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INTRODUCTION |
Thromboxane A2, a potent vasoconstrictor and inducer
of platelet aggregation, is known to be involved in many
physiologic and pathologic processes including
hemostasis/thrombosis, cardiovascular disease (1, 2), pulmonary
diseases (e.g. bronchial asthma, pulmonary hypertension) (3,
4), and glomerulonephritis (5). Thromboxane synthase
(TXAS),1 an endoplasmic
reticulum membrane protein, catalyzes the conversion of prostaglandin
H2 to thromboxane A2 (6, 7). TXAS is a member
of the cytochrome P450 superfamily and is expressed in lung, liver,
kidney, and blood cells, including megakaryocytes and monocytes (8).
TXAS cDNA was first isolated from human (9-11), with the mouse
(12), rat (13), and porcine (14) cDNAs being cloned subsequently.
The complete human TXAS gene contains 13 exons and spans 193 kb of
contiguous DNA in chromosome 7 (15); the genomic structure was recently
confirmed by the Celera human genomic sequence (16).
The transcriptional start site of the human TXAS gene was mapped at
nucleotide 137 upstream from the ATG translation site (15). The
sequence of TXAS initiator (Inr) resembles the weakly conserved Inr
element for RNA polymerase II. An upstream sequence between nucleotides
90 and 56 relative to the TXAS transcriptional start site was shown
to be important for TXAS promoter activity in a megakaryotic cell line.
Further detailed analysis identified transcription factor NF-E2 binding
at the nucleotide region 83/77 as being responsible for TXAS
transactivation (17, 18). The NF-E2 site also plays an important role
in activation of the mouse and rat TXAS promoters (19, 20).
Transcription factor NF-E2 is a basic leucine zipper heterodimer
consisting of p45 and p18 subunits, which belong to the CNC protein
family and the small Maf protein family, respectively (21-24).
Expression of p45 NF-E2 is primarily restricted to hematopoietic cells
(22), whereas small Maf (p18) appears to be ubiquitously expressed
(24). p45 NF-E2 is a major enhancer mediating globin gene expression in developing and maturing erythroid cells (Ref. 25 and references therein). However, mice lacking p45 NF-E2 develop relatively normal erythropoiesis but suffer from abnormal megakaryocytic development and
fail to produce platelets (26). Megakaryocytes from p45 NF-E2-null mice
do not express TXAS mRNA, whereas other tissues, such as fetal
liver and bone marrow, have normal levels of TXAS mRNA (18). It
should also be noted that the transactivation mechanism of NF-E2
participates, at least in part, in the chromatin remodeling (27-29).
Binding of the NF-E2 to the chromatin template removed nucleosomes from
the 
- or 
-globin promoter in both in vivo and in
vitro studies (30, 31).
Previous studies describing transcriptional regulation of the TXAS
promoter have primarily been carried out by transient transfection assays in which the template DNA is nonreplicating and is unlikely to
be assembled into organized nucleosomes (Ref. 32 and references therein). Stably transfected DNA templates, however, represent replicating chromatin and are assembled into repeated arrays of nucleosomes. Considering that transcriptional activities may depend on
the template state, we examined TXAS promoter activities from naked
DNA, nonreplicating chromatin, and replicating chromatin templates. Our
results led us to propose a model in which NF-E2 induces nucleosomal
alteration in the vicinity of the TXAS promoter, thereby allowing the
basic transcriptional machinery to bind to the core promoter.
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EXPERIMENTAL PROCEDURES |
Materials--
The G-free cassette vectors, pC2AT
and pMLC2AT190, were kindly provided by Drs. Michele
Sawadogo and Ming-Jer Tsai (33, 34). Luciferase reporter vectors,
pGL3-Basic (promoterless), pGL3-Control (luciferase reporter gene
driven by SV40 promoter/enhancer), and the luciferase assay kit were
from Promega (Madison, WI). The -galactosidase reporter vector
pCMV--Gal (-galactosidase reporter gene driven by cytomegalovirus
immediate early promoter/enhancer) and the luminescent
-galactosidase assay kits were from CLONTECH (Palo Alto, CA). p220.2, an Epstein-Barr virus vector containing oriP, EBNA-1, and the hygromycin resistance gene, was
a generous gift from Dr. Bill Sugden (35). Lipofectin reagent, PLUS
reagent, OptiMEM, LipofectAMINE 2000, and hygromycin B were from
Invitrogen, and FuGENE6 was from Roche Molecular Biochemicals.
Micrococcal nuclease (MNase) was purchased from Worthington. Antibodies
against p45 NF-E2, Nrf1, and Nrf2 were obtained from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA).
Cell Culture--
The human megakaryoblastic cell line (MEG-01)
and the human erythroleukemia cell line (HEL) were maintained in
suspension in RPMI 1640 medium, and HeLa human cervical cancer cells
were cultured in modified Eagle's medium. All of the media were
supplemented with 10% heat-inactivated fetal bovine serum (Hyclone,
Logan, UT), 2 mM glutamine, 100 units/ml penicillin,
and 100 µg/ml streptomycin. Cells were grown at 37 °C in a 5%
CO2 humidified incubator.
Plasmid Construction--
DNA fragments containing the TXAS
promoter sequences 36/+3 and 248/+3 (relative to the
transcriptional start site) were obtained by PCR amplification and were
inserted into the Ecl136II site of a G-free cassette vector,
pC2AT, to obtain both the correct orientations
(pC2AT-TX36(+) and pC2AT-TX248(+)) and the
reverse orientations (pC2AT-TX36() and
pC2AT-TX248()). Mutations in the TATA-box (TATA to
TCCC; underline indicates mutation) and Inr (CACATT to
CACGTG) were introduced by PCR-based
site-directed mutagenesis. Plasmid pGL3-248/+3, which has the 248/+3
TXAS promoter fragment linked to a luciferase reporter vector, was
constructed first by cloning the 248/+3 PCR fragment into the
EcoRV site of pBluescript II SK (Stratagene) and then
cloning the SacI/HindIII fragment of the
resultant pBluescript into pGL3-Basic. The correct orientation was
confirmed by PCR. A series of 5'-deletion mutants in the TXAS promoter
linked to pGL3 (i.e. pGL3280/+137, 190/+137, 140/+137,
90/+137, 56/+137, and 38/+137) and 3'-deletion mutations of
pGL3280/+90, 280/+75, 280/+52, and 280/+3 were constructed in a
similar fashion. The numberings of the constructs indicate the
nucleotide positions relative to the transcriptional start site. The
minichromosome constructs, pMC280/+137, 190/+137, 90/+137, and
56/+137, were created by first inserting the
XbaI/BamHI fragment of pGL3-Basic that contains
the SV40 poly(A) signal into the XbaI/BamHI site
of p220.2. Subsequently, the XbaI fragments of
pGL3280/+137, 190/+137, 90/+137, and 56/+137, containing the
luciferase gene and the corresponding TXAS promoter sequence, were each
inserted into the XbaI site of the p220.2-SV40 poly(A) construct. A point mutation at the NF-E2 binding site (TGATTCA to
GGATCCA) in pMC280/+137, called
pMC280/+137-mNF-E2, was generated by PCR. NF-E2 cDNA was obtained
by reverse transcription-PCR from total MEG-01 cellular RNA (17) and
first subcloned into TA vector and then into the pcDNA3.1
eukaryotic expression vector (Invitrogen) at the
BamHI/HindIII site. All constructs generated by
PCR were confirmed by DNA sequencing.
In Vitro Transcription Assay--
HeLa cell nuclear extracts
were obtained as described (36). Optimal assay conditions were defined
in preliminary studies. Typically, reactions (25 µl) contained 20 mM HEPES, pH 7.9, 10% glycerol, 45 mM KCl, 3 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 1 µg of test template DNA, 0.5 µg of
control adenovirus major late promoter template pMLC2AT190,
10 units of RNase T1, 20 units of RNA guard (Amersham Biosciences), 3 µl of HeLa nuclear extract, and the nucleotide mix (0.4 mM ATP, 0.4 mM TTP, 1 mM
3'-O-methyl-GTP (Amersham Biosciences), 0.016 mM
UTP, and 10 µCi of [
-32P]UTP (800 Ci/mmol)).
Reactions were initiated by adding the nucleotide mix, incubated for 60 min at 30 °C, and terminated by adding 150 µl of stop solution
containing 0.3 M Tris, pH 7.4, 0.3 M sodium acetate, 0.5% SDS, 2 mM EDTA, and 0.45 µg of yeast tRNA.
Products were then extracted with phenol/chloroform/isoamyl alcohol and precipitated with ethanol. The pellets were washed with 80% ethanol, dried, and resuspended in 50% formamide and 5 mM EDTA. The
samples were heated at 95 °C for 2 min before they were separated on
a 5% polyacrylamide gel containing 7 M urea and visualized
by autoradiography.
Transient Transfection--
The cationic lipids used for
transient transfection were Lipofectin reagent and PLUS reagent for
HeLa cells and LipofectAMINE 2000 for MEG-01 cells. Transfection was
initially performed following the manufacturer's instructions and then
optimized in later experiments. Briefly, 1 day before transfection,
1 × 106 MEG-01 cells were seeded in six-well plates.
MEG-01 cells were exposed to 10 µl of LipofectAMINE 2000, 2 µg of
pGL3-TXAS reporter vector DNA, and 1 µg of pCMV--Gal in 2 ml of
OptiMEM. For HeLa cells, 1 × 105 cells were
transfected by 0.5 ml of OptiMEM containing 2 µg of pGL3-TXAS
reporter vector DNA, 1 µg of pCMV--Gal, 20 µl of Lipofectin reagent, and 10 µl of PLUS reagent after washing with serum-free medium. The medium was replaced with complete medium 4 h after transfection for HeLa cells. After a 48-h incubation without changing the medium, the cells were harvested and lysed in reporter lysis buffer
(Promega). Luciferase assays were performed with 20 µl of lysate and
100 µl of luciferase assay reagent using a MiniLumat Luminometer
LB9506 (Berthold, Wildbad, Germany). -Galactosidase activities were
determined using a chemiluminescence assay kit.
Stable Transfection--
Stable transfection of HEL cells
(1 × 106 cells in 2 ml) and HeLa cells (1 × 105 cells in 2 ml) was carried out using 6 µl of FuGENE6
and 2 µg of the minichromosome DNA. One day after transfection, cells
were subcultured into 20 wells of 96-well plate (HEL) or a 100-mm dish (HeLa) and selected with 400 µg/ml of hygromycin B. Several clones of
each construct were obtained. Copy numbers of each clone were determined by Southern blot analysis.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts of HEL cells were prepared by the method described previously
(37) and stored at 70 °C until use. EMSA was performed based on a
previously described method (38). Briefly, 5 µg of nuclear extracts
were incubated with a 32P-end-labeled probe (TXAS promoter
nucleotides 121 to +5) in a binding reaction buffer containing 20 mM HEPES, pH 7.9, 0.2 M EDTA, 15% glycerol, 1 µg of poly(dI-dC), 100 mM KCl, 2.5 mM MgCl2, and 1 mM dithiothreitol for 20 min at
room temperature. For supershift assay, nuclear extracts were incubated
with antibodies for 30 min before the labeled probe was added. The
mixtures were electrophoresed in a 4.5% polyacrylamide gel and
autoradiographed overnight at 70 °C.
Chromatin Immunoprecipitation (ChIP) Assay--
ChIP and duplex
PCR were carried out as previously described (39). HEL cells (2.4 × 107) maintaining minichromosome pMC280/+137 were fixed
with 1% formaldehyde for 10 min. Formaldehyde was neutralized by
incubation with 125 mM glycine for 10 min and then washed
twice with ice-cold PBS. Cells were lysed for 5 min on ice in 500 µl
of buffer containing 50 mM Tris, pH 8.0, 2 mM
EDTA, 0.1% Nonidet P-40, 10% glycerol supplemented with protease
inhibitors. Nuclei were centrifuged at 3000 rpm for 3 min and
resuspended in 200 µl of 50 mM Tris, pH 8.0, 5 mM EDTA, 1% SDS, and then sonicated five times for 5 s each time. The nuclei preparations were cleared by centrifugation and
diluted 10 times in 50 mM Tris, pH 8.0, 0.5 mM
EDTA, 0.5% Nonidet P-40, 0.2 M NaCl. Extracts were
precleared for 2 h with 60 µl of a 50% suspension of protein
A-Sepharose 4B beads (Sigma), 40 µg of sonicated salmon sperm DNA,
and 80 µg of bovine serum albumin. An aliquot of precleared chromatin
was removed, heat-treated to reverse protein-DNA interaction, and
precipitated with isopropyl alcohol (referred to as input) and
used in the subsequent duplex PCR analysis. The remainder of chromatin
sample was then separated into two tubes for different antibody
treatments. Immunoprecipitations were carried out at 4 °C for 6 h by adding 10 µg of p45 NF-E2 antibody or irrelevant rabbit serum
with 20 µl of 50% protein A-Sepharose 4B beads (presaturated with
salmon sperm DNA). The beads were washed five times with 1 ml of wash
buffer (20 mM Tris, pH 8.0, 0.1% SDS, 2 mM
EDTA, 1% Nonidet P-40, 150 mM NaCl) and three times with
10 mM Tris, pH 8.0, 1 mM EDTA. Protein-DNA
complexes were eluted by three successive treatments with 150 µl of
1% SDS, 100 mM NaHCO3. NaCl was then adjusted
to 0.3 M, and 1 µl of RNase A (10 mg/ml) was added.
Protein-DNA cross-links were reversed by heating at 65 °C overnight,
and DNA was extracted by adding 20 µl of 1 M Tris, pH
7.5, 10 µl of 0.5 M EDTA, and 2 µl of proteinase K (20 mg/ml) at 50 °C for 30 min. After phenol/chloroform extraction and
isopropyl alcohol co-precipitation with 30 µg of glycogen, the pellet
was resuspended in 30 µl of H2O. For the duplex PCR, 2 µl of ChIP DNA was used as template in a total volume of 10 µl of
PCR buffer containing 0.5 µM each of the primer pair for TXAS promoter (corresponding to the promoter sequences 90/72 and
+118/+137) and 0.5 µM each of the primer pair for the
TXAS 3'-untranslated region (corresponding to the cDNA sequences
1581/1600 and 1901/1920 downstream from the translational start site),
7.5 µCi of [-32P]dCTP, 0.2 mM dNTP and
Taq polymerase. After amplification, PCR products were
separated by a 4.5% polyacrylamide gel before autoradiography.
Chromatin-DNA Cross-linking, Preparation of Nuclei, and
MNase-coupled Southern Blot Analysis--
Nuclei from HEL cell clones
were prepared according to a modification of published procedures (30).
Briefly, 3 × 107 cells were washed with
phosphate-buffered saline, and chromatin-DNA was cross-linked by
incubation of cells with 1% formaldehyde at room temperature for 20 min (40) before being washed twice with ice-cold phosphate-buffered
saline and resuspended in 6 ml of suspension buffer (10 mM
Tris, pH 7.4, 1 mM EDTA, 0.1 mM EGTA, 15 mM NaCl, 50 mM KCl, 0.15 mM
spermine, and 0.5 mM spermidine) containing 0.2% Nonidet
P-40 and 5% sucrose. After a 5-min incubation on ice, the suspension
was centrifuged (15 min, 150 × g, 4 °C) through a
3.5-ml cushion of suspension buffer containing 10% sucrose. Nuclei
were gently resuspended in 3 ml of suspension buffer containing 8.5%
sucrose, and aliquots of 1 ml were digested with 10, 50, or 150 units
of MNase in the presence of 1 mM CaCl2 for 7 min at room temperature. The reactions were stopped by adding stop buffer at final concentrations of 0.5 M NaCl, 10 mM EDTA, 1% SDS, and 0.1 mg/ml proteinase K and then
incubated at 37 °C overnight. The cross-linking of DNA-protein was
reversed by heating at 60 °C for 5 h (41). The DNA was purified
by multiple phenol/chloroform extractions, analyzed by gel
electrophoresis, and subjected to Southern blot analysis using probes
that were labeled by random primers.
For indirect end-label analysis, the procedures for sample preparation
including nuclei preparation, chromatin-DNA cross-linking, MNase
digestion, and DNA isolation, were the same as described above except
that 0, 10, and 30 units of MNase were used. Purified DNAs were
digested with XbaI and BclI, separated by gel
electrophoresis after phenol/chloroform extraction, and analyzed by
Southern blotting using the 5'-end of the luciferase cDNA (cut with
HindIII and BclI from the pGL3-basic) as the probe.
MNase-coupled Ligation-mediated Polymerase Chain Reaction
(LM-PCR) Analysis--
MNase-treated nuclei were prepared, and total
DNAs were isolated as described above. LM-PCR was carried out following
a standard method (38). Briefly, the DNA sample (5 µg) was
phosphorylated with T4 polynucleotide kinase (New England Biolabs) in
the presence of 1 mM ATP and ligated to the unidirectional
linker (prepared by annealing the two oligonucleotides, linker 1 (5'-GCGGTGACCCGGGAGATCTGAATTC-3') and linker 2 (5'-GAATTCAGATC-3'))
with T4 DNA ligase (Invitrogen). The linker-ligated DNA was extracted
with phenol/chloroform and precipitated with ethanol. DNA
samples (0.3-0.9 µg; based on the copy number of minichromosomes)
were used as templates for 22 cycles of PCR amplification. The PCR
mixture (40 µl) contained 0.4 µl of Taq DNA polymerase,
0.4 µl of 10× Expand HF PCR buffer with 15 mM
MgCl2 (Roche Molecular Biochemicals), 0.8 µl of 10 mM dNTP, and 1.6 µl each of 10 µM linker 1 oligonucleotide and TXAS-specific primer P1. The reaction was carried
out with the following conditions: first cycle, denature for 5 min at
95 °C, with the remaining cycles for 1 min at 95 °C, 2 min
at optimal annealing temperature (54-56 °C, empirically determined
for each primer set), 5 min at 72 °C, and an additional 5-min
extension at 72 °C. The PCR product (5 µl) was added to 15 µl of
a fresh PCR mixture containing 0.1 µl of Taq DNA
polymerase, 1.5 µl of 10× Expand HF PCR buffer with 15 mM MgCl2, 0.4 µl of 10 mM dNTP solution, and 1.0 µl of 1 µM 32P-end
labeled TXAS-specific primer P2. The second amplification was carried
out for 11-18 cycles (empirically determined for each primer set) of
linear PCR amplification consisting of denaturation for 5 min at
95 °C for the first cycle, with the remaining cycles for 1 min at
95 °C, 2 min at optimal annealing temperature (56-58 °C,
empirically determined for each primer set), 5 min at 72 °C, and an
additional 5-min extension at 72 °C. After the second amplification, the reaction products were separated on 6% polyacrylamide, 7 M urea-sequencing gels. The sequence of the TXAS-specific
oligonucleotides were as follows: 3' side promoter primers, P1
(5'-TCCTCTGAGACCCCAGAGTG-3') and P2
(5'-GATCTCGCCGCCCTTATGGGAAG-3'); 5' side promoter primers, P1
(5'-GTACAAGTCCGTGGTTACAACC-3') and P2 (5'-GTTTCTCAGCAAACATGGGGAAG-3'). The locations of the primers in the TXAS promoter are shown in Fig.
1.
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Fig. 1.
Nucleotide sequence of the 5'-flanking region
of the human TXAS gene. Locations of putative cis-elements for
transcription factors are boxed. Nucleotides are numbered on
the right in bp relative to the transcription start site,
and the translation start site is underlined. The specific
5' and 3' primer sets for LM-PCR are indicated as 3'-P1, 3'-P2, 5'-P1,
and 5'-P2.
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RESULTS |
In Vitro Transcription of the Human TXAS Gene Promoter--
In
initial experiments, we used the core promoter (nucleotides 36 and
+3) of the TXAS gene linked to a 377-bp G-free cassette vector,
pC2AT, as a template (pC2AT-TX36(+)) to examine
TXAS gene expression. Transcription of this template was carried out in HeLa nuclear extracts as described under "Experimental Procedures." The test template yielded a 380-nucleotide transcript (Fig.
2A, lane
1), consistent with the predicted length of transcript
accurately initiated from the TXAS transcriptional start site. The
promoter sequence from the adenovirus major late gene (400 to +10)
inserted into a 180-bp G-free pC2AT vector
(pMLC2AT190) was used as control template and resulted in
correct initiation and a 190-nucleotide transcript as well as
"read-through" transcripts giving a 200-nucleotide transcript after
digestion by the G-specific ribonuclease T1 (Fig. 2A,
lane 2). The reverse orientation of the TXAS core
promoter inserted into the pC2AT vector
(pC2AT-TX36()), however, showed no promoter activity
above that for the internal promoter template pMLC2AT190
(Fig. 2A, lane 3). We conclude that
the 39 bp of TXAS core promoter is sufficient to activate correct
initiation of transcription in a direction-dependent manner
in the cell-free system.
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Fig. 2.
In vitro transcription
of the TXAS core promoter (A) and proximal promoter
(B). The arrows indicate the correctly
initiated transcripts from the designated templates. The sizes of
markers are indicated on the left.
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To optimize concentrations of nuclear extract, templates, KCl, and
MgCl2 for the TXAS in vitro transcription, we
carried out assays at different final concentrations of these
components. Increasing the amount of nuclear extract from 1 to 4 µl
(the protein concentration was ~12 mg/ml) gave a linear increase in
correctly initiated transcripts from both pC2AT-TX36(+) and
pMLC2AT190 templates (data not shown). We thus used 3 µl
of HeLa nuclear extract for the subsequent studies. Template levels of
1.0 µg for pC2AT-TX36(+) and 0.5 µg for
pMLC2AT190, a KCl concentration of 45 mM, and a MgCl2 level of 3 mM were also found to maximize
the in vitro transcription activities (data not shown).
These conditions are similar to those used for analyzing other native
and artificial promoters (42, 43).
Effects of TXAS Gene Upstream Promoter Sequence on in Vitro
Transcription--
To test whether the upstream region is important in
cell-free transcription, we linked TXAS promoter nucleotides between
248 and +3 to the 377-bp G-free cassette vector
(pC2AT-TX248(+)) and compared its promoter strength with
that of pC2AT-TX36(+). Both pC2AT-TX36(+) and
pC2AT-TX248(+) showed strong signal intensity at the
position of the expected 380-nucleotide transcript, with no significant
difference between the two (Fig. 2B, lanes
3 and 4). In fact, the signal from
pC2AT-TX36(+) was 30-fold higher than that from
pC2AT-TX248(+) when normalized to signals from pMLC2AT190. As negative controls, constructs with reversed
core promoter (pC2AT-TX36()) and upstream promoter
(pC2AT-TX248()) were also examined. Both constructs
produced very weak transcriptional activities (Fig. 2B,
lanes 1 and 2). Because HeLa cells do
not express TXAS (44), nuclear extracts of HEL cells (which
constitutively express TXAS) were added to the HeLa nuclear extract.
However, supplementation with HEL nuclear extract did not enhance the
transcriptional activity of pC2AT-TX248(+) (data not
shown). These results suggest that the core promoter of the TXAS gene,
introducing as a naked DNA template in the cell-free system, is
sufficient for transcription, and the upstream region is not critical
for transcription.
Role of TATA Box and Initiator of TXAS Promoter in the in Vitro and
in Vivo Transcription--
We further examined the functional elements
of TXAS core promoter that elicit transcriptional activity in
vitro. Two putative elements, the TATA box and Inr, are present at
nucleotides 29/26 and 3/+3, respectively (Fig. 1). The sequence
of the pyrimidine-rich TXAS Inr, CACA+1TT, closely
resembles a weak Inr consensus sequence,
PyPyA+1N(T/A)PyPy (A is the transcriptional start
site and Py is pyrimidine) (45), and is similar to the adenovirus major
late Inr (CTCACT) and the -globin Inr (CTTACA) (42). We first
introduced single mutations in either the TATA box or in the Inr and a
double mutation in pC2AT-TX36(+). As shown in Fig.
3A, a single mutation in
either the TATA box (lane 2) or in Inr
(lane 3) and the double mutation (lane
4) each dramatically reduced the TXAS transcriptional
activity, indicating that these elements are functional and crucial in
directing the transcriptional activity in vitro. In a
separate experiment, double-stranded oligonucleotide composed of
wild-type TATA and the Inr region (36/+3) was used as a competitor in
the in vitro transcription assay. However, no competition
was observed at a 500-fold molar excess of competitor over the template
pC2AT-TX36(+) (data not shown). We suspect that the
oligonucleotide competitor is too short for the basic transcription
machinery to bind and thereby fails to compete with the template
plasmid.
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Fig. 3.
Functional analysis of TATA box and Inr
elements of TXAS promoter using in vitro transcription
and in vivo transient transfection assays.
A, in vitro transcription using
pC2AT-TX36(+) wild type (lane 1),
TATA mutation (lane 2), Inr mutation
(lane 3), and double TATA/Inr mutation
(lane 4) as templates. B, effects of
the mutations at TATA box and/or Inr on the TXAS promoter activity in
MEG-01 cells. Schematic illustration of the constructs is shown on the
left. The promoter constructs of the TXAS gene mutated at
the TATA box and/or Inr are indicated as mTATA,
mInr, and mTATA/Inr. The numbers
indicate the positions of the start and end points of the TXAS promoter
fragments, and crosses indicate the mutations. The promoter
activity of each construct is indicated on the right
(means ± S.D. of at least three independent transfections).
Details are described under "Experimental Procedures."
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We then tested whether the TATA and Inr elements were also functional
in vivo. As described below, the TXAS core promoter alone
yielded very low transcriptional activity in vivo. We thus included the upstream promoter region and linked the nucleotides between 248 and +3 into the pGL3-Basic luciferase reporter vector as
the wild-type construct. Mutations were then made in this construct at
either TATA and/or Inr sites. These reporter vectors were transiently co-transfected into MEG-01 cells with pCMV--Gal serving as an internal control. In addition, we used pGL3-Control as a positive control and the promoterless pGL3-Basic as a negative control. Promoter
activities were normalized to those with the pGL3-Control and corrected
for variations in transfection efficiency using the ratio of luciferase
to -galactosidase activity. In contrast to the in vitro
assays, mutation at the TATA box alone had no effect on promoter
activity, and mutation at Inr showed only a 30% decrease in activity
(Fig. 3B). However, a significant decrease in promoter
activity was observed in the double mutant construct. These data
suggest that both the TXAS TATA box and the Inr are essential for
transcription in vivo and, as discussed below, that they may
act as compensatory elements.
In Vivo Transcription of the TXAS Gene Promoter by Transient
Transfection--
To further dissect the cis-elements involved in TXAS
expression, we made 5'- and 3'-deletion constructs of the TXAS promoter and linked them to the pGL3-Basic vector. The parent construct in this
study (pGL3280/+137) contained nucleotides between 280 and +137,
which includes two putative GATA sites near nucleotide 250
(Fig. 1). For the 5'-deletion mutation analysis, DNA fragments containing nucleotides 190/+137, 140/+137, 90/+137, 56/+137, and 38/+137 were generated by PCR and linked to pGL3-Basic. These reporter constructs were transiently co-transfected into MEG-01 cells
along with the pCMV--Gal plasmid. As shown in Fig.
4A, the promoter activity of
pGL3280/+137 was potent and was about 125% of that of pGL3-Control.
Deletion of nucleotides between 280 and 90 did not much affect the
promoter activity, since the pGL3190/+137, 140/+137, and 90/+137
constructs showed similar levels of activity. However, the removal of
nucleotides between 90 and 56 in pGL356/+137 reduced the promoter
activity by 90% as compared with that of pGL3280/+137, indicating
the presence of important elements in this segment.
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Fig. 4.
Transcriptional analyses of TXAS promoter
in vivo. A, deletion analysis of TXAS promoter by
transient transfection in MEG-01 cells. The diagram on the
left illustrates the constructs, and numbers
indicate the positions of the start and end points of the TXAS promoter
fragments. The diagram on the right shows the
promoter activities of each construct. B, promoter activity
in stably transfected HEL cells harboring minichromosomes with various
5'-deletions of the TXAS gene. A schematic illustration of the
constructs is on the left, and promoter activities monitored
by luciferase activity are shown on the right (means ± S.D.). The promoter activity of the longest construct is assigned as
100%.
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Many promoters contain functionally important sequences in the
5'-untranslated region, sequences that are referred to as downstream promoter elements (46, 47). Any such downstream promoter elements of
TXAS cannot be studied by our in vitro transcriptional assay using the G-free cassette. To further examine the role of the 5'-untranslated region, a series of 3'-deletion mutants of TXAS promoter construct were made in the +1/+137 segment. Luciferase reporter vectors containing DNA fragments corresponding to TXAS sequences 280/+137, 280/+90, 280/+75, 280/+52, and 280/+3 (referred to as pGL3280/+137, pGL3280/+90, and so forth) were generated. A mutation at the putative Sp1 site near +80, the
only recognizable site in the 5'-untranslated region, was also made (GGGCGGG to GAAAGGG) in the pGL3280/+137 construct. These
vectors were transiently co-transfected into MEG-01 cells with
pCMV--Gal. Promoter activities were found to gradually decrease as
the 5'-untranslated region became shorter (data not shown). Although
the difference in promoter activities between the strongest
(pGL3280/+137) and the weakest (pGL3280/+52) was about 2-fold, no
remarkable reduction was observed between any two successive
constructs. Furthermore, mutation at the putative Sp1 site caused only
about 20% reduction in promoter activity. These results demonstrate
that the effect of TXAS 5'-untranslated sequence on the promoter
strength, albeit weak, is additive and is proportional to the length of
this region. It has been speculated that, in many eukaryotic mRNAs,
lengthening of the 5'-untranslated region can lead to a proportional
increase in translational efficiency by increasing the load of the 40 S ribosomal subunits on the longer 5'-untranslated region (48). It should
be noted that many downstream promoter elements are located within 50 nucleotides downstream of the transcriptional start site (47), and
removal of these sequences drastically reduces the promoter activities
in vivo and in vitro. Such a phenomenon is not
observed in the TXAS 5'-untranslated region, since the pGL3280/+52
and pGL3280/+3 exhibited no significant variation in promoter activities.
In Vivo Transcriptional Studies of the TXAS Promoter Using
Chromosomal Reporter Constructs--
The transient transfection
experiments indicated that the TXAS sequence between nucleotides 90
and 56 was important for transcriptional activity (Fig.
4A), but in vitro assay implied that this region
was not critical for promoter activity (Fig. 2B). We
suspected that the discrepancy was a result of differences in the
template state; one is a naked DNA and the other an uncharacterized, nonreplicating nucleosomal DNA. To investigate the role of natural chromatin structure in TXAS transcription, we inserted various sizes of
TXAS promoter/luciferase reporter constructs into a minichromosome containing the Epstein-Barr virus origin for self-replication. Initial
attempts to develop MEG-01 cell lines harboring TXAS promoter/reporter constructs were unsuccessful. We subsequently transfected HEL cells,
which constitutively express TXAS, with a series of 5'-deletion mutations corresponding to nucleotides 280/+137, 190/+137,
90/+137, and 56/+137 of the TXAS gene (designated as pMC280/+137,
190/+137, 90/+137, and 56/+137, respectively). These episomal
minichromosomes were stably maintained in HEL cells after selection by
hygromycin B for 3-4 weeks. Quantitation of the expression levels of
the minichromosomal reporter gene controlled by these TXAS promoter fragments revealed that cis-elements between 280/190 and 90/56 are important for transcription (Fig. 4B). These results are
consistent with those obtained from transient transfection experiments
except that the effects exerted by these elements are more pronounced in the stably transfected cells, more than a 150-fold difference between 90/+137 and 56/+137 in the stably transfected cells (Fig.
4B) as compared with an 8-fold difference between the
corresponding constructs in the transient transfection (Fig.
4A). The results indicate that cis-element(s) between 90
and 56 is important in regulating TXAS promoter activity in the
native chromosomal structure context.
MNase-coupled Southern Blot Analyses of TXAS
Promoter/Reporter Minichromosomes--
To investigate
how the transcriptional activities of pMC-TXAS promoter constructs were
affected by changes in nucleosomal structures, we chose the two clones
carrying pMC90/+137 or pMC56/+137 for MNase-coupled Southern blot
analysis, because they exhibited the largest difference in
transcriptional activities (Fig. 4B). Partial digestion of
chromatin by MNase, which cleaves linker DNA between nucleosome cores,
produces an array of DNA fragments corresponding to various multiples
of the length of nucleosomal DNA monomer. Ethidium bromide-stained
total nuclear DNA digested by different concentrations of MNase showed
the expected laddering pattern for bulk DNA (Fig.
5B). The positions of probes
used for structural analysis in the TXAS and downstream regions of the minichromosomes are indicated in Fig. 5A. When Southern
blotting was performed using TXAS promoter nucleotides 90/+137 as a
probe, a ladder of fragments with a repeat of about 150 bp indicated that this part of the minichromosome of pMC56/+137 was assembled into
nucleosomes (Fig. 5C). On the other hand, a less distinct ladder and poorer hybridization were observed for the same region of
the pMC90/+137 minichromosome, particularly at the highest MNase
concentration. The results indicated that the promoter region of
pMC90/+137 was less compact and protected than that of pMC56/+137. We next examined the nucleosomal structure of luciferase gene by
hybridizing the same membrane with a probe containing a segment of the
luciferase gene. As shown in Fig. 5D, the luciferase segment in pMC56/+137 adopts a nucleosomal structure, and its MNase digestion pattern is very similar to that observed for the TXAS promoter region
(cf. Fig. 5, C and D). However, the
luciferase gene region in pMC90/+137 reveals less nucleosomal
structure (Fig. 5D), similar to that seen in the promoter
region (Fig. 5C), again suggesting a disrupted nucleosomal
structure. Finally, the DNA region downstream from the luciferase gene
was examined by rehybridizing the same membrane with a probe of p220.2
DNA fragment. Both pMC90/+137 and pMC56/+137 showed a similar
nucleosomal ladder in this downstream region (Fig. 5E). It
should be emphasized that mononucleosomes can be clearly seen in both
pMC90/+137 and pMC56/+137, suggesting that the region downstream
from the reporter gene is well protected in both minichromosomes. Taken
together, these results indicate that the disruption of nucleosomes
occurs at the promoter and luciferase gene region in the minichromosome
and TXAS is also actively transcribed when nucleotides 90/56 are
present. In contrast, the minichromosome maintains its intact
nucleosomal structure and loses its promoter activity when the
90/56 is deleted. These results suggest that the cis-element(s)
located in the 90/56 region of the TXAS promoter is involved, at
least in part, in the disruption of nucleosomes.
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Fig. 5.
Nucleosomal structure of
minichromosomes. Nuclei from HEL cells carrying minichromosome
constructs, pMC90/+137 and pMC56/+137, were digested by increasing
amounts of MNase (10, 50, and 150 units/ml; indicated by the
wedges). Total DNAs were isolated, separated on a 2%
agarose gel, and subjected to Southern blot analysis. A,
linear map of minichromosome indicating the location of the
PCR-generated probes, as shown in rectangles
below the map. P indicates the TXAS
promoter. B, ethidium bromide-stained gel. The amounts of
total DNA loaded were as follows: pMC90/+137, 30-35 µg/lane;
pMC65/+137, 75-85 µg/lane (the relative copy number of
pMC90/+137:pMC65/+137 was 2.5:1). C, hybridization with
a 227-bp promoter probe extending from TXAS promoter nucleotides
between 90 and +137. D, hybridization with a 250-bp
luciferase probe extending from nucleotide +586 to +835 (relative to
the TXAS transcriptional start site) of the luciferase gene.
E, hybridization with a 240-bp p220.2 probe extending from
nucleotide +2184 to +2423 (relative to the TXAS transcriptional start
site).
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Role of NF-E2 Site in TXAS Gene Transcription in a Native Chromatin
Context--
In transient transfection experiments, the 300/40
TXAS upstream promoter sequence, particularly at the NF-E2 site between 83 and 77, is crucial for transcriptional activity in several types
of cells (15, 17, 18, 20). To test whether NF-E2 activates the TXAS
gene in a natural chromatin context, we generated a mutation at the
NF-E2 site in the pMC280/+137 minichromosome (pMC280/+137-mNF-E2)
and stably transfected it into HEL cells. Cells harboring the wild-type
TXAS promoter exhibited 14-fold higher luciferase activity than those
harboring mutant NF-E2 promoter (Fig.
6A), indicating that an intact
NF-E2 site dramatically increases TXAS transcriptional activity under
the native chromosomal conditions.
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Fig. 6.
Effect of NF-E2 on the TXAS promoter in the
stably transfected cells. A, promoter activities of
TXAS gene from HEL cells carrying wild type and NF-E2 mutated
minichromosomes were stably transfected in HEL cells. Promoter
activities were determined by normalization of luciferase activity,
protein concentration, and copy number of minichromosome. Data
represent means ± S.D. of the relative luciferase activities, as
compared with that obtained from the wild type. B,
stimulation of TXAS promoter activity in stably transfected HeLa cells
by forced expression of p45 NF-E2. HeLa cells carrying wild type and
NF-E2 mutated minichromosomes were transiently transfected with (+) or
empty () p45 NF-E2 expression vector (pCMV-NF-E2). Data represent
means ± S.D. of the relative luciferase activities, as compared
with that obtained from the wild type transfected with p45 NF-E2
vector.
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Next, we asked whether p45 NF-E2 could activate TXAS gene expression in
HeLa cells, which do not express TXAS. We first carried out transient
transfection experiments using the same series of 5'-deletion reporter
plasmids described in the legend to Fig. 4A. As expected,
the promoter activities of all constructs transfected into HeLa cells
were very low, reaching at most only ~2% of the pGL3-Control (data
not shown). No significant change was observed when the 90/56
region was deleted. We also compared the promoter activities of stably
transfected pMC280/+137 and pMC280/+137-mNF-E2 in HeLa cells.
Luciferase activities were again very low from both wild type and
mutant constructs. However, transient transfection with a p45 NF-E2
expression vector strongly stimulated luciferase activity in the cells
containing pMC280/+137 (150-fold increase) as compared with those
containing pMC280/+137-mNF-E2 (20-fold increase) (Fig.
6B). These results clearly demonstrated an important role
for the NF-E2 site in controlling the TXAS gene expression under native
chromosomal conditions.
In Vitro and in Vivo Binding of p45 NF-E2 to the TXAS
Promoter--
To further demonstrate that p45 NF-E2 is involved in the
binding of TXAS promoter, we carried out EMSA and ChIP assay. EMSA results indicated that the nuclear extracts from HEL cells contained factor(s) capable of binding DNA fragments corresponding to the TXAS
promoter sequences 121/+5 (Fig.
7A). In a separate experiment, we performed reverse transcription-PCR experiments and found out that
HEL cells expressed Nrf1, Nrf2, and Nrf3 as well as p45 NF-E2 (data not shown). Supershift analyses were therefore conducted with
antibodies against p45 NF-E2, Nrf1, and Nrf2. As shown in Fig.
7A, a supershift of protein-DNA complex was observed with the addition of antibodies against p45 NF-E2 but not with Nrf1 or
Nrf2 antibodies. We also carried out competition experiments using a 20-bp oligonucleotide encompassing the NF-E2 site. The shift
band was completely diminished by the addition of a 10-fold molar
excess of wild type oligonucleotide but was not affected by even a
100-fold molar excess of oligonucleotide with mutated NF-E2 (data not
shown).
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Fig. 7.
In vitro and in vivo
binding of p45 NF-E2 to TXAS promoter. A, EMSA
reactions were carried out by incubation of HEL nuclear extracts and
end-labeled TXAS promoter nucleotides 121/+5. Antibodies against
Nrf1, Nrf2, and p45 NF-E2 were incubated with nuclear extracts
before the addition of the probe. The reaction mixtures were then
separated by electrophoresis. The positions of NF-E2·DNA complex and
the supershifted band are indicated. B, autoradiogram of PCR
products. ChIP and duplex PCR assays were carried out as described
under "Experimental Procedures." One primer pair amplifies the TXAS
3'-untranslated region (3'-UTR), and the other
pair amplifies the promoter region (Promoter). PCR templates
from input DNA and ChIP DNAs treated with anti-p45 NF-E2, irrelevant
serum, and no chromatin are indicated.
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ChIP experiments were performed using the stably transfected HEL cells
harboring ~10 copies of pMC280/+137. The enriched chromatin was
cross-linked and sonicated to generate fragments averaging less than
500 bp. Antibodies specific for p45 NF-E2 were used to
immunoprecipitate protein-DNA complexes, and a duplex PCR was carried
out with one primer pair specific for the TXAS promoter sequence
encompassing the NF-E2 site and a second pair specific for the TXAS
3'-untranslated region to generate the 227- and 340-bp PCR products,
respectively. The 3'-untranslated region is 193 kb away from the TXAS
promoter in the human chromosome 7 and does not contain the NF-E2
consensus sequence (15), thus serving as a negative control for NF-E2
binding. Duplex PCR assay was carried out under conditions of linear
amplification for both products and results of 28 cycles of PCR were
shown in Fig. 7B. Immunoprecipitation with anti-p45 NF-E2
antibody resulted in the recovery of considerably more promoter
fragment than the irrelevant serum control or the control with no
chromatin added to the protein A-Sepharose beads. In contrast, the
3'-untranslated region, which lacks the NF-E2 binding site, was not
detectable with anti-p45 NF-E2 antibody or control samples in the same
experimental conditions. In comparison with input DNA sample, p45 NF-E2
binding to TXAS promoter was significantly enriched relative to an
irrelevant locus, i.e. 3'-untranslated region. Taken
together, these results demonstrate that p45 NF-E2 binds TXAS promoter
in vitro and in vivo.
Indirect End Label and MNase-coupled LM-PCR Analyses of TXAS
Promoter/Reporter Minichromosomes--
To examine the nucleosomal
organization of the TXAS promoter encompassing the NF-E2 region in more
detail, we carried out MNase-coupled LM-PCR using the minichromosomal
constructs in stably transfected HEL cells. For the pMC56/+137
minichromosome, MNase digestion revealed two prominent sites at
nucleotide positions 130 and 280, indicating a 150-bp nucleosome core
particle (Fig. 8A). A weaker
band was also seen at nucleotide position 105, suggesting a 25-bp
linker. MNase digestion of the pMC90/+137 minichromosome, however,
revealed several additional cleavage sites not observed with
pMC56/+137, namely at nucleotide positions 185, 180, and 147 (Fig.
8A). These results are consistent with the MNase-coupled Southern blot analysis in which intact nucleosomes were readily seen in
the promoter of pMC56/+137 but not in pMC90/+137, indicating a
different nucleosomal structure when nucleotides 90/56 were present.
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Fig. 8.
Nucleosome structure analysis of TXAS
promoter by MNase-coupled LM-PCR. Nuclei from HEL cells carrying
stably transfected minichromosomes were digested by 30 or 60 units/ml
MNase as indicated by the wedges. A, LM-PCR
analysis using 3'-P1 and 3'-P2 primer set from the nuclei stably
transfected with pMC90/+137 or pMC56/+137. The thick
vertical line and thin line
indicate TXAS promoter and vector DNA, respectively, and X
indicates the NF-E2 site. The positions of MNase cleavage sites are
shown by the arrows. The oval at the
right and the dotted oval at the
left represent nucleosome 1 (N1) and remodeled
nucleosome N1, respectively. L1 and L2 indicate
linker regions and are bracketed. B, LM-PCR
analysis using the 3'-P1 and 3'-P2 primer set from nuclei of cells
stably transfected with pMC280/+137 (wild type) and
pMC280/+137-mNF-E2 (mutant). The bent arrow
indicates the transcription start site. C, LM-PCR analysis
5'-P1 and 5'-P2 primer set from nuclei of cells stably transfected with
pMC280/+137 (wild type) and pMC280/+137-mNF-E2 (mutant).
N, naked DNA; M, HpaII-digested pBR322
size marker.
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To ascertain whether binding at the NF-E2 site was associated with the
nucleosomal remodeling, we examined the nuclei harboring the
pMC280/+137 and pMC280/+137-mNF-E2 minichromosomes by indirect end
label analysis. The minichromosomes were partially digested with MNase,
cleaved with XbaI and BclI to obtain the 1.03-kb
parent fragment, and then subjected to Southern blot analysis using the 0.58-kb 5'-end luciferase gene as a probe (Fig.
9). MNase-sensitive sites were observed
in the wild type minichromosome. The positions of MNase cleavage sites
of the wild type minichromosome were mapped approximately between the
size of 900 and 750 bp, corresponding to TXAS nucleotides 180 and
30. Mutation of NF-E2 site, however, resulted in loss of the
MNase-hypersensitive sites (Fig. 9). We thus conclude that an intact
NF-E2 site is associated with nucleosomal disruption.
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Fig. 9.
MNase hypersensitivity analysis of TXAS
promoter by indirect end labeling. Nuclei from HEL cells carrying
stably transfected pMC280/+137 and pMC280/+137-mNF-E2 were digested
by 0, 10, or 30 units of MNase as indicated by the wedges.
After purification of the DNA and digestion with XbaI and
BclI, the products (7-15 µg of DNA) were separated on a
1.2% agarose gel and analyzed by Southern blotting using a
HindIII/BclI probe. HS, X,
and the horizontal arrow indicate hypersensitive
site to MNase, the NF-E2 site, and the transcription start site,
respectively. M, 1-kb size marker.
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To further map the MNase-hypersensitive sites in this region,
MNase-coupled LM-PCR analysis using the nested primer set 3'-P1 and
3'-P2 (Fig. 1) was carried out. LM-PCR defined a nucleosome core
particle for both wild type and mutant minichromosomes between nucleotide positions 130 to 280 and a linker at nucleotide positions 130-105 (Fig. 8B), which correspond to the TXAS promoter
nucleotides 40 to 190 and 40 to 15, respectively. This position
of the nucleosome particle identified by LM-PCR is consistent with that by indirect end label analysis (Fig. 9). In this nucleosome particle, MNase-hypersensitive sites at nucleotide positions 230, 188, 184, and
172 (corresponding to TXAS promoter nucleotides 140, 98, 94, and
82, respectively) were found in pMC280/+137 minichromosome but not
in pMC280/+137-mNF-E2 minichromosome or naked pMC280/+137 (Fig.
8B). Furthermore, many MNase cleavage sites were observed near the transcriptional start site (nucleotide position 90 in Fig.
8B) of pMC280/+137 and were absent in
pMC280/+137-mNF-E2. We subsequently examined the nucleosomal
structure from the 5'-end of TXAS promoter using the nested primer set
5'-P1 and 5'-P2 (Fig. 1). As shown in Fig. 8C, MNase
cleavage sites at nucleotide position 96 and the 68-74 region
(corresponding to the TXAS promoter nucleotide 81 and the 103 to
108 region, respectively) were more pronounced in the wild type
minichromosome than in the mutant. In contrast, two cleavage sites at
nucleotide positions 152 and 135 were seen at similar levels of
intensity for both wild type and mutant minichromosomes. These two
sites corresponding to TXAS promoter nucleotides 25 and 42 define a
linker region, in agreement with LM-PCR using the nested primer set
3'-P1 and 3'-P2. Moreover, these MNase cleavage sites were not found
using the naked wild type minichromosome DNA (Fig. 8, B and
C).
The results of LM-PCR thus place the NF-E2 site at one nucleosome
particle (N1 in Fig. 8, A-C), TATA at a linker (L2), and Inr at another nucleosome particle (N2). When the NF-E2 site is occupied, both nucleosomes N1 and N2 are disrupted (Fig. 8,
B and C). It should be noted that in Fig.
5C, significant nucleosomal changes at the promoter region
were observed at 150 units of MNase but were less evident at 50 units
of MNase. Because the MNase concentrations used in the LM-PCR and
indirect end labeling assays were at the range of 10-60 units, gross
nucleosomal changes were not seen, since the linker regions were still
prominent (Fig. 8, A and B). However, the
MNase-hypersensitive sites were clearly detected at this concentration
of MNase. Taken together, these results demonstrated that NF-E2 binding
was associated with the disruption of the nucleosomal structure of TXAS
promoter, particularly in the NF-E2 region.
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DISCUSSION |
The strength of promoter and accuracy of initiation have been
shown, in general, to largely depend on the sequence and relative distance of TATA and Inr elements in many genes driven by RNA polymerase II (Ref. 49 and references therein). Although the consensus
Inr sequence is loosely defined, a pyrimidine at 1, an A at +1, and a
T or A at +3 are the most critical nucleotides for determining the
strength of an Inr (50). These critical nucleotides are conserved in
the TXAS Inr, CA+1TT. In addition, the distance of 25 bp
between TATA and Inr, also the case in the TXAS core promoter, was
found to give the highest level of in vitro transcription
from the artificial templates (51). These factors may thus contribute
to a strong TXAS core promoter activity in vitro. Lee
et al. identified the transcription start site and TATA box
of the TXAS gene at the positions corresponding to our nucleotides 98
and 127/124, respectively (52), whereas Miyata et al.
(53) identified the transcription start site corresponding to our
nucleotide 2. However, no functional analysis was reported with
respect to the Inr and TATA elements. We showed here that mutation of
either element considerably reduced the promoter strength in the
cell-free transcription but did not affect the accuracy of initiation,
suggesting that the TATA and Inr elements played similar roles in
determining the transcription initiation site and directing the
transcription machinery to the TXAS promoter. In contrast to in
vitro studies, mutation of either the TATA or Inr element in the
presence of upstream sequence had little effect on promoter activity,
whereas mutation of both elements abolished TXAS promoter activity.
These findings suggested that the TATA and Inr elements of the TXAS
promoter were functionally redundant in vivo. This is
consistent with the notion that a preinitiation complex can be formed
through either a TATA or an Inr element, followed by a common step
leading to the gene activation (54).
Our results support the model that the upstream activator
(i.e. NF-E2) alters the nucleosomal structure and opens up
the core promoter. Because NF-E2 lacks the chromatin-modifying
activity, NF-E2 activation upon TXAS promoter is probably aided by
recruiting other proteins. In this aspect, evidence for NF-E2 involved
in chromatin disruption has been extensively studied in the globin genes (30, 31). The mechanism by which NF-E2 alters nucleosomal structures was elucidated from several studies. In the glutathione S-transferase pull-down experiments, p45 NF-E2 was shown to
interact directly with CBP/p300, a protein that possesses histone
acetyltransferase activity (55). Transient transfection experiments
showed that NF-E2 transactivation activity was enhanced by expression
of CBP/p300 and was inhibited by E1A, an inhibitor of CBP/p300 (56).
Interestingly, both subunits of NF-E2 were acetylated by CBP, and the
DNA binding and transcriptional activities were hence increased (57).
It is noteworthy that, despite the evidence of direct interaction between NF-E2 and histone-modifying enzymes in the globin gene transactivation, no evidence of this direct interaction is presented for the TXAS promoter. Furthermore, although our hypothesis favors the
model in which NF-E2 induces the nucleosomal changes of the TXAS
promoter, it is also plausible that NF-E2 increases transcription of
the TXAS gene by recruiting the transcriptional machinery before the
nucleosomal structures are altered.
The importance of NF-E2 in TXAS expression can probably explain why
TXAS is expressed at high levels in blood cells but much lower levels
in the nonblood cells (58). It should be noted that considerable
functional redundancy has been found among NF-E2 sites. The NF-E2 site
is recognized not only by p45 NF-E2 but also by other family members,
such as Nrf-1 (59), Nrf-2 (60), Nrf-3 (61), Bach1, and Bach2 (62).
Small Maf proteins include MafK, MafG, and MafF (23). Both MafK and
MafG could activate globin gene expression (63, 64). A combination of
these factors with others, such as zinc finger transcription factors,
may thus account for the diversity of TXAS expression in different
cells (65). For example, Rat macrophage TXAS is suppressed by
peroxisome proliferator-activated receptor 
ligands through
an interaction of Nrf2 and peroxisome proliferator-activated
receptor binding at the NF-E2 site (20). p18 NF-E2 can form a
heterodimer with Fos to act as a repressor for NF-E2 activity (66).
Furthermore, the gene regulation can also be controlled by the
localization of NF-E2 transcription factors. A recent work showed that
p18 NF-E2 was located in the centromeric heterochromatin compartment and bound globin gene as a homodimer in the repressed stage. Upon induction, p18 NF-E2 was relocated to the euchromatin compartment and
formed a heterodimer with p45 NF-E2 (67). We are currently studying the
TXAS transcriptional activity in the nonblood cells. Preliminary
results indicated that the NF-E2 site is also important for TXAS
promoter activity in these
cells.2 It is therefore
plausible that through the NF-E2 site TXAS is regulated not only for
certain activators or inhibitors but also for the cell preference expression.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Div. of Hematology,
Dept. of Internal Medicine, University of Texas- Houston, 6431 Fannin,
Houston, TX 77030. Tel.: 713-500-6794; Fax: 713-500-6810; E-mail:
lee-ho.wang@uth.tmc.edu.
