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Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232
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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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locus (2). Several lines of evidence suggest that the specificity of rearrangement events is regulated at the level of gene segment accessibility to a common recombinase complex. Consistent with this model, activation of germline transcription at nearly all Ig and TCR loci temporally correlates with activation of locus recombination (1, 3, 4, 5). More recent studies have demonstrated an important role for cis-acting elements in the dual control of transcription and V gene segment accessibility (6, 7, 8, 9, 10). For example, both germline transcription and rearrangement of a TCRß minilocus substrate requires the inclusion of an active transcriptional enhancer (6, 7). Furthermore, targeted deletion of Ig or TCR enhancers dramatically impairs V region gene assembly at the mutated alleles (11, 12, 13, 14, 15). Together, these studies indicate that changes in enhancer activity coordinately affect transcription and recombinational accessibility of V gene segments during lymphocyte development.
Despite these advances, it has been difficult to experimentally dissect the individual contributions of enhancer activity and germline transcription in promoting efficient rearrangement of V gene segments. For example, sterile transcription of germline gene segments is a highly conserved feature of lymphocyte development, suggesting an important role for promoter/enhancer interactions in the mechanisms that target recombinase to specific clusters of gene segments. However, the IgH enhancer (Eµ) can establish areas of accessible chromatin spanning distances of up to 1 kilobase in transgenic constructs (16). Because this long-range accessibility in pre-B cells does not require transcription of neighboring gene segments, enhancer-mediated alterations in chromatin may be sufficient to regulate the recombination potential of associated gene segments.
To further understand the function of germline transcription in the control of TCRß locus accessibility, we have identified an enhancer-responsive promoter located directly upstream from the Dß1 gene segment. Biochemical and mutational analyses indicate that maximal activity of this promoter, termed PDß, requires DNA binding sites for SP1 as well as the T cell-specific transcription factor, GATA-3. Furthermore, PDß drives expression of germline gene segments in thymocytes that are poised to undergo DßJß rearrangement. When linked to its cognate enhancer (Eß), PDß supports high levels of reporter gene expression specifically in precursor T cells. Together, these findings suggest that the stage-specific interplay between PDß and Eß may serve to differentially modulate DßJß accessibility during thymocyte ontogeny.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymphocyte cell lines were cultured at 37°C/5%
CO2 in RPMI 1640 media supplemented with 10% FCS, 2 mM
L-glutamine, 0.01% penicillin/streptomycin, and 50 µM
ß-mercaptoethanol. The cell lines T3 and T7 were transformed by the
Abelson murine leukemia virus (6) and are classified as pre-T cells
based on their expression of specific genetic markers (e.g., RAG-1 and
RAG-2, data not shown). The EL4 and BW5147 cell lines do not express
RAG gene products (data not shown) and previously have been classified
as mature T cell thymomas based upon the rearrangement status and
expression of their TCR and ß loci (17, 18, 19, 20).
Transient transfection of all cell lines was accomplished by electroporation (250 V/960 µF) in serum-free medium (400 µl) containing the test construct (15 µg), and a control construct encoding placental alkaline phosphatase (pSV2PAP, 10 µg). Protein extracts were harvested 24 h posttransfection, with the exception of M12 cells, which were harvested at 48 h. Luciferase and PAP activities were quantitated using commercially available kits (Analytical Luminescence Laboratory, Ann Arbor, MI; and Phospha-Light, Tropix, Bedford, MA). All transfections were performed in duplicate or triplicate with two independent preparations of each reporter construct.
Plasmid constructs
For localization of promoter activity (Fig. 2
), PDß fragments
were generated by PCR amplification of a plasmid containing the
Dß1/Jß gene segments and 2.5 kb of 5' flanking sequences. The
oligonucleotides used for amplification of specific promoter regions
are as follows: p698 (upstream 5'-GTA GGT ACC CTA ATT GAA AAG ACT TCA
G-3' and downstream 5'-GAA GAT CTC CCC ACA ATG TTA CAG C-3'), p377
(upstream 5'-GTA GGT ACC GAC GCA CAG CCT TAG GG-3'), p377/3'
(downstream 5'-GTA GAG CTC GGC CTT GGG ACA GAC AG-3'). Amplified
fragments were digested with KpnI/SacI and
introduced into corresponding sites in the pGL2-Enhancer reporter
plasmid (Promega). Constructs p377
3' and p47 were
generated by deletion of either the AccI/SacI or
the AccI/KpnI fragments from p377/3'. For studies
of promoter specificity, either Eß (400 bp
HpaI-NcoI fragment) or Eµ (670 bp
EcoRI-XbaI fragment) was inserted into the
BamHI site of pGL2-Basic or pGL2-Promoter (Promega, Madison,
WI). The SV2PAP construct has been described previously
(21).
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S1 nuclease protection assays
Total cellular RNA was prepared from B cell transfectants or
murine tissues using the lithium chloride method. To generate antisense
RNA probes, the AccI/BglII fragment, spanning
from -47 to +358 relative to the Dß1-coding sequence, was subcloned
into the EcoRI site of pBluescript (Stratagene, La
Jolla, CA). The resulting plasmid was linearized (KpnI) and
subjected to an in vitro transcription reaction (T7 polymerase) in the
presence of [-32P]CTP to produce a single-stranded RNA
probe containing 404 bases of Dß1 sequence and 110 bases of
polylinker sequence. The radiolabeled probe was hybridized to cellular
RNAs (80°C/70 min) as described previously (23). The resulting
hybridization mixture was digested with S1 nuclease (800 U) at 37°C
(60 min.) in a buffer containing 33 mM NaOAC, 50 mM NaCl, and 0.03 mM
ZnSO4 (pH 4.5). Digested duplexes were separated on a 5%
polyacrylamide sequencing gel and visualized by autoradiography.
Gelshift analyses
Nuclear extracts for electrophoretic mobility shift analysis
were prepared by a high salt extraction method in the presence of an
extensive protease inhibitor mixture (24). Oligonucleotide probes
corresponding to all transcription factor binding sites were prepared
by klenow fill-in reactions of partially overlapping double-stranded
DNA ends using [-32P]dCTP and
[-32P]dATP radionucleotides. DNA binding reaction
mixtures (20 µl) consisted of 20 µg nuclear extract, ds poly(dI-dC)
(2 µg), and BSA (10 µg) buffered in 20 mM HEPES (pH 7.9), 5%
glycerol, 1 mM EDTA, 1% Nonidet P-40, and 5 mM DTT (25). The resultant
nucleoprotein complexes were separated by electrophoresis through a 5%
polyacrylamide gel in 1x TBE buffer (190 V/2.5 h), and detected by
autoradiography. Upper-strand sequences of the SP1 and GATA
oligonucleotides are as follows: SP1 5'-AAA ATT TGA GAA GGG CGG ATA CAA
GAG GGA-3'; SP1 HIV LTR (26); GATA(a) 5'-TGT CAC CTT CCC TTA TCT
TCA ACT CCC C-3'; GATA(b) 5'-GCA GCT TAT CTG GTG GTT TCT TCC A-3', and
GATA-Eß (27). For supershift analyses, 100 ng of either the GATA-3
mAb, SP1 pAb, PU.1 mAb (Santa Cruz Biochemicals, Santa Cruz, CA)
or the p-50 pAb (28) were added to nuclear extracts 30 min before
initiation of binding reactions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One hallmark of T lineage commitment is the activation of TCRß
germline transcription, which originates within the endogenous
Dß1/Jß and Dß2/Jß clusters (12, 13, 29). Similar germline
transcripts are detected in cells expressing a transgenic TCRß
minilocus that harbors only Dß1 and its two most proximal Jß gene
segments (7, 9). These findings suggest the presence of germline
promoters within each of the DßJß clusters. However, neither the
precise location of these promoters nor the factors that contribute to
their function have been defined. As an initial step toward mapping the
position of the Dß1 germline promoter, we characterized
transcriptional initiation sites within the unrearranged TCRß
minilocus using an S1 nuclease protection assay and a 404-bp antisense
probe that spans sequences -47 to +357 relative to the Dß1-coding
region (Fig. 1A). Consistent
with previous findings (7), stable B cell transfectants containing an
enhancerless TCRß minilocus did not express DßJß germline
transcripts (Fig. 1B, lane 3). In contrast,
the 404-bp probe protected two major and several less abundant
transcripts in total RNA derived from a stable transfectant of the
TCRß/E
minilocus, which contains the Ig intronic enhancer
(lane 4). Judging from the sizes of these protected
species (approximately 350 and 330 bp), we conclude that a majority of
germline transcription initiates in TCRß miniloci at two major sites,
positioned within the Dß1-coding sequence or within its 3' RS (± 5
nucleotides, Fig. 1A).
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B, identical patterns of Dß1 probe protection were
observed with RNAs harvested from either RAG-2-/-
thymocytes or TCRß/E transfectants (lanes
4 and 5). In control experiments, the Dß1 probe
was not protected significantly by TCRß transcripts expressed in
wild-type thymocytes (lane 6) or splenocytes
(lane 7). In these tissues, the majority of T lineage
cells have deleted Dß1 probe sequences via DßJß or VßDßJß
rearrangements. However, prolonged exposure of the gel revealed an
identical pattern of probe protection in wild-type thymocyte but not in
splenocyte RNA. These data are consistent with the numbers of T
lymphocytes that harbor germline TCRß loci in either thymus
(approximately 5%) or spleen (less than 1%). Together, our findings
strongly suggest that expression of accessible Dß1/Jß gene segments
is driven by an enhancer-responsive promoter located directly upstream
from Dß1-coding sequences. Localization of the Dß1 germline promoter
To gain further insights into the functional architecture of
PDß, we performed computer analyses on the DNA sequences located
within 2.5 kb upstream from the Dß1 gene segment. These analyses
revealed a consensus TATA element situated within the nonamer of the
5'Dß1 RS (Fig. 2A).
Consistent with our S1 nuclease mapping studies, the PDß TATA motif
is positioned approximately 20 to 40 bp upstream from the major
transcriptional initiation sites mapped in RAG 2-/-
thymocytes (Fig. 1B). In addition, Dß1 proximal sequences
contained potential binding sites for the SP1, ETS, and GATA
transcription factor families (Fig. 2A). Thus, sequences
located upstream from the Dß1 gene segment exhibit hallmarks of a
functional promoter element.
Prior studies have shown that enhancer-dependent expression of germline
Dß/Jß elements occurs at both the
CD4-/CD8- and
CD4+/CD8+ stages of thymocyte development (5, 9, 12, 19). As such, we restricted initial analyses of PDß function
to a pre-T cell line (T7) that expresses high levels of endogenous
TCRß transcripts (6). To monitor promoter function, we tested the
ability of Dß1 upstream sequences to drive expression of luciferase
reporter constructs containing the SV40 transcriptional enhancer (Fig. 2A). Transient transfection experiments demonstrated that
DNA sequences spanning a 700-bp region 5' to Dß1 supported
substantial levels of luciferase expression in T7 cells (Fig. 2B, p698). Deletion of sequences upstream from the consensus
SP1 site had only modest effects on this promoter activity (p377).
Furthermore, the addition of Dß1 3' flanking sequences failed to
enhance PDß activity in our reporter gene assay (p377/3'). This
latter finding is fully consistent with the continued expression of
rearranged DßJß elements in pre-T cells that have deleted their
corresponding downstream sequences (29). In contrast, removal of the
Dß1 element, which spans the consensus TATA motif, significantly
impaired PDß function (p377/3'). Finally, to test whether the TATA
element was sufficient to mediate full PDß activity, we generated the
p47/3' construct, which contained only the Dß1 element and its
associated RS. Relative to constructs containing putative factor
binding sites, this core promoter supported only minimal levels of
reporter gene expression (Fig. 1B). Based on these
functional assays, we conclude that sequences spanning a 377-bp region
upstream from Dß1 constitute an active promoter element in this pre-T
cell line.
SP1 and GATA-3 bind to sites within PDß
Maximal PDß activity is restricted to a 377-bp fragment that
spans putative binding sites for ubiquitous and tissue-specific
transcription factors (Fig. 2). To determine whether these sites are
competent for transcription factor binding, we performed a gel shift
survey using nuclear extracts from staged lymphocyte cell lines.
Previous studies have clearly demonstrated that ETS family proteins are
expressed in both B and T lineage cells (31, 32). Indeed, a
radiolabeled probe corresponding to the PDß-ETS motif specifically
bound the ETS family member PU.1 in mature B cell extracts (data not
shown). However, under a variety of conditions, this probe failed to
form nucleoprotein complexes with extracts from T lineage cells, which
express high levels of the ETS-1 protein (31, 32).
Binding sites for the ubiquitous transcription factor SP1 are present
in a broad spectrum of eukaryotic promoters and contribute
significantly to their basal activities (26). As shown in Figure 3, a probe spanning the putative SP1 site
in PDß bound to multiple nuclear proteins expressed by the pre-T cell
line, T7 (lane 1, complexes I-IV). The specificity of
these proteins for the SP1 site was confirmed by a series of
competition experiments. All of the major nucleoprotein complexes (I,
III, and IV) were effectively competed with unlabeled SP1
oligonucleotides derived from either PDß (lane 2)
or the HIV-LTR (lane 3). Furthermore, mutation of the
SP1 motif within the PDß oligonucleotide severely impaired its
ability to compete for binding with each of these specific complexes
(lane 4).
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, complexes I, III, and IV contained proteins that were reactive with an
SP1-specific Ab (lane 5) but were not supershifted by
an irrelevant Ab specific for the p50 subunit of NF-B
(lane 6). Furthermore, only complex I formed at high
levels with nuclear extracts from all cell lines tested
(lanes 7–10). Although the composition of specific
complexes III and IV remains unclear, control experiments revealed no
evidence for protein degradation in any of the nuclear extracts (data
not shown). As such, these complexes may contain related members of the
SP transcription factor family, which also bind to GC-rich sequences
and share common epitopes with SP1 (33). Together, these biochemical
results indicate that the SP1 transcription factor binds to an upstream
site in the germline Dß promoter.
In addition to the SP1-binding motif, PDß contains a pair of
consensus sites for GATA transcription factors that are situated within
a 24-bp region (Fig. 2A). The GATA transcription factor
family includes six proteins that are each expressed in a
tissue-specific manner (34). Of special note, the GATA-3 protein is
expressed in all T lineage cells (35), and targeted disruption of the
GATA-3 gene blocks T cell development at the
CD4-/CD8- pro-T cell stage (36). As shown in
Figure 4, a radiolabeled probe that spans
the 5'-GATA site in PDß (GATA-a) forms a single nucleoprotein complex
in pre-T cell nuclear extracts (lane 1). This binding
activity was specifically competed by sequences spanning each of the
GATA motifs in PDß, or a consensus GATA-3 site present in the human
TCRß enhancer (lanes 2–4). In contrast, specific
mutation of the GATA-a or GATA-b sites in the relevant PDß
oligonucleotides significantly impaired their abilities to compete with
the wild-type GATA-a probe (lanes 5 and
6). The presence of GATA-3 proteins in the observed complex
was confirmed by supershift assays using a GATA-3-specific mAb
(lane 7). Consistent with prior studies, the GATA-a
probe detected GATA-3-binding activity in both pre-T and mature T cell
lines (Fig. 4B, lanes 1–4) but failed to
recognize nuclear proteins in any of the B cell lines examined
(lanes 5 and 6; and data not shown).
Collectively, these findings indicate that PDß encompasses a pair of
functional binding sites for the T cell-specific transcription factor
GATA-3.
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In addition to the studies described above, we performed gel shift
analyses with a panel of probes that spanned the entire Dß1 promoter
region. In pre-T cell extracts, this survey revealed specific factor
binding only to probes containing the SP1 or GATA motifs (Figs. 3 and 4). To assess the precise contribution of identified sites to PDß
function, we generated a series of luciferase reporter constructs
harboring mutations that eliminate factor binding to each motif.
Consistent with the lack of detectable protein-binding activity at the
native ETS site, mutation of this motif only modestly reduced PDß
activity. Relative to the wild-type p377/3' reporter plasmid, the
promoter activities of SP1, GATA-a, GATA-b, or TATA mutants were each
impaired approximately two- to fourfold in the T7 pre-T cell line (Fig. 5). In contrast, neither of the GATA
mutations affected PDß activity in the pre-B cell line 38B9, which
lacks GATA-3 protein (data not shown). To determine whether the GATA-3
sites additively contribute to PDß function in pre-T cells, we
introduced mutations at both GATA sites in p377/3'. This doubly mutated
vector supported levels of luciferase expression that did not
significantly differ from those observed for the single GATA-b mutant
(Fig. 5). These results are qualitatively similar to the effects of
multiple GATA mutations on TCRß enhancer activity (27) and indicate
that, similar to Eß, GATA-3 regulates PDß via a cooperative
mechanism.
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Prior enhancer targeting studies have demonstrated that transcription and rearrangement of the TCRß locus critically depend on the presence of an active enhancer linked in cis (12, 13). Consistent with the lineage specificity of Eß, germline expression of endogenous Dß/Jß gene segments is restricted to pre-T lymphocytes. However, targeted replacement of Eß with Eµ redirects germline Dß/Jß transcription to both the B and T cell compartments (8, 12). Although these findings indicate that PDß can be dominantly regulated by linked enhancers, the independent contribution of this promoter to the tissue- and stage-specific expression of germline TCRß loci has not been addressed.
To determine whether PDß provides an additional level of regulation
for germline TCRß transcription, we transiently transfected a panel
of lymphocyte cell lines with an enhancerless version of the p377/3'
reporter construct. Relative to control constructs that lacked a
promoter, PDß stimulated only modest levels of luciferase activity in
most cell lines tested (Fig. 6A). Consistent with the
enhancer dependence of germline Dß/Jß expression, PDß activity
was significantly elevated in pre-T cells when linked to Eß. In
addition, PDß supported reporter gene expression in the mature B cell
line M12 upon inclusion of the Ig heavy chain enhancer element (Eµ).
Surprisingly, Eß failed to stimulate significant PDß function in
either the BW5147 or EL4 mature T cell lines (Fig. 6A),
which both express fully rearranged TCR and TCRß transcripts
(17, 18, 19, 20). This differential reporter gene activity was not due to a
lack of Eß function in mature T cells, since control experiments
demonstrated that Eß can stimulate a heterologous promoter (PSV)
approximately three- to sixfold in the BW5147 and EL4 cell lines (Fig. 6B). Likewise, when linked to the SV40 enhancer, PDß
directed significant levels of reporter gene expression in all T
lineage cell lines (data not shown). Together, these studies indicate
that PDß can function in most cell types when linked to an active
enhancer. However, this germline TCRß promoter is refractory to Eß
stimulation at more mature stages of T cell development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enhancers indicate that multiple
regulatory elements contribute to the recombinational accessibility of
these loci (11, 15). In contrast, targeted deletion of Eß abrogates
germline transcription and rearrangement of TCRß gene segments (12, 13). This strict enhancer dependence is preserved in TCRß minilocus
substrates using several experimental systems, including transgenic
mice and recombinase-inducible cell models (6, 7, 8, 9). Furthermore,
germline transcription and rearrangement of these substrates correlate
temporally with the activity profiles of test enhancer elements (9). As
such, TCRß miniloci have served as physiologically relevant models to
study the role of transcriptional control elements in the developmental
regulation of variable region gene assembly. Despite these advances,
attempts to mechanistically link transcription of gene segments with
their recombination potential have yielded conflicting results (1).
These disparities are due, at least in part, to a lack of information
regarding the localization and regulation of germline promoters. We now
report the identification and functional characterization of a
stage-specific promoter that directs germline transcription of DßJß
gene segments in developing thymocytes.
Although germline promoters have been identified for other Ig and TCR
loci (38, 39, 40, 41, 42), the transcription factors that regulate their activities
have not been established. Our biochemical and sequence analyses
demonstrate that PDß possesses a functional architecture shared by
many eukaryotic promoters, including a consensus TATA element and
binding sites for the ubiquitous SP1 activator. In addition, the
presence of two binding sites for the T cell-specific transcription
factor GATA-3 supports a potential regulatory role for PDß during T
lymphopoiesis. Prior studies have shown that GATA-3 binding contributes
to the function of all TCR enhancer elements, as well as the germline
promoter associated with J germline transcription (27, 35, 38, 39).
Likewise, our mutational analyses revealed that the GATA-3 sites
present in PDß contribute in a cooperative manner to its promoter
function (Fig. 5). Consistent with these findings, ablation of the
murine GATA-3 gene in mice blocks thymocyte development at a stage
before DßJß rearrangement (36). Thus, GATA-3 may be an important
component of the program that regulates levels of Ag receptor gene
expression and locus accessibility during T cell development.
A critical role for PDß in TCRß locus regulation is further
supported by its homology to other Dß-flanking regions. Specifically,
murine PDß1 exhibits 83% and 61% identities with sequences upstream
from the human Dß1 and murine Dß2 gene segments, respectively (29, 43). Furthermore, both the human Dß1- and murine Dß2-flanking
sequences harbor multiple GATA motifs situated 5' to a consensus TATA
element. Coupled with previous studies of IgH loci (41, 42), our
findings demonstrate that the positioning of competent transcriptional
promoters immediately upstream from J-proximal D elements is a highly
conserved feature of Ag receptor loci. In the context of the TCRß
locus, we have shown that one function of PDß is to drive expression
of unrearranged Dß1 gene segments in both TCRß miniloci and in
pro-T cells derived from RAG-/- mice (Fig. 1). In
addition, Northern blot analyses of TCRß minilocus transfectants
harboring a preformed DßJß1 rearrangement reveal continued high
level expression of Jß-hybridizing transcripts (data not shown).
Significantly, the decrease in transcript size observed for these
transfectants is consistent with the loss of Dß-Jß intervening
sequences, suggesting that PDß also directs expression of rearranged
DßJß gene segments. Given the link between transcriptional control
and recombination, it is likely that PDß participates not only in the
initial targeting of Dß gene segments but may be required to maintain
accessibility of DßJß joins for subsequent recombination
with Vß gene segments.
Our functional analyses of PDß in staged lymphocyte cell lines
recapitulate numerous regulatory aspects of germline TCRß expression
in vivo. First, the enhancer dependence of PDß in luciferase reporter
assays (Fig. 6) correlates with the observed block in germline DßJß
transcription at TCRß loci lacking active enhancer elements (6, 12, 13). Second, the isolated PDß element is active in precursor T cell
lines when linked to Eß. Likewise, DßJß transcripts are expressed
at high levels in RAG-/- pro-T cells, as well as pre-T
cells derived from RAG-/- animals complemented with a
functionally rearranged TCRß gene (12 and 19). Third, when coupled to
Eµ, PDß drives efficient reporter gene expression in mature B cell
lines (Fig. 6). The relevance of these data is supported by experiments
in which replacement of the endogenous TCRß enhancer with Eµ (12),
or inclusion of Eµ in transgenic miniloci (6, 8), redirects DßJß
expression to B lineage cells. Together, these studies indicate that
PDß function is not sufficient to restrict Dß/Jß germline
expression to precursor T cells. Rather, PDß may be dominantly
regulated by the activities of linked enhancer elements in most
lymphoid cells.
An unexpected finding from our reporter gene analyses was the complete
lack of PDß function in mature T cells when linked to Eß (Fig. 6A),
despite the ability of Eß to stimulate the activity of other
promoters in these cell lines (Fig. 6B; 20 . This
developmental uncoupling of PDß from Eß control cannot be explained
solely by alterations in promoter function, since SP1 and GATA-3 are
expressed at high levels in both pre-T and mature T cells (Figs. 3 and 4). Furthermore, no striking disparity in PDß activity was observed
in pre-T and mature T cells when this promoter was linked to a
heterologous enhancer (data not shown). Instead, the mechanisms that
prohibit functional interaction between Eß and PDß must be stage
specific and restricted to these particular elements. Differential
regulation of PDß during T cell development is further supported by
studies using Eß-TCRß minilocus transgenes. Specifically, the
levels of minilocus germline transcripts expressed by
CD4+/CD8+ thymocytes were significantly higher
than those measured in either CD8+ or CD4+
mature T cells (9).
Why might stage-specific constraints be imposed on the Eß/PDß
interaction? In precursor T lymphocytes, activation of DßJß
germline transcription and rearrangement would require a productive
interplay between these regulatory elements. However, in mature T
cells, Eß must activate Vß promoters to drive expression of
functional TCRß transcripts. Due to the tandem nature of DßJßCß
cassettes within this locus, a distinct population of mature T
lymphocytes will retain a single PDß element (Fig. 7). Thus, we propose that enhancer
function is redirected in these mature T cells to mediate normal
expression of functional TCRß transcripts at the expense of remaining
PDß elements (Fig. 7). The results presented here provide a framework
to test this model and to further dissect the role of transcriptional
control elements in providing recombinational accessibility to TCRß
loci.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eugene Oltz, Department of Microbiology and Immunology, Vanderbilt University Medical School, A4203 MCN, 1161 21st Avenue South, Nashville, TN 37232. E-mail address:
3 Abbreviations used in this paper: RS, recognition sequence; PAP, placental alkaline phosphatase; LTR, long terminal repeat.
Received for publication December 24, 1997. Accepted for publication March 31, 1998.
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M. M. Hughes, R. E. Tillman, T. D. Wehrly, J. M. White, and B. P. Sleckman The B12/23 Restriction Is Critically Dependent on Recombination Signal Nonamer and Spacer Sequences J. Immunol., December 15, 2003; 171(12): 6604 - 6610. [Abstract] [Full Text] [PDF]
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 J. Syken and C. J. Shatz Expression of T cell receptor {beta}locus in central nervous system neurons PNAS, October 28, 2003; 100(22): 13048 - 13053. [Abstract] [Full Text] [PDF]
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 P. F. van Loo, P. Bouwman, K.-W. Ling, S. Middendorp, G. Suske, F. Grosveld, E. Dzierzak, S. Philipsen, and R. W. Hendriks Impaired hematopoiesis in mice lacking the transcription factor Sp3 Blood, August 1, 2003; 102(3): 858 - 866. [Abstract] [Full Text] [PDF]
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X. O. Yang, R. T. Doty, J. S. Hicks, and D. M. Willerford Regulation of T-cell receptor D{beta}1 promoter by KLF5 through reiterated GC-rich motifs Blood, June 1, 2003; 101(11): 4492 - 4499. [Abstract] [Full Text] [PDF]
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M. L. Sikes, A. Meade, R. Tripathi, M. S. Krangel, and E. M. Oltz Regulation of V(D)J recombination: A dominant role for promoter positioning in gene segment accessibility PNAS, September 17, 2002; 99(19): 12309 - 12314. [Abstract] [Full Text] [PDF]
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R. Tripathi, A. Jackson, and M. S. Krangel A Change in the Structure of V{beta} Chromatin Associated with TCR {beta} Allelic Exclusion J. Immunol., March 1, 2002; 168(5): 2316 - 2324. [Abstract] [Full Text] [PDF]
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P. Sieh and J. Chen Distinct Control of the Frequency and Allelic Exclusion of the V{beta} Gene Rearrangement at the TCR{beta} Locus J. Immunol., August 15, 2001; 167(4): 2121 - 2129. [Abstract] [Full Text] [PDF]
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F. Chen, L. Rowen, L. Hood, and E. V. Rothenberg Differential Transcriptional Regulation of Individual TCR V{{beta}} Segments Before Gene Rearrangement J. Immunol., February 1, 2001; 166(3): 1771 - 1780. [Abstract] [Full Text] [PDF]
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 N. Mathieu, W. M. Hempel, S. Spicuglia, C. Verthuy, and P. Ferrier Chromatin Remodeling by the T Cell Receptor (Tcr)-{beta} Gene Enhancer during Early T Cell Development: Implications for the Control of Tcr-{beta} Locus Recombination J. Exp. Med., September 5, 2000; 192(5): 625 - 636. [Abstract] [Full Text] [PDF]
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M. Essand, G. Vasmatzis, U. Brinkmann, P. Duray, B. Lee, and I. Pastan High expression of a specific T-cell receptor gamma transcript in epithelial cells of the prostate PNAS, August 3, 1999; 96(16): 9287 - 9292. [Abstract] [Full Text] [PDF]
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R. T. Doty, D. Xia, S. P. Nguyen, T. R. Hathaway, and D. M. Willerford Promoter Element for Transcription of Unrearranged T-Cell Receptor beta -Chain Gene in Pro-T Cells Blood, May 1, 1999; 93(9): 3017 - 3025. [Abstract] [Full Text] [PDF]
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M. L. Sikes, C. C. Suarez, and E. M. Oltz Regulation of V(D)J Recombination by Transcriptional Promoters Mol. Cell. Biol., April 1, 1999; 19(4): 2773 - 2781. [Abstract] [Full Text] [PDF]
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