.
Induction of early B cell factor
(EBF) and multiple B lineage genes by the basic helix-loop-helix transcription factor E12.
J. Exp. Med.
188:
699-713
|
35. | Choi, J., C.-P. Shen, H. Radomska, L. Eckhardt, and T. Kadesch. 1996. E47 activates the Ig-heavy chain and TdT loci in non-B cells. EMBO (Eur. Mol. Biol. Organ.) J. 15: 5014-5021 [Abstract]. |
36. | Schlissel, M., A. Voronova, and D. Baltimore. 1991. Helix loop helix transcription factor E47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrangement in a pre-T-cell line. Genes Dev. 5: 1367-1376 [Abstract]. |
37. | Sigvardsson, M., M. O'Riordan, and R. Grosschedl. 1997. EBF and E47 collaborate to induce expression of the endogenous immunoglobulin surrogate light chain genes. Immunity. 7: 25-36 [Medline]. |
38. |
Boismenu, R., and
W. Havran.
1994.
Modulation of epithelial cell growth by intraepithelial ![]() ![]() |
39. |
Livak, F.,
H. Patrie,
I. Crispe, and
D. Schatz.
1995.
In-frame
TCR ![]() ![]() ![]() ![]() ![]() |
40. |
Asarnow, D.,
W. Kuziel,
M. Bonyhadi,
R. Tigelaar,
P. Tucker, and
J. Allison.
1988.
Limited diversity of ![]() ![]() |
41. | Asarnow, D., T. Goodman, L. LeFrancois, and J. Allison. 1989. Distinct antigen receptor repertoires of two classes of murine epithelium-associated T cells. Nature. 341: 60-62 [Medline]. |
42. | Roth, D., C. Zhu, and M. Gellert. 1993. Characterization of broken DNA molecules associated with V(D)J recombination. Proc. Natl. Acad. Sci. USA. 90: 10788-10792 [Abstract]. |
43. | Schlissel, M., A. Constantinescu, T. Morrow, M. Baxter, and A. Peng. 1993. Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev. 7: 2520-2532 [Abstract]. |
44. |
Heemskerk, M.,
B. Blom,
G. Nolan,
A. Stegmann,
A. Bakker,
K. Weijer,
P. Res, and
H. Spits.
1997.
Inhibition of T
cell and promotion of natural killer cell development by the
dominant negative helix loop helix factor Id3.
J. Exp. Med.
186:
1597-1602
|
45. |
Petrie, H.,
R. Scollay, and
K. Shortman.
1992.
Commitment
to the T-cell receptor ![]() ![]() ![]() ![]() |
46. |
Godfrey, D.,
J. Kennedy,
T. Suda, and
A. Zlotnik.
1993.
A
developmental pathway involving four phenotypically and
functionally distinct subsets of CD3![]() ![]() ![]() |
47. |
Dudley, E.,
M. Girardi,
M. Owens, and
A. Hayday.
1995.
![]() ![]() ![]() ![]() |
48. |
Raulet, D.,
D. Spencer,
Y.-H. Hsiang,
J. Goldman,
M. Bix,
N.-S. Liao,
M. Zijlstra,
R. Jaenisch, and
I. Correa.
1991.
Control of ![]() ![]() |
49. |
Carding, S.,
S. Kyes,
E. Jenkinson,
R. Kingston,
K. Bottomly,
T. Owen, and
A. Hayday.
1990.
Developmentally
regulated fetal thymic and extrathymic T-cell receptor ![]() ![]() |
50. |
Lafaille, J.,
A. DeCloux,
M. Bonneville,
Y. Takagaki, and
S. Tonegawa.
1989.
Junctional sequences of T cell receptor ![]() ![]() ![]() ![]() |
51. |
McVay, L.,
S. Carding,
K. Bottomly, and
A. Hayday.
1991.
Regulated expression and structure of T cell receptor ![]() ![]() |
52. |
Elliot, J.,
E. Rock,
P. Patten,
M. Davis, and
Y. Chien.
1988.
The adult T-cell receptor ![]() |
53. | Heilig, J., and S. Tonegawa. 1986. Diversity of murine gamma genes and expression in fetal and adult T lymphocytes. Nature. 322: 836-840 [Medline]. |
54. |
Nakajima, P.,
J. Menetski,
D. Roth,
M. Gellert, and
M. Bosma.
1995.
V-D-J rearrangements at the T cell receptor ![]() ![]() ![]() |
55. | Zhu, C., M. Bogue, D.-S. Lim, P. Hasty, and D. Roth. 1996. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell. 86: 379-389 [Medline]. |
56. |
Cortes, P.,
F. Weis-Garcia,
Z. Misulovin,
A. Nussenzweig,
J.-S. Lai,
G. Li,
M. Nussenzweig, and
D. Baltimore.
1996.
In
vitro V(D)J recombination: signal joint formation.
Proc. Natl.
Acad. Sci. USA.
93:
14008-14013
|
57. | Lewis, S.. 1994. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv. Immunol. 56: 27-150 [Medline]. |
58. | Eastman, Q., T. Leu, and D. Schatz. 1996. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature. 380: 85-88 [Medline]. |
59. | Blackwell, T., M. Moore, G. Yancopoulos, H. Suh, S. Lutzker, E. Selsing, and F. Alt. 1986. Recombination between immunoglobulin variable region gene segments is enhanced by transcription. Nature. 324: 585-590 [Medline]. |
60. | Ferrier, P., B. Krippl, T.K. Blackwell, A.J.W. Furley, H. Suh, A. Winoto, W.D. Cook, L. Hood, F. Costantini, and F.W. Alt. 1990. Separate elements control DJ and VDJ rearrangement in a transgenic recombination substrate. EMBO (Eur. Mol. Biol. Organ.) J. 9: 117-125 [Abstract]. |
61. |
Lennon, G., and
R. Perry.
1990.
The temporal order of appearance of transcripts from unrearranged and rearranged Ig
genes in murine fetal liver.
J. Immunol.
144:
1983-1987
|
62. |
Martin, D.,
R. Huang,
T. LeBien, and
B. VanNess.
1991.
Induced rearrangement of ![]() ![]() ![]() |
63. | Schlissel, M.S., and D. Baltimore. 1989. Activation of immunoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription. Cell. 58: 1001-1007 [Medline]. |
64. | Yancopoulos, G., and F.W. Alt. 1985. Developmentally controlled and tissue-specific expression of unrearranged VH gene segments. Cell. 40: 271-281 [Medline]. |
65. | Yancopoulos, G., T. Blackwell, H. Suh, L. Hood, and F. Alt. 1986. Introduced T cell receptor variable region gene segments recombine in pre-B cells: evidence that B and T cells use a common recombinase. Cell. 44: 251-259 [Medline]. |
66. |
Henthorn, P.,
M. Kiledjian, and
T. Kadesch.
1990.
Two distinct transcription factors that bind the immunoglobulin enhancer µE5/![]() |
67. |
Ho, I.-C.,
L.-H. Yang,
G. Morle, and
J. Leiden.
1989.
A
T-cell-specific transcriptional enhancer element 3' of C![]() ![]() |
68. | Murre, C., P.S. McCaw, and D. Baltimore. 1989. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 56: 777-783 [Medline]. |
69. |
Takeda, J.,
A. Cheng,
F. Mauxion,
C.A. Nelson,
R.D. Newberry,
W.C. Sha,
R. Sen, and
D.Y. Loh.
1990.
Functional
analysis of the murine T-cell receptor ![]() |
70. |
Lauzurica, P., and
M. Krangel.
1994.
Temporal and lineage-specific control of T cell receptor ![]() ![]() ![]() ![]() |
71. |
Capone, M.,
F. Watrin,
C. Fernex,
B. Horvat,
B. Krippl,
L. Wu,
R. Scollay, and
P. Ferrier.
1993.
TCR![]() ![]() |
72. |
Okada, A.,
M. Mendelsohn, and
F. Alt.
1994.
Differential activation of transcription versus recombination of transgenic T
cell receptor ![]() |
73. |
Roberts, J.,
P. Lauzurica, and
M. Krangel.
1997.
Developmental regulation of VDJ recombination by the core fragment of the T cell receptor ![]() |
74. |
Mombaerts, P.,
A. Clarke,
M. Rudnicki,
J. Iacomini,
S. Itohara,
J. Lafaille,
L. Wang,
Y. Ichikawa,
R. Jaenisch,
M. Hooper, and
S. Tonegawa.
1992.
Mutations in T-cell antigen receptor genes ![]() ![]() |
75. |
Mallick, C.,
E. Dudley,
J. Viney,
M. Owen, and
A. Hayday.
1993.
Rearrangement and diversity of T cell receptor ![]() ![]() |
76. | Shinkai, Y., S. Koyasu, K. Nakayama, K. Murphy, D. Loh, E. Reinherz, and F. Alt. 1993. Restoration of T-cell development in RAG-2 deficient mice by functional TCR transgenes. Science. 259: 822-825 [Medline]. |
77. |
Redondo, J.,
S. Hata,
C. Brocklehurst, and
M. Krangel.
1990.
A T cell specific transcriptional enhancer within the
human T cell receptor ![]() |
78. |
Redondo, J.,
J. Pfohl, and
M. Krangel.
1991.
Identification
of an essential site for transcriptional activation within the
human T cell receptor ![]() |
79. | McMurry, M., C. Hernandez-Munain, P. Lauzurica, and M. Krangel. 1997. Enhancer control of local accessibility to V(D)J recombinase. Mol. Cell. Biol. 17: 4553-4561 [Abstract]. |
80. | Clausell, A., and P. Tucker. 1994. Functional analysis of the V gamma 3 promoter of the murine gamma delta T-cell receptor. Mol. Cell. Biol. 14: 803-814 [Abstract]. |
81. | Zhuang, Y., P. Cheng, and H. Weintraub. 1996. B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB. Mol. Cell. Biol. 16: 2898-2905 . |