From the
Previous studies suggested that transcription enhancer factor-1
(TEF-1) was involved in mediating the human chorionic somatomammotropin
(hCS) gene enhancer (CSEn) function (Jiang, S.-W., and
Eberhardt, N. L.(1994) J. Biol. Chem. 269, 10384-10392).
We now show that an unrelated protein (CSEF-1) found in BeWo and COS-1
cells binds to the GT-IIC enhanson in CSEn and is correlated with CSEn
activity in these cells. TEF-1 and CSEF-1 were distinguished by
differential migration as GT-IIC complexes, thermal stability,
molecular mass, and cross-reactivity with chicken TEF-1 antibodies.
TEF-1 and CSEF-1 bound to the GT-IIC and Sph-I/Sph-II enhansons with
identical binding properties, and in vitro generated TEF-1
competed with CSEF-1 binding to the GT-IIC motif, suggesting that their
actions might be mutually exclusive. Up- and down-regulation of TEF-1
levels by expression systems and antisense oligonucleotides
demonstrated that TEF-1 inhibited the hCS promoter in a manner
independent of the enhancer or a known TEF-1 DNA binding site. The data
suggest that TEF-1 may provide a counter-regulatory stimulus to the
actions of CSEF-1, which may be involved in mediating enhancer
stimulatory activity.
Previous studies indicate that the human chorionic
somatomammotropin enhancer (CSEn)
The hCS enhancer is composed of multiple
DNA elements that are homologous to several SV40 enhansons
(Jiang and Eberhardt, 1994; Jacquemin et al., 1994). Both the
SV40 GT-IIC and Sph-I/Sph-II enhanson motifs present in CSEn
are protected by placental cell nuclear proteins in DNase I
footprinting assays, and mutation of these elements results in marked
loss of enhancer activity (Jiang and Eberhardt, 1994; Jacquemin et
al., 1994). Transcription enhancer factor-1 (TEF-1) has been shown
to bind to the GT-IIC and Sph-I/Sph-II enhansons (Davidson et
al., 1988; Xiao et al., 1991) and a few variant elements
with the consensus 5`-TGTGG(T/A)(T/A)(T/A)G-3` (Kariya et al.,
1993). Accordingly, TEF-1 has been implicated in the mechanism of CSEn
action. TEF-1 is a widely distributed transcription factor that appears
to be involved in mediating the regulation of a diverse set of genes,
including SV40 enhancer function (Davidson et al.,
1988; Xiao et al., 1991; Hwang et al., 1993), human
papillomavirus-16 E6 and E7 oncogene transcription
(Ishiji et al., 1992), cardiac and skeletal muscle-specific
gene expression (Stewart et al., 1994; Farrance et
al., 1992; Kariya et al., 1993, 1994), early gene
expression in mouse development (Melin et al., 1993), and
hCS gene tissue-specific expression (Jacquemin et
al., 1994; Jiang and Eberhardt, 1994; Lytras and Cattini, 1994).
The mechanism of TEF-1 action appears to be complex. Overexpression
of TEF-1 in a variety of cells results in squelching of transcriptional
activity (Xiao et al., 1991; Ishiji et al., 1992;
Hwang et al., 1993), suggesting that other limiting
transcription factors or adapters are required for TEF-1 function (Xiao
et al., 1991). Additionally, co-repressors may be involved in
repressing stimulation by TEF-1 (Chaudhary et al., 1994).
Since such co-activators or co-repressors have not been isolated or
cloned, we do not understand how TEF-1 mediates positive enhancer
function. In the current studies we sought to establish whether TEF-1
was involved in CSEn function and provide information about its
mechanism of action. We establish the presence of TEF-1 in the BeWo
choriocarcinoma cell line and demonstrate that a 30-kDa protein
(CSEF-1) competes for TEF-1 binding to the GT-IIC and Sph-I/Sph-II
enhansons with identical binding specificity. CSEF-1 is distinguishable
from TEF-1 on the basis of molecular mass, thermal stability and
absence of cross-reactivity with antibodies to TEF-1. Overexpression of
TEF-1 in BeWo cells resulted in marked inhibition of basal hCS promoter activity without affecting the relative enhancer-mediated
stimulation of transcription. In contrast, down-regulation of TEF-1
expression in BeWo cells resulted in increased basal promoter activity
and increased enhancer-mediated stimulation. Also, the enhancer was
very active in COS-1 cells, a cell line that contained very low levels
of TEF-1 but contained a CSEF-1-like protein. Thus CSEF-1 may be
involved in the stimulatory properties of CSEn. The data suggest
alternate models for TEF-1 action, including the possibility that it
mediates negative enhancer functions.
Cell
harvesting, transfection by electroporation, and luciferase assays have
been described in detail (Jiang and Eberhardt, 1994). Sense and
antisense TEF-1 phosphorothioate oligonucleotides (35 mM,
Molecular Biology Core Facility, Mayo Clinic) () were
introduced into BeWo cells via electroporation.
Complementary oligonucleotides (Molecular Biology Core Facility,
Mayo Clinic) GT-IIC
TEF-1 was also expressed and purified from E. coli HB101.
TEF-1 coding sequences were amplified from pXJ40-TEF-1A by PCR
using primers GST-TEF_1 and GST-TEF_2 (). The DNA fragments
were digested with EcoRI and SalI and ligated to
pGEX-4T-3 vector (Pharmacia) digested by the same enzymes.
Bacterial lysates were prepared by ultrasonic lysis and TEF-glutathione
S-transferase fusion proteins were bound to
glutathione-Sepharose 4B (Pharmacia) and cleaved by thrombin (Sigma).
Cleavage products were immediately stored at -70 °C and used
in gel shift assays without further purification.
Southwestern
blotting experiments were performed on proteins transferred onto
membrane that were denatured/renatured with guanidine hydrochloride as
described by Vinson et al.(1988). Nonspecific DNA binding
activities were eliminated by incubation in 0.5
UV
cross-linking was performed essentially as described in detail
previously (Jiang et al., 1995). Authentic in vitro generated TEF-1 (5 µl) and 15 µg of BeWo nuclear extract
were incubated with the GT-IIC
TEF-1 involvement in CSEn function has been considered
likely, since mutation of the CSEn GT-IIC enhanson abolished enhancer
activity (Jiang et al., 1994; Jacquemin et al.,
1994). In the present studies we sought to establish whether TEF-1
operating through the GT-IIC and/or Sph-I/Sph-II enhansons was involved
in mediating CSEn stimulatory function. Our finding of a rapidly
migrating GT-IIC
One important issue is whether CSEF-1
and TEF-1 are structurally related. The proteins have different
molecular weights by Southwestern analysis (Fig. 5C) and
exhibit differential heat stabilities (Fig. 3). CSEF-1 can be
chromatographically separated from the bulk of TEF-1 binding activity
(Fig. 4), although it does co-elute with at least one putative
TEF-1 binding isoform. Introduction of TEF-1 antisense oligonucleotides
into BeWo cells resulted in dramatic changes in the intracellular
concentration of TEF-1 without affecting CSEF-1 levels (Fig. 8).
Finally, a rabbit antibody raised against chicken TEF-1 (Kariya et
al., 1993) only cross-reacted with TEF-1, but not CSEF-1
(Fig. 5A). Since mixed synthetic peptides corresponding
to the entire chicken TEF-1 polypeptide were used to raise the
polyclonal antibody and the antibodies cross-react with human TEF-1
(Kariya et al., 1993), which is 76% identical to chicken
TEF-1, CSEF-1 does not appear to be closely related to TEF-1.
Nevertheless, since their binding specificities to the GT-IIC and
Sph-I/Sph-II enhansons are nearly identical (Fig. 2, A and B), it is possible that CSEF-1 is a member of the
family of transcription factors containing the TEA DNA-binding domain
(Bürglin, 1991). This domain has been identified in a diverse set
of regulatory genes, including the scalloped gene, a
neuronal-specific Drosophila gene that is 68% identical to
TEF-1 (Campbell et al., 1992), the abaA gene of
Aspergillus nidulans that regulates differentiation of asexual
spores (Mirabito et al., 1989), and yeast TEC1 that regulates
transposon Ty1 enhancer activity in Saccharomyces
cerevisiae (Laloux et al., 1990).
Our finding that
CSEn is a potent enhancer in COS-1 cells (Fig. 7, A and
B) indicates that CSEn or a related activity may not be
restricted to the placenta. Of interest, with respect to the potential
role of TEF-1 in CSEn action, is the almost complete absence of this
factor in COS-1 cells and the presence of a GT-IIC-protein complex,
which co-migrates with CSEF-1 (Fig. 1). Thus in COS-1 cells CSEn
stimulatory activity is correlated with CSEF-1, but not TEF-1 binding
activity, suggesting that CSEF-1 mediates the positive enhancer
function in these cells. This conclusion is strengthened by the absence
of CSEn activity in HeLa cells (Fig. 7, A and
B), which contain abundant TEF-1 binding activity but lack
CSEF-1 binding activity (Fig. 1).
The finding that GT-IIC
multimers introduced upstream of the CS promoter resulted in
significant positive stimulation of promoter activity suggests that
this enhanson plays a major role in mediating CSEn stimulatory activity
(Fig. 7, C and D). Interestingly, these
constructs are much more potent enhancers in COS-1 cells than in BeWo
cells, suggesting that BeWo cells might contain factors that
down-regulate enhancer responsiveness. This view is supported by
studies with the TEF-1 antisense oligonucleotide ( Fig. 8and
Fig. 9
), which demonstrate that down-regulation of TEF-1 levels
in BeWo cells results in a significant stimulation (
Our data appear to
parallel somewhat those found with the myosin heavy chain
(MHC) promoter. Shimizu et al.(1993) have reported
that both the ubiquitous mouse (m) TEF-1 (99% amino acid sequence
homology to hTEF-1; Blatt et al.(1993)) and a distinct
muscle-specific factor A1 bind to the GT-IIC variant motif, M-CAT,
present in the MHC promoter. The binding specificities of A1
and mTEF-1 are indistinguishable. Over expression of mTEF-1 had no
effect on MHC promoter activity, implicating a role for A1 in
mediating MHC muscle-specific gene expression. Shimizu et
al.(1993) suggested that stimulation might involve the
heterodimerization of A1 and mTEF-1; however, our data do not support
such a mechanism, since down-regulation of TEF-1 levels in BeWo cells
resulted in an increase in CS promoter and enhancer activity
(Fig. 9, A and B) and the enhancer functioned
in COS-1 cells which lack high levels of TEF-1 (Fig. 7). In
addition, no evidence for an interaction between TEF-1 and CSEF-1 was
observed in the gel shift experiments where in vitro generated
TEF-1 was added to a TEF-1-depleted cell extract containing CSEF-1
(Fig. 6). The simplest model to explain our data would be that
down-regulation of TEF-1 levels results in increased occupancy of the
GT-IIC domain by CSEF-1, which acts as a positive modulator of enhancer
activity.
It is important to emphasize that our findings do not
exclude the possibility that TEF-1 and a co-activator as proposed
initially for the SV40 and HPV enhancers (Xiao et
al., 1991; Ishiji et al., 1992; Hwang et al.,
1993) account for CSEn function. For example, the endogenous TEF-1
levels in BeWo cells may exceed the available co-activator
concentration such that ``endogenous squelching'' occurs.
Accordingly, down-regulation of endogenous TEF-1 levels could result in
the net liberation of a co-activator and concomitant activation. Since
the modulation of TEF-1 levels in BeWo cells affects the hCS promoter (Fig. 9A), herpes simplex thymidine kinase
and Rous sarcoma virus promoters as well as a minimal hCS promoter containing only a TATA box and initiator
element,(
Recently,
Stewart et al.(1994) cloned a novel TEF-1 isoform, TEF-1B
containing 13 extra amino acids immediately downstream of the TEA
domain, that is probably derived from TEF-1 pre-mRNA by alternative
splicing. Interestingly, the TEF-1B, but not the TEF-1A,
transactivation domain was capable of stimulating transcription when
fused to a GAL4 DNA binding domain in a GAL4-dependent reporter gene in
muscle cells. Thus different TEF-1 isoforms may be involved in
mediating positive enhancer functions. Given the apparent heterogeneity
in TEF-1 binding isoforms in BeWo cells (Fig. 4), we cannot
exclude the possibility that one of these TEF-1 isoforms plays a role
in mediating positive CSEn functions. However, since the TEF-1
antisense oligonucleotide was directed toward the 5`-end of the mRNA,
this should deplete all isoforms of TEF-1 except those containing a
different translation start codon.
The mechanisms that control the
cell-specific character of enhancer activity appear to depend on their
multipartite structure. Single copies of individual enhansons usually
lack or have very weak enhancer activity; however, when homogeneously
multimerized, the individual enhansons can work independently and
generate significant stimulatory activity. An interesting feature of
these homogeneous enhancers is that they exhibit relatively restricted
cell type dependence (Schirm et al., 1987; Ondek et
al., 1987; Forsberg and Westin, 1991). Schirm et
al.(1987) demonstrated that two related SV40 core
consensus motifs (GT-IIC, GTGGAATGT; TC-II, GTGGAAAGT), displayed
markedly different behavior in a variety of cells. These data could be
explained by multiple TEF-1 isoforms that recognize the distinct
enhansons as supported by the muscle cell expression studies of Stewart
et al.(1994). Our data indicate that the GT-IIC multimer is
sufficient in mimicking the wild-type CSEn activity in BeWo and COS-1
cells, emphasizing the important role that this enhanson plays in
mediating positive enhancer function and suggest that CSEF-1 may play a
role in mediating cell-specific enhancer function.
We express our appreciation to Drs. Pierre Chambon and
Irwin Davidson for providing the pXJ40-TEF-1 expression
plasmid, Drs. Charles Ordahl and Iain K. G. Farrance for the
anti-chicken TEF-1 antibodies, Dr. Drew Arnold (Endocrine Research
Unit, Mayo Clinic/Mayo Foundation) for advice with data analyses, Dr.
Whyte Owen for helpful discussions of the studies involving
characterization of the CSEF-1 protein, Dr. Miguel Trujillo for careful
reading and criticism of the manuscript, and Nicole Henry for
secretarial and editorial help.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is involved
in regulating the placental-specific expression of the human chorionic
somatomammotropin genes (hCS-1 or hCS-B and hCS-2 or hCS-B; or placental lactogen, hPL) and the
growth hormone variant gene (hGH-2 or hGH-V). CSEn
was first identified by Saunders and co-workers (Rogers et
al., 1986; Walker et al., 1990). Since the enhancer was
localized nearest to the hCS-2 gene, which is expressed at
high levels and is the 3`-most member in the hGH/hCS gene
cluster on chromosome 17q22-24 (Miller and Eberhardt, 1983; Hirt
et al., 1987; Chen et al., 1989), it was implicated
in mediating hCS cell-specific expression (reviewed by Walker et
al.(1991)). Three copies of the enhancer are present within the
hGH/hCS chromosomal locus and are located downstream of the
hCS-5 (putative pseudogene; Hirt et al., 1987),
hCS-1, and hCS-2 genes; however, only the enhancer
downstream of the hCS-2 gene appears to have significant
enhancer activity (Jacquemin et al., 1994). Cell-specific
expression of the hCS genes does not depend on the enhancer
alone, since we recently demonstrated that an initiator element
downstream of the TATA box contributes significantly to the basal- and
enhancer-stimulated hCS promoter activity (Jiang et
al., 1995).
Cell Transfection and Luciferase Assays
COS-1
and HeLa cells (ATCC) were maintained in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.); BeWo cells (ATCC) were
maintained in RPMI 1640 (Life Technologies, Inc.). All media were
supplemented with 10% fetal bovine serum (Whittaker), 100 units/ml
penicillin (Life Technologies, Inc.), 100 µg/ml streptomycin (Life
Technologies, Inc.) and 2 mML-glutamine (Life
Technologies, Inc.). Cells were maintained at 37 °C in an
atmosphere containing 5% CO and 100% humidity.
Data Analysis
Data were subjected to analysis of
variance (ANOVA). For experimental groups satisfying the initial ANOVA
criterion (p < 0.05), comparisons were performed using
Student's t tests and a Bonferroni inequality to
compensate for the multiple comparisons (Snedecor and Cochran, 1980).
When the variances were not equivalent, logarithmically transformed
(log) data were analyzed.
Plasmid Constructions
The plasmids
pALUC (Maxwell et
al., 1989); EnA_CSp.LUC, containing the CSEn and
the hCS-1 5`-flanking region (493 bp); and
EnA_EnAP_CSp.LUC, containing an additional copy of
CSEn inserted in EnA_CSp.LUC at the PstI
site (nucleotide -300 relative to the transcription initiation
site) have been described previously (Jiang and Eberhardt, 1994).
D(+) and
GT-IIC
D(-) (), containing tandem copies
of the SV40 GT-IIC enhanson (OGT2-56; Xiao et al. (1991)) were synthesized to construct reporter plasmids containing
multiple GT-IIC binding sites. Single-stranded oligonucleotides were
phosphorylated with T4 polynucleotide kinase (New England BioLabs),
annealed and ligated with T4 DNA ligase (Boehringer Mannheim). The
ligation products were resolved on a 2.5% agarose gel, and 4-mer
oligonucleotides containing 8 copies of the GT-IIC site were selected
by eluting the 120-bp DNA fragment. Following purification (GeneClean),
BglII linkers were added and the DNA fragments were cloned
into CSp.LUC at the BglII site, to yield
(GT-IIC)
CSp.LUC. Additionally, the 120-bp
(GT-IIC)
fragments were blunt-ended by T4 dNA Polymerase
treatment, ligated to PstI linkers, and inserted in the
PstI site of (GT-IIC)
CSp.LUC to
yield (GT-IIC)
CSp.LUC and
(GT-IIC)
CSp.LUC.
Nuclear Extracts
Large scale nuclear extracts were
isolated from cultured cells according to the method of Dignam et
al. (1983). For microscale extracts, 1 10
transfected cells were plated onto 6-cm dishes. After 20 h, cells
were harvested by scraping in 1.5 ml of phosphate-buffered saline and
centrifugation (800
g for 1 min). After aspirating the
phosphate-buffered saline, cells were resuspended in 3 packed cell
volumes of 20 mM HEPES (pH 7.9), 20% glycerol, 0.5 M
KCl, 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mM DTT
at room temperature. The cell suspension was rapidly frozen on dry ice
and thawed in a 37 °C water bath, centrifuged (30 s at 14,000
g), and the supernatant was diluted with 3.5 volumes
of 20 mM HEPES (pH 7.9), 20% glycerol, 0.2 mM EDTA,
0.5 mM PMSF, and 0.5 mM DTT. Protein concentration
was determined by Coomassie binding, and nuclear extracts were stored
at -70 °C.
Gel Shift Assays
The synthetic oligonucleotides
used in gel shift assays () were labeled with
[-
P]ATP (Amersham Corp.), and the
conditions for DNA-protein binding and gel shift analyses were
performed as described in detail by Jiang et al.(1995).
In Vitro Translation and Escherichia coli Expression of
TEF-1
The pXJ40-TEF-1A plasmid (Xiao et al.,
1991) was generously provided by Dr. Pierre Chambon (Pasteur Institute,
Paris) and was used to generate TEF-1 protein by in vitro translation (TNT, Promega). The reaction was performed
per the manufacturer's protocol using 3.0 µg of
double CsCl gradient-purified pXJ40-TEF-1A plasmid
DNA and 50 µl of TNT reticulocyte lysate in a 100-µl reaction
volume. Typically 1.5 µl of in vitro translational product
was used in each gel shift reaction. Product from a mock translation
with 3.0 µg of pBluescript was used as negative control.
Western and Southwestern Blotting and UV
Cross-linking
The rabbit anti-chicken TEF-1 antibody was
generously provided by Drs. Charles Ordahl and Iain Farrance
(University of California, San Francisco). For blots, 10 µl of
in vitro translation product and 80 µg of BeWo or HeLa
cell nuclear extracts were resolved on a 9% SDS-polyacrylamide gel.
Proteins were electro-transferred onto 0.2 µM
nitrocellulose membrane (Trans-Blot, Bio-Rad) with 48 mM Tris
(pH 9.2), 39 mM glycine, 20% methanol, and 1.3 mM
SDS, using a semidry electrophoretic transfer cell (Trans-Blot SD,
Bio-Rad). After 1 h of blocking in 3% gelatin in 20 mM Tris
(pH 7.5), 500 mM NaCl, TEF-1 antibody diluted at 1:2500 in 20
mM Tris (pH 7.5), 500 mM NaCl, and 0.05% Tween 20 was
applied. Following alkaline phosphatase-conjugated antibody binding,
color development was carried out using the Immun-Blot kit (Bio-Rad)
according to the manufacturer's specifications.
Dignam buffer
D, 5% nonfat dried milk, 10 mM MgCl
, 0.5
mM PMSF, and 0.5 mM DTT at 4 °C for 90 min. The
filters were incubated with 5` end-labeled GT-IIC
probe
(), which was diluted to 2
10
cpm/ml in
0.5
Dignam buffer D, 0.2% nonfat dried milk, 10 mM
MgCl
, 0.5 mM PMSF, 0.5 mM DTT, and 50
µg/ml sonicated salmon sperm DNA at 4 °C for 180 min. The
filters were then washed in the same buffer and exposed to Kodak x-ray
film with intensifying screens at -70 °C for 48 h.
probe (50,000 cpm), and the
complexes were resolved on a 10% SDS-PAGE gel.
Heparin-Sepharose Chromatography
A 0.5-ml
heparin-Sepharose (L-6B, Pharmacia, Sweden) column prepared in a 1.5-ml
Pasteur pipette was washed with 5 ml of distilled water and
equilibrated with 5 ml of Dignam buffer D containing 10 mM
MgCl (Catala et al., 1989; Ristiniemi and
Oikarinen, 1989; Hatamochi et al., 1993). Crude BeWo cell
nuclear extract (200 µl, 5 µg/µl) was applied, and the
column was washed with 5 ml of loading buffer. The bound proteins were
then step-eluted with 1 ml of buffer D containing 0.2, 0.3, 0.4, 0.5,
0.6, and 0.7 M KCl. Four 250-µl fractions were collected
for each elution and stored immediately at -70 °C. The
protein concentration was measured by Coomassie dye binding, and GT-IIC
DNA binding activities were detected by gel shift assay.
Proteins Present in BeWo and COS-1 Cells Other than
TEF-1 Bind to the GT-IIC Motif
Previous footprint analyses of
CSEn with nuclear extracts from a variety of cells were
indistinguishable, suggesting that the factors that bound to CSEn might
be ubiquitously distributed (Jiang et al., 1994). To determine
if different proteins might bind to the various enhanson motifs in
CSEn, we compared the binding of nuclear proteins from BeWo, COS-1, and
HeLa cells along with in vitro generated TEF-1 to the
GT-IIC enhanson. Three distinct complexes were formed with
nuclear proteins from these cell types (Fig. 1). Nonspecific
binding was distinguished with the GT-IIC
MUT, containing a
mutated GT-IIC core consensus (). No differences were
observed in the specific binding patterns using the SV40
(GT-IIC
) or CS (GT-IIC
) TEF-1
binding motifs (data not shown). The slowest migrating complex was
tentatively identified as containing TEF-1, since it was the only
complex observed in HeLa cell extracts and it co-migrated with
authentic in vitro generated TEF-1. BeWo extracts appeared to
contain significant quantities of a TEF-1-like binding activity as well
as a protein, designated CSEF-1, which formed a more rapidly migrating
complex with the GT-IIC
enhanson. COS-1 cell extracts
produce a complex that migrates identically to that of the CSEF-1
complex, produce a unique, more rapidly migrating complex and appear to
contain only minor amounts of TEF-1. Since the CS enhancer is
very active in COS-1 cells (discussed below), the data suggest that the
protein forming the CSEF-1 complex might be involved in mediating CSEn
function.
Figure 1:
BeWo and COS-1 cells
contain nuclear factors in addition to TEF-1 that form distinct,
specific complexes with the GT-IIC enhanson. E. coli
expressed TEF-1 (50 ng) or 3.0 µl of in vitro generated TEF-1 translation products (TNTTEF) were
compared with 7.0 µg of BeWo, COS-1, and HeLa nuclear extracts in
the gel shift assay as described under ``Materials and
Methods.'' The various extracts were incubated with 15,000 cpm of
P-labeled GT-IIC
WT or GT-IIC
MUT
probes and 1.5 µg of poly(dI-dC) as a nonspecific competitor in
each reaction. Probe GT-IIC
MUT contains mutations in the
``core consensus,'' but identical flanking sequences as the
wild-type GT-IIC
probe (Table I). No significant DNA
binding activity was present in the mock translation products (TNT)
using pBluescript plasmid as template to provide negative
control. The TEF-1 complex appeared to be absent in COS-1 extracts,
very abundant in BeWo extracts, and present in only minor amounts in GC
cell extracts. The rapidly migrating complex that is abundantly present
in BeWo and COS-1 cells is designated
CSEF-1.
Both CSEF-1 and TEF-1 Recognize the GT-IIC and
Sph-I/Sph-II Motifs
To further compare the properties of CSEF-1
and TEF-1, we examined their ability to bind the Sph-I/Sph-II enhanson.
TEF-1 is known to bind to both the Sph-I/Sph-II and GT-IIC motifs
(Davidson et al., 1988). Oligonucleotides containing the
wild-type GT-IIC (Fig. 2A) and Sph-I/Sph-II
(Fig. 2B) motifs compete for the binding of TEF-1 and
CSEF-1 about equally, whereas neither the GT-IIC or Sph-I/Sph-II mutant
oligonucleotides competed for TEF-1 or CSEF-1 binding to the GT-IIC
wild-type DNA (Fig. 2, A and B). Both TEF-1 and
CSEF-1 bind the GT-IIC enhanson with relatively higher affinity than
the Sph-I/Sph-II enhanson. Thus TEF-1 and CSEF-1 have identical binding
specificities and raise the question whether these two proteins might
be related.
Figure 2:
CSEF-1 and TEF-1 bind to the GT-IIC and
Sph-1/Sph-II enhansons with nearly identical binding specificity. The
DNA binding specificity of CSEF-1 was examined by a gel shift
competition assay. Gel shift assays were performed under the same
conditions as those of Fig. 1 using 15,000 cpm of
P-labeled GT-IIC
WT oligonucleotide as the
probe. Increasing amounts of GT-IIC
WT or
GT-IIC
MUT (A) and Sph
WT or
Sph
MUT oligonucleotides (B) were included in the
reactions.
CSEF-1 and TEF-1 Are Distinguished by Differential
Thermal Stability
To assess the relatedness of CSEF-1 and TEF-1,
BeWo (Fig. 3A) and COS-1 (Fig. 3B)
nuclear extracts were subjected to heat treatment at various
temperatures for 2 min. The data demonstrate that the CSEF-1-containing
complex in both BeWo and COS cells is very heat-stable, since it
survived 100 °C treatment. In contrast, the TEF-1-containing
complex in BeWo cells (Fig. 3A) was virtually absent
after 2 min at 70 °C. This latter behavior was identical to
authentic in vitro generated TEF-1 (data not shown). Thus
CSEF-1 and TEF-1 may be unrelated proteins that independently recognize
the same DNA elements. Additionally, the CSEF-1 binding activity in
BeWo and COS cells appears to be due to a very similar or identical
heat-stable protein.
Figure 3:
CSEF-1 and TEF-1 exhibit differential
thermal stabilities. BeWo (A) and COS-1 (B) cell
nuclear extracts were heated at different temperatures, and the ability
of the proteins to bind P-labeled GT-IIC
WT
DNA was evaluated in gel shift assays (see ``Materials and
Methods''). Incubation at 70 °C for 2 min destroyed almost all
the TEF-1 binding activity. In contrast, CSEF-1 was resistant to
heating at 100 °C for 2 min.
TEF and CSEF-1 Exhibit Partially Different
Heparin-Sepharose Chromatography Profiles
To further
characterize the potential relationship of CSEF-1 and TEF-1,
heparin-Sepharose chromatography of BeWo nuclear extracts was
performed. Crude BeWo nuclear extract was loaded onto a
heparin-Sepharose column equilibrated with Dignam buffer D (0.1
M KCl). After extensive washing with the same buffer, DNA
binding activities were stepwise eluted with buffer D containing
0.2-0.7 M KCl. The flow-through and salt-eluted
fractions were collected and DNA binding activities characterized by
gel shift assay (Fig. 4). Only very weak or nonspecific DNA
binding activities were detected in the flow-through and 0.2 M
eluate, which contained about 65% of the initial protein. CSEF-1 eluted
in a sharp peak at 0.4 M KCl, whereas apparent TEF-1 binding
activity was eluted broadly as two separate peaks of activity with 0.3
and 0.4 M KCl. The bulk of the TEF-1 binding activity eluted
at 0.3 M KCl. Accordingly TEF-1 binding activity may be
composed of different proteins, multiple isoforms, or differentially
modified forms of TEF-1. Thus the profiles of the two proteins can be
partially distinguished, supporting the concept that the two proteins
may be distinct.
Figure 4:
CSEF-1 can be separated from the bulk of
TEF-1 binding activity with heparin-Sepharose chromatography. BeWo
nuclear proteins were separated on a heparin-Sepharose column as
described under ``Materials and Methods.'' The flow-through
and salt-eluted fractions were analyzed by gel shift analysis to
P-labeled GT-IIC
WT oligonucleotide. Most
nonspecific DNA binding activity was eluted in the 0.2 M KCl
eluate. The bulk of the TEF-1 binding activity eluted in the 0.3
M KCl eluate. CSEF-1 and a portion of the TEF-1 binding
activity were co-eluted with 0.4 M KCl. The specific and
nonspecific (GT-IIC
MUT) DNA binding activities from the
original extract are shown to the right of the
figure.
TEF-1 Is Present in BeWo Cells and Is Distinguished from
CSEF-1 by Its Molecular Mass and Immunoreactivity
To further
characterize TEF-1 and CSEF-1 binding activities in BeWo and COS-1
cells, Western and Southwestern analyses were performed. As
demonstrated in Fig. 5A, an antibody to chicken TEF-1
(Kariya et al., 1993), which cross-reacts with human TEF-1,
recognizes a major protein species with mass of 55 kDa and a minor
species with mass of
57 kDa in both HeLa and BeWo nuclear
extracts. TEF-1 was not detected by Western analysis in COS-1 cells
(Fig. 5B). The finding of two immunoreactive TEF-1
species of differing molecular mass may help to explain the complexity
of the heparin-Sepharose profile (Fig. 4). These two
immunoreactive proteins exhibit identical electrophoretic properties as
TEF-1 prepared by in vitro translation, demonstrating that
BeWo cells contain TEF-1 (Fig. 5A). In addition, these
data indicate that BeWo and HeLa cell nuclear extracts contain
comparable levels of TEF-1, a finding supported by the previous gel
shift analyses (Fig. 1). The antibody did not appear to recognize
any other proteins in BeWo, HeLa, or COS cells with electrophoretic
migration characteristics different from TEF-1, indicating that TEF-1
and CSEF-1 are unrelated proteins. In addition, although the chicken
TEF-1 antibody was able to supershift TEF-1-DNA complexes containing
two copies of the GT-IIC motif, no supershift of CSEF-1-containing
complexes was observed with the same DNA in the presence of
heat-treated BeWo cell nuclear extract (data not shown).
Figure 5:
CSEF-1 is a 30-kDa protein that is
immunologically distinct from TEF-1. 50 µg of HeLa or BeWo cell
nuclear extracts and 5 µl of in vitro generated TEF-1
were resolved by SDS-PAGE, transferred to nitrocellulose membranes and
detected by either antibody (A and B) or
P-labeled GT-IIC
WT probe (C and
D) as described under ``Materials and Methods.'' A and B, TEF-1 resolved in a band with an apparent
molecular mass of 55 kDa on the basis of cross-reactivity with antibody
raised against chicken TEF-1 (Kariya et al., 1993). The 55-kDa
TEF-1 band was observed in BeWo and HeLa cells, but was not detected in
COS cell extracts. C, a 55-kDa band was also observed in
Southwestern blots of in vitro generated TEF-1 using the
labeled GT-IIC probe. D, Southwestern analysis of BeWo, HeLa,
and COS cell extracts using the GT-IIC probe revealed the presence of a
55-kDa band corresponding to TEF-1 in BeWo and HeLa cells, whereas a
30-kDa factor that bound the probe was detected in BeWo and COS cells
but not HeLa cells. The 30-kDa factor was not recognized by the TEF-1
antibody (C). E, UV cross-linking analysis of the
TEF-1 and CSEF-1-GT-IIC
complexes present in BeWo nuclear
extracts (Fig. 1).
To
determine the relative molecular mass of CSEF-1, Southwestern analysis
was done comparing the binding of in vitro generated TEF-1
(Fig. 5C) and BeWo, HeLa, and COS nuclear proteins
(Fig. 5D) to the wild-type GT-IIC motif.
The 55- and 57-kDa proteins present in BeWo and HeLa cells bind the
GT-IIC probe (Fig. 5D) as does in vitro generated TEF-1 (Fig. 5C). In contrast, a protein
with mass of
30 kDa present in BeWo and COS-1 nuclear extracts
binds the GT-IIC motif (Fig. 5D). UV cross-linking
indicated that the CSEF-1-GT-IIC
complex migrates with a
size of about 33 kDa (Fig. 5E), indicating that the
30-kDa protein (Fig. 5D) very likely corresponds to
CSEF-1. Thus CSEF-1 appears to be present in BeWo and COS-1 cells and
can be distinguished from TEF-1 on the basis of mass and the lack of
common epitopes for binding the polyclonal antibody to chicken TEF-1.
CSEF-1 and TEF-1 Compete for Binding to the GT-IIC
Motif
To establish whether TEF-1 and CSEF-1 compete for binding
to the same site, we added in vitro generated TEF-1 to
heat-treated BeWo nuclear extracts, which lack TEF-1 (Fig. 3),
and measured TEF-1 and CSEF-1 binding to the GT-IIC enhanson. With
varying amounts of TEF-1, increasing amounts of the heat-treated BeWo
nuclear extract result in diminished TEF-1 binding and a corresponding
increase in CSEF-1 binding to the GT-IIC enhanson (Fig. 6). Thus
TEF-1 and CSEF-1 compete for the same binding site, suggesting that
both proteins may be involved in enhancer function through mutually
exclusive mechanisms.
Figure 6:
TEF-1 and CSEF-1 compete for the same DNA
motif in vitro. TEF-1 was produced by in vitro translation. Boiled BeWo cell nuclear extracts were used as source
of CSEF-1 that lacked detectable TEF-1 binding as shown in Fig. 3.
Varying amounts of in vitro generated TEF-1 and heat-treated
BeWo cell nuclear extract were mixed and assayed by gel shift analysis
using P-labeled GT-IIC
WT DNA as a probe. The
increase in the relative amount of the TEF-1-GT-IIC complex with
increasing amounts of in vitro generated TEF-1 was accompanied
by a corresponding decrease in CSEF-1-GT-IIC complex
formation.
The Stimulatory Effects of CSEn and Artificial Enhancers
Containing GT-IIC Multimers Correlate with the Presence of CSEF-1, but
Not TEF-1
The relative roles of TEF-1 and CSEF-1 in mediating
CSEn activity were assessed by functional assays with constructs
containing multimers of the GT-IIC enhanson to determine if the
activity of enhancers could be correlated with the levels of CSEF-1
and/or TEF-1 in HeLa, COS-1, and BeWo cells. Initially, plasmids
containing one or two copies of CSEn were transfected into the three
cell lines (Fig. 7, A and B). As previously
demonstrated (Jiang and Eberhardt, 1994), one or two copies of CSEn
significantly increased CS promoter activity in BeWo cells,
but failed to stimulate the CS promoter in HeLa cells. CSEn
was as potent a stimulator of the CS promoter in COS-1 cells
as it was in BeWo cells. This stimulation does not depend on the
expression of the SV40 T antigen in COS-1 cells, since CSEn
also stimulated CS promoter activity in the parent CV-1 cell
line (data not shown). Thus CSEn function is not restricted to
placental cells as had been previously thought. Since COS-1 cells
contain only minor TEF-1 binding activity and contain a protein that
produces similar GT-IIC complexes as CSEF-1, the presence of CSEF-1 is
positively correlated with enhancer stimulatory activity.
Figure 7:
The hCS enhancer and multiple
copies of the GT-IIC enhanson functioned efficiently in BeWo and COS-1
cells, but was inactive in HeLa cells, providing a negative correlation
of TEF-1 binding activity with enhancer function. Cells (5
10
) were transfected by electroporation using 15 µg of
luciferase reporter plasmids as described under ``Materials and
Methods.'' Reporter activity was normalized to protein
concentration and expressed as light units/µg of protein ±
S.E. (A and C) or as -fold stimulation ± S.E.
over the basal activity (B and D) to provide a
measure of the relative enhancer activity. A, comparison of
hCSp.LUC (openbars), EnA_CSp.LUC (stripedbars), and EnA_EnP_CSp.LUC (solidbars) gene activities in BeWo, COS-1, and
HeLa cells. Separate ANOVA analyses for the effect of the enhancer in
BeWo and COS-1 cells were significant at the p = 0.001
and 0.0004 levels, respectively. There was no effect of the enhancer in
HeLa cells (ANOVA analysis, p > 0.05). B, -fold
stimulation of CSp.LUC constructs containing one (stripedbars) or two (solidbars) copies of
CSEn. ANOVA analyses for the effect of the enhancer in BeWo, COS-1, and
HeLa cells were p = 0.0038, 0.0043, and >0.05,
respectively. C, effect of inserting multiple copies of the
SV40 GT-IIC enhanson upstream of the CSp.LUC gene.
Eight (stripedand stippledbars),
16 (checkeredbars), or 24 (solidbars) copies of the GT-IIC enhanson were inserted
upstream of the CSp.LUC gene in either the syn (A alignment,
striped, checkered, and solidbars)
and/or anti (B alignment, stippledbars) orientation
with respect to the promoter and the relative activities were assessed
in BeWo, COS-1, and HeLa cells. ANOVA analyses for the effect of the
enhanson in all of these cells were significant at the p = 0.0001 level. D, -fold stimulation of the
CSp.LUC gene containing 8, 16, or 24 copies of the GT-IIC
enhanson in BeWo, COS-1, and HeLa cells. ANOVA analyses for the effect
of the enhanson in BeWo, COS-1, and HeLa cells were p =
0.0307, 0.0062, and 0.017, respectively. Although constructs containing
16 or 24 copies of the enhanson were about 2-3 times higher than
those containing 8 copies, none of the individual t test
comparisons were significant when corrected for multiple comparisons
using the Bonferroni correction factor.
To study
more directly the function of GT-IIC binding factors in the absence of
participation by other enhansons, artificial enhancers containing
multiple GT-IIC enhansons were constructed and tested (Fig. 7,
C and D). Significant enhancer activity was observed
with GT-IIC 8-, 16-, and 24-mers when introduced into BeWo and COS-1
cells. Neither the trend of increased enhancer activity with an
increasing number of GT-IIC enhansons nor the small increase in
activity induced by the GT-IIC 24-mer in HeLa cells was statistically
significant. Interestingly, in contrast to the wild-type enhancer
(Fig. 7, A and B), the GT-IIC multimers were
much more active in COS-1 cells than in BeWo cells (Fig. 7, C and D), suggesting that BeWo cells might contain a factor
that down-regulates enhancer activity through the GT-IIC enhanson.
These data are consistent with the concept that the GT-IIC site is a
major contributor to enhancer stimulatory activity and suggest that
TEF-1 might account for the reduced GT-IIC multimer activity in BeWo
cells.
Modulation of TEF-1 Levels in BeWo Cells Resulted in
Dramatic Alterations of Basal CS Promoter and Enhancer
Function
To study the influence of TEF-1 on CSEn function
directly, we analyzed the effects of up- and down-regulation of TEF-1
expression on basal and CSEn- or GT-IIC-stimulated CS promoter
activity in BeWo cells. Up-regulation of TEF-1 expression was
accomplished by transient transfection of a TEF-1 cDNA expression
plasmid, and down-regulation was accomplished by co-transfection of
TEF-1 antisense oligonucleotides. We first verified by gel shift and
Western analysis that these manipulations modulated TEF-1 expression in
the appropriate manner. Increasing amounts of co-transfected TEF-1
expression plasmid resulted in an increase in the amount of
GT-IIC-TEF-1 complex and co-transfection of the TEF-1
antisense oligonucleotide decreased the levels of this complex
(Fig. 8A). The levels of the CSEF-1-GT-IIC
complex serves as an internal control. Similar changes in the
levels of TEF-1 were observed in Western blots
(Fig. 8B). Analysis of these data by scanning
densitometry indicated that TEF-1 levels could be up- and
down-regulated by at least 2-fold.
Figure 8:
TEF-1 levels are up- and down-regulated
respectively in BeWo cells transfected with a TEF-1 expression plasmid
or antisense oligonucleotides. BeWo cells (1 10
)
were transfected with increasing amounts of the TEF-1A expression
vector or 35 mM antisense TEF-1 oligonucleotide. After 20 h
whole cell soluble proteins were extracted from the cells with the
modified mini-prep protocol as described under ``Materials and
Methods.'' In vitro translated TEF-1 was used as control.
A, GT-IIC DNA binding activities were tested using 25 µg
of whole cell extracts and 30,000 cpm of
P-labeled
GT-IIC
probe. The TEF-1 levels, reflected by the
intensities of the gel shift bends, were selectively up- or
down-regulated, while the CSEF-1 concentration was relatively
unchanged, serving as an internal control of protein concentration.
B, nuclear extract samples (60 µg) were resolved on
SDS-PAGE, transferred onto nitrocellulose membranes, and detected by
the chicken TEF-1 antibody (Kariya et al., 1993) to provide an
alternate measure of the modulation of TEF-1 levels in transfected BeWo
cells.
We next examined the effects of
co-transfecting the TEF-1 expression plasmid and antisense
oligonucleotide on the basal and CSEn- and
(GT-IIC)-stimulated CS promoter activity
(Fig. 9, A and B). The plasmid pBluescript and
the TEF-1 sense oligonucleotide served as negative controls. We
utilized the EnA_EnP_CSp.LUC and
(GT-IIC)
CSp.LUC constructs for these
studies, since these have the highest enhancer activities and should be
the most sensitive toward sensing changes of TEF-1 concentrations on
enhancer activity. Interestingly, up-regulation of TEF-1 resulted in a
7-fold decrease in basal CS promoter activity and
down-regulation of TEF-1 produced a 3.1-fold increase in basal promoter
activity (Fig. 9A). Virtually identical effects were
observed with the EnA_EnP_CSp.LUC and
(GT-IIC)
CSp.LUC constructs
(Fig. 9A). As shown in Fig. 9B,
down-regulation of TEF-1 levels did result in a significant 2.7-fold
increase in the relative enhancer activity, whereas up-regulation of
TEF-1 levels had no effect. These data do not support the concept that
TEF-1 mediates the positive stimulatory effects of CSEn in BeWo cells
and suggest that TEF-1 is a negative modulator of CSEn and basal
promoter function. The data provide additional evidence that factors
other than TEF-1 that recognize the GT-IIC motif are required for
positive CSEn function.
Figure 9:
The
modulated levels of TEF-1 in BeWo cells are inversely proportional to
the activities of the CSp.LUC, EnA_EnP_CSp.LUC, and
(GT-IIC)24_CSp.LUC genes in co-transfected BeWo cells in a
GT-IIC site-independent manner. The reporter plasmids,
CSp.LUC, EnA_EnP_CSp.LUC, and
(GT-IIC)24_CSp.LUC, together with 5 µg each of
pBluescript control DNA (openbars), the CMV promotor-driven expression plasmid TEF-1A (stripedbars), 35 mM TEF-1 antisense oligonucleotide
(stippledbars), or 35 mM TEF-1 sense
oligonucleotide (solidbars) were co-transfected into
BeWo cells (see ``Materials and Methods''). Reporter activity
was normalized to protein concentration and expressed as light
units/µg of protein ± S.E. (A) or as -fold
stimulation ± S.E. over the basal activity (B) to
provide a measure of the relative effect of manipulating TEF-1 levels
on the activities of the various reporter genes. ANOVA analyses for the
different treatment groups with the CSp.LUC, EnA_EnP_CSp.LUC and (GT-IIC)24_CSp.LUC genes' basal activity
(A) were significant at the p = 0.0001 level.
ANOVA analyses for the -fold stimulation (B) of the
EnA_EnP_CSp.LUC and (GT-IIC)24_CSp.LUC genes were
significant at the p = 0.001 and 0.001 levels,
respectively.
complex in BeWo nuclear extracts confirms
the studies of Jacquemin et al.(1994), who reported a pattern
of complexes with HeLa, JEG-3, and placental tissue extracts that is
very similar to that observed with HeLa and BeWo cells (Fig. 1).
We have demonstrated that the slower migrating complex, which appears
to be present in all of these cell types, corresponds to TEF-1. The
more rapidly migrating complexes CSEF-1 (Fig. 1) and f (Jacquemin
et al., 1994) appear to be identical based on their relative
migration and similar placental cell distribution. Since the GT-IIC
enhanson is essential for CSEn function (Jiang et al., 1994;
Jacquemin et al., 1994), the finding of multiple
GT-IIC-binding proteins raises the question of the exact role of TEF-1
in mediating CSEn activity.
3-fold) of
either wild-type CSEn- or (GT-IIC)
-mediated activity.
Nevertheless, since modulation of TEF-1 levels results in significant
effects on basal promoter activity in a manner independent from the
GT-IIC enhanson (Fig. 9A), the data suggest that TEF-1
squelching does not require binding to the GT-IIC enhanson.
Accordingly, the increased effectiveness of CSEn or the GT-IIC enhanson
in COS-1 cells is correlated with the relative absence of TEF-1 in
these cell types, supporting the concept that TEF-1 mediates negative
but not positive effects on enhancer function.
)
the putative co-activator could be a
component of the normal basal transcription apparatus.
Table:
Oligonucleotide use in studies
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.