By
From the Department of Genetics and the Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115
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Abstract |
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The pre-T cell receptor (pT
) protein is a critical component of the pre-T cell receptor
complex in early thymocytes. The expression of the pT
gene is one of the earliest markers of
the T cell lineage and occurs exclusively in pre-T cells. To investigate the molecular basis of
thymocyte-specific gene expression, we searched for the genomic elements regulating transcription of the mouse pT
gene. We now report that expression of the pT
gene is primarily
controlled by an upstream genomic region, which can drive thymocyte-specific expression of a
marker gene in transgenic mice. Within this region, we have identified two specific DNase-hypersensitive sites corresponding to a proximal promoter and an upstream transcriptional enhancer. The pT
enhancer appears to function preferentially in pre-T cell lines and binds
multiple nuclear factors, including YY1. The enhancer also contains two G-rich stretches homologous to a critical region of the thymocyte-specific lck proximal promoter. Here we show
that these sites bind a common nuclear factor and identify it as the zinc finger protein ZBP-89.
Our data establish a novel experimental model for thymocyte-specific gene expression and suggest an important role for ZBP-89 in T cell development.
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Introduction |
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The development of a diverse and functional lymphocyte repertoire is achieved through the precise regulation of gene expression in various lymphoid lineages and at different developmental stages (1). As a powerful approach to the study of gene regulation in the immune system, genomic elements controlling the expression of lymphocyte-specific genes have been identified and used to isolate transcription factors involved in lymphocyte development (2).
The regulatory mechanisms underlying gene expression during the specification of the T cell lineage and its subsequent effector differentiation are being rapidly uncovered. Transcriptional regulation of many T cell-specific genes, particularly the TCR genes (3), has been described in great detail. Several transcription factors, including common lymphoid factors such as PU.1 and Ikaros and T cell-specific factors, such as GATA3 and TCF1/LEF1, were found to be critical for T cell development by gene targeting techniques (4). In contrast, our understanding of stage-specific gene expression in T cell development is still limited. Although several stage-specific regulatory elements such as alternative lck promoters (5), a CD4 silencer (6, 7), and CD8 enhancers (8) have been identified, their mode of action is not fully understood. In particular, molecular mechanisms that bring about gene expression specifically in pre-T cells remain to be elucidated.
The pre-TCR- (pT
)1 protein pairs with a newly synthesized TCR-
chain to form the pre-TCR, which transduces an obligatory survival signal to developing T cells at
the so-called "
selection" checkpoint (11). As an essential
component of the pre-TCR, pT
is critical for the efficient generation of T cells with productive TCR-
rearrangements, allelic exclusion at the TCR-
locus, and lineage commitment of
/
T cells (12). Consistent with its function at the early stages of T cell development, the pT
gene is expressed in pre-T cells in the thymus and extrathymic T cell maturation sites but not in mature thymocytes,
peripheral T cells, or other cell types (13, 14). Moreover,
pT
appears to be one of the earliest molecular markers of
the T cell lineage, as its expression was detected in T cell
progenitors in the mouse bone marrow and fetal blood (14),
as well as in adult human blood (15). In view of its pre-T
cell-specific expression, the pT
gene may provide an attractive model system to study the transcriptional regulation
of early T cell development. To this end, we set out to characterize regulatory regions within the mouse pT
locus.
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Materials and Methods |
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DNA Constructs.
Mouse pTCells.
A panel of T cell lymphomas, either derived in this laboratory from various knockout mouse strains or obtained from the American Type Culture Collection, was screened for the expression of pTDNase Hypersensitivity Assay.
Cells were harvested and lysed in reticulocyte standard buffer (10 mM Tris/HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl2) with 0.5% NP-40. Nuclei were washed and resuspended in reticulocyte standard buffer and treated with twofold dilutions of DNase I (Roche Molecular Biochemicals) for 5 min at room temperature. The reaction was stopped with a buffer containing 1% SDS, 50 mM EDTA, and proteinase K, and DNA was extracted, digested with appropriate enzymes, and analyzed by Southern hybridization. For the comparison between different cell lines, the amount of DNase was calibrated for each line and three concentrations were chosen: (i) the minimal DNase concentration producing a visible downshift of DNA fragments after restriction digest, (ii) a twofold lower concentration, and (iii) no DNase. The following genomic probes were used: a 0.46-kb PstI-NcoI fragment encompassing pTTransgenic Mice.
The construct containing a 9-kb pTRNase Protection Assay.
An NcoI fragment spanning 1.6 kb 5' of the pTTransfections.
Cells were transferred into 6-well plates and transfected with 1-2 µg DNA/well in duplicate, using lipid reagents according to the manufacturers' instructions. Cell lines LR1, BW5147, MEL, and NIH3T3 were transfected using Fugene 6 reagent (Roche Molecular Biochemicals); cells from cell lines LR2 and 642 were transfected using Superfect reagent (Qiagen). LacZ reporter vectors containing no promoter (pElectrophoretic Mobility Shift Assay.
For crude nuclear extract preparation, cell nuclei were isolated by hypotonic lysis, and proteins were extracted with a buffer containing 0.4 M NaCl. Each binding reaction (15 µl) contained 10 fmol (105 cpm)
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Results |
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Because regulatory genomic regions often display increased
sensitivity to DNase I treatment, we searched for such
DNase-hypersensitive sites (DHS) in the pT locus of T
cell lymphomas manifesting or lacking pT
expression. We
first used a probe corresponding to pT
exon 2 in conjunction with several restriction digests shown in Fig. 1 A. No
strong DHS correlating with pT
expression were detected
within or downstream of the pT
gene (EcoRV and NheI digests in Fig. 1 B and data not shown). A nonspecific DHS
was detected 4 kb downstream of the last exon in all cell
lines tested, confirming the validity of the analysis (NheI digest, Fig. 1 B). In contrast, a specific site (DHS 1) was found
immediately upstream of the first exon in pT
-positive but
not pT
-negative T cell lines (BamHI digest, Fig. 1 B).
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To examine the regions further upstream of the pT
gene, we initially used a probe detecting a 10-kb BglII
fragment 5' of the gene (data not shown). This preliminary
analysis suggested the presence of a specific DHS within
the region; however, a nonspecific DHS immediately 3' to
the probe precluded more precise mapping. To better localize the potential second DHS, we used a more downstream fragment as a probe (probe 2, Fig. 1 A). As shown in
Fig. 1 C, these experiments revealed the presence of two
closely located DHS, collectively referred to as DHS 2, ~4-4.5 kb upstream of the first pT
exon specifically in
pT
-positive cell lines. Thus, the genomic region 5' of
pT
harbors at least two specific DHS and is likely to play
a major role in the regulation of pT
expression.
To verify that putative regulatory
regions upstream of the pT gene were sufficient for pre-T
cell-specific gene expression, we created transgenic mice
carrying the marker gene LacZ under the control of a pT
5' fragment. The transgene contained 9 kb of pT
5' region, including both DHS and a part of the known 5' UTR, upstream of LacZ and SV40 intron and Poly(A) signal. The heterozygous progeny of transgenic founders were
analyzed for transgene expression by Northern hybridization with a LacZ probe.
Of six transgenic lines analyzed, two lines manifested detectable LacZ expression in the thymus. Both lines expressed LacZ in the thymus but not in the spleen; the line
carrying fewer copies of the transgene (four copies) was analyzed in more detail. As shown in Fig. 2, the marker gene
was expressed exclusively in the thymus but not in the
spleen, lymph nodes, or other organs, with a pattern and
abundance comparable to that of the endogenous pT
gene. Thus, a 9-kb 5' genomic fragment of the pT
gene
supported thymus-specific transgene expression in two
transgenic mouse lines. We therefore conclude that this
fragment contains all information necessary for pT
expression in thymocytes.
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Because of
its position 5' of the first exon, the first DHS (DHS 1) was
likely to reflect the activity of a proximal promoter. To
confirm this notion, we analyzed the 5' transcriptional start of the pT gene by RNase protection assay, using a probe
spanning 0.4 kb immediately upstream of the translation
start site (Fig. 3 A). Fig. 3 B shows the presence in T cell
lymphomas of heterogeneous pT
transcripts initiated
within 100-200 bp of ATG, with the most abundant transcript corresponding to a short 5' UTR of ~124 bp. This
position is only 5 bp 5' of the longest pT
cDNA clone
(16) and might represent a major transcription start site.
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To examine the promoter function of the sequences adjacent to the identified transcription start site, a 1.3-kb fragment spanning this region was subcloned upstream of a
LacZ (-galactosidase) reporter gene and transfected into
the T cell line LR1. This early passage cell line, derived
from an atm
/
rag-2
/
thymoma, has an immature T cell
phenotype (Thy-1hiCD4loCD8loCD2loCD25
CD44
TCR-
pT
+) and can be transfected using lipid reagents. As
shown in Fig. 3 C, the pT
upstream fragment manifested
a relatively weak orientation-dependent promoter activity.
Using 5' deletions of the fragment, the promoter function
was localized to a 0.15-kb PpuMI-PstI fragment containing 115 bp 5' of the putative transcription start site. Although
the low activity of pT
promoter precluded the analysis of
its function in other T cell lines, the promoter was inactive
in an erythroleukemia cell line, MEL (Fig. 3 C) and in
NIH3T3 fibroblasts (not shown). Thus, DHS 1 apparently
corresponds to a short proximal promoter that might be
specific at least for lymphoid cells.
To search
for the possible distal enhancers in the pT locus, we examined the effect of larger pT
genomic fragments in a transient transfection assay with LR1 T cells. As shown in
Fig. 4, a 9-kb 5' pT
fragment, used in the transgene as
described above, was more active than a 0.5-kb 5' fragment
containing the core promoter. Using in-vector deletions of
the 9-kb region, we first localized the enhancer activity to a
2.8-kb XbaI fragment. By creating a series of nested 5' and
3' deletions of this fragment (data not shown), this activity
was further mapped to a 0.35-kb region between BstEII
and NarI sites, 4 kb upstream of the pT
promoter. Fig. 4
demonstrates that this fragment and a smaller 0.25-kb
BstEII-MluNI fragment were fully active as transcriptional enhancers. In contrast, 5' or 3' truncations at a PstI site
within this region significantly reduced the enhancer function. Importantly, these mapping data are consistent with
the location of DHS 2, suggesting that the described pT
enhancer is at least one regulatory region corresponding to
DHS 2.
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Fig. 4 also demonstrates that pT enhancer increased
transcription when placed in either orientation and at a
variable distance from the promoter. To examine its activity on heterologous promoters, the larger 4.3-kb and the
smaller 0.35-kb fragments containing the pT
enhancer
were subcloned upstream of the SV40 early promoter. As
shown in Fig. 5 A, both pT
enhancer fragments significantly increased the activity of the SV40 promoter in LR1
T cells. The activity of pT
enhancer in LR1 cells was
stronger than that of previously described T cell-specific
enhancers of CD3
(Fig. 5 A) and CD2 (not shown) genes
and was also observed with another strong heterologous
promoter, the CMV immediate early promoter (data not
shown). Thus, the identified upstream enhancer of the pT
gene appears as a powerful, bona fide transcriptional enhancer, functioning irrespective of distance, orientation, or
the corresponding promoter (22).
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To examine the cell and stage specificity of the pT enhancer, the enhancer/SV40 promoter constructs were introduced into a pT
-negative T cell line, BW5147. As
shown in Fig. 5 A, the large enhancer fragment was completely inactive, whereas the small, 0.35-kb fragment produced only a minor increase similar to the CD3
enhancer.
Similar results were obtained with smaller enhancer fragments (not shown). This was not due to a limiting transfection efficiency, as the CMV promoter produced a strong
reporter activity in these cells. A similarly low activity of
the pT
enhancer was observed in nonlymphoid MEL and
NIH3T3 cells (data not shown). In another series of experiments, the same reporter constructs were introduced into
pT
-positive (642) or -negative (LR2) T cell lines. These
early passage T cell lymphomas could be transfected with a
comparable low efficiency by the same protocol. Fig. 5 B
demonstrates that the pT
enhancer increased the promoter activity in 642 cells but was scarcely functional in
LR2 cells compared with the control CD3
enhancer. Altogether, these data suggest, but do not prove, that the pT
enhancer is preferentially active in pre-T cells as compared
with mature T cells or nonlymphoid cells.
The sequence of the 0.25-kb BstEII-MluNI enhancer fragment was determined and is shown in Fig. 6 A. Nested 5' and 3' deletions of this region were produced by PCR using the indicated primers, subcloned into the SV40 promoter/ LacZ reporter vector, and assessed for their activity in LR1 cells (Fig. 6 B). This analysis revealed a core enhancer of 149 bp, defined by primers F2 and R2. Further deletions in this region significantly decreased the enhancer activity; the regions between F2/F3, R2/R3, and R4/R5 appeared particularly important. These data are consistent with the deleterious effect of truncations at the PstI site within the core enhancer region (Fig. 4).
Next, we examined nuclear proteins binding to the pT
enhancer by electrophoretic mobility shift assay (EMSA)
using nuclear extracts from several pT
-positive or -negative T cell lines, a B cell line, and an erythroleukemia cell
line. As probes, we used six double-stranded oligonucleotides spanning the core enhancer (Fig. 6 A) or larger promoter and enhancer DNA fragments. This analysis revealed
multiple distinct nuclear factors interacting with the core
enhancer; however, we were unable to detect any DNA-
protein complexes appearing specifically in pT
-expressing T cell lines.
The core enhancer sequence contained a potential binding site (CCAT; reference 23) for the transcription factor
YY1. Indeed, probe 3, containing the putative YY1 site,
formed a complex, found in all cells examined, that could
be specifically competed by YY1 consensus oligonucleotide (Fig. 7). Furthermore, Fig. 7 shows that the factor
binding to both probes could be supershifted by anti-YY1
Ab, thus confirming its identity as YY1. To explore the functional role of this interaction, the YY1 binding site was mutated so that the mutant sequence was unable to compete with YY1 binding (not shown). As shown in Fig. 6 C,
the YY1 site mutation resulted in a minor, but consistent,
increase in enhancer activity. Thus, the core pT enhancer
appears to interact with YY1 transcription factor, which
might contribute to the repression of its activity.
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It was
reported previously that the thymocyte-specific lck proximal promoter contains a G-rich site that appears critical for
the promoter function and binds a T cell nuclear factor
(factor B) (20). Because the pT enhancer sequence features two similar C-rich stretches, we examined the factors
binding to these sites and their possible relation to factor B
of the lck promoter. Enhancer probes 2, 5, and 6 produced
a similar pattern of nuclear complexes and effectively competed with each other for binding (not shown); we therefore concluded that the 5' and 3' C-rich sites bind the same
nuclear factors. Fig. 8 demonstrates that enhancer probe 2, encompassing the 5' C-rich site, formed two distinct complexes in T cell nuclear extracts. The upper complex was
specifically competed out by an Sp1 consensus probe,
whereas the lower complex was competed out by the
G-rich B site of lck promoter (lckB); moreover, the labeled
lckB probe formed a single complex of similar size. Antibody supershift experiments confirmed that the upper complex consisted of Sp1 and Sp3 transcription factors
(Fig. 8, center panel). Thus, the C-rich enhancer regions
bind Sp1 family proteins and another protein identical to
the B complex of lck promoter.
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A recently cloned zinc finger transcription factor, ZBP-89 (BFCOL1, BERF-1), was shown to bind long, G-rich
stretches in several promoters (24) and, therefore, represented a good candidate for the observed binding activity.
We tested this possibility and found that anti-ZBP-89 Ab
specifically blocked the formation of the lower complex
with probe 2 and of the major complex with lckB probe
(Fig. 8, right panel). Thus, ZBP-89 appears to interact with
two sites within the pT enhancer core and with a critical site of the lck proximal promoter. These observations suggest a possible role for ZBP-89 in thymocyte-specific gene expression.
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Discussion |
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This study was aimed at establishing a model to study the regulation of pre-T cell-specific gene transcription. The regulation of genes expressed at the early stages of B cell development has been extensively studied, and stage-specific transcription factors such as EBF (early B cell factor) have been identified (27). In contrast, the mechanisms of stage-specific gene expression in T cells are less well understood. Certain transcription factors appear to regulate specific stages of T cell development, as illustrated by the role of LKLF (lung Kruppel-like factor) in the maintenance of mature T cell quiescence (28). A clear example of reciprocal transcriptional regulation in early versus mature T cells is the alternative promoter usage at the lck tyrosine kinase gene (5). In particular, the lck proximal promoter was proven to function specifically in immature thymocytes (20) and has been extensively used to target transgene expression to early T cells. In another case, a thymocyte-specific enhancer was found in the third intron of Thy-1 gene (29). Furthermore, stage-specific silencers and enhancers were described in the CD4 (6, 7) and CD8 (8) loci, respectively. Despite this progress, the molecular basis of an apparently common pattern of pre-T cell- specific transcription is obscure.
The expression of the pT gene has been examined in
great detail and was shown to occur specifically in pre-T cells
(13, 14). Recently, the expression of an alternatively spliced
pT
isoform (lacking the extracellular Ig domain) was described (30, 31) and proposed to occur in mature T cells as
well as in pre-T cells (30). This isoform was also observed in
the original analysis of pT
expression but was not detected
in mature T cells (14). Similarly, we could not detect its
expression in the spleen or in pT
-negative T cell lines
(Reizis, B., unpublished results). Therefore, the expression
of a shorter pT
isoform in mature T cells is likely to occur,
if at all, only in a specialized minor population of T cells or at
extremely low levels. Thus, by and large, the pT
gene appears to be expressed in early T cells and as such represents a
valuable model for stage-specific T cell gene expression.
Although the mouse and human pT genes have been
extensively mapped and sequenced (16, 31), the regulation
of pT
gene expression has not been studied. We now report that mouse pT
gene transcription is regulated primarily by an upstream genomic region. It is possible, however, that additional genomic elements within, downstream
of, or farther upstream of the gene might contribute to its
regulation. In particular, possible locus control regions conferring position-independent transgene expression remain
to be identified in the pT
locus; the nonspecific DHS 5'
and 3' of the gene are candidates for such elements. Another likely regulatory region is the recently described sequence in the first pT
intron, which is conserved between
mouse and human genes (31). We found that a fragment
containing this sequence lacked any detectable enhancer
activity in a transient transfection assay (data not shown);
thus, it might function as a silencer or an element regulating chromatin accessibility. Further studies are required to
delineate the complete hierarchy of pT
transcriptional
regulation. In any case, our data indicate that the cell and
stage specificity of pT
expression are fully determined by
upstream elements.
Within the pT upstream region, we have identified a
proximal promoter and an enhancer located 4 kb 5' of the
promoter. As in many T cell-specific genes, the promoter
appeared relatively weak and is most likely insufficient for
pT
expression. The pT
enhancer, on the other hand,
manifested high activity in transient transfection assays and
appeared to function preferentially in pT
-positive pre-T
cell lines. It is possible, however, that additional elements
in the vicinity of the enhancer contribute to its specificity. In this regard, it is noteworthy that DHS 2 actually consists of two sites separated by ~0.3-0.4 kb; the precise nature of
these sites is currently under investigation. In addition, the
enhancer may require its cognate pT
promoter to achieve
full specificity.
Our analysis of the nuclear factors interacting with the
core pT enhancer (Figs. 7 and 8, and Reizis, B., unpublished data) suggests a preliminary model of its architecture
(Fig. 9). We could detect at least two distinct factors binding to the 5' end of the sequence, and these interactions
appear important for the enhancer function, as evidenced
by the F2/F3 deletion (Fig. 6). The two 5' E boxes appear
to have formed identical complexes, whereas the 3' E box
represents a consensus binding site for bHLH-ZIP transcription factors and might bind a distinct set of proteins. The enhancer features a perfect Ikaros binding site, and probe 4, spanning this site, formed a nuclear complex that was specifically inhibited by Ikaros consensus probe IK-BS2 (19) and
vice versa; however, we were unable to confirm the identity of this factor using anti-Ikaros antiserum. Nevertheless,
Ikaros protein isoforms or Ikaros-related factors might interact with this site in vivo and contribute to the repression
of the enhancer by recruiting it to centromeric heterochromatin in pT
-negative cells (32, 33). In addition, transcription factor YY1 binds to the middle portion of the enhancer and appears to repress its activity. YY1 is a
multifunctional factor implicated in, among other things, the repression of tissue-specific genes such as Ig and globin genes, possibly due to the recruitment of corepressors such
as histone deacetylases (34). Thus, the pT
enhancer may
be subject to the complex regulation at the level of chromatin modification.
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The pT enhancer contains two C-rich sites, and deletion of the 3' site compromised the enhancer function (Fig.
6). We found that these sites bind Sp1 and Sp3, the related
ubiquitous transcriptional activator and repressor proteins,
respectively (35). The true role of Sp proteins in the regulation of the pT
enhancer might be difficult to establish;
indeed, the expression of many genes thought to be regulated by Sp1 was not affected in its absence (35). It should
be noted, however, that Sp1 is abundantly expressed in the
thymus (36) and therefore might play a specific role in thymocyte gene expression.
In addition, these C-rich sites, as well as an important
G-rich site in the proximal lck promoter, bind a common nuclear factor identified here as ZBP-89. The truncated clone
of ZBP-89, ht, was originally cloned as a zinc finger protein binding to the CACCC box in the human TCR-
promoter (37). Recently, the full length protein ZBP-89/
BFCOL1/BERF-1 was cloned as a protein binding to
long, G-rich stretches in various promoters (24, 38,
39). This protein appears to be capable of both transcriptional activation (25, 26, 37) and repression (24, 26, 38),
possibly depending on the DNA context and cell type; the
precise function of ZBP-89 on the pT
enhancer remains
to be established. Despite its apparently ubiquitous expression, we have found that ZBP-89 mRNA is expressed in
the thymus at significantly higher levels than in any other
organ tested, including the spleen (Reizis, B., unpublished
results). Indeed, six out of eight cDNA clones corresponding to ZBP-89 in the mouse expressed sequence tag database are derived from the thymus library. In addition, the
ZBP-89 complex was easily detectable by EMSA in T cell
lines but not in nonlymphoid cells such as MEL. Together
with the observed binding of ZBP-89 to the regulatory elements active specifically in early T cells, these observations
suggest an important role of ZBP-89 in T cell development.
A proposed model of the pT core enhancer differs significantly from the core enhancers of TCR genes (3); in
particular, it lacks obvious binding sites for T cell-specific
transcription factors such as GATA3 or TCF1/LEF1. Furthermore, we were unable to detect enhancer-binding nuclear factors directly correlating with pT
expression in T
cell lines. This may be due to the technical limitations of
our approach, or it might reflect the requirement for additional regulatory sites as discussed above. Another possibility, however, is the existence of protein cofactors interacting with the enhancer-binding proteins and conferring cell
and stage specificity on the enhancer. Indeed, the bHLH
proteins, YY1 and Sp1, are known to be involved in complex interactions with other proteins, which are essential
for their function on a particular regulatory region. Similarly, ZBP-89 is likely to undergo cell type-specific regulation by other proteins. Future studies using this and other
models should delineate the precise mechanism of stage-specific gene expression in T cells.
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Footnotes |
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Address correspondence to Philip Leder, Dept. of Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: 617-432-7667; Fax: 617-432-7944; E-mail: leder{at}rascal.med.harvard.edu
Received for publication 9 March 1999.
B. Reizis is supported by the Cancer Research Institute Postdoctoral Fellowship.We thank Anne Harrington for oocyte injections, Christoph Westphal and Cathy O'Hara for cell lines, and Juanita Merchant for anti-ZBP89 antiserum. We are grateful to Mark Bedford for his crucial advice and support and to Robert Weiss, Jennifer Michaelson, Kevin Fitzgerald, and Yasumasa Ishida for many helpful discussions.
Abbreviations used in this paper
DHS, DNase-hypersensitive site;
EMSA, electrophoretic mobility shift assay;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
pT, pre-TCR-
;
UTR, untranslated region.
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