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Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and
Institute for Genetics, University of Cologne, Cologne, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and
ß-chains, respectively. The Ag-binding domains of Ig and TCR chains
are encoded by variable gene segments, V, D, and J, for IgH and
TCR ß-chains, and V and J for IgL and TCR -chains. Assembly
of the variable gene segments through V(D)J recombination is required
before the Ag receptor genes can be expressed (1, 2, 3). Because
vertebrates are diploid and one-third of V(D)J recombination events
result in the expression of functional protein chains (1, 4, 5),
one-fifth of lymphocytes are expected to express any given Ag receptor
chain from both alleles. However, the majority of lymphocytes express
Ag receptors from only one of the two alleles. This phenomenon is known
as allelic exclusion (reviewed in Refs. 6 and 7). Allelic exclusion was
first observed in rabbits using allotypic markers for Ig heavy chains.
Individual B cells were found to express Ig heavy chain from only one
of the two alleles (8, 9). Similar studies with human and mouse B cells
yielded the same results. Using Vß-specific Abs, 99% of T cells were
also found to express TCR ß-chain from only one of the two alleles
(10, 11). Compared with the IgH, IgL, and TCR-ß genes, allelic
exclusion at the TCR- gene locus is less stringent (12, 13).
Over the years, many studies were performed in an attempt to elucidate
the mechanisms that underlie the control of allelic exclusion. The
presence of DJ rearrangements at the second excluded IgH locus in
mature B cells and at the TCR-ß locus in mature T cells strongly
suggests that allelic exclusion is achieved through a mechanism
regulating V(D)J recombination (5, 14). According to current
understanding, each differentiating lymphocyte has two chances of
assembling a productive VDJ rearrangement. Because DJ rearrangements
occur on both alleles, the regulated step is at the V to DJ
rearrangement step. If the first VDJ rearrangement is productive, then
V to DJ joining on the other allele is inhibited. If the initial VDJ
rearrangement is nonproductive, then V to DJ rearrangement can proceed
on the second allele. Feedback inhibition of further rearrangement by a
prior productive recombination event has been demonstrated by studies
with transgenic and gene-targeted mice. Expression of the
membrane-bound, but not the secreted µ heavy chain inhibits
VH to DJH rearrangements at the endogenous IgH
loci (15, 16, 17, 18). Targeted deletion of µm exons also
abolishes IgH gene allelic exclusion (19). Similarly, expression of a
TCR-ß transgene blocks rearrangements of the endogenous loci at the
DßJß stage (20, 21). Complete rearrangement and expression of the
endogenous alleles occur only when the TCR-ß transgene is deleted
(22). In pro-B cells, the µ heavy chains form pre-B cell receptors by
associating with surrogate light chains, 
5 and VpreB,
and Ig/Igß heterodimers
(pre-BCR)4 (23, 24). Signals
initiated from the pre-BCR mediate not only IgH gene allelic exclusion,
but also pro-B to pre-B cell differentiation and expansion of pre-B
cells (6, 7, 25, 26). In an analogous manner, TCR-ß chains associate
with the pre-TCR -chains (pT) and CD3 components to form pre-TCRs
(pre-TCR) (27, 28), which signals for TCR-ß gene allelic exclusion as
well as DN to DP thymocyte differentiation and the expansion of DP
thymocytes (29, 30, 31). Thus, allelic exclusion at IgH and TCR-ß loci is
mediated through a feedback inhibition of V to DJ rearrangement,
occurring during pro-B to pre-B cell and DN and DP thymocyte
differentiation, respectively.
The feedback signal initiated from pre-BCR and pre-TCR could effect
allelic exclusion by regulating recombinase activity as well as
modulating substrate gene segment accessibility. It is known that pro-B
to pre-B cell and DN to DP thymocyte differentiation is accompanied by
a transient down-regulation of RAG-1 and RAG-2 gene transcription (32, 33). Furthermore, RAG-2 protein is degraded before cells enter the S
phase, as occurring in rapid proliferating cells during pre-B cell and
DP thymocyte expansion (34, 35, 36). Although the down-regulation of RAG
expression following the feedback signaling has been postulated to
prevent further V to DJ rearrangement on the second allele, thereby
enabling the establishment of allelic exclusion (37), coupling of V(D)J
recombination to the cell cycle is not essential for allelic exclusion
(36, 38). RAG genes are re-expressed in pre-B cells and DP thymocytes
for rearranging IgL and TCR- genes, respectively (39, 40, 41). To
prevent V to DJ rearrangement on the second IgH and TCR-ß allele in
pre-B cells and DP thymocytes, respectively, an additional mechanism is
required to specifically maintain their allelic exclusion. Because
V(D)J recombinations at all Ig and TCR loci are mediated by conserved
recombination signal sequences (1, 3), it seems unlikely that the
control is achieved solely by modifying the recombinase so that it no
longer utilizes IgH or TCR-ß alleles as substrates. Rather, it seems
more likely that allelic exclusion is maintained by controlling the
accessibility of the involved gene segments to the recombinase (6, 7).
In this model, signals from pre-BCR and pre-TCR induce the
differentiation and proliferation of pre-B cells and DP thymocytes as
well as stimulate chromatin structural remodeling at the IgH and
TCR-ß loci, resulting in inaccessibility to the recombinase.
Supporting this model, exogenously added RAG-1 and RAG-2 core proteins
can introduce dsDNA breaks at the recombination signal sequences
flanking DJH and VH gene segments in nuclei of
pro-B cells, but not pre-B cells (42). However, chromatin changes
associated with allelic exclusion, their molecular nature, and the
underlying cis-regulatory elements involved still need to be
defined to prove this model.
Changes in DNase I hypersensitive sites, DNA methylation, and
transcription have traditionally been used as indicators of changes in
chromatin structure (43, 44, 45, 46, 47, 48). V(D)J recombination activity is
associated with increased DNase I hypersensitivity, hypomethylation,
and transcription. Before recombination, IgH and 
locus becomes
hypersensitive to DNase I treatment, hypomethylated, and actively
transcribed (49, 50, 51, 52). Hypomethylation of recombination substrates also
promotes their rearrangements in both cell lines and transgenic mice
(53, 54). In addition, transcription enhancers such as Eµ, E,
Eß, and E are all known to promote recombination of their
respective genomic loci and minilocus recombination substrates,
although their mechanism of action remains elusive (41, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64).
According to current understanding, allelic exclusion should be
associated with a decrease in HS, hypermethylation, and diminished
transcription at the IgH and TCR-ß locus, all indicators of an
inaccessible locus.
To characterize the chromatin changes associated with allelic exclusion at the DP stage of thymocyte development and to identify the cis-regulatory elements involved, we assayed for DNase I hypersensitive sites, DNA methylation, and transcription in DN and DP thymocytes in a 100-kb region of the TCR-ß locus starting 20 kb upstream of Dß1 and ending 50 kb downstream of Vß14. Contrasting the current understanding of the control of V(D)J recombination, our data suggest that DNase I hypersensitivity, DNA methylation, and transcription in general may not be reliable molecular indicators of inaccessibility to the recombinase during allelic exclusion. In addition, we demonstrate that three of the DNase I hypersensitive sites identified are not critical for TCR-ß gene allelic exclusion. These findings suggest that alternative cis-regulatory elements within the assayed region such as Eß enhancer and/or in the upstream Vß region are likely to play a dominant role in allelic exclusion control.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RAG-1- and RAG-2-deficient mice were obtained from Drs. Susumu
Tonegawa (Massachusetts Institute of Technology, MA) and Fred Alt
(Harvard Medical School, MA), respectively (65, 66). Activated
lck transgenic mice were from Dr. Roger Perlmutter
(University of Washington, WA) (67). Two different TCR-ß transgenic
mouse strains, one from Dr. Mark Davis (Stanford University School of
Medicine, CA) and one from Dr. Eugenia Spanopoulou (Mount Sinai School
of Medicine, NY), were used in the present studies. The former was
constructed from a genomic TCR-ß transgene, and the latter from a
cDNA TCR-ß transgene (68, 69). The two strains of TCR-ß transgenic
mice are essentially the same in our assays, except that Jß2-Cß2
intronic probes did not hybridize to the cDNA TCR-ß transgene, and
thus simplified Southern blot hybridization patterns in this assay. The
same pattern of DNase I hypersensitive sites was detected in DN
thymocytes from either RAG-1- or RAG-2-deficient mice (data not shown).
Similarly, the same pattern of DNase I hypersensitive sites was
detected in DP thymocytes from anti-CD3
Ab-treated RAG-1- or
RAG-2-deficient mice (data not shown), using a protocol developed by
Shinkai and Alt (70), and later experiments were conducted with
RAG-2-deficient mice only. TCR-ß and lck transgenes were
also on the RAG-2-deficient background. Cre transgenic deleter mouse
was from Klaus Rajewsky (University of Cologne, Germany) (71). NZW mice
were from The Jackson Laboratory (Bar Harbor, ME). The genomic TCR-ß
transgene was also introduced into the NZW mice and mice harboring a
targeted deletion of HS1 (see below). Mice were maintained under
specific pathogen-free condition in the animal facilities at
Massachusetts Institute of Technology (Cambridge, MA).
Probes and genomic clones
Two cosmid clones containing Dß1, Jß1, Cß1, Dß2,
Jß2, Cß2, Vß14, and 30 kb downstream of Vß14 were kindly
provided by Dr. Marie Malissen (Centre d’Immunologie, INSERM-CNRS de
Marseille-Luminy, France) (72). A genomic clone extending 20 kb further
downstream of the existing clones was identified by PCR from a
Stratagene (La Jolla, CA) AKR mouse SuperCos library and by filter
hybridization using probe H (Table I
and
Fig. 2A). Probes isolated from the cosmid clones are
summarized in Table I and in Figure 2A. The probes used to
assay for homologous recombination in ES cells and for allelic
exclusion in thymocytes are as follows: probe 1, 0.6-kb
HindIII-PstI fragment; probe 2, 0.9-kb
EcoRV-PstI fragment; and probe 3 is the same as
probe H. Probes used for Northern hybridization were provided by Dr.
Susumu Tonegawa and are as follows: Cß2, a 430-bp cDNA fragment;
Vß8, a 190-bp EcoRI-PstI fragment; and Vß14,
a 668-bp AccI genomic fragment.
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Single cell suspensions of thymocytes were prepared for the
isolation of DNA and RNA. Because RAG-deficient thymus contains only a
few million cells compared with a hundred million cells in normal
thymus (Table II), larger numbers of
thymus were pooled to obtain enough cells for DNA and RNA isolation.
Special caution was taken to remove thymic stromal cells by filtering
through a nylon mesh. Thymocyte preparations were assayed for purity by
FACS staining for CD4 and CD8. In general, thymocytes contained at
least 95% DN cells from RAG mice and 95% DP cells from
TCR-ß/RAG, lck/RAG, and CD3/RAG mice. Thymocytes of
TCR-ß mice contained 80% DP cells.
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Nuclei from DN and DP thymocytes were prepared according to Forrester et al. (73). Briefly, 1 x 108 cells were washed in cold PBS, centrifuged at 1000 rpm for 5 min, and resuspended into 10 ml reticulocyte standard buffer (RSB) containing 10 mM Tris-Cl, pH 8, 10 mM NaCl, and 10 mM MgCl2. After slowly adding an equal volume of cold RSB containing 0.2% Nonidet P-40, the mixture was kept on ice for 10 min and centrifuged at 900 rpm for 5 min, and the resulting nuclei were resuspended in 2 ml of RSB. Two-hundred-microliter aliquots of nuclei were then treated with a range of DNase I (0.125–15 µg/ml) at 37°C, and the digestion was stopped by adding 200 µl of 2x lysis buffer containing 1.2 M NaCl, 20 mM Tris-Cl, pH 8, 10 mM EDTA, and 1% SDS. The lysates were treated with proteinase K overnight at 55°C, and DNA isolated by phenol-chloroform extraction.
Southern and Northern hybridization
For Southern analysis, 10 to 15 µg of DNA from DN and DP
thymocytes were digested with specific enzymes and were fractionated on
a 0.9% agarose gel. After denaturation and neutralization, DNA was
transferred to Z-probe filters and hybridized with specific probes
labeled with [-32P]dCTP. For Northern analysis,
10 or 20 µg of RNA from DN and DP thymocytes were fractionated on a
1% formamide-agarose gel. RNA was transferred onto Z-probe filters and
hybridized with specific probes labeled with
[-32P]dCTP. Filters were washed twice for 30 min in
2x SSC and 0.1% SDS at 65°C. Hybridization signals were detected by
phosphor imaging and autoradiography.
Targeted mutagenesis of HS1
Mice harboring a targeted deletion of HS1 were constructed by
targeted mutation in embryonic stem (ES) cells and then deriving a
mouse strain from the mutant ES cells. The targeting vector was
constructed by replacing a 780-bp XcmI-BglII
fragment containing HS1 with a phosphoglycerate kinase promoter-driven
neomycin-resistance (neo) gene flanked by loxP sites (Fig. 5). The
targeting vector contained 5.9-kb and 8.2-kb homologous sequences at
the 5' and 3' ends, respectively, and the thymidine kinase gene was
inserted just outside of the 3' homologous sequence. The targeting
vector was transfected into J1 ES cells, and doubly resistant clones
were picked from three independent transfections. Homologous
recombinants were identified by EcoRI digestion of DNA and
hybridization with a 0.6-kb HindIII-PstI fragment
that does not hybridize to randomly integrated constructs. Among 258
clones analyzed, 31 yielded the expected hybridizing fragments at 10
and 7.4 kb from the normal and the targeted alleles, respectively. The
correct targeting of the 31 positive ES cell clones was confirmed by
Southern blotting using the deleted 780-bp fragment as a probe, and
then a 0.9-kb BglII-NcoI fragment containing the
Eß as a probe. ES cells from 5 of the 31 clones were injected into
C57BL/6 blastocysts. Chimeras with more than 95% agouti coat color
were generated from all five clones and were bred with cre transgenic
deleter mice for germline transmission and deletion of the introduced
neo gene at the same time (71). Heterozygous mutant mice were interbred
to obtain homozygous mutant mice, and the genomic TCR-ß transgene was
bred onto the mutant background. Data shown are from littermates and/or
age-matched mice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify chromatin changes associated with allelic exclusion,
we chose the TCR-ß locus for study because it fulfilled two
requirements (Fig. 1). First, the DNA
region in which we sought to characterize differences in DNase I
hypersensitive sites, DNA methylation, and transcription could be
narrowed down to a manageable size. The IgH locus, which spans several
megabases (74), is too large for such a study. In contrast, the entire
TCR-ß locus is within 600 kb (75, 76). Although it is still not
feasible to analyze the entire TCR-ß locus, we postulated that the
chromatin changes associated with TCR-ß allelic exclusion would most
likely occur in a 100-kb region, starting 20 kb upstream of Dß1 and
ending 50 kb downstream of Vß14 (Fig. 1, see
Discussion). Second, DN and DP thymocytes
representing the two developmental stages before and after allelic
exclusion were readily available (77). Although the normal thymocyte
population is a mixture containing approximately 5% DN, 80% DP, and
15% CD4 or CD8 single-positive (SP) thymocytes, a relatively pure
population of DN and DP thymocytes can be obtained easily from several
mutant/transgenic mouse strains (Table II
). In RAG-2-deficient mice
(referred to as RAG mice), thymocytes are all DN due to a block in
V(D)J recombination (66). These DN thymocytes are arrested at the
CD44-CD25+ stage at which TCR-ß gene
recombination normally occurs (78, 79). Thus, the status of DNase I
hypersensitivity, DNA methylation, and transcription in the region most
likely represents the chromatin structure that is accessible to the
V(D)J recombinase. On the other hand, introduction of a functionally
assembled TCR-ß transgene or an activated form of protein tyrosine
kinase lck transgene onto the RAG-2-deficient background
(referred to as TCR-ß/RAG and lck/RAG mice, respectively),
or injection of anti-CD3 Abs into RAG-2-deficient mice (referred
to as CD3/RAG mice) restores the differentiation of DP thymocytes to
normal levels (Table II) (70, 80, 81, 82). Thymocytes in TCR-ß/RAG,
lck/RAG, and CD3/RAG mice do not undergo further
differentiation into SP T cells due to their inability to rearrange the
TCR- locus and express a functional TCR, and thus are more than 95%
DP. As a result of signaling through the pre-TCR pathway (28), the
endogenous TCR-ß loci in these DP thymocytes are likely to have
undergone chromatin changes associated with allelic exclusion, even
though they are still in the germline configuration. Nevertheless, to
more closely mimic the normal situation, we also analyzed DNase I
hypersensitive sites in thymocytes (80% DP) from TCR-ß transgenic
mice (RAG+) (referred to as TCR-ß mice) in which the
endogenous TCR-ß loci undergo DßJß rearrangement (Table II) (20, 21). If the same pattern of DNase I hypersensitive sites, methylation,
and transcription is observed in DP thymocytes from TCR-ß,
TCR-ß/RAG, lck/RAG, and CD3/RAG mice, these changes most
likely represent the chromatin structure that is inaccessible to the
V(D)J recombinase.
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To begin our investigation, DNase I hypersensitive sites at the
TCR-ß locus were assayed in DN and DP thymocytes. Thymocytes
were harvested from all five mutant/transgenic mice (Table II).
Aliquots were analyzed by flow cytometry for CD4 and CD8 expression.
Thymocytes were 98% DN from RAG mice; 97% DP from TCR-ß/RAG,
lck/RAG, and CD3/RAG mice; and 3% DN, 80% DP, and 17% SP
from TCR-ß mice (data not shown). Nuclei were prepared from the
remaining thymocytes and treated with different amounts of DNase I (see
Materials and Methods). DNA isolated was assayed for
DNase I hypersensitive sites by Southern blotting using specific probes
isolated from the 100-kb region. Figure 2A shows the strategy of the
analysis and summarizes probes used and their hybridizing fragments,
and the latter are described in more detail in Table I. Southern
blotting using probes B, C, E, F, and K detected DNase I hypersensitive
sites. As an example, the identification of hypersensitive site 1 to 4
with probe E is detailed below. Southern blotting using probes A, D, G,
H, I, J, L, and M did not detect any hypersensitive sites, and these
assays will not be described further.
DNA was digested by BamHI plus BglI, and the
blots were hybridized with probe E, a 1.6-kb
HindIII-BglI fragment from 5' of the Vß14 gene
segment (Table I and Fig. 2A). In addition to the
expected 6.7-kb fragment, with increased DNase I concentrations, four
smaller hybridizing fragments representing HS1, HS2, HS3, and HS4 were
detected at 4.4, 3, 1.2, and 0.8 kb, respectively (Fig. 2B). The minor fragments (HS3 and HS4) were not
hybridization to sequences between HS1 and HS2, because probe E does
not extend to the region (Fig. 2A). Furthermore, they
were detected by probe E when NcoI that cuts in between HS2
and HS3 was used to digest DNAs (data not shown). HS2 and HS3 were
detected in all five DNA samples, and therefore are present in both DN
and DP thymocytes. In contrast, HS1 was only faintly detectable in DNA
from DN thymocytes and was strongly induced in DNAs from DP thymocytes.
Similarly, HS4 appeared to be present only in DP thymocytes from
CD3/RAG, lck/RAG, TCR-ß/RAG, and TCR-ß mice (Fig. 2B).
Based on the fragment sizes, HS1 corresponds to a previously identified DNase I hypersensitive site found in pre-T cell lines, and HS2 corresponds to Eß (83, 84, 85, 86). This was further confirmed by digesting DNA with the same enzymes and hybridization with the same probes as in previous studies (data not shown) (85). In addition, DNA was digested with BamHI plus one of the following enzymes, BglI, EcoRI, NcoI, PstI, XbaI, or HpaI, which cut at different positions successively downstream of HS1. Hybridization of the filter with probe D from the BamHI (5') end of the fragment gave rise to different sizes of the endogenous fragments (data not shown). Comparing the positions of HS1 and HS2 with the endogenous fragments enabled a more precise determination of the locations of HS1 and HS2 and showed that HS1 and HS2 colocalize with the previously identified hypersensitive sites.
In summary, we have assayed for DNase I hypersensitive sites in a
100-kb region, starting 20 kb upstream of Dß1 and ending 50 kb
downstream of Vß14, and have found a total of 11 DNase I
hypersensitive sites (Fig. 2). All of the identified sites are located
within a 30-kb region, from 3 kb upstream of Dß1 to 3 kb downstream
of Vß14. Eight of the sites (HS2, HS3, HS5–8, HS10, and HS11) are
present in both DN and DP thymocytes; three (HS1, HS4, and HS9) are
strongly induced in DP thymocytes. These data show that in DP
thymocytes, the region of the TCR-ß locus that we have analyzed
remains accessible, and in some regions there are increases in
accessibility to factor-binding activities.
Analysis for DNA methylation
To further characterize chromatin changes at the TCR-ß
locus, DNA methylation status was assayed in DN and DP thymocytes.
Within the 100-kb region in which DNase I hypersensitive sites were
assayed, 46 kb containing all of the identified DNase I hypersensitive
sites have been sequenced (L. Rowen and L. Hood, personal
communication). Within this 46-kb region, there are 23
MspI/HpaII sites and 19 HhaI sites,
most of them clustered in two subregions, one surrounding Jß2 and the
other downstream of the Vß14 gene segment (Fig. 3C). The methylation
status of these sites in DN and DP thymocytes was determined by
Southern blotting using methylation-sensitive enzymes.
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B). Sequence data showed
the presence of one MspI/HapII site and three
HhaI sites in the 5.1-kb region. EcoRI and
MspI digestion yielded the two expected size fragments, one
at 3.8 kb and the other at 1.3 kb in all of the samples (Fig. 3A, lanes 1, 4, 7, and
10). EcoRI and HpaII digestion
of DNA from DN thymocytes of RAG mice yielded only the 5.1-kb fragment
(Fig. 3A, lane 2), indicating that
HpaII site is methylated in these cells. In contrast, DNA
from DP thymocytes of CD3/RAG, lck/RAG, and to a lesser
extent, TCR-ß/RAG mice yielded the 5.1-kb as well as the 3.8- and
1.3-kb fragments (Fig. 3A, lanes 5, 8,
and 11). Quantitation of the fragments showed that 5
to 20% of the DNAs are demethylated at the HpaII site in DP
thymocytes.
EcoRI and HhaI digestion of DNA from DN
thymocytes generated 2.6- and 2.4-kb hybridizing fragments at equal
intensities (Fig. 3A, lane 3). Since there
are three HhaI sites in the fragment, the only possibility
for this hybridization pattern is that the first HhaI site
is unmethylated and the other two sites are completely methylated (Fig. 3B). Similarly, this HhaI site is also
unmethylated in DP thymocytes from TCR-ß/RAG, CD3/RAG, and
lck/RAG mice because the largest and dominant hybridizing
fragments are at 2.6 and 2.5 kb (Fig. 3A, lanes
6, 9, and 12). In addition, two
smaller hybridizing fragments of 1.7 and 1.1 kb were detected at
submolar levels in DNA from DP thymocytes. (A 0.6-kb fragment was also
visible on the original autoradiogram. The 0.6-kb fragment was expected
to hybridize least because the entire 5.1-kb fragment was used as
probe.) These patterns were generated when the other two
HhaI sites were partially digested due to their partial
demethylation. Quantitation of the fragments suggests 5% of the DNA is
demethylated at the other two sites. Together, these data suggest that
among the four detectable methylation sites in the 5.1-kb
EcoRI fragment, one is unmethylated in both DN and DP
thymocytes, and the other three sites are methylated in DN thymocytes
and partially demethylated in DP thymocytes.
We have determined the methylation status of most of the
MspI/HpaII and HhaI sites in the 46-kb
region. The results are summarized in Figure 3C. First, most
of the sites in and around Jß2 gene segments are unmethylated in both
DN and DP thymocytes. Three of the sites in the region become
completely unmethylated in DP thymocytes. Second, among the four sites
immediately downstream of the Vß14 gene segment, one site is
unmethylated in both DN and DP thymocytes, and the other three become
partially demethylated during DN to DP thymocyte differentiation.
Third, most sites farther downstream of the Vß14 gene segment,
including the cluster of nine HhaI sites, are methylated in
both DN and DP thymocytes. These findings suggest that DN to DP
thymocyte differentiation is associated with demethylation in the
TCR-ß locus.
Analysis for germline transcription
Chromatin changes at the TCR-ß locus were also assayed by
comparing germline transcripts from the locus in DN and DP thymocytes.
Northern blot hybridization was performed with total RNA (10 µg) from
thymocytes of RAG, CD3/RAG, lck/RAG, and TCR-ß/RAG mice.
As controls, RNA from the EL4 T cell line and a thymoma derived from a
DN thymocyte of a p53 and RAG-1 double-knockout mouse were used.
Hybridization with a 430-bp Cß2 cDNA probe revealed the presence of
1- and 1.6-kb germline transcripts from DN thymocytes that comigrated
with those from the control thymoma (Fig. 4A). These germline
transcripts were the same sizes as those identified previously from the
TCR-ß locus in DN thymocytes from normal and mutant mice (30, 78, 87). The unique 1.3-kb transcript present in RNA from TCR-ß/RAG
thymocytes comigrated with the mature TCR-ß transcript from the EL4 T
cell line and was most likely derived from the transgene in these mice.
The hybridization signal larger than 28S was not informative, as it may
have resulted from trace genomic DNA contamination in the RNA
samples.
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B). These transcripts represent germline
transcription from the endogenous Vß14 gene segment, as the TCR-ß
transgene in the TCR-ß/RAG mice utilizes the Vß3 gene segment. To
determine the transcription status of 5' Vß gene segments in DN and
DP thymocytes, the same two filters were stripped and hybridized with a
190-bp Vß8 probe or other Vß probes, including Vß6, 7, 9, and 13.
In contrast to Vß14, the level of Vß8 germline transcript
(including Vß8.1, 8.2, and 8.3) was much higher in DN thymocytes from
RAG mice than in DP thymocytes from CD3/RAG, lck/RAG, and
TCR-ß/RAG mice (Fig. 4C). No RNA transcripts were
detected when Vß6, 7, 9, or 13 probes were used individually (data
not shown). Together, these data suggest that germline transcription in
Cß-Vß14 region is increased in DP thymocytes, indicative of
increased accessibility in this region, whereas germline transcription
in the upstream Vß region is nonexistent or decreased in DP
thymocytes, suggesting a region with decreased accessibility. Analysis of the role of HS1 in TCR-ß gene allelic exclusion
We next focused our investigation on determining whether
cis-regulatory elements contained within HS1 control TCR-ß
gene allelic exclusion. HS1 was interesting because it was the most
prominent chromatin structural change identified in the TCR-ß locus
between DN and DP thymocytes, and because it was only 400 bp upstream
of Eß. Considering that cis elements within Eß have a
dominant role in promoting TCR-ß VDJ recombination (41, 55, 63, 64),
it was possible that factors binding to cis elements in HS1
might interact with and modulate those binding to Eß and effectively
suppress recombination at the entire locus, thereby mediating allelic
exclusion. To test this possibility, a 780-bp
XcmI-BglII fragment containing HS1 was replaced
with a floxed neo gene by homologous recombination in ES cells, and
chimeric mice were produced for germline transmission of the alteration
(Fig. 5; Materials and
Methods). To minimize effects from the introduced neo gene,
it was deleted by Cre/loxP-mediated recombination by breeding mutant
mice with deleter mice, in which Cre is expressed at the two-cell stage
of embryonic development (71). After Cre-mediated recombination, a
single 34-nucleotide loxP site remains that is unlikely to influence
the phenotype resulting from the HS1 deletion. The resulting mutant
allele is detected as a 7.4-kb EcoRI fragment by Southern
hybridization using probe 1 (Fig. 5B). Homozygous
mutant mice with or without the neo gene deleted yielded normal numbers
of thymocytes and peripheral T cells in spleen and lymph nodes (see
below and data not shown). A comparison of DNase I hypersensitivity of
thymocyte nuclei from normal and homozygous mutant mice, in which the
neo gene was deleted, confirmed that HS1 was deleted successfully in
mutant mice (Fig. 5C). HS2, HS3, and HS4 remained in
the mutant mice despite the deletion of HS1, indicating that these
DNase I hypersensitive sites form independently of HS1.
Phenotypic analyses of cells from spleen, lymph node, and thymus by
FACS showed that T cell development and TCR-ß expression in the
mutant mice were indistinguishable from that of normal mice (Fig. 6). Additional FACS analyses of lymph
node T cells stained with a panel of Abs specific for individual Vßs
revealed no significant difference in Vß repertoire usage between
mutant and normal mice (data not shown). In addition, amplification of
thymocyte DNA from normal and mutant mice by PCR to detect Dß1 to
Jß1.1 through Jß1.5, and Dß1 to Jß2.1 through Jß2.5
rearrangements revealed no differences between the normal and mutant
mice in D to J rearrangement (data not shown). Thus, the deletion of
HS1 does not grossly affect TCR-ß gene rearrangement, expression, and
T cell development.
|
A). In normal mice,
the level of hybridization to the 2.2-kb PstI fragment was
almost undetectable in thymocyte DNA (Fig. 7B). In
the presence of the TCR-ß transgene, the levels of hybridization were
the same in DNA from thymocytes and kidney, indicating the occurrence
of allelic exclusion. The retention of the signal in mice expressing
the TCR-ß transgene is not due to differences in DNA loading, as
indicated by the similar level of simultaneous hybridization of probe 3
to a 10-kb PstI control fragment located downstream of
Vß14 (Fig. 7, A and B). If the deletion
of HS1 abolished allelic exclusion, we expected that Vß to DßJß
recombination would persist in the presence of the TCR-ß transgene
and result in a lower level of retention of the 2.2-kb PstI
fragment located upstream of Dß1. However, the level of hybridization
was the same in the mutant samples as in the normal mice (Fig. 7B). The same results were also obtained when DNA
isolated from purified lymph node T cells of mutant and normal mice
were used for the assay (data not shown). In addition,
ligation-mediated PCR measuring the levels of dsDNA break at the 5' of
Dß1 and at the 3' of Vß14 gene segment in thymocytes failed to
detect any consistent difference between the normal and homozygous
mutant mice (data not shown). Therefore, these combined data show that
cis elements within HS1 are not required for TCR-ß gene
allelic exclusion.
|
We next considered the possibility that cis-regulatory
elements within HS7 and/or HS8 might play a role in controlling allelic
exclusion because these sites are maintained after Dß to Jß
recombination (Fig. 2). In NZW mice, a natural deletion of 8.8 kb of
DNA in the TCR-ß locus has removed Cß1, Dß2, and Jß2 gene
segments, as well as HS7 and HS8 (88). Therefore, to assess the
requirement of cis-regulatory elements associated with HS7
and HS8 for TCR-ß gene allelic exclusion, we bred a functional
TCR-ß transgene onto the NZW mice, isolated thymocyte DNA, and then
measured for the extent of allelic exclusion by Southern hybridization
of probe 2 to the 2.2-kb PstI fragment located upstream of
Dß1. As shown in Figure 7C, Vß to DßJß rearrangement
of TCR-ß in NZW mice was inhibited effectively by expression of the
TCR-ß transgene, as indicated by the increased hybridization signal
to the 2.2-kb PstI fragment compared with normal NZW mice.
Therefore, deletion of HS7 and HS8 does not grossly affect TCR-ß gene
allelic exclusion. FACS analyses of lymph node T cells from NZW mice
stained with a panel of Abs to endogenous Vßs to detect dual TCR-ß
expression confirmed that TCR-ß allelic exclusion was intact in the
NZW mice (data not shown). These data demonstrate that
cis-regulatory elements within HS7 and HS8 are not required
for TCR-ß gene allelic exclusion.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Studies to identify all potential chromatin changes of the
TCR-ß locus are inherently difficult due to its immense size of
600 kb (Fig. 1) (75, 76). In our study, we first considered the
following facts about the TCR-ß locus to narrow down the region of
analysis. Because allelic exclusion is regulated at the V to DJ
rearrangement step, it is unlikely that cis elements
involved in maintaining allelic exclusion reside between Dß1 and
Jß2.7. Therefore, we focused our effort on identifying chromatin
changes occurring in a 100-kb region from 20 kb upstream of Dß1 to
nearly 70 kb downstream of Jß2. This represents the most
comprehensive study ever undertaken for an Ag receptor locus. Second,
we did not study beyond 20 kb upstream of Dß1 because of the presence
of a trypsinogen gene that, in our thinking, may separate upstream
Vßs from the downstream gene segments and associated regulatory
elements. Third, it was impractical to study chromatin changes in the
entire upstream Vß region due to its immense size. In addition,
previous studies using TCR-ß minilocus substrates have suggested that
a functional Vß promoter is not required for allelic exclusion and
that the signal for allelic exclusion may be mediated by sequences 3'
of the Jß2.2 gene segment (89). Therefore, we focused our study on
the DNA flanking the unique Vß14 gene segment, which is located
downstream of the Dß, Jß, and Cß gene segments and seems to be
subjected to the same allelic exclusion control as all other Vß gene
segments. The Vß14 gene segment is also interesting because of its
inverted transcriptional orientation relative to all other TCR-ß gene
segments and the conservation of this configuration in human, mouse,
and chicken. Because Vß14 rearranges to the DßJß complex by
inversional joining, this configuration ensures that DNA between Vß14
and the DßJß complex is retained after rearrangement, suggesting
that the Jß2 to Vß14 intervening region may contain critical
cis-regulatory elements required for TCR-ß gene
recombination and/or transcription.
We identified a total of 11 DNase I hypersensitive sites in the 100-kb
region. Three of the sites, HS1, HS4, and HS9, are unique or strongly
induced in DP thymocytes, whereas the other eight sites identified are
present in both DN and DP thymocytes (Fig. 2). Six of the sites are
located between the Jß2-Vß14 intervening region, consistent with
the importance of this region for TCR-ß gene regulation. A general
correlation between transcriptional activity of the TCR-ß locus and
the presence of the DNase I hypersensitive sites is apparent. For
example, HS2 maps to the previously identified Eß enhancer (83, 84, 86), and is present in both DN and DP thymocytes (Fig. 2), consistent
with the enhancer being active at both stages of development. Indeed,
we detected germline transcripts (1 and 1.6 kb) containing Cß
sequences in both DN and DP thymocytes (Fig. 4), and most
MspI/HpaII and HhaI sites in the Cß
region are unmethylated (Fig. 3), a state usually associated with
active transcription. In DP thymocytes, demethylation of three
MspI/HpaII sites in the Jß2 region correlated
with increased levels of Cß-containing transcripts and the induction
of HS1 and HS9. HS9 is located immediately upstream of Dß1, and
analogous to Ig and TCR- loci (51, 52, 90), may serve as the
promoter for germline transcription through the Dß, Jß, and Cß
regions. Consistent with this notion, a germline transcript initiated
immediately upstream of Dß1 was identified recently in human fetal
intestine (91). HS1 is 400 bp upstream of Eß and has been implicated
to influence transcription, perhaps by modulating Eß activity (see
below). Another site, HS5, maps to the Vß14 transcriptional promoter.
Although HS5 is present in both DN and DP thymocytes, Vß14 germline
transcripts are only detectable in DP thymocytes (Fig. 4). Vß14
transcripts correlate with the partial demethylation of three
MspI/HpaII sites located upstream of the Vß14
gene segment in DP thymocytes. These observations suggest that if the
factors that cause HS5 are involved in transcriptional activity, their
binding alone is not sufficient for the induction of Vß14 germline
transcription. Additional factors or signals induced during the DP
thymocyte stage are also most likely required for the induction of
Vß14 transcription. In summary, our findings suggest that chromatin
structure in the 100-kb region of the TCR-ß locus is dynamic and
accessible in both DN and DP thymocytes, as reflected by the presence
of multiple DNase I hypersensitive sites, hypomethylation, and active
transcription. We did not find evidence supporting a model of allelic
exclusion, wherein accessibility of the TCR-ß locus is decreased
dramatically during the DP stage of thymocyte development.
Chromatin changes and TCR-ß gene allelic exclusion
Our studies reveal that none of the chromatin changes at the
TCR-ß locus in DN and DP thymocytes is indicative of a typical
inaccessible locus. In DP thymocytes, 3 of 11 identified DNase I
hypersensitive sites are either unique or strongly induced, 6 of 33
HpaII/HhaI sites show partial to complete
demethylation, and increased germline transcription initiated from the
Dß-Cß region and Vß14 is evident. Moreover, all DNase I
hypersensitive sites, hypomethylation, and transcription observed in DN
thymocytes are also maintained in DP thymocytes. The observed changes
most likely represent genuine chromatin alterations that are induced by
pre-TCR signaling because the same changes are observed in DP
thymocytes by either the expression of a TCR-ß or an activated
lck transgene, or by anti-CD3 treatment. Because
pre-TCR also signals for TCR-ß gene allelic exclusion (28), the
observed chromatin changes are either coincident or associated with
allelic exclusion (38). Our data show that the TCR-ß locus is
accessible in DP thymocytes to the various enzymes and factors that
bind DNA sequences and mediate the transcription and demethylation of
the locus. However, in spite of this, the locus remains inaccessible to
the V(D)J recombinase because the TCR-ß gene is excluded from further
rearrangement in DP thymocytes (28). This observation is significant
for several reasons. First, although chromatin changes have been
postulated to block recombinase accessibility or to retarget the
recombinase for achieving allelic exclusion, the underlying mechanism
is unknown (92). Our findings have eliminated some possibilities by
showing that the mechanism does not involve the general shutdown of the
locus, which would be reflected by an absence of transcription and
active hypermethylation of the locus, and perhaps the disappearance of
DNase I hypersensitive sites. Second, and contrary to our expectations,
the locus becomes hypomethylated and more actively transcribed. This
suggests that while transcription and hypomethylation may be required
for the initiation of V(D)J recombination (6, 7), and may be used to
measure general chromatin changes and accessibility, the status of
transcription and methylation may not be indicative of the
recombination inaccessibility associated with the excluded TCR-ß
allele. Third, new DNase I hypersensitive sites are induced at the
excluded TCR-ß locus. Therefore, if cis-regulatory
elements associated with these new DNase I hypersensitive sites are
involved in the control of allelic exclusion, it seems likely that the
associated factors would function as repressors of V to DJ
recombination.
HS1, HS7, and HS8 are not required for TCR-ß gene allelic exclusion
HS1 is 400 bp upstream of Eß and maps to a previously
identified DNase I hypersensitive site found in precursor T cell lines
(Fig. 2) (85). Although HS1 was shown to correlate with TCR-ß
expression in cell lines (85), findings from our more physiologic
experimental system demonstrate that HS1 is primarily present in DP
thymocytes induced in RAG-deficient mice by expression of a TCR-ß or
activated lck transgene or by anti-CD3 Ab treatment.
Importantly, HS1 was also detected in DP thymocytes from normal TCR-ß
transgenic mice, in which the endogenous TCR-ß loci underwent
DßJß rearrangement and were excluded from Vß to DßJß
rearrangement. Therefore, the data provide strong support that HS1 is
induced by pre-TCR engagement during DN to DP thymocyte differentiation
and could represent a chromatin change associated with TCR-ß gene
allelic exclusion.
Based on the close proximity of HS1 to Eß, we hypothesized that
factors binding to HS1 during the DP thymocyte stage might suppress
Eß function specifically in recombination, but not transcription, and
consequently would promote allelic exclusion. To test this hypothesis,
we deleted a 780-bp DNA fragment encompassing HS1 from the endogenous
TCR-ß locus by targeted mutagenesis. We found that while HS1 was
deleted by this mutation, HS2, HS3, and HS4 remained (Fig. 5),
suggesting that these sites may function independently of HS1. Compared
with normal mice, homozygous mutant mice displayed normal TCR-ß gene
recombination, expression, and allelic exclusion either with or without
a TCR-ß transgene (Figs. 6 and 7). Therefore, our data show that HS1
is not required for the control of allelic exclusion. This finding
suggests the possibility that other cis-regulatory elements
in the locus may have a primary role in allelic exclusion control. If
HS1 does participate in the process, it may do so in cooperation with
other more essential and dominant elements. Although we do not
currently know the function of HS1, its role in transcription has been
suggested both by its proximity to Eß and by the presence of several
conserved sequence motifs homologous to known transcriptional elements
(85). Octamer, c-myb, AP-1, and enhancer core sequences of
SV40 and polyoma virus are present within two subregions that are most
conserved between human and mouse (85 and 95% homology). Nevertheless,
HS1 has no enhancer activity in both transient transfection assays and
transgenic mice (83, 84) (Erik Selsing and Jim Miller, personal
communication). Furthermore, our current biochemical evidence suggests
that in DP thymocytes, factor binding to HS1 occurs in a region
different from that described by the above consensus motifs and may
function to repress TCR-ß transcription (unpublished data).
Additional studies are required to understand the function of the
nuclear factors binding to HS1.
HS7 and HS8, located within the Jß2-Cß2 intron, were detected
previously in T cell lines, and nuclear factor-B and additional
factors were found to interact with specific sequences in the region
(93, 94). We found that HS7 and HS8 were present in both DN and DP
thymocytes, suggesting a role in transcription and/or recombination of
the TCR-ß locus at both stages of development. Interestingly, in mice
harboring a targeted replacement of 15 kb of the locus encompassing
Jß1.3 to Cß2, recombination of the remaining Dß1, Jß1, and Vß
gene segments did not occur. Although the recombination defect could be
caused by the removal of other unknown elements within the region
important for recombination, or due to the insertion of a neomycin gene
cassette that has been shown to perturb recombination in other loci
(56, 57, 61, 62, 95), we considered the possibility that the deletion
of HS7 and HS8 might be responsible for the observed defect. In
addition, HS7 and HS8 might also function in allelic exclusion control.
NZW mice have naturally deleted 8.8-kb region encompassing Cß1,
Dß2, and Jß2 gene segments as well as HS7 and HS8 (88). From the
literature, there was no evidence of a defect in the surface expression
of TCR-ß on NZW T lymphocytes, suggesting that transcription and
recombination of the locus are normal. However, whether allelic
exclusion was normal in these mice remained to be tested, especially
considering that (NZW x NZB)F1 mice have a high
incidence of autoimmune disease characterized by high levels of
autoantibodies in the circulation (96, 97, 98, 99, 100), and that this disease
state might be influenced indirectly by a loss of TCR-ß gene allelic
exclusion control. However, our analyses from Southern blotting and
FACS demonstrated no apparent defect in the control of TCR-ß gene
allelic exclusion in NZW mice after introduction of a TCR-ß transgene
(Fig. 7 and data not shown), indicating that cis-regulatory
elements within HS7 and HS8 are not required for the process.
Therefore, it seems that the loss of HS7 and HS8 in NZW mice is
unlikely to be a contributing factor to the development of autoimmune
disease in (NZB x NZW)F1 mice.
Cis-regulatory elements involved in TCR-ß gene allelic exclusion
DNase I hypersensitive sites are generated when nucleosomes are
replaced by specific trans-acting factors that competitively
bind to their cognate sequences. The presence of hypersensitive sites
usually implies the presence of cis-regulatory elements and
their associated factors. Although targeted deletion of HS1 or natural
deletion of HS7 and HS8 has no apparent effect on TCR-ß gene
recombination, expression, or allelic exclusion, it remains possible
that in normal circumstances these sites may contribute to any of these
processes in cooperation with other more dominant regulatory elements
such as Eß in the assayed region. The Eß enhancer was identified
initially as a transcription enhancer (83, 84); it is also critical for
the recombination of the TCR-ß gene. Targeted deletion of a 0.5-kb
region containing Eß from the endogenous locus in mice results in a
complete absence of Dß to Jß rearrangement and therefore a block of
ß T cell development (63, 64). Inclusion of Eß in a minilocus
recombination substrate readily promotes Vß to DßJß rearrangement
in transgenic mice (41, 58). Thus, an effective mechanism of
suppressing recombination of the entire TCR-ß locus may be through
the specific suppression of Eß function in recombination. It should
be emphasized that our data suggest that such a mechanism could not
operate through decreasing transcription, as we observed increased
transcriptional activity and associated demethylation of the TCR-ß
gene in DP thymocytes under conditions of allelic exclusion. Our data
are consistent with a mechanism wherein Eß may contain independent
and separable cis elements that function to regulate either
transcription or recombination/allelic exclusion of the TCR-ß
gene (101).
If the mechanism of TCR-ß gene allelic exclusion does involve a
shutdown in chromatin accessibility to the recombinase, our data are
also consistent with the possibility that the mechanism may involve
cis elements residing completely outside of the 100-kb
region of DNA that we have analyzed, perhaps in the upstream Vß
region. Indeed, we did observe a significant down-regulation of Vß8
germline transcription in DP thymocytes compared with DN thymocytes,
suggesting decreased accessibility to the transcriptional machinery. In
this scenario, allelic exclusion of the downstream Vß14 gene segment
may involve a different mechanism since we observed increased germline
transcription of this gene segment and demethylation near its promoter
in DP thymocytes. However, it remains possible that germline
transcription of Vß gene segments may not be a reliable indicator of
accessibility to the recombinase. This is consistent with previous
studies utilizing minilocus recombination substrates, in which a
correlation between transcription and recombination has not always been
found (58, 101, 102). Interestingly, Alvarez et al. showed that while
deletion of the decamer motif of a Vß gene segment abolished
transcription to below detectable levels, the rearrangement and allelic
exclusion of the Vß gene segment remained unaffected (89). Therefore,
allelic exclusion of each Vß gene segment may not be controlled
individually, but by dominant cis-regulatory elements that
control allelic exclusion of all Vß gene segments (except Vß14). If
such dominant cis elements are present, they do not reside
in the 100-kb region encompassing Vß5.2 to Vß9, because this 100 kb
is deleted in SJL mice, and these mice have no apparent defect in
TCR-ß gene allelic exclusion (75, 76). Alternatively, allelic
exclusion could be mediated by multiple local cis-regulatory
elements, analogous to the T early element for the rearrangement of
the 5' J gene segments (90).
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 The first two authors contributed equally to the work.
3 Address correspondence and reprint requests to Dr. Jianzhu Chen, Center for Cancer Research, Massachusetts Institute of Technology, E17-128, 40 Ames Street, Cambridge, MA 02139. E-mail address:
4 Abbreviations used in this paper: BCR, B cell receptor; DN, double negative; DP, double positive; Eß, T cell receptor-ß gene enhancer; ES, embryonic stem; HS, DNase I hypersensitive site; neo, neomycin-resistance gene; NZB, New Zealand Black; NZW, New Zealand White; RAG, recombination activating gene; RSB, reticulocyte standard buffer; SP, single positive.
Received for publication August 5, 1997. Accepted for publication October 21, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, 
, and µ heavy polypeptide chains and to allotypic markers Aa1 and Aa2. J. Exp. Med. 123:547.[Abstract]
genes but uses one ß heterodimer for allorecognition and self MHC-restricted antigen recognition. Cell 55:49.[Medline]
chains: dual receptor T cells. Science 262:422. genes in a transgenic mouse line. Nature 314:330.[Medline]
- and ß-genes. Nature 334:156.[Medline]
gene in development of ß but not 
T cells. Nature 375:795.[Medline]
gene. EMBO J. 14:4641.[Medline]
chain in T cell development and the allelic exclusion at the T-cell receptor ß locus. Proc. Natl. Acad. Sci. USA 93:2169. gene enhancers confer tissue- and stage-specificity on V(D)J recombination events. EMBO J. 12:4335.[Medline]
light chain intronic enhancer/matrix attachment region impairs but does not abolish VJ rearrangement. Immunity 4:377.[Medline]
3' enhancer influences the ration of Ig versus Ig B lymphocytes. Immunity 5:241.[Medline]
ß T-cell development. Proc. Natl. Acad. Sci. USA 93:7877.ß versus ß T-cell receptor transgenic mice undergoing negative slection. Nature 340:559.[Medline]
-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCR ß chain expression. Int. Immunol. 6:995.. Proc. Natl. Acad. Sci. USA 90:11401./ lymphocytes. Eur. J. Immunol. 21:1529.[Medline]
following targeted deletion of T early (TEA): implications for TCR locus accessibility. Immunity 5:331.[Medline]
B binding site in the murine T cell receptor ß2 locus. J. Exp. Med. 170:1737. chain intron enhancer abolishes chain gene rearrangement in cis but not chain gene rearrangement in trans. EMBO J. 12:2329.[Medline]
gene enhancer acts independently on rearrangement and on transcription. Eur. J. Immunol. 23:1917.[Medline]
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