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INTRODUCTION |
The glycoprotein hormone
(GPH)1 family consists of
four members, chorionic gonadotropin, luteinizing hormone (LH),
follicle-stimulating hormone (FSH), and thyroid-stimulating hormone
(TSH). These hormones are heterodimers, sharing a common
-subunit but having unique 
-subunits that are thought to confer
their biological specificity. LH, FSH, and TSH are produced in the
anterior pituitary, whereas chorionic gonadotropin is synthesized in
the developing placenta. Thus, it is significant that the isolated -
or -subunits are also synthesized by a variety of tumors (1-5) and
tumor-derived cell lines (6-17). The free subunits are secreted by
cell lines established from both trophoblastic (e.g. JAr,
JEG-3) and nontrophoblastic (e.g. HeLa, ChaGo, CBT) tumors
(16, 18). In the latter instance, they are considered to be ectopic
proteins, i.e. characteristic of a cell type other than that
from which the tumor was derived. An active role for -subunit at
some stage of the tumorigenic process is supported by reports showing
direct correlations between -subunit production and tumor formation
in nude mice (19, 20) and the anchorage-independent growth of tumor
cell lines in vitro (8, 20).
The molecular mechanisms controlling human -subunit gene expression
in the placenta have been studied extensively. It has been shown that
multiple elements in the first 300 bp of DNA upstream from the
transcription start site (+1) are necessary to regulate the gene in a
tissue-specific manner (21). These are illustrated in Fig. 1. Basal
promoter elements include consensus TATA and CAAT boxes residing at
29 and 89, respectively. Cyclic AMP-responsive elements (CREs)
occur within two tandemly repeated 18-bp sequences that extend from
146 to 111 (22-24). The core CRE sequence is an 8-bp palindrome
(TGACGTCA) that is also found in several other cAMP-responsive genes
(25-30). Adjacent to the CREs in the GPH gene resides a
tissue-specific enhancer located from 180 to 150 that stimulates
basal levels of GPH gene expression in placental choriocarcinoma
cells (22, 31-33). The trophoblast-specific enhancer is a composite
element, containing adjacent and overlapping DNA-binding domains for at
least three proteins. The upstream enhancer requires the CRE to impart
its effects on transcription (22, 32, 33). The distal and central
domains are referred to as TSE (trophoblast-specific element) and
upstream regulatory element, respectively (21, 31-33); and the
proximal region between 161 and 142 contains a GATA binding motif,
originally identified as -activation element, which is able to
respond to cAMP (34). Cellular proteins have been identified that
interact specifically with these distinct regulatory elements (22, 23,
31, 32, 34, 35).
Control of cell-specific expression of the -subunit gene in the
pituitary differs from that in the placenta (21, 32-34, 36-38).
Regulation of GPH gene expression in the JEG-3 choriocarcinoma cell
line and in the T3-1 pituitary gonadotrope cell line is dependent
on the CREs (38-40). However, the trophoblast-specific enhancer seems
not to be involved, and no TSE binding activity could be detected in
gonadotrope or thyrotrope cell lines (38). In contrast, several regions
upstream of the TSE have been implicated for expression in pituitary
cells (40-42). The best characterized motif, called the
gonadotrope-specific element (GSE), is located in the human gene from
223 to 197 and is conserved in all mammalian -subunit genes
examined thus far (38). A protein that binds this element was detected
in T3-1 cells but not in thyrotrope cells and was identified as
steroidogenic factor-1 (SF-1) (36). The regions (480 to 417, 254
to 177, and 177 to 120) that confer thyrotrope-specific
expression to the mouse -subunit gene do not contain homology to the
GSE (41, 42). The sequence between 344 and 300 is referred to as
the pituitary glycoprotein basal element (PGBE), and an imperfect
palindrome centered between 342 and 329 has been shown to bind a
LIM homeodomain transcription factor (LH-2) (43). This suggests that a
LIM homeodomain protein can stimulate expression of one of the earliest
markers of pituitary differentiation. Regions comparable with the
murine PGBE have yet to be identified in the human gene. In addition,
two potential basic-helix-loop-helix protein binding sites (E-boxes)
are located in the GPH promoter just downstream (21 to 16) and
just upstream (50 to 45) of the TATA box. These are referred to as
EB2 and EB1, respectively, and their mutation reduces basal
activity of the promoter 60-80% in pituitary cells (44).
Despite the identification and characterization of these basal and
enhancer elements that confer tissue-specific activation of the GPH
gene in trophoblasts, gonadotropes, and thyrotropes, the molecular
basis for its expression in nonendocrine cell types remains poorly
understood. In HeLa cervical carcinoma cells, the GPH -subunit is
ectopically expressed and secreted at levels comparable with those in
the eutopic expressing JEG-3 choriocarcinoma cells (7). However,
previous studies have suggested that the -subunit gene proximal
promoter is not as active in HeLa cells as it is in JEG-3 cells and
that HeLa cells do not have the requisite binding protein to interact
with the TSE (22, 38). To investigate the mechanisms controlling
ectopic production of the gonadotropin subunits in nonendocrine tumors,
the regulation of GPH gene expression was studied in HeLa cells. In
a detailed analysis of the GPH gene promoter sequence, an imperfect
inverted repeat centered at the transcription start site was noted. It
is demonstrated in this report that the palindromic cap site sequence
constitutes a negative cis-acting element that
differentially contributes to promoter activity in a variety of cell
types depending on the level of a nuclear factor that demonstrates
specific binding to the cap site sequence. The element and its cognate
trans-acting factor are referred to as the cap site diad
element (CSDE) and cap site-binding protein (CSBP), respectively.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
HeLa cervical carcinoma cells, HT-29 colon
carcinoma cells, Panc-1 pancreatic carcinoma cells, and
GH4C1 murine pituitary cancer cells were
maintained in minimum essential medium supplemented with 5% bovine
calf serum. JEG-3 and JAr choriocarcinoma cells, U-2 OS osteosarcoma
cells, and MCF-7 breast carcinoma cells were grown in RPMI 1640 medium
supplemented with 10% fetal calf serum. The MCF-10A breast cell line
was maintained in Dulbecco's modified Eagle's medium/F-12 medium with
5% equine serum, 0.1 µg/ml cholera toxin, 10 µg/ml insulin, 0.5 µg/ml amphotericin B, 0.5 µg/ml hydrocortisone, and 0.02 µg/ml
epidermal growth factor. All media were also supplemented with
glutamine (0.06%), penicillin (100 units/ml), and streptomycin (100 µg/ml). Cells were grown as monolayer cultures and maintained in
T-flasks at 37 °C in a humidified atmosphere consisting of 95% air
and 5% CO2.
Plasmid Constructions--
(a) A 0.89-kbp fragment
extending from 846 to +48 was liberated from a GPH genomic clone
(45) by digestion with BglII and BamHI. The
pBLCAT3 vector (46) was linearized with BamHI and ligated to the BglII/BamHI fragment with T4
DNA ligase, placing the chloramphenicol acetyltransferase (CAT)
reporter gene under control of the wild-type -subunit gene promoter,
thereby generating p(846/+48)CAT. (b) To construct
p(846/+3)CAT, the p(846/+48)CAT vector was digested with
PstI, and the liberated fragment was isolated and subcloned
into the polylinker PstI site of pBLCAT3. (c) To introduce point mutations into the cap site sequence,
a DNA fragment extending from 1637 to +48 of the GPH genomic clone (45) was subcloned into M13, and uracil-containing single-stranded DNA
was isolated to serve as a template for mutagenesis (47, 48). Mutagenic
primers changed bases at 5 to 2 from TAAC to ATTG (m-1) and bases
at 4, 3, +4 and +6 from A, C, G, and T to T, G, A, and C,
respectively (m-2). The fragments extending from 846 to +48
containing the point mutations were liberated from M13 and subcloned
into pBLCAT3. (d) A synthetic oligonucleotide (CSDE, Table I) extending from 13 to +15 with BamHI
overhangs was inserted in both forward and reverse orientation into the BamHI site of p(846/+3)CAT to generate p(+3-CSDE)CAT
and p(+3-EDSC)CAT, respectively. (e) To fabricate HCAP
and HPAC, p(+3-CSDE)CAT and p(+3-EDSC)CAT vectors were cut with
PstI, and the larger fragments were gel-purified and
recircularized. Appropriate recombinants were identified by PCR or
restriction fragment analysis. Sequences across the cap site
(underlined) were verified: HCAP
(5'-GACTTCATTAACTGCAGTTACTGAGAAC-3') restores the imperfect
palindrome truncated at +15; HPAC
(5'-GACTTCATTAACTGCAGTTAATGAAGTC-3') creates a perfect
palindrome. All mutations and the orientation and number of inserts in
these reporter constructs were verified by dideoxy sequencing (49).
DNA-mediated Transient Expression Assay and Construction of
Stable Transfectants--
Transfections were performed in duplicate
using DNA-calcium phosphate co-precipitates (50) containing 10 µg of
the appropriate pCAT expression plasmid and 5 µg of the internal
control plasmid pCMVlacZ, which places the Escherichia coli
-galactosidase gene under control of the cytomegalovirus (CMV)
promoter. Cells were harvested 48 h after glycerol shock, and cell
lysates were assayed for CAT activity as described by Gorman et
al. (51, 52) and for -galactosidase activity as described by
Maniatis et al. (50). CAT activity was normalized to
-galactosidase activity for each sample. The aliquots assayed for
CAT were heated at 65 °C for 10 min; those to be assayed for
-galactosidase activity were left unheated. Protein concentration of
cell extracts was determined relative to a bovine serum albumin
standard by the method of Bradford (53). In separate experiments,
relative CAT activities for a given vector generally varied by no
more than ± 20%.
Stable transfectants were established by incubating HeLa SR3 cells with
calcium phosphate-DNA precipitates containing 20 µg of either
p(846/+48)CAT or p(846/+3)CAT plus 5 µg of
pSV2neo. Twenty-four hours after the glycerol shock, cells
were trypsinized and subcultured from a confluent T-25 flask into a
T-75 flask in medium containing 600 µg/ml G418 sulfate. Colonies
present after 14 days were pooled and subcultured in medium containing 200 µg/ml G418.
Preparation of Nuclear Extracts--
Crude nuclear extracts from
HeLa, JEG-3, HT-29, Panc-1, GH4C1, U-2 OS, JAr,
MCF-7, and MCF-10A cells were prepared as follows. Confluent cells in a
150-cm2 flask were washed and collected in a cold solution
containing 50 mM potassium phosphate (pH 7.4), 150 mM NaC1, 0.5 mM EDTA, and 0.5 mM
EGTA. The cell pellet was resuspended in 10 ml of cold nuclear wash
buffer containing 10 mM HEPES (pH 8.0), 50 mM
NaCl, 15% (w/v) sucrose, 0.1 mM EDTA, 0.5% (v/v) Triton
X-100, 1 mM dithiothreitol (DTT), 5 mM
MgCl2, and 1 mM phenylmethylsulfonyl fluoride
(PMSF). After incubation for 10 min on ice, the cell lysates were
underlaid with the above solution, omitting the Triton X-100 and
increasing the sucrose concentration to 30%, and centrifuged at 2,000 rpm for 30 min at 4 °C. Nuclear pellets were resuspended in 150 µl
of cold 10 mM Tris-Cl (pH 7.4) plus 1 mM EDTA
(TE buffer) and then incubated for 60 min on ice after adding 150 µl
of a solution containing 20 mM HEPES (pH 8.0), 1 M NaCl, 20 mM MgCl2, 0.2 mM EDTA, 2 mM DTT, 10 mM
spermidine, and 2 mM PMSF. After pelleting the nuclei,
protein extracts were dialyzed against 100 volumes of a buffer
containing 200 mM HEPES (pH 7.9), 20% (v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, and
1 mM PMSF. After dialysis for 4 h, the buffer was
replaced with an additional 100 volumes of fresh buffer, and dialysis
was continued another 4 h. Protein concentration in nuclear
extracts was determined as described by Bradford (53).
Electrophoretic Mobility Shift Analysis
(EMSA)--
Oligonucleotides used as binding probes and competitors
are summarized in Table I. EMSA was performed as described by Carthew et al. (54) with modification. Binding reactions were
carried out in a final volume of 25 µl containing 10 µg of crude
nuclear extract protein, 10% (v/v) glycerol, 20 mM HEPES
(pH 7.9), 100 mM KCl, 3 mM MgCl2, 4 mM spermidine, and 0.5 mM DTT.
Poly(dI-dC)-poly(dI-dC) (2 µg) was added to eliminate nonspecific
protein binding. For competition analysis, the reaction mixtures were
supplemented with 50-200 ng of unlabeled oligonucleotide, which
provided up to a 1000-fold excess of the competitor relative to the
probe. After a 10-min preincubation at 22 °C, 10,000 cpm of
32P-labeled oligonucleotide (~0.5 ng) was added, and
incubation was continued for 30 min. DNA-protein complexes were
resolved on 6.5% nondenaturing polyacrylamide gels in 1× TBE buffer
(90 mM Tris base, 64.6 mM boric acid, and 2.5 mM EDTA) and visualized by autoradiography of the
dried gel.
Methylation Interference Analysis--
Sense and antisense
oligonucleotides (13/+22, Table I) were individually end-labeled with
[
-32P]ATP and annealed with the appropriate unlabeled
complementary strand in excess. Double-stranded oligonucleotide probes
were purified over a 20% polyacrylamide gel, eluted, precipitated with ethanol, and resuspended in TE buffer as described above. The probes
were partially methylated by incubating 106 cpm of DNA in
5-10 µl of TE for 10 min at room temperature with 1-2 µl of
dimethyl sulfate in 200 µl of buffer containing 50 mM sodium cacodylate (pH 8.0) and 1 mM EDTA (pH 8.0). The
reaction was stopped by adding 40 µl of a solution containing 1.5 mM NaOAc (pH 7.0), 1 M 2-mercaptoethanol, and
10 µg of tRNA. The methylated probe was purified by ethanol precipitation.
The methylated probe was subjected to standard EMSA, and bands
corresponding to DNA-protein complex and free probe were excised and
electroeluted into 0.1× TBE. The eluates were supplemented with 10 µg of tRNA, extracted with phenol/chloroform (1:1), and cleared of
nucleic acid by ethanol precipitation. Precipitates were rinsed with
70% ethanol, air-dried, and resuspended in 30 µl of 0.5 M piperidine. The DNA was hydrolyzed at 90 °C for 30 min
and then precipitated twice with ethanol. The pellet was rinsed twice
with 70% ethanol, air-dried, resuspended in 10 µl of a solution containing 80% (v/v) deionized formamide, 50 mM Tris
borate (pH 8.3), 1 mM EDTA, 0.1% (w/v) xylene cyanol, and
0.1% (w/v) bromphenol blue, and boiled for 2 min. Equal amounts of
radioactivity derived from bound and free probe were subjected to
electrophoresis on a 10% polyacrylamide sequencing gel containing 7 M urea.
Isolation of Total Cytoplasmic RNA--
Cells that were stably
transfected with p(846/+48)CAT or p(846/+3)CAT were harvested
from confluent flasks by scraping into ice-cold Tris-buffered saline
(50 mM Tris-Cl (pH 7.4) and 0.15 M NaCl) and
washed twice in the same solution by centrifugation (1200 × g; 5 min; 4 °C). The cells were resuspended in ice-cold TE buffer and lysed by addition of 0.4% (v/v) Nonidet P-40. After removing nuclei by centrifugation, total cytoplasmic RNA was prepared from the postnuclear supernatant by addition of 1% sodium dodecyl sulfate and phenol extraction as previously described (7).
Primer Extension--
The oligonucleotide for specific priming
of CAT reverse transcripts was CAT-REV2
(5'-GAGCTTGGCGAGATTTTCAGGAGCTAAGGAAGC-3'), which is located at 36 to
4 relative to the CAT gene translation start site. This primer (10 pmol) and dephosphorylated, HinfI-digested 
X174 DNA (250 ng) were end-labeled using T4 polynucleotide kinase and
[-32P]ATP. After labeling, reactions were heated to
90 °C for 2 min to inactivate the T4 kinase. For primer extension, 1 µl of end-labeled primer was added to 15 µg of total RNA and 5 µl
of 2× buffer, which contained 100 mM Tris-Cl (pH 8.3), 100 mM KCl, 20 mM MgCl2, 20 mM DTT, 2 mM each dNTP, and 1 mM
spermidine. The primer and RNA were annealed by heating the tubes at
58 °C for 20 min followed by cooling at room temperature for 10 min.
Reaction mixtures for extension were constructed by adding 5 µl of
2× avian myeloblastosis virus primer extension buffer, 1.4 µl of 40 mM sodium pyrophosphate, 1 unit of avian myeloblastosis
virus reverse transcriptase, and 1.6 µl of H2O to the
annealing mixture. Incubation was at 42 °C for 30 min. The products
were supplemented with 20 µl of loading dye and heated at 90 °C
for 10 min. A sample aliquot of 10 µl and 1 µl of labeled X174
DNA marker were loaded onto a 10% polyacrylamide, 7 M urea
sequencing gel. The gel was run at 13 watts in 0.6× TBE buffer for
about 4 h and subjected to autoradiography.
| |
RESULTS |
Identification of a Regulatory Element Located at the Transcription
Start Site of the Glycoprotein Hormone -Subunit Gene--
The level
of promoter activity for many genes can be defined by the array of
cis-acting elements in the proximal promoter upstream of +1
and by the interactions of their corresponding DNA-binding proteins. In
addition, a number of regulatory factors have also been identified that
bind at, or slightly downstream from, the transcription start site and
make significant contributions to promoter activity (55-59). As
described in the Introduction, the GPH gene promoter is complex
(Fig. 1), containing multiple cis-acting elements in the proximal 5'-flanking DNA that
interact with nuclear proteins isolated from HeLa cervical carcinoma
cells (60), JEG-3 choriocarcinoma cells (21-23, 31, 32, 61), and
T3-1 pituitary gonadotropes (40-43). However, the activity of the
GPH gene promoter extending from 846 to +48 (i.e.
p(846/+48)CAT) was previously reported to be extremely low in HeLa
cells relative to the activity expressed in JEG-3 cells (22). To gain
an understanding of the mechanisms leading to low level expression of
the GPH promoter in nonplacental and nonpituitary cell types such as
HeLa, it was important to identify regulatory elements that account for
this low promoter activity.
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Fig. 1.
Schematic diagram of the
GPH promoter regulatory elements and CSDE
sequence. The GPH gene extending from 350 to +50 contains
multiple cis-acting elements. In the human GPH promoter,
a murine PGBE sequence is conserved at 333 to 320; it may serve as
a pituitary-specific element in thyrotropes for production of TSH. A
GSE at 223 to 197 is important for basal activation of the GPH
gene for FSH and LH synthesis in pituitary gonadotropes. The region
from 180 to 150 consists of three overlapping protein binding
subdomains, the TSE, the upstream regulatory element (URE),
and the -activation element (-ACT). The
cis-elements play a critical role in placenta-specific
expression. The CRE is composed of two 18-bp tandem repeats that are
located between 146 and 111. An element defined as the junctional
regulatory element (JRE) is located downstream of the CREs
(120 to 100) and overlaps a negative androgen response element
(ARE). The GPH gene also contains TATA and CCAAT basal
promoter elements located at position 29 and 89, respectively. Two
E-boxes (EB1 and EB2) flank the TATA motif,
and a negative thyroid hormone response element (T3RE) is
situated between the TATA box and the downstream E-box
(EB2). The CSDE sequence is indicated below
the map. Stars above and below the
bases mark diad symmetry of the GPH cap site. The start site of
transcription is indicated by +1. See the Introduction for
appropriate references.
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Because much of the earlier work had examined the first few hundred
base pairs lying immediately upstream of the cap site, this study was
undertaken to examine downstream sequences for potential regulatory
elements. Taking advantage of a PstI restriction site
centered at the +1 position of the GPH gene, a vector was generated
that terminated at +3 on the 3' end (i.e.
p(846/+3)CAT). Whether the first 45 bp of the 5'-untranslated
region of the -subunit gene affected promoter activity could be
assessed by comparing reporter gene expression in HeLa cells
transfected with vectors p(846/+48)CAT and p(846/+3)CAT. As
seen in Fig. 2, acetyltransferase activity from p(846/+3)CAT was increased 6-7- fold relative to
that from p(846/+48)CAT. These results suggest the presence of a
negative regulatory element located in the first exon of the GPH
gene. The element is located at, or downstream of, the transcription
start site, as the promoter shows increased activity by truncation from
+48 to +3. In analyzing the GPH gene in this region, an imperfect
inverted repeat was identified (Fig. 1). It is located with the diad
center at the transcription start site (i.e. +1) and will be
referred to as the CSDE.
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Fig. 2.
Activation of the GPH
promoter by 3'-truncation. The CAT reporter plasmids
containing DNA from 846 to +48 or from 846 to +3 relative to the
GPH transcription start site (+1) were transiently transfected into
HeLa cells in duplicate. After 48 h, cells were harvested and
assayed for CAT activity as described under "Experimental
Procedures." Values represent percent conversion of
[14C]chloramphenicol to acetylated derivatives after
normalization to -galactosidase activity introduced via
cotransfection of pCMVlacZ; they are the means obtained from at least
three independent experiments.
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Negative Influence of CSDE on GPH Transcription--
To firmly
establish a role for the CSDE in expression of the GPH gene,
clustered point mutations were introduced into the cap site to disrupt
the diad symmetry. Two mutants were constructed using classical
methodologies based on M13. Mutation m-1 extends from 5 to 2 and
converts TAAC to ATTG, which disrupts the diad left arm; and mutation
m-2 has substitutions at 4, 3, +4, and +6 to change A, A, G, and T
to T, G, A, and C, respectively, which alters both diad arms (Fig.
3A). The DNA fragments
extending from 846 to +48 containing mutated cap site sequence were
released from M13 DNA and engineered into pBLCAT3. The
reporter plasmids p(846/+48)CAT (wild type), m-1, m-2, and
p(846/+3)CAT were transiently cotransfected with pCMVlacZ into
HeLa cells. Acetyltransferase activity was normalized to
-galactosidase activity to account for differences in transfection
efficiency. As shown in Fig. 3B, CAT activity from m-1, m-2,
and p(846/+3)CAT (abbreviated as +3) was increased 3-, 4-, and 6.5-fold, respectively, relative to that from the
p(846/+48)CAT (abbreviated as +48) vector set at 1.0. Because the
mutant promoters were more active than the wild-type promoter, these
results indicate that CSDE acts as a negative regulatory element.
Furthermore, both halves of the diad contribute to the element's
activity, as point mutations in m-1 inactivate the upstream arm, and
truncation at +3 inactivates the downstream arm.
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Fig. 3.
Transient transfection of HeLa cells with
wild-type and mutant expression vectors. A, two cap
site mutants were constructed as described under "Experimental
Procedures." The boxed bases indicate the clustered
mutations. One mutant (m-1) had four base pair changes from 5 to 2
and the other (m-2) contained point mutations at 4, 3, +4, and +6.
Presented are the confirmatory dideoxy sequencing gels for the two
mutants and a wild-type cap site. B, the DNA fragments
extending from 846 to +48 and containing either wild-type or mutated
cap site sequence were subcloned from coliphage M13 into the
pBLCAT3 expression vector. DNA sequence in the
boxes indicate the mutated bases (lowercase).
HeLa cells were transfected in duplicate with 10 µg of the CAT
reporter gene constructs and 5 µg of pCMVlacZ. After 48 h, cells
were harvested, and CAT and -galactosidase activities were measured.
Acetylated chloramphenicol was revealed by autoradiography, and CAT
activity relative to -galactosidase activity in the same extracts is
indicated by the bar graph. The autoradiogram is
representative of a single experiment, and the quantitative values
represent the mean activity obtained from three independent
experiments.
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Site of Transcription Initiation in the 3'-Truncated and Wild-type
Promoters--
The possibility was considered that the high level of
expression exhibited by p(846/+3)CAT might arise from a change in the transcription start site as a result of the extensive 3' deletion. Consequently, primer extension analysis was used to map the 5' ends of
mRNA transcribed from the wild-type (p(846/+48)CAT) and
deletion mutant (p(846/+3)CAT) vectors. A diagram of the relevant
portion of these plasmids is presented in Fig.
4B. If the GPH gene cap
site is used, the predicted sizes of the primer extension products are
98 nucleotides for the wild-type transcript and 71 nucleotides for the
3'-deletion transcript. These were determined by summing the length of
CAT-REV2 primer (33 nucleotides), plasmid backbone (either 17 or 35 nucleotides for wild-type and mutant, respectively), and exon I (either
48 or 3 nucleotides for wild-type and mutant, respectively). The
results presented in Fig. 4A show that primer extension
products generated from p(846/+48)CAT (lane
3) and p(846/+3)CAT (lane 4) RNAs
were the sizes predicted in Fig. 4B, indicating that both
transcripts terminated at the same nucleotide, which corresponds to +1
of the GPH gene. It is also noted that the abundance of
p(846/+48)CAT mRNA was significantly lower than that of
p(846/+3)CAT mRNA, further indicating that the activity
increase produced by the 3'-deletion mutant is at the level of
transcription.
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Fig. 4.
Determination of transcription start sites by
primer extension. HeLa cells were stably transfected with either
p(846/+48)CAT or p(846/+3)CAT. Total RNA was isolated from
G418-resistant cells cultured in minimum essential medium supplemented
with 5 mM D-mannose, 3 mM sodium
butyrate, and 1 mM theophylline (77, 78). Total RNA (15 µg) was subjected to primer extension with a 32P-labeled
CAT gene-specific oligonucleotide primer as described under
"Experimental Procedures" and depicted in panel
A. Arrows indicate the major extended products,
98 nucleotides for p(846/+48)CAT and 71 nucleotides for
p(846/+3)CAT. As a positive control, kanamycin RNA and primer
(Clontech) were used to generate a product of the
expected size (87 nucleotides). The schematics in panel
B depict the p(846/+48)CAT and p(846/+3)CAT
vectors, the CAT-REV2 primer (33 nucleotides), and the calculated
distance from the primer 5'-terminus to the predicted transcription
start sites (indicated by +1).
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CSDE Forms a Distinct Complex with HeLa Cell Nuclear
Proteins--
The effects of site-directed mutations (m-1 and m-2) in
the CSDE on transcriptional activity of the -subunit gene promoter suggested the possibility that this site interacts with distinct nuclear proteins to repress GPH gene transcription. To examine this
possibility, EMSA was performed. A 28-bp oligonucleotide that extends
from 13 to +15 relative to the transcription start site (+1) of the
-subunit gene was radiolabeled and incubated with HeLa cell nuclear
extract (Fig. 5). Analysis of the binding mixtures by electrophoresis through native polyacrylamide gels revealed
a DNA-protein complex migrating slower than the free CSDE probe (Fig.
5, lane 1). This complex was eliminated by the addition of excess, unlabeled homologous (CSDE)
oligonucleotide (lane 3), but it was not affected
by the addition of excess, unlabeled heterologous (Het)
oligonucleotide (lane 2). Additionally, 1.7-kbp GPH DNA fragments (extending from 1637 to +48), which contain wild-type or mutated cap site sequence, were used as competitors of
protein binding to the 32P-labeled CSDE
oligonucleotide. Because the competitors were significantly different
in length (28 versus 1685 bp), the levels of DNA added are
indicated in Fig. 5 as picomoles. As seen, the complex was eliminated
(lanes 4-6) with increasing amounts of wild-type
competitor, whereas the addition of excess unlabeled fragment
containing a clustered mutation at 5 to 2 (changing TAAC to ATTG)
did not significantly interfere with the formation of complex even at the highest concentration tested (lanes 7-9).
Thus, elimination of the DNA-protein complex was dependent on an intact
CSDE motif. The formation of a distinct DNA-protein complex with a
probe representing the GPH cap site diad suggests that the CSBP
represents a previously undefined binding activity for the GPH
promoter and may act as a repressor to affect promoter activity.
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Fig. 5.
Identification of CSDE binding activity in
HeLa nuclear extracts by EMSA. Binding assays were performed using
a HeLa cell nuclear extract and 32P-labeled CSDE
oligonucleotide (lane 1) and were analyzed by
electrophoresis through 6.5% polyacrylamide gels in 1× TBE buffer.
The free probe and the retarded complex are indicated. The complex was
challenged with 0.561 pmol of unlabeled heterologous (CRE,
5'-CGGCAAATTGACGTCATGGTAAGCCC-3') (lane 2) or
homologous (lane 3) oligonucleotides. The complex
was also competed with 0.045, 0.312, and 0.579 pmol of unlabeled
1.7-kbp DNA fragments that extend from 1637 to +48 of the GPH gene
and contain wild-type (lanes 4-6) or mutated
(lanes 7-9) cap site sequence. The clustered
point mutation corresponds to m-1 (Fig. 3A). Nuclear
extracts (10 µg of protein) were preincubated with the indicated
competitors for 10 min at room temperature before addition of the CSDE
probe (10,000 cpm, ~0.022 pmol).
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The Regions from 5 to 2 and +4 to +11 Are Critical for Binding
of CSBP to CSDE--
To better localize the binding site for this
factor, a series of oligonucleotides containing clustered substitution
mutations that collectively span the region of diad symmetry (Table
I) were synthesized and used in
competition EMSA. As a first approach, the wild-type CSDE(13/+15) and
mutant oligonucleotides (M-1, M-2, M-4, M-5, and +3) were used as
competitors for protein binding to a 32P-labeled CSDE
probe. A single complex was generated (Fig.
6A). This complex was
inhibited 97% by coincubation with increasing amounts of homologous,
nonradioactive CSDE oligonucleotide, whereas unlabeled mutant
oligonucleotides M-1, M-2, M-4, M-5, and +3 were inefficient as
competitors, reducing complex formation by only 30% at the highest
concentration tested (Fig. 6, A and B). Thus, the
regions from 5 to 2 and +5 to +10 in the CSDE are important for
interactions with CSBP. Because p(846/+3)CAT had better promoter
activity than m-1 and m-2 (Fig. 3B), it was considered that
bases farther downstream may also be important for binding (see
"Discussion"). To examine this possibility, another series of
longer oligonucleotides containing substitution mutations on either
side of, as well as within, the diad sequence was generated (Table I),
and competition binding assays were performed. The wild-type
oligonucleotide (13/+22) and mutant oligonucleotides (M-8, M-1L, M-7,
and M-6) were used as competitors for protein binding to a
32P-labeled 13/+22 probe. Again, one complex was observed
(Fig. 6C). This complex was eliminated by increasing amounts
of homologous, nonradioactive 13/+22 oligonucleotide, suggesting that
the complex represents specific protein-DNA interactions. Mutations in
both M-1L and M-7 severely inhibited binding, increasing by 9- and 14-fold, respectively, the level of oligonucleotide required to inhibit
complex formation by 50% (Fig. 6, C and D). In
contrast, mutations in M-8 and M-6 had much less effect on binding, as
they were equivalent to 13/+22 at concentrations only 2-3- fold
higher. Taken together with the results in Fig. 6 (A and
B), these data indicate that the regions from 5 to 2 and
+4 to +11 are the most critical for binding of HeLa CSBP, and that
bases farther upstream (from 11 to 8) and downstream (from +15 to
+19) may also contribute to complex formation but at a much reduced
level.
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Table I
Sequences of the wild-type and mutant cap site oligonucleotides used as
probes or competitors in DNA binding assays
Uppercase indicates the wild-type sequences, and bold lower
case identifies mutated bases. For M-4, the inserted hexamer is shown
in bold lowercase and the diad left and right arms are shown in
uppercase, even though a direct comparison reveals that many of the
downstream positions are mutated in the strictest sense.
Oligonucleotide sequences are aligned at the transcription start site
indicated by the underlined G at +1.
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Fig. 6.
Competition of gel shift complexes with
oligonucleotides having clustered point mutations that collectively
span the GPH cap site. This analysis was
carried out to define the boundaries of CSBP-CSDE interactions.
A, protein (10 µg) from HeLa nuclear extracts was
incubated at room temperature for 20 min with 10,000 cpm of
radiolabeled CSDE oligonucleotide after a 20-min preincubation with 2 µg of poly(dI-dC)-poly(dI-dC) and unlabeled competitors as identified
in the figure. The quantity of competitor DNA used in each reaction is
shown at the top of the autoradiogram. B, the
DNA-protein complex intensity was determined by densitometry and
plotted as a function of competitor DNA. C, HeLa nuclear
protein (10 µg) was incubated as described for panel
A with 10,000 cpm of 32P-radiolabeled 13/+22
oligonucleotide and 2 µg of poly(dI-dC)-poly(dI-dC) after
preincubation of protein with the indicated competitors. The quantity
of competitor DNA used in each reaction is shown at the top
of the autoradiogram. D, competition curves were determined
by quantitative densitometry. Values in arbitrary integrator units for
samples receiving no competitor were set at 100%. Sequences of the
various oligonucleotides are summarized in Table I.
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Interactions between CSBP and CSDE as Determined by Methylation
Interference--
To identify specific base contacts that contribute
to CSBP binding, HeLa nuclear extracts were incubated with dimethyl
sulfate-treated oligonucleotide (13/+22). In this assay, methylation
of specific guanines (m7G) in contact with the protein, but
not at guanines outside the binding site, will reduce DNA-complex
formation. The results presented in Fig.
7 show that methylated guanines at +2 and
+8 on the antisense (noncoding) strand were significantly diminished in
the bound DNA, as were methylated guanines at +1 and +4 on the sense
(coding) strand. These are indicated with large
arrows in panel B. Methylation of
guanines at +10 on the sense strand and at 2, 8, and 11 on the
antisense strand showed less interference with CSBP binding, and this
is indicated by small arrows in Fig.
7B. No interference was observed by methylation of G at +12
on the sense strand. These results provide strong support of the
mutagenesis study to confirm the diad sequence as a protein binding
motif.
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Fig. 7.
Methylation interference analysis of HeLa
CSBP. A, oligonucleotides comprising either the
antisense (lanes 1 and 2) or sense
(lanes 3 and 4) strand of the GPH
gene and spanning bases from 13 to +22 were 5'-end-labeled with T4
polynucleotide kinase. Duplex DNA was partially methylated with
dimethyl sulfate as described under "Experimental Procedures."
Modified DNA was incubated with HeLa nuclear extract, and the complexed
(B) and free (F) DNAs were separated by EMSA as
shown in the previous figures. Six replicate samples were made. The
individual bands of protein-bound (lanes 1 and
3) and unbound (lanes 2 and
4) DNAs were isolated from the gel, combined, cleaved with
piperidine (1:10), and resolved by electrophoresis at 12 watts for
3 h on a 10% polyacrylamide gel containing 7 M urea.
G residues are identified to the left and right
of the autoradiogram. B, sequence of the 13/+22
oligonucleotide. Arrowheads indicate bases whose methylation
reduce complex formation; large and small
arrows denote the degree of interference by the G
nucleotides indicated.
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Analysis of CSDE Activity When Reintroduced Downstream of the
p(846/+3)CAT Promoter--
Because mutation of the
CSDE produced a GPH promoter that was more robust than the wild-type
promoter, it was of interest to determine whether the diad element
could restore repression to the 3'-truncated mutant, and if it could,
whether it was effective in an orientation-dependent or an
orientation-independent manner. To investigate this question, one copy
of the CSDE was inserted downstream of the p(846/+3)CAT promoter
in both forward and reverse orientations to generate vectors
p(+3-CSDE)CAT and p(+3-EDSC)CAT. These constructs were mixed with
pCMVlacZ and transfected into HeLa cells. The CAT activity relative to
-galactosidase activity is shown in Fig.
8A. Expression levels for
p(+3-CSDE)CAT and p(+3-EDSC)CAT were approximately 38 and 65% of
those for p(846/+3)CAT. Thus, the downstream CSDE in either
orientation could repress activity of the -subunit gene promoter,
but somewhat greater inhibition was provided by CSDE in the forward
direction. Moreover, the promoter activity of both constructs remained
higher than that of p(846/+48)CAT, suggesting either that full
activity of the negative element may require additional sequences
outside the cap site diad or that the downstream location may reduce
interactions between CSBP and other factors in the transcription
initiation complex.
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Fig. 8.
Reintroduction of the CSDE into the truncated
GPH promoter. A, the diad
element was inserted downstream of the GPH promoter (extending from
846 to +3) in both the forward (CSDE) and reverse (EDSC) orientations
to generate p(+3-CSDE)CAT and p(+3-EDSC)CAT, respectively. The
constructs were cotransfected into HeLa cells with pCMVlacZ so that
-galactosidase activity could be used to normalize for differences
in transfection efficiency. Expression vectors are diagramed at the
left; a representative autoradiogram showing acetylated
derivatives of chloramphenicol (CM) is presented in the
center; and the ratio of CAT to -galactosidase activity
obtained with the CSDE- and EDSC-containing vectors relative to the
ratio obtained with the parental p(846/+3)CAT vector is shown at
the right; values are the average of duplicate plates in two
separate assays. B, the constructs HCAP and HPAC were
generated from p(+3-CSDE)CAT and p(+3-EDSC)CAT as outlined under
"Experimental Procedures." Along with p(846/+48)CAT and
p(846/+3)CAT, they were cotransfected into HeLa cells with
pCMVlacZ. The presentations are as described in panel
A.
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The above constructs place the diad center of the CSDE insert 35 bp
downstream of the original cap site. To test CSDE(13/+15) function in
its native position, without the downstream sequence provided by
p(846/+48)CAT, the p(+3-CSDE)CAT and p(+3-EDSC)CAT vectors
were digested with restriction endonuclease PstI; and the
larger fragments were religated to generate two constructs, HCAP and
HPAC, named for the inclusion of the right half (H) of the cap site
from CSDE(+3/+15) and EDSC (+15/+3), respectively. The PstI
sites lie at the transcription start of the GPH promoter and at the
diad center 35 bp downstream in the inserted CSDE (and EDSC).
Religation of the large fragment eliminates 35 bp and restores an
intact CSDE in both the wild-type and inverted orientation with
elimination of sequences farther downstream (i.e. between +15 and +48). Derivative HPAC contains mutations at +8, +12, +13, and
+14 and forms a perfect palindrome. When p(846/+48)CAT, p(846/+3)CAT, HCAP, and HPAC were transfected into HeLa cells, the
CAT activity from HCAP and HPAC was 2-3-fold higher than that from
p(846/+48)CAT but ~2-fold lower than that from p(846/+3)CAT (Fig. 8B). These results suggest that additional downstream
sequence (i.e. from +15 to +48) of the GPH gene first
exon may also contribute to negative regulation of the chromosomal
gene, and that converting the CSDE to a perfect palindrome does not
increase its repression activity.
Relationship between CSDE Action and Levels of CSBP--
Because
tumors originating in a variety of tissues produce the GPH -subunit
(4, 9, 12, 13, 15, 16, 62, 63), it was of interest to examine the
activity of wild-type and mutant promoters in some of these cell types.
Consequently, the p(846/+48)CAT, m-1, m-2, and p(846/+3)CAT
vectors were co-transfected with pCMVlacZ into several different cell
lines, including HT-29 (colon carcinoma), Panc-1 (pancreatic
carcinoma), JEG-3 and JAr (choriocarcinoma), U-2 OS (osteosarcoma),
GH4C1 (pituitary carcinoma), MCF-7 (breast carcinoma), and MCF-10A (normal breast). CAT levels were normalized to
those of -galactosidase in the same extracts. The values reported in
Table II show that the CAT activities
derived from mutants m-1, m-2, and p(846/+3)CAT were relatively
higher than those from the wild-type p(846/+48)CAT in most of the
cell types. For example, HeLa and Panc-1 cells showed a strong
preference for the mutated promoter, whereas the MCF-10A cell line
showed little or no ability to discriminate among the wild-type and
mutant expression vectors, and other lines were intermediate in
their relative expression levels.
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Table II
Determination of wild-type and mutant promoter activity and the
relative level of CSBP in a variety of tumor cell lines
Cells were transfected as described under "Experimental Procedures"
with the expression vectors indicated (wild-type, m-1, m-2, +3) and
assayed for CAT and -galactosidase 48 h after transfection. The
values represent CAT activity normalized to -galactosidase activity
in aliquots of the same extract; they are means ± standard
deviation determined for duplicate flasks in three separate
experiments. The last column represents the relative binding activity
of CSBP in nuclear extracts prepared from the indicated cell lines. The
amount of CSBP-CSDE complex was determined by laser densitometry of
standard EMSA autoradiograms. The arbitrary integrator units were
normalized to HeLa extracts set at 100. Values are the average of two
experiments using different nuclear extract preparations.
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Because the CSDE provided a strong negative influence on promoter
activity in transient expression assays and demonstrated the capacity
to form a specific DNA-protein complex in gel shift assays, it was
reasoned that the variability of CSDE action in the cell lines examined
might result from differences in the levels of CSBP that are
characteristic of each cell type. To test this possibility, DNA-protein
interactions were examined in the collection of cell lines by EMSA.
When the CSDE was used as a labeled probe, a single DNA-protein complex
was generated from nuclear extracts prepared from each of the cell
lines listed above (Fig. 9), but their
autoradiographic intensities were notably different. Competition with
heterologous and homologous oligonucleotides showed that the complex in
each cell type represented specific DNA-protein interactions (data not
presented). The amount of complex was quantified by densitometry, and
the arbitrary integrator units for each cell line, relative to that of
HeLa cells set at 100, are listed in Table II. Similar results were
obtained with multiple nuclear extract preparations and with varied
DNA:protein ratios (data not presented). It appears unlikely that an
inhibitor of CSBP binding is present in nuclear extracts from cell
lines with low binding activity, as the amount of complex generated by
mixtures of HeLa and MCF-10A extracts (10 µg of protein each) was
equal to the sum of the levels produced by each extract alone (data not
presented). Additional control experiments suggested that the variable
levels of CSBP were not a reflection of extract quality, as other
DNA-binding proteins either did not show significant variation in
binding activity, or the binding activity fluctuated in a manner
distinct from that of CSBP (e.g. levels in MCF-10A were
greater than those in HeLa and MCF-7).
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Fig. 9.
CSBP activity in nuclear extracts prepared
from a variety of cell lines. Ten µg of nuclear protein from the
indicated cell lines was incubated with 10,000 cpm of
32P-radiolabeled CSDE oligonucleotide. Free DNA and
DNA-protein complex (indicated by arrows) were resolved by
nondenaturing polyacrylamide gel electrophoresis. The DNA-protein
complex levels were quantified by laser densitometry, and average
values from two experiments with different extract preparations are
reported in Table II after normalizing to values for HeLa
extracts.
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The relative effectiveness of the most active (p(846/+3)CAT) and
least active (p(846/+48)CAT) promoters was evaluated in each cell
line by determining the -fold increase in CAT/-galactosidase activity for the mutant compared with the wild-type promoter; this
comparison is indicated as the +3/+48 ratio. By comparing the ratio of
CAT levels produced from the two plasmids, rather than absolute levels,
any intrinsic differences in the ability of specific cell lines to
transcribe the GPH promoter would be eliminated except for the
contribution derived from the CSDE or from sequence downstream of +3.
Normalization of CAT activity to that of -galactosidase accounts for
any inherent differences in their transfection efficiency. In Fig.
10, the ratio of promoter activities
(i.e. deletion mutant/wild-type) in each cell line is
plotted against their corresponding level of CSDE binding activity. As
seen, there is a direct correlation (r = 0.96, p < 0.0001) between these parameters. That is, cells
with the highest levels of CSBP activity (e.g. HeLa and
Panc-1) showed a proportionately greater increase in transcription from
p(846/+3)CAT relative to that from p(846/+48)CAT. Similarly,
MCF-10A cells, which exhibited the lowest levels of CSBP activity,
produced comparable levels of CAT activity from expression vectors
driven by the wild-type (+48) and truncated promoters (+3). These
results suggest that the increase in activity of the mutant promoter
relative to the wild-type promoter is dependent, at least in part, on
the levels of CSBP activity in these cell lines.
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Fig. 10.
Relationship between the levels of CSBP and
the relative activities of promoters containing a mutated or wild-type
CSDE. The CAT activity produced from p(846/+48)CAT and
p(846/+3)CAT vectors in a variety of cell lines was determined and
listed in Table II. For each cell line, the -fold increase in CAT
activity produced from the truncated promoter (+3) relative to the wild
type promoter (+48) was calculated and plotted against the CSDE binding
activity in nuclear extracts from the same cell type (normalized to
that of HeLa cells). The ratio of promoter activities is used to
eliminate inherent differences in the ability of different cell lines
to transcribe the transfected GPH-CAT chimera. The line
represents a least squares fit to the data points, with correlation
coefficient of 0.96.
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DISCUSSION |
Expression of the glycoprotein hormone -subunit gene is
controlled by a complex promoter that contains multiple, distinct regulatory elements that interact via specific, nuclear DNA-binding proteins (21, 23, 31, 32, 37). Regulatory elements controlling transcription are generally located upstream of the transcription start
site. For example, upstream regulatory elements that interact with
placenta-specific factors involved in basal transcription of the GPH
gene in JEG-3 choriocarcinoma cells are located between nucleotides
180 and 150 relative to the transcription start site (+1). However,
it has become increasingly evident that a large number of both cellular
and viral genes utilize elements for transcriptional regulation that
are located at, or downstream from, the cap site (55-59). Examples of
such regulatory elements include those located in introns (64, 65), in
3'-flanking DNA (66), and in both untranslated (67) and translated
exons (68). They can function as either enhancers or silencers.
Transient expression of reporter gene constructs carrying the GPH
gene promoter was previously reported to be significantly lower in HeLa
cells than in JEG-3 cells (22), suggesting that the
trans-acting factor(s) necessary for expression in JEG-3
cells is not present in HeLa cells (38). As shown in Fig. 2, however,
levels of CAT activity produced from a GPH 3'-deletion mutant, which
contains 5'-flanking DNA extending from 846 to +3, were significantly greater than those produced from the plasmid containing GPH promoter DNA extending farther downstream to +48. This result argues that a
heretofore unrecognized motif, comprising the cap site and/or downstream sequence, constitutes a cis-regulatory element
responsible, at least in part, for low level transcription of the
GPH promoter in transfected HeLa cells.
Support for the above assertion comes from the analysis of two
site-directed mutants, m-1 and m-2. Together with the 3'-deletion mutant, they demonstrate that proximal sequence both upstream and
downstream of the transcription start site contributes to the
repression activity of the diad element centered at +1 (Fig. 3B). The CSDE is classified as a negative regulatory element
for the GPH promoter because of the greater activity of the mutants relative to that of the wild-type. The possibility that the increase in
CAT production is caused either by changes in the translational efficiency of the CAT transcript via a possible change in mRNA secondary structure (69) or by the use of a novel, more act
§,
TOP
ABSTRACT
INTRODUCTION
