From INSERM U.474, Hôpital Henri Mondor, 94010 Créteil, France
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ABSTRACT |
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The GATA-3 transcription factor is required for
development of the T-cell lineage and Th2 cytokine gene expression in
CD4 T-cells. We have mapped the DNase-I-hypersensitive (HS) regions of
the human GATA-3 gene in T-cells and non-T-cells and
studied their transcriptional activities. HS I-III, located 5' from
the transcriptional initiation site, were found in hematopoietic and non-hematopoietic cells, whereas HS IV-VII, located 3' from the transcriptional start site, were exclusively observed in T-cells. Among
these hypersensitive sites, two transcriptional control elements were
found, one in the first intron of the GATA-3 gene and the
other between 8.3 and 5.9 kilobases 5' from the GATA-3 transcriptional initiation site. The first intron acted as a strong transcriptional activator in a position-dependent manner
and with no cell-type specificity. The upstream regulatory element
could confer T-cell specificity to the GATA-3 promoter
activity, and analysis of this region revealed a 707-base pair silencer
that drastically inhibited GATA-3 promoter activity in
non-T-cells. Two CAGGTG E-boxes, located at the 5'- and 3'-ends of the
silencer, were necessary for this silencer activity. The 3'-CAGGTG
E-box could bind USF proteins, the ubiquitous repressor ZEB, or the basic helix-loop-helix proteins E2A and HEB, and we showed that a
competition between ZEB and E2A/HEB proteins is involved in the
silencer activity.
Lineage commitment and differentiation of multipotent
hematopoietic stem cells occur throughout life and are mostly regulated at the transcriptional level (1). Multiple studies have now shown that
lineage-restricted expression of a subset of transcription factors is
essential to achieve proper development of all the hematopoietic
lineages, and therefore, the knowledge of the mechanisms involved in
the regulation of the expression of these lineage-restricted transcription factors is of considerable importance for further understanding of hematopoiesis.
Transcription factors of the GATA family are related by their conserved
zinc-finger motif that binds to the consensus DNA sequence
5'-(A/T)GATA(A/G)-3' (2). Among the GATA factors, GATA-1, -2, and -3 are necessary for hematopoiesis, as gene disruption of any one of these
factors results in major hematopoietic defects (3-6). GATA-1, -2, and
-3 display different lineage-restricted patterns of expression in
hematopoietic cells. GATA-1 is abundant in the erythrocytic,
mastocytic, and megakaryocytic lineages and is also present at a lower
level in multipotential progenitors (7-9); GATA-2 is mostly expressed
in uncommitted hematopoietic progenitors, immature erythroid cells, and
proliferating mast cells (10); and GATA-3 is expressed in very immature
hematopoietic progenitors and then only in the T-cell and natural
killer cell lineages (11, 12).
GATA-3 was first shown to be abundantly expressed in T-lymphocytes,
natural killer cells, and embryonic brain (13, 14). More detailed
studies have shown that GATA-3 gene expression occurs in
numerous sites during development: placenta, kidney, and adrenal gland;
the embryonic central and peripheral nervous systems; and embryonic
liver and thymus (14). Contrasting with this wide expression during
development, GATA-3 mRNAs are mostly detected in
thymocytes and T-lymphocytes and in the central nervous system in the
adult. This gene expression pattern, regulated during development and
cellular differentiation, might be mediated by a complex array of
cis-acting elements, as shown in the regulation of
transcription factor genes in invertebrates (15). As for hematopoiesis,
an extinction of GATA-3 gene expression in stable cell
hybrids formed by fusion of cell lines representing the erythrocytic
and the T-lymphocytic lineages indicated that GATA-3 might
be repressed in hematopoietic non-T-cells (16), a kind of regulation
already shown for the CD4 gene, another T-cell-specific gene (17).
The human, mouse, and chicken GATA-3 genes have been cloned,
and sequence analysis of their promoters revealed that they are embedded within a CpG island and share structural features often found
in promoters of housekeeping genes (14, 18, 19). Transfection experiments of reporter genes controlled by the mouse or chicken GATA-3 promoter have failed to show any appropriate
T-cell-regulated expression, which indicates that the GATA-3
regulatory elements lie 5' and/or 3' from the GATA-3
promoter (14, 19). To understand the transcriptional controls that
regulate GATA-3 gene expression in T-cells, we have mapped
the DNase-I-hypersensitive
(HS)1 regions of the human
GATA-3 gene in T-cells and in hematopoietic non-T-cells, and
we have studied the role of these regions in GATA-3 gene
expression in T-cells.
Nuclei Preparation and DNase-I Treatment
Approximatively 109 cells were washed twice in
phosphate-buffered saline; resuspended in 20 ml of homogenization
buffer (10 mM Tris (pH 7.4), 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, and 5%
sucrose) containing 0.05% (Jurkat), 0.2% (K562), or 0.6% (HeLa)
Nonidet P-40; and broken by five strokes of a Dounce homogenizer.
Nuclei were purified by centrifugation over a sucrose gradient (10%
sucrose in homogenization buffer), washed twice in wash buffer (15 mM Tris (pH 7.4), 15 mM NaCl, 60 mM
KCl, 0.15 mM spermine, 0.5 mM spermidine, and
10% sucrose), and subjected to increasing concentrations of DNase-I (0.1-15 mg/ml; Worthington) in wash buffer plus 1 mM
MgCl2 for 10 min at 37 °C. The reaction was stopped by
the addition of proteinase K (0.1 mg/ml final concentration), SDS (1%
final concentration), and EDTA (10 mM final concentration).
DNA Extraction and Southern Hybridization
DNA was extracted by proteinase K digestion (0.1 or 0.2 mg/ml)
at 56 °C overnight, followed by phenol/chloroform extraction and
ethanol precipitation. 10 µg of DNA were digested to completion with
the indicated restriction enzymes, electrophoresed on 0.8% agarose
gels, and transferred to nitrocellulose membranes (Hybond C Extra,
Amersham Pharmacia Biotech) by Southern blotting. Hybridization was
performed with random-primed 32P-labeled probes at 65 °C
in 5× SSC (0.6 M NaCl and 0.06 M sodium citrate (pH 7)), 1× Denhardt's solution, 20 mM
NaPO4 (pH 6.7), and 10% dextran sulfate. Highest
stringency washes consisted of 0.1× SSC and 0.1% SDS at 65 °C. The
genomic probes used for the DNase-I studies were a 582-bp
BglII-ClaI fragment and a 441-bp SspI-SstI fragment.
Construction of Plasmids
Constructs Used to Delimit the Human GATA-3 Promoter--
The
Constructs Used to Characterize a Human GATA-3 Gene-regulating
Element--
A human placental DNA library
(CLONTECH) was screened with a 5'-probe obtained
from a cosmid previously cloned, and a phage containing 12 kb of DNA 5'
from the human GATA-3 transcriptional initiation site was
isolated. A 2.4-kb BamHI-BamHI DNA fragment containing HS I and HS II (
All the constructs were finally cloned 5' from the CAT reporter gene
using the pBL-CAT-3 vector (20). All these constructs were sequenced
before use. Sequence analysis was performed using a Taq
DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems, Inc.,
Foster City, CA) and an automatic sequencer (Applied Biosystems Model 373A).
Construct Used to Overexpress ZEB--
ZEB cDNA
was cloned by reverse transcription-polymerase chain reaction using
oligonucleotides that encompass the ATG initiation codon and the TAA
stop codon. The polymerase chain reaction product was sequenced and
cloned into the pECE vector (21), where the inserted cDNA is driven
by the SV40 promoter and enhancer.
Cell Culture and Transfection
Human cell lines were grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum (HeLa) or in RPMI 1640 medium supplemented with 10% fetal calf serum (Jurkat and K562),
L-glutamine, penicillin, and streptomycin (Life
Technologies, Inc.). For transient transfection, 107 cells
were transfected by electroporation (Bio-Rad Gene Pulser; 960 microfarads, 200-250 V) in a volume of 175-250 µl using the same
molarity of reporter gene plasmids together with 2 µg of pRSV-Luc
plasmid. pBluescript was added to bring the total amount of DNA to 10 µg.
Cells lysates were prepared 24 h after transfection by the
freeze-thaw procedure (22). Luciferase assays were used to determine transfection efficiency, and CAT assays were performed using amounts of
extract normalized for transfection efficiency (23). The percentage of
acetylation for each extract was determined by quantification of the
14C-acetylated chloramphenicol on thin-layer chromatography
plates using a Molecular Dynamics PhosphorImager. Stable transfections were performed like transient transfections, except that the cells were
electroporated with 20 µg of linearized plasmid together with 1 µg
of plasmid containing the SV40 promoter-driven
neomycin-phosphotransferase gene. After selection on G418, three
independent pools of transfected cells were assayed for CAT activity.
Nuclear Extracts and DNA Binding Assays
Nuclear extracts were prepared from HeLa and Jurkat cell lines
(24), and DNA binding assays were performed essentially as described
(25). Poly(dI·dC) was used as nonspecific competitor, and in
competition assays, 50 ng of unlabeled competitor DNA was preincubated
with the nuclear extract for 5 min before the addition of the labeled
probe (0.5 ng). The µE5 and MEF1m oligonucleotides used have
been previously described (26), and the 3'-CAGGTG E-box oligonucleotide
is 5'-AGCTTTTTACCAGGTGGTCTCTA-3'. Antibodies against HEB and E2A
proteins were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Antibodies against human USF1 and USF2 were a kind gift from Dr. Michel
Raymondjean (INSERM U.129, Paris, France), and anti-ZEB antiserum was a
gift from Dr. H. Kondoh (Osaka University, Osaka, Japan).
Mapping of the DNase-I-hypersensitive Sites of the Human GATA-3
Locus--
Hypersensitivity of chromatin to DNase-I digestion has been
used to identify regulatory elements of numerous genes (27, 28). To
determine which regions of the human GATA-3 locus might be
implicated in the regulation of its expression in T-cells, we mapped
the DNase-I HS sites in 35 kb of human genomic DNA encompassing the
human GATA-3 transcription unit in a T-cell line (Jurkat) that expresses human GATA-3 and in a non-hematopoietic cell line (HeLa)
and an erythrocytic cell line (K562) that do not express human GATA-3.
Fig. 1A shows the mapping of
DNase-I HS sites after EcoRV-BglII digestion and
hybridization with a 5'-BglII-ClaI probe. In
addition to the 12-kb germ line fragment, two fragments of 7 and 2.5 kb
were observed exclusively in the Jurkat cell line, and a fragment of
1.1 kb was detected in the three cell lines studied. The 2.5-kb
fragment results from DNase digestion around the transcriptional
initiation site of the human GATA-3 gene; the 7-kb fragment
located an HS site in intron 3; and the 1.1-kb fragment identified a
region located 1.5 kb 5' from the transcriptional initiation site of
the human GATA-3 gene. Using SalI digestion and a
3'-SspI-SstI probe, we then mapped two HS sites,
located between exons 5 and 6 and found only in Jurkat T-cells (Fig.
1B). Using other digests, we finally mapped the seven HS
sites that are shown in Fig. 1C. Interestingly, the 5'-HS
sites were found in all the cell lines tested, whereas the 3'-HS sites
were found only in the Jurkat cell line.
Human GATA-3 Contains a Minimal Promoter That Extends in the First
Intron--
To look for any regulatory function of the HS sites
previously mapped, we first delimited the human GATA-3
minimal promoter. A segment of the human GATA-3 gene
encompassing the presumptive minimal promoter, from nucleotides Characterization of a DNA Fragment That Confers T-cell Specificity
to the Human GATA-3 Minimal Promoter--
All the DNA fragments that
encompassed the different HS sites mapped in the human
GATA-3 locus were cloned 5' from the HS I Acts as a Strong Silencer in Non-T-cells--
To characterize
the function of HS I and HS II, a 966-bp DNA fragment ( A 5'- and a 3'-Element Are Necessary for HS I Silencing--
5'-
and 3'-deletion analysis of this silencer revealed two
cis-acting elements that were necessary for efficient
silencing of the human GATA-3 promoter activity. The
5'-element was located between The Two CAGGTG E-boxes Are Necessary for HS I Silencing
Activity--
As the The 3'-CAGGTG E-box Could Bind ZEB, USF, and E2A Proteins--
To
characterize the proteins that can bind the 3'-CAGGTG E-box, we first
performed gel-shift analysis using Jurkat or HeLa nuclear extract. As
shown in Fig. 8A, two
complexes (C1 and C2) were obtained with Jurkat or HeLa nuclear
extract, whereas a third complex (C3) was obtained only with Jurkat
nuclear extract. To define the proteins present in these three
complexes, we first used competition with oligonucleotides known to
bind E2A or related proteins (µE5 oligonucleotide) or the ubiquitous
ZEB repressor (MEF1m oligonucleotide) (26). These experiments showed
that the C1 complex had the same migration as a ZEB complex (Fig.
8B, lanes 4 and 7) and that
the 3'-CAGGTG E-box oligonucleotide efficiently competed the ZEB
binding on the MEF1m oligonucleotide (lanes 7 and
8). The C3 complex migrated like the E2A complex bound on the µE5 oligonucleotide (Fig. 8B, lanes
3 and 4), and the 3'-CAGGTG E-box and the µE5
oligonucleotides efficiently competed each other (lanes
2 and 3 and lanes 4 and
6, respectively).
To characterize the proteins present in the C1, C2, and C3 complexes,
we used antibodies against ZEB, E2A, HEB, USF1, and USF2 for supershift
assays. As shown in Fig. 8C, the addition of anti-ZEB
antibodies supershifted the C1 complex, whereas the addition of control
serum had no effect on this complex (compare lanes 1 and
2). Increasing amounts of anti-ZEB antibodies completely abolished the formation of the C1 complex, but also abolished the
supershift shown in Fig. 8C (lane 2) (data not
shown). To identify the proteins present in this C3 complex, antibodies
against E2A or HEB protein were used for supershift assays, and both
antibodies completely supershifted the C3 complex, indicating that this
complex contains a heterodimer of HEB and E2A proteins (Fig.
8D, lanes 2 and 3).
Finally, antibodies against human USF1 or USF2 basic helix-loop-helix
protein showed that the C2 complex contained homo- and heterodimers
consisting of these two proteins (Fig. 8E, lanes
2-4). In conclusion, the 3'-CAGGTG E-box oligonucleotide could bind ZEB and USF proteins in HeLa cells, whereas it bound ZEB,
USF, and an E2A/HEB heterodimer in Jurkat T-cells.
ZEB and E2A/HEB Regulate the Silencing Activity of HS I--
To
demonstrate that ZEB was involved in human GATA-3 gene
repression, we cotransfected a ZEB expression vector together with the
human GATA-3 promoter linked to the
We then performed several point mutations of the 3'-CAGGTG E-box, which
can bind ZEB, E2A/HEB, and USF proteins. We transformed this 3'-E-box
to a MEF1m sequence, i.e. a sequence that can weakly bind
ZEB and neither E2A/HEB nor USF (Fig. 8B, lane 7)
(26); to the sequence AGTTCAGGTGTGTT located at Among the GATA transcription factors, GATA-3 displays a peculiar
expression, as it is expressed in many different tissues (i.e. central and peripheral nervous systems and embryonic
liver, kidney, and adrenal medulla) during development and only in the placenta, the central nervous system, very immature hematopoietic cells, and T-cells in the adult. To define regulatory sequences that
control GATA-3 gene expression in T-cells, we first
investigated the DNase-I hypersensitivity of the human
GATA-3 gene as DNase-I hypersensitivity has been associated
with a wide range of cis-regulatory sequences and is usually
indicative of protein-DNA interactions. Although no T-cell-specific HS
site was found 5' from the human GATA-3 gene initiation
site, three T-cell-specific HS sites were discovered in the
3'-direction. We assayed these different T-cell-specific HS sites,
linked to the endogenous or to a heterologous promoter, in both
transient and stable transfections, but we were unable to show any
function of these HS sites in transcriptional regulation. These results
indicate a possible requirement for other regulatory elements or that
the assays we used could not detect the function of these DNase-I HS sites.
We first determined the cis-acting sequences, located near
the transcriptional initiation site and involved in the human
GATA-3 gene activation. We did not find any sequence
involved in the T-cell-specific activity of the human GATA-3
gene, but mapped a sequence, located in the first intron between +475
and +598, required for high transcriptional activity. This sequence did not display classical enhancer activity, as it functioned, in both
sense and antisense orientations, only when located 3' from the
GATA-3 minimal promoter. Similar results have been obtained using the mouse GATA-3 promoter (14), and comparison of the mouse and human sequences located at the end of the first intron showed
little homology except at their 3'-ends, where the sequence 5'-CAGGTCTC(C/T)-3' lies (in the human and murine introns) one base 5'
from the 3'-splicing sequence.
We then studied the effects of the 5'-HS sites on the transcriptional
activity of the human GATA-3 promoter and found that a DNA
fragment, located between ZEB and E2A/HEB have been shown to regulate genes during hematopoiesis
(26, 42), but no competition between these proteins for DNA binding has
yet been reported. ZEB is an active transcriptional repressor that has
been shown to regulate both muscle differentiation and
We have previously shown that a cosmid that contains the human
GATA-3 transcription unit extended by 4 kb of 3'-flanking
sequence and 3 kb of 5'-flanking sequence is sufficient for directing
T-cell expression (18). This cosmid does not contain the silencer
described in this paper, and thus, the human GATA-3 gene
seems to be under the control of discrete regulatory elements that can
enhance and/or silence the transcriptional activity of a minimal
promoter. Furthermore, a recent publication has characterized an
enhancer sequence that regulates mouse GATA-3 gene
expression in the brachial arch (44). This enhancer, located between
nucleotides
INTRODUCTION
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Abstract
Introduction
References
MATERIALS AND METHODS
96/+598 DNA fragment was cloned from a cosmid that contained the
human GATA-3 gene by BamHI-BstEII
digestion, followed by electrophoresis and fragment purification. The
96/+44 DNA fragment was obtained by an XmnI digest of the
BamHI-BstEII fragment, followed by purification
of the BamHI-XmnI fragment. Mutants of the
96/+598 DNA fragment were obtained by double restriction digests of
the
96/+598 DNA fragment, followed by fill in with Klenow polymerase
and blunt-end ligation. The constructs used for orientation and
position dependence of the 3'-activating element were obtained by
cloning a RsaI-BstEII DNA fragment containing the
3'-activating element 5' or 3' from the
96/
44 DNA fragment.
8300/
5900 fragment) was subcloned into
pBSK, and the various constructs shown in this study were obtained by
subsequent digests of the BamHI-BamHI DNA
fragment. All point mutations were obtained by polymerase chain
reaction and subsequent cloning.
RESULTS
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Fig. 1.
A and B, mapping of the
DNase-I HS sites in the human GATA-3 locus after
BglII-EcoRV or SalI digestion,
respectively. The map shows the human GATA-3 gene, the
BglII-ClaI and SspI-SstI
probes used, and the resulting fragments observed after DNase-I
digestion together with the 12-kb BglII-EcoRV or
the 17-kb SalI-SalI germ line fragment. HS sites
are depicted by arrows. HS V, located at the beginning of
intron 3, could not be distinguished from the germ line fragment in the
SalI digestion. DNA was extracted from nuclei treated with
increasing amounts of DNase-I, digested with BglII and
EcoRV or SalI, electrophoresed, blotted, and
hybridized with the indicated probe. Jurkat cells (a T-cell line) express GATA-3,whereas HeLa cells (a non-hematopoietic
cell line) and K562 cells (an erythrocytic cell line) do not express
GATA-3. The different fragments observed are indicated by
arrows. M indicates the molecular mass marker.
C, summary of the DNase-I HS sites of the human
GATA-3 locus. The diagram shows the DNase-I HS sites of the
human GATA-3 locus ( ) and their location within this
locus. All the exons are indicated.
, noncoding sequences;
,
coding sequences. The bent arrow indicates the
transcriptional start site of the human GATA-3 gene. The
cell lines studied are indicated, and + and
indicate the
presence or absence of the HS sites in the cell line studied.
96 to
+44, was first studied (Fig.
2A). After transient
transfection into a T-cell line (Jurkat) and into two cell lines that
do not express human GATA-3 (HeLa and K562), we detected a
weak transcriptional activity that did not display any cell-type
specificity (Fig. 2A). By primer extension, we showed that
the transfected constructs were correctly initiated in the three cell
lines (data not shown). The addition of 5'-sequence (up to
2500) did
not change the transcriptional activity of the
96/+44 DNA fragment or
bring any T-cell specificity to the constructs used (data not shown).
However, the addition of 554 nucleotides located 3' from the
94/+44
DNA fragment resulted in a 6-10-fold enhancement of transcriptional
activity in the three cell lines (Fig. 2A). This
554-nucleotide fragment contained both the first exon and most of the
first intron of the human GATA-3 gene and, together with the
96/+44 DNA fragment, defined the
96/+598 human GATA-3
minimal promoter. To delimit the regions involved in the activity of
this promoter, we subjected this fragment to 3'-deletion analysis. As
shown in Fig. 2B, this analysis defined a 123-bp DNA
fragment, located at the end of the first intron, as necessary for
efficient activity of the human GATA-3 promoter. We then
looked for any position dependence of this 3'-element. A 390-bp DNA
fragment containing the human GATA-3 promoter 3'-element was
cloned in both orientations 5' or 3' from the
96/+44 DNA fragment
(Fig. 2C). Only the constructs that contained the 390-bp DNA
fragment 3' from the
96/+44 DNA fragment were transcriptionally
active in the three cell lines tested, indicating that the 3'-element acted as a strong transcriptional activator in a
position-dependent manner and with no cell-type
specificity.
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Fig. 2.
A, characterization of the human
GATA-3 promoter. The CAT reporter gene was transcriptionally
directed by the 96/+44 or
96/+598 DNA fragment. These constructs
were transfected into a T-cell line (Jurkat;
) that expresses
GATA-3 and into a non-hematopoietic cell line (HeLa;
)
and an erythrocytic cell line (K562;
) that do not express
GATA-3. After normalization, CAT activities were detected
and quantified using a PhosphorImager. The values represent the average
of at least three independent transfections, and 1 represents the CAT
activity obtained with the empty pBL-CAT-3 vector. The bent
arrows indicate the human GATA-3 transcriptional
initiation site. The white boxes indicate the first exon of
the human GATA-3 gene. B, an activating
cis-acting sequence is located within the first intron of
the human GATA-3 gene. Sequential 3'-deletions of the
96/+598 human GATA-3 DNA fragment were cloned 5' from the
CAT reporter gene as indicated under "Materials and Methods" and
transfected in the three cell lines previously described. C,
the sequence located at the 3'-end of the first human GATA-3
intron acts in a position-dependent manner. The +208/+598
3'-activating sequence was cloned in both orientations upstream or
downstream from the
96/+44 DNA fragment, and the resulting constructs
were transfected in the three cell lines studied. The indicated values
represent the average of three independent transfections.
96/+598 human
GATA-3 promoter, and the resulting constructs were stably transfected into Jurkat, K562, and HeLa cell lines. None of the 3'-T-cell-specific HS sites provided any specificity to the
96/+598 human GATA-3 promoter (data not shown), and thus, we focused
our study on the 5'-region of the human GATA-3 gene.
Starting from a construct that contained all the 5'-HS sites, we
performed deletion analysis to study, by stable transfections, the
three 5'-HS sites (Fig. 3). The 8.3-kb
DNA fragment that contained all the 5'-HS sites displayed a T-cell
specificity. The 6.5-kb DNA fragment containing only HS II and HS III
showed a higher transcriptional activity in Jurkat cells than in HeLa
or K562 cells, but the difference between these three cell lines was
smaller than that observed with the 8.3-kb DNA fragment, as the
transcriptional activity of this 6.5-kb DNA fragment was present in
HeLa and K562 cells (Fig. 3). The 3-kb DNA fragment that contained only
HS III displayed no cell-type specificity and was as active as the
human GATA-3 minimal promoter in all three cell lines (Fig.
3). To demonstrate that HS I and HS II were sufficient for T-cell
specificity, a 2.1-kb DNA fragment that contained these two HS sites
was cloned 5' from the human GATA-3 minimal promoter, and
this construct was stably transfected into Jurkat, K562, and HeLa
cells. As shown in Fig. 3, this 2.1-kb DNA fragment conferred T-cell
specificity to the human GATA-3 minimal promoter, indicating
that HS I and HS II were necessary and sufficient for T-cell
specificity of the human GATA-3 minimal promoter in the
assay we used.
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Fig. 3.
HS I and HS II confer T-cell specificity to
the human GATA-3 promoter. Sequential
5'-deletions of the 8025/+598 DNA fragment that contained HS I-IV
were fused to the CAT reporter gene and stably transfected into cells
lines that express (Jurkat T-cell line;
) or do not express (HeLa
non-hematopoietic (
) and K562 erythrocytic (
) cell lines) human
GATA-3. CAT activities were detected and quantified using a
PhosphorImager. The values indicated represent the average of three
independent pools of transfected cells, and 1 represents the CAT
activity obtained with the empty pBL-CAT-3 vector. The bent
arrows indicate the human GATA-3 transcriptional
initiation site. The white boxes indicate the first exon of
the human GATA-3 gene. The vertical arrows
indicate the location of HS I-IV.
8025 to
7059) containing only HS I was cloned 5' from the human
GATA-3 promoter, and the resulting construct was stably
transfected into HeLa, K562, and Jurkat cells. This fragment repressed
human GATA-3 promoter activity in the HeLa and K562 cell
lines, but not in the Jurkat T-cell line. The repression level was
identical to the one obtained with the HS I-HS II DNA fragment (Fig.
4), and interestingly, this silencer did
not reduce the transcriptional activity of the human GATA-3
minimal promoter in T-cells (data not shown). The orientation
dependence of this negative regulatory element was tested by inserting
the 966-bp DNA fragment in reverse orientation 5' from the human
GATA-3 promoter, and indeed, the transcriptional inhibition
obtained with this construct was the same, indicating that this
silencer was orientation-independent (Fig. 4). We then cloned a
1.1-kb DNA fragment (
7059 to
5900) containing only HS II 5' from
the human GATA-3 minimal promoter and stably transfected the
resulting construct into HeLa, K562, and Jurkat cells. We obtained a
5-6-fold decrease in the transcriptional repression in HeLa and K562
cells, and we got a 2-3-fold increase in the transcriptional activity
of the human GATA-3 promoter in Jurkat cells (Fig. 4). These
results indicate that HS I contains a major regulatory element that
confers T-cell specificity to the human GATA-3 minimal
promoter. We thus performed a detailed analysis of this silencer.
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Fig. 4.
Characterization of a non-T-cell
silencer. The 8025/
5900 DNA fragment containing HS I and HS II
was cut to generate a
8025/
7059 DNA fragment that contained only HS
I and a
7059/
5900 DNA fragment that contained only HS II. These two
DNA fragments were cloned 5' from the human GATA-3 promoter,
and all these constructs were cloned 5' from the CAT reporter gene. The
different constructs were stably transfected into T-cell (Jurkat;
),
non-hematopoietic (HeLa;
), and erythrocytic (K562;
) cell lines,
and the CAT activities were quantified using a PhosphorImager. The
values represent the average of three independent pools of transfected
cells, and 1 represents the CAT activity obtained with the pBL-CAT-3
vector. The bent arrows indicate the human GATA-3
transcriptional initiation site. The white boxes represent
the first exon of the human GATA-3 gene.
7828 and
7746, and the 3'-element
was located between
7197 and
7121 (Fig.
5). This analysis was done by stable
transfection, and we next examined whether these elements were
sufficient for efficient repression. We cloned the various deleted
regions 5' from the human GATA-3 minimal promoter and
transfected these constructs into HeLa or Jurkat cells, but never
obtained any efficient repression of the human GATA-3
promoter (data not shown), suggesting that the silencer identified
requires multiple elements to be functional. The sequence of the 707-bp
DNA fragment (
7828 to
7121) containing HS I is shown in Fig.
6. The 3'-DNA fragment necessary for
efficient repression in non-T-cells contained a YY1-binding site
adjacent to a CAGGTG E-box and a TCCTCCT motif already shown to be
required for neuronal expression of the zebrafish gata-2
gene (29), and the 5'-DNA fragment characterized also contained a
CAGGTG E-box. The presence of this same motif in the 5'- and 3'-regions
of the silencer prompted us to analyze its function.
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Fig. 5.
Deletion analysis of the HS I silencer.
5'- and 3'-deletions of the 8025/
7059 DNA fragment that contained
HS I were linked to the
96/+598 human GATA-3 promoter and
cloned upstream from the CAT reporter gene. The different constructs
were stably transfected into T-cell (Jurkat;
), non-hematopoietic
(HeLa;
), and erythrocytic (K562;
) cell lines, and the CAT
activities were quantified using a PhosphorImager. The values represent
the average of three independent pools of transfected cells, and 1 represents the CAT activity obtained with the pBL-CAT-3 vector. The
bent arrows indicate the human GATA-3
transcriptional initiation site. The white boxes represent
the first exon of the human GATA-3 gene.
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Fig. 6.
Sequence of the human GATA-3
gene silencer. The 7828/
7121 DNA fragment containing the
human GATA-3 gene silencer was sequenced on both strands.
The potential DNA-binding sites present in the 5'- and 3'-elements
defined by the deletion analysis of the HS I silencer are indicated in
boldface letters. The nucleotide sequence has been submitted
to the GenBankTM/EBI Data Bank with accession number AJ 131811.
7828/
7121 DNA fragment has the same
transcriptional activity as the initial
8025/
7059 repressor, the
role of the two CAGGTG E-boxes in HS I silencing activity was tested in
this DNA fragment beginning with the 3'-region. Mutations that deleted or disrupted the 3'-CAGGTG E-box were shown by gel-shift analysis to
prevent binding of any protein (data not shown), and stable transfections of the resulting constructs showed that they were completely unable to silence the human GATA-3 promoter
activity in non-T-cells and did not modify this promoter activity in
T-cells (Fig. 7A). A similar
mutation was performed on the 5'-CAGGTG E-box, and stable transfections
of the mutated silencer showed that it could not repress the human
GATA-3 promoter activity in non-T-cells (Fig.
7B). These results indicate that the HS I silencing activity needs these two CAGGTG E-boxes.
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Fig. 7.
The CAGGTG E-boxes present 5' and 3' from the
HS I silencer are necessary for efficient repression of the human
GATA-3 promoter activity. A, deletion
or point mutation of the 3'-CAGGTG E-box was performed on the
7828/
7121 DNA fragment, and the resulting constructs were linked to
the
96/+598 human GATA-3 promoter and cloned upstream from
the CAT reporter gene. The different constructs were stably transfected
into T-cell (Jurkat;
), non-hematopoietic (HeLa;
), and
erythrocytic (K562;
) cell lines, and the CAT activities were
quantified using a PhosphorImager. The values represent the average of
three independent pools of transfected cells, and 1 represents the CAT
activity obtained with the pBL-CAT-3 vector. The bent arrows
indicate the human GATA-3 transcriptional initiation site.
The white boxes represent the first exon of the human
GATA-3 gene. B, a point mutation of the 5'-CAGGTG
E-box was performed on the
7828/
7121 DNA fragment, and the mutated
fragment was analyzed as described for A.
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Fig. 8.
A, cell-type specificity of the
3'-CAGGTG E-box complexes. HeLa (lanes 1 and 2)
and Jurkat (lanes 3 and 4) nuclear extracts were
tested for binding activity with the end-labeled 3'-CAGGTG E-box
oligonucleotide probe in an electrophoretic mobility shift assay. The
three specific complexes (C1, C2, and C3) are indicated by
arrows. B, mapping of the 3'-CAGGTG protein-DNA
complexes. µE5 (lane 3), 3'-CAGGTG E-box (lane
4), and MEF1m (lane 7) oligonucleotides were used as
probes in an electrophoretic mobility shift assay with Jurkat nuclear
extract. Cold competition of µE5 binding with µE5 (lane
1) or the 3'-E-box (lane 2), of 3'-E-box binding with
the 3'-E-box (lane 5) or µE5 (lane 6), and of
MEF1m binding with the 3'-E-box (lane 8) is shown. The
specific C1, C2, and C3 complexes are indicated. C, anti-ZEB
antibodies induce supershift of the C1 complex. The 3'-CAGGTG E-box
oligonucleotide was end-labeled and incubated with a nuclear extract
from HeLa cells in the presence of a preimmune (lane 1) or
an immune (lane 2) serum against ZEB. Similar results were
obtained with a nuclear extract from Jurkat cells, but are not shown as
the C3 complex migrated at the same position as the anti-ZEB supershift
complex. The C1 and C2 complexes are indicated by arrows.
D, supershift of the C3 complex with an anti-E12/E47 or
anti-HEB antibody. The end-labeled 3'-CAGGTG E-box oligonucleotide was
incubated with a nuclear extract from Jurkat cells in the presence of a
preimmune serum (lane 1) or antibodies against E12/E47
(lane 2) or HEB (lane 3). The C1, C2, and C3
complexes are indicated by arrows. E, supershift
of the C2 complex with anti-USF1 or anti-USF2 antibodies. The 3'-CAGGTG
E-box oligonucleotide was end-labeled and incubated with a nuclear
extract from Jurkat cells in the presence of a preimmune (lane
1) or immune (lane 4) serum against USF1 or USF2
(lane 3) or a mixture of the two immune sera (lane
2). The preimmune serum used in this experiment drastically
decreased the C3 complex. The specific C1 and C2 complexes are
indicated by arrows.
7828/
7121 repressor or to the mutated
7828/
7121 fragment that could not bind ZEB (mutant CAGGTG
CAGGCC described in Fig. 7A).
Overexpression of ZEB resulted in significantly reduced transcriptional
activity of the
7828/
7121 construct in Jurkat T-cells (65%
repression), whereas the mutated version of this DNA fragment was not
sensible to this ZEB overexpression (Fig.
9A). These results indicate
that ZEB is involved in the repression obtained with the
7828/
7121 DNA fragment through the 3'-CAGGTG E-box.
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Fig. 9.
A, overexpression of ZEB in Jurkat
T-cells results in 7828/
7121 fragment-mediated repression of the
human GATA-3 promoter. The pECE/ZEB expression vector was
cotransfected with the human GATA-3 promoter, the
7828/
7821 human GATA-3 promoter, or a 3'-mutated
7828/
7821 human GATA-3 promoter into HeLa (
) and
Jurkat (
) cells. The two
7828/
7821 constructs differ only by the
CAGGTG
CAGGCC mutations that impaired ZEB binding to the 3'-E-box.
After normalization, CAT activities were detected and quantified using
a PhosphorImager. The values represent the average of three independent
transfections, and 1 represents the CAT activity obtained with the
empty pBL-CAT-3 vector cotransfected with pECE/ZEB. The bent
arrows indicate the human GATA-3 transcriptional
initiation site. The white boxes indicate the first exon of
the human GATA-3 gene. B, transformation of the
3'-CAGGTG E-box to a ZEB-only or a ZEB/USF-binding site results in
repression of the human GATA-3 promoter in Jurkat T-cells,
whereas transformation of this 3'-E-box to a ZEB/E2A/HEB site does not
change the transcriptional activity of the
7828/
7121 DNA fragment.
Point mutations that transformed the 3'-CAGGTG E-box to a ZEB-only, a
ZEB/USF-, or a ZEB/E2A/HEB-binding site were performed on the
7828/7121 DNA fragment, and the mutated fragments were linked to the
96/+598 human GATA-3 promoter and cloned upstream from the
CAT reporter gene. These three constructs, together with the control
plasmid, were stably transfected into T-cell (Jurkat;
) and
non-hematopoietic (HeLa;
) cell lines, and the CAT activities were
quantified using a PhosphorImager. The values represent the average of
three independent pools of transfected cells, and 1 represents the CAT
activity obtained with the pBL-CAT-3 vector. The bent arrows
indicate the human GATA-3 transcriptional initiation site.
The white boxes represent the first exon of the human
GATA-3 gene.
361 in the human
4-integrin promoter and shown to bind only ZEB and USF
proteins (30, 31); or to a
E2 sequence known to bind only ZEB and
E2A/HEB proteins (32). Stable transfections of the resulting constructs
in HeLa and Jurkat T-cells showed that the mutated
E2 sequence had
the same effect as the wild type 3'-E-box, whereas the MEF1m or
361 sequence induced a repression of the human GATA-3 promoter
in Jurkat T-cells (Fig. 9B). These results indicate that the
E2A/HEB heterodimer, but not the USF proteins, can relieve the
repression mediated by ZEB on the 3'-E-box.
DISCUSSION
8025 and
5900, could confer T-cell
specificity to the human GATA-3 promoter. This T-cell
specificity is mainly mediated by a strong silencer active in
non-T-cells and located between
7828 and
7121. Previous studies
have shown an extinction of the GATA-3 gene in both murine
and human erythroid × T-cell hybrids (16) and during the
commitment of Th0-like cells to Th1 effector cells (33). These
extinctions could be direct silencing or loss of positive regulators,
and our data strongly support the first hypothesis. Transcriptional
regulation by negative regulatory elements is now well documented, and
studies using transgenic mice or transfection assays have identified
silencers of transcription in numerous tissues and developmentally
specific genes (34-37). The position of silencers, like the position
of enhancers, varies depending on the gene studied and can be located adjacent to or far away from the regulated promoter (38, 39). The human
GATA-3 silencer was found 7 kb 5' from the promoter and thus
belongs to the second type of silencer. The molecular mechanisms that
mediate transcriptional silencing are not completely understood, but
several DNA-binding proteins, like COUP-TF or YY1 (40, 41), seem
clearly implicated in silencing. Deletion and point mutation analyses
of the human GATA-3 silencer revealed two CAGGTG E-boxes
that are necessary but not sufficient for efficient silencing. Point
mutation that destroyed the 5'-CAGGTG E-box resulted in an absence of
silencing, indicating the importance of this DNA-binding motif in human
GATA-3 gene regulation. Electrophoretic mobility shift
assays done with this E-box as a probe revealed two specific complexes
present in Jurkat, HeLa, and K562 cells (data not shown). These
complexes did not contain ZEB, USF, or E2A protein, as no supershift
could be obtained with antisera against all members of the USF, E2A,
and ZEB proteins (data not shown). Further characterizations of these
complexes are now needed to understand this 5'-CAGGTG E-box function.
Electrophoretic mobility shift assays done with the 3'-CAGGTG E-box
revealed three specific complexes, two present in T- and non-T-cells
(C1 and C2) and one present only in T-cells (C3). These three complexes
contained the ubiquitous repressor ZEB (C1), the USF basic
helix-loop-helix proteins (C2), and an E2A/HEB heterodimer (C3).
Overexpression of ZEB in T-cells showed that ZEB acted as a repressor,
and several point mutations indicated that E2A and HEB, but not USF
proteins, were able to relieve ZEB repression in T-cells.
4-integrin gene expression during hematopoiesis (30,
31). Interestingly, ZEB seems to compete with muscle-specific basic helix-loop-helix proteins for the regulation of muscle differentiation (31), whereas it impairs the myb-ets synergy
during hematopoiesis (30). As for human GATA-3 gene
regulation, our results suggest that ZEB represses the human
GATA-3 promoter activity and that the E2A/HEB heterodimer
can displace ZEB in T-cells. This E2A/HEB heterodimer also positively
regulates CD4 gene expression by regulating the activity of its
enhancer (43), and thus, E2A/HEB heterodimers seem to regulate early
and late genes activated during T-cell differentiation. Interestingly,
HEB or E2A mutant homozygous mice seem to have a developmental delay in
the transition from the double-negative stage to the double-positive
stage (42), and studies on chimeras indicated that the differentiation
of GATA-3
/
T-cells is also blocked at the
double-negative stage of development (6). These results, together with
the data presented in this paper, strengthen the link between HEB and
E2A proteins and GATA-3 gene regulation. Furthermore, as
E2A, HEB, and ZEB proteins are not T-cell-specific, their relative
amounts might switch on or switch off the human GATA-3 gene,
and thus, these non-lineage-specific proteins might be involved in
T-lymphoid determination.
2832 and
2642, does not confer any T-cell specificity,
and thus, the GATA-3 gene seems to be regulated by discrete
regulatory elements required for its complex expression pattern. Such a
modular cis-regulatory organization has been described for
many genes encoding transcription factors in Drosophila (15)
and shows the actual complexity of cis-regulatory elements
that regulate these genes.
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ACKNOWLEDGEMENTS |
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We thank the members of INSERM U.474 for constant support during this work and Anne-marie Dulac for preparation of the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from INSERM, the Association pour la Recherche sur le Cancer, and the Fondation de France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ 131811.
Supported by a grant from the Ligue Nationale contre le Cancer.
§ To whom correspondence should be addressed. Tel.: 33-1-49-81-35-26; Fax: 33-1-49-81-28-95.
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ABBREVIATIONS |
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The abbreviations used are: HS, hypersensitive; bp, base pair; kb, kilobase(s); CAT, chloramphenicol acetyltransferase.
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REFERENCES |
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