Human Chorionic Somatomammotropin Enhancer Function Is Mediated by Cooperative Binding of TEF-1 and CSEF-1 to Multiple, Low-Affinity Binding Sites
Shi-Wen Jiang,
Miguel A. Trujillo and
Norman L. Eberhardt
Endocrine Research Unit (S.-W.J., M.A.T., N.L.E.) Departments
of Medicine and Biochemistry/Molecular Biology (N.L.E.) Mayo
Clinic Rochester, Minnesota 55905
 |
ABSTRACT
|
---|
The human chorionic somatomammotropin gene
enhancer (CSEn) is composed of multiple enhansons (Enh) that share
sequence similarities with those of the simian virus, SV40 enhancer
(SVEn). The sequence homology includes two GT-IIC-like (Enh1 and Enh4)
and three SphI/II-like enhansons (Enh2, Enh3, and Enh5). We previously
showed that transcription enhancer factor 1 (TEF-1) and a 30-kDa
placental-specific factor, chorionic somatomammotropin enhancer factor
1 (CSEF-1), bind to Enh4, which plays an essential role in enhancer
function. In this study, we demonstrate that TEF-1 and CSEF-1 bind
specifically to all the other GT-IIC- and SphI/II-like elements within
CSEn with a broad range of binding affinities that vary between 0.005
and 0.15 that of Enh4. Each individual concatenated enhanson was able
to stimulate hCS promoter activity in an
orientation-independent manner in choriocarcinoma cells (BeWo) with an
observed stimulation that was directly proportional to its relative
binding affinity for TEF-1 and CSEF-1. These results indicate that CSEn
function results from the cooperative interaction of TEF-1 and/or
CSEF-1 binding to multiple, low-affinity GT-IIC- and SphI/II-like
enhansons within the enhancer.
 |
INTRODUCTION
|
---|
The human chorionic somatomammotropin (hCS) genes are
members of the GH and PRL gene families. The multiple hGH
and hCS genes (Fig. 1A
) arose by relatively
recent duplication of the hGH gene (1). These homologous,
nearly identical genes are located within a short 50-kbp span of DNA on
chromosome 17q22-q24 and yet are expressed in a strict cell-specific
pattern whereby the hCS-1 and hCS-2 genes are
expressed exclusively in placental syncytiotrophoblasts (1, 2). A
chorionic somatomammotropin enhancer (CSEn) found 2 kb downstream of
the hCS-2 gene (Fig. 1A
) participates in the cell-specific
control of hCS gene expression (3, 4, 5, 6). A minimal enhancer
(Fig. 2B
) is contained within a 240-bp element that
stimulates hCS promoter activity in human placental BeWo and
JEG-3 and monkey kidney COS-1 cells, but not in HeLa or pituitary GC
cells (6, 7). The choriocarcinoma cell lines BeWo and JEG-3 express low
levels of hCS-1, hCS-2, and hGH-2 mRNAs, but not hGH-1 mRNA (8, 9) and
have served as the dominant model for studying cell-specific expression
of the hCS genes (3, 4, 5, 6, 7, 8, 9). The minimal enhancer has been shown
to contain several enhansons (individual DNA response elements
comprising a modular enhancer) that are homologous to the GT-IIC and
SphI/SphII enhansons in the SV40 enhancer, which are binding sites for
transcription enhancer factor-1 (TEF-1) (3, 5, 6, 7, 10). We and others
have demonstrated that the 53-kDa TEF-1 and a 30-kDa factor, chorionic
somatomammotropin enhancer factor 1 (CSEF-1) that is present in
placental and COS-1 cells, binds to the major GT-IIC-like enhansons in
CSEn (5, 7, 10). Positive enhancer activity is correlated with the
binding of CSEF-1 (7), whereas the binding of TEF-1 appears to be
associated with inhibition of enhancer activity as well as basal
promoter activity (11). The inhibition that occurs through TEF-1 is
correlated with its ability to interact with the TATA-binding protein,
TBP, and the resultant inability of the TEF-1-TBP complex to bind to
the TATA element (11).

View larger version (29K):
[in this window]
[in a new window]
|
Figure 1. Schematic Diagrams of the hGH/hCS
Chromosomal Locus (A), the 240-bp Minimal Human Chorionic
Somatomammotropin Enhancer, CSEn2 (B), and the Individual GT-IIC- and
SphI/SphII-Like Enhansons (Enh1-Enh5) That Comprise the Enhancer (C)
The hGH/hCS locus contains five genes, including the
pituitary-specific hGH-1 and placental-specific
hCS-5 or hCS-like (a putative
pseudogene), hCS-1, hCS-2, and hGH-2 or
hGH-variant. The location of the two other enhancers, CSEn5 and
CSEn1, that are related ( 97.5% nucleotide sequence identity) to the
CSEn2 enhancer are shown, and the more detailed structure of the
minimal CSEn2 enhancer is depicted (B). Arrows indicate
the orientation of the GT-IIC-related (black squares)
and SphI/II-related (shaded circles) enhansons.
Open rectangles (FP-1 to FP-5) indicate the extent of
DNaseI-protected regions in the presence of BeWo, HeLa, and GC cell
nuclear extracts and the location of the 6- to 8-bp block mutations
(EM1-EM8) that were used in the initial analysis of the enhancer
structure (6 ). Sequences of the individual enhansons were aligned using
the GCG PILEUP program (Genetics Computer Group, Madison, WI) (C). Enh5
is included in both the GT-IIC and SphI/SphII comparisons because a
reasonable fit was found for both consensus types. M-CAT is the
designation for the muscle-specific regulatory element that is related
to the GT-IIC enhanson.
|
|

View larger version (109K):
[in this window]
[in a new window]
|
Figure 2. Gel Shift Analysis of the Binding of in
vitro-Generated TEF-1 to the GT-IICSV and Enh1-Enh5
Enhansons
In vitro control reactions (TNT) and TEF-1-programmed
reactions (TEF) were carried out as described (Materials and
Methods). A mutated form (MUT) of the GT-IICSV
enhanson was included as an additional nonspecific DNA binding
control.
|
|
An essential GT-IIC enhanson, Enh4, is located within a region (FP-3,
Fig. 1B
) that is protected from deoxyribonuclease I (DNaseI) digestion
in the presence of BeWo cell nuclear proteins (3, 5, 6). However,
several lines of evidence indicate that additional sequences are also
involved in the regulation of CSEn activity. First, footprinting
analyses with nuclear extracts from placental cells detected four
additional DNaseI-protected regions, FP-1, FP-2, FP-4, and FP-5 (Fig. 1B
), within the 240-bp fragment (5, 6). Second, mutations of sequences
within these footprinted regions resulted in significant reduction of
CSEn activity (EM1-EM8, Fig. 1B
), indicating that these sites are
functional (5, 6). Finally, although a single copy of a CSEn enhancer
harboring a mutation in the central Enh4 enhanson (EM5, Fig. 1B
) is
virtually devoid of activity, a construct containing two copies of the
enhancer carrying the Enh4 mutation restored enhancer activity in
placental cells (S.-W. Jiang and N. L. Eberhardt, unpublished results).
These findings support the concept that functional DNA elements other
than Enh4 are required for CSEn activity.
In the current studies we have performed further analysis of the
additional GT-IIC- and SphI/II-like enhansons (Enh1-Enh5) within CSEn.
Utilizing gel shift assays, gel supershift experiments with TEF-1
antibodies, and UV cross-linking techniques, we demonstrate that TEF-1
and CSEF-1, generated in vitro or from nuclear cell
extracts, specifically bind to each of these enhansons. Interestingly,
competition experiments revealed differences of as much as 85-fold in
the relative binding affinities of the different enhansons.
Nevertheless, each of the individual concatenated enhansons was capable
of stimulating hCS promoter activity in an
orientation-independent manner in transfected placental choriocarcinoma
cells. These results suggest that full CSEn function depends on the
cooperative binding of TEF-1 and CSEF-1 to multiple low-affinity
binding sites.
 |
RESULTS
|
---|
We have demonstrated that five regions of CSEn, designated FP-1 to
FP-5 (Fig. 1B
) (6), are protected by nuclear extracts from GC, HeLa,
and placental BeWo cells. Mutations of individual GT-IIC- and
SphI/SphII-like sequences in an otherwise intact enhancer within FP-2
(EM3 and EM4, Fig. 1B
) and FP-3 (EM5, Fig. 1B
) reduced enhancer
activity dramatically (70100%), whereas mutations within FP-1 (EM1),
FP-4 (EM6), and FP-5 (EM7 and EM8) resulted in less dramatic, but
significant (ca. 3050%) reductions in CSEn function (6).
Thus, factors binding to FP-1, FP-4, and FP-5 may contribute to the
overall enhancer activity. With the exception of sequences within FP-5,
the other four regions share extensive similarities with GT-IIC and
SphI/II enhansons (Fig. 1C
), suggesting that the binding of a common
factor to these regions might account for a majority of CSEn functional
activity. We therefore sought to determine whether these sequences are
recognized by TEF-1 and/or CSEF-1. To test this possibility, we
performed gel shift assays using the 5'-end labeled oligonucleotides
(Table 1
) with in vitro-generated TEF-1 and
BeWo nuclear cell extracts containing both TEF-1 and CSEF-1.
TEF-1 and CSEF-1 Binding to Enh1-Enh5
In vitro-generated TEF-1 binding to Enh1-Enh5
oligonucleotides is shown in Fig. 2
. All of the double-stranded Enh
oligonucleotides showed a pattern of migration similar to that of the
SV40 GT-IIC (GT-IICSV) oligonucleotide, whereas no retarded
band is observed in unprogrammed extracts (TNT, Fig. 2
). Thus, the
shift in mobility appears to be specifically produced by TEF-1.
Although the same amount of in vitro-generated TEF-1 and
radioactivity were applied in each case, the intensity of the TEF-1-DNA
complexes was quite different. Since the specific activity of all the
probes was similar, we infer that the differences in intensity indicate
differences in the relative binding affinities of TEF-1 for each of the
enhansons, Enh1-Enh5. The relative binding affinity appears to be
GT-IICSV
Enh4
Enh5
Enh1
Enh3
Enh2.
We previously showed that nuclear extracts from BeWo cells contain an
additional protein that recognizes the GT-IIC- and SphI/SphII-like
motifs (7). This protein was designated CSEF-1 and was shown to have a
molecular mass of approximately 30 kDa, which produces a much more
rapidly migrating complex with the GT-IIC oligonucleotide in gel shift
analyses. As shown in Fig. 3
, this more rapidly
migrating complex was observed in gel shift analyses with all of the
Enh enhansons in the presence of BeWo nuclear cell proteins. Moreover,
the general pattern of intensities with the various Enh1-Enh5
oligonucleotides was similar to that observed with the TEF-1 and CSEF-1
complexes in BeWo nuclear extracts (Fig. 3
) as with in
vitro-generated TEF-1 (Fig. 2
).

View larger version (64K):
[in this window]
[in a new window]
|
Figure 3. Gel Shift Analysis of the Binding of BeWo Cell
Nuclear Extracts to the GT-IICSV and Enh1-Enh5 Enhansons (A
and B)
Experiments were performed as described in Materials and
Methods. Nonspecific DNA binding activity was assessed by
inclusion of a mutated GT-IICSV enhanson (MUT). The
identities of TEF-1- and CSEF-1-containing complexes have been
described in detail in Jiang and Eberhardt (7 ). The gel in panel A was
exposed for 3 days to visualize the weaker interactions, resulting in
overexposure of lanes containing the high-affinity enhansons. The lanes
shown in panel B are from the same gel exposed for 6 h; however,
in this case only the lanes including the GT-IICSV- and
Enh4-protein complexes are shown.
|
|
Characterization of the Factors That Bind to the CSEn Enhanson
To verify that the complexes formed with each of the enhansons
corresponded to TEF-1 and CSEF-1 binding, we characterized the
complexes further. We previously demonstrated that TEF-1 is heat
sensitive whereas CSEF-1 is heat resistant. Gel shift experiments with
heat-treated BeWo cell extracts revealed a single, more rapidly
migrating complex typical of that formed with the CSEF-1 complex (data
not shown), indicating that the more slowly migrating band corresponded
to TEF-1. To corroborate that TEF-1 was the factor present in the slow
migrating band, a TEF-1-specific antibody was used in a supershift
assay. In this experiment, the CSEF-I band cannot be clearly observed
because minimal amounts of nuclear extracts were mixed with large
amounts of DNA. These conditions were chosen to achieve maximal TEF-1
occupancy of its cognate site to optimize the observation of the
supershifted complex. The TEF-1 antisera, but not the preimmune
antisera, clearly produced a supershifted band that was observed with
all of the Enh probes (Fig. 4
). In addition, the
relative amount of supershifted TEF-1-DNA complex, as judged by the
relative intensity of the band, was in the same relative order as
observed in simple gel shift experiments (Enh4
Enh5
Enh1
Enh3
Enh2). Taken together, these results
indicate that the more highly retarded band corresponds to TEF-I.

View larger version (97K):
[in this window]
[in a new window]
|
Figure 4. Gel Supershift Experiment with a Chicken TEF-1
Antibody (AB) or Nonimmune Serum (NI) in Reactions Containing BeWo Cell
Nuclear Proteins and Enhansons Enh1-Enh5
Reaction conditions were modified (Materials and
Methods) to maximize TEF-1 binding to the various
oligonucleotides.
|
|
CSEF-1 has not been cloned, and specific antibodies are not yet
available. However, UV cross-linking characterized CSEF-I as a factor
migrating with an apparent molecular mass of 30 kDa (7). Using the
GT-IICSV oligonucleotide, the cross-linked factor from the
more rapidly migrating complex has an apparent molecular mass of 30 kDa
after electrophoresis in SDS gels (Fig. 5
). A factor
with the same molecular mass was cross-linked to each of the Enh1-Enh5
oligonucleotides (Fig. 5
), and the relative binding affinity, as judged
by the intensity of the band, was similar to that observed in the
previous experiments (
Figs. 24

). These data are consistent with the
concept that CSEF-1 binds to each of the Enh1-Enh5 enhansons with an
affinity similar to that of TEF-1 binding.

View larger version (45K):
[in this window]
[in a new window]
|
Figure 5. UV Cross-Linking of CSEF-1 to the
GT-IICSV and Enh1-Enh5 Enhansons
The enhanson-CSEF-1 complexes migrate with an apparent molecular mass
of 30 kDa. A negative GT-IICSVMUT control
oligonucleotide was included in the gel shift experiment that was
generated by complexation with BeWo cell nuclear proteins and was
subjected to UV cross-linking; however, because of the absence of a
shifted band (see Fig. 3 ), it was not eluted for SDS gel
electrophoresis.
|
|
Relative Binding Affinities of TEF-1 and CSEF-1 for the GT-IIC and
SphI-Like Elements
To measure more precisely the relative binding affinities of TEF-1
and CSEF-1 for Enh1-Enh5, we performed competition experiments. In this
case we used a labeled GT-IICSV oligonucleotide, and
competition was effected by including increasing amounts of cold Enh
oligonucleotides (Enh1-Enh5, 0.06600 nM) in gel shift
assays. The intensity of bands resulting from complexes with both TEF-I
and CSEF-I binding were scanned by densitometry, and competition curves
were generated (Fig. 6
). The relative affinities of each
enhanson was estimated from the data in Fig. 6
at the point at which
50% of the binding was inhibited, and the relative binding affinities
are shown in Table 2
. TEF-1 has an almost 2-fold higher
binding affinity for each of the enhansons than CSEF-1, and the order
of binding affinities is identical to that observed in the earlier
experiments (
Figs. 25


).

View larger version (24K):
[in this window]
[in a new window]
|
Figure 6. Competition Analysis to Establish the Relative
Binding Affinities of TEF-1 (A) and CSEF-1 (B) to the Individual
Enhansons Enh1-Enh5
BeWo cell nuclear extracts were incubated with the labeled
GT-IICSV probe, and unlabeled competitor DNA was included
at concentrations ranging from 0.06600 nM. Enh Mut
represents GT-IICSV MUT. Gel shift assays are described in
Materials and Methods. The intensities of the TEF-1- and
CSEF-1-DNA complexes were measured by densitometry analysis of the
autoradiograms using NIH IMAGE software.
|
|
The Concatenated Individual Enhansons (Enh1-Enh5) Stimulate hCS
Promoter Transcription
To evaluate the ability of individual enhansons to stimulate
transcription, a series of constructs containing concatenated
individual enhansons were cloned upstream of the hCSp.LUC
gene. Tandem repeats (three to nine copies) in both orientations were
placed 800 bp upstream of the hCS promoter, and their
activity was assessed in transfected BeWo cells (Fig. 7
). Almost all of these constructs exhibited significant
stimulation of hCS promoter activity (1.5- to 4-fold). To
evaluate the relative activities of each of the enhansons, the
activities of constructs containing five copies of the individual
enhansons in both orientations were averaged and plotted against the
relative binding affinity as shown in Fig. 8
. There was
a linear relationship between the relative activity and the relative
binding affinity for TEF-1 and CSEF-1. These results, along with our
previous mutational analyses (6), strongly support the concept that, in
addition to Enh4, Enh1, Enh3, Enh5, and possibly Enh2 contribute to
CSEn activity by acting as relatively weak binding sites for TEF-1 and
CSEF-1. The data suggest that the cooperative interaction of multiple
TEF-1 and/or CSEF-1 molecules accounts for CSEn activity.

View larger version (37K):
[in this window]
[in a new window]
|
Figure 7. Enhancer Activity Associated with Concatamers of
the Individual Enhansons When Cloned Upstream of the
hCSp.LUC Gene
Concatamers containing three to nine repeats were cloned into the
vector in both orientations (+ [syn] and - [anti] relative to
the hCS promoter), and the constructs were transfected
into BeWo cells. Luciferase activity was measured as described
(Materials and Methods), and the fold activation was
plotted. Data were analyzed by multivariate ANOVA (P 0.0001) and by post hoc Bonferroni
t tests (asterisks indicate P
< 0.05 compared with the control hCSp.LUC
activity).
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Figure 8. Relationship of Relative Stimulatory Activity and
Relative Binding Affinity of the Enh1-Enh5 Enhansons
The relative binding affinities (Table 2 ) were plotted against the
combined functional data (Fig. 7 ) for the constructs containing five
copies of each of the enhansons. For this analysis the data for both
orientations were included. Linear regression equations and
r2 values are indicated for both CSEF-1 binding and TEF-1
binding.
|
|
 |
DISCUSSION
|
---|
The CSEn is a typical enhancer with a modular structure that is
related to the SV40 enhancer (3, 4, 5, 6, 7, 10). In previous studies we found
that two factors, TEF-1 and CSEF-1, bind to the central, high affinity
GT-IIC-like enhanson (Enh4) of CSEn and demonstrated that this enhanson
is essential for enhancer function (6, 7). The two proteins compete
with each other for this site in a mutually exclusive manner. Several
lines of evidence suggest that TEF-1 represses, whereas CSEF-1 induces,
CSEn activity. First, CSEn is active in BeWo and COS-1 cells that
express relatively large amounts of CSEF-1 compared with TEF-1, whereas
in GC and HeLa cells that only express TEF-1, CSEn lacks enhancer
activity (7). Furthermore, cotransfection of TEF-1 expression
constructs in BeWo cells only results in CSEn inhibition.
Cotransfection with a TEF-1 antisense oligonucleotide, which inhibits
TEF-1 expression, up-regulates enhancer activity in CSEF-1-expressing
cells (7). Finally, we have shown that TEF-1 binds to the TATA binding
protein, TBP, and inhibits its ability to bind to the TATA element
(11), providing a possible mechanism by which to understand
TEF-mediated inhibition of the hCS promoter activity.
DNaseI footprinting studies of the 240-bp minimal enhancer with nuclear
extracts from a variety of cells has revealed five protected regions
(5, 6). Four of these DNaseI-protected regions contain GT-IIC- and
SphI/SphII-like sequences (Enh1-Enh5, Fig. 1A
and B). Enh2 does not
reside within a previously recognized DNaseI-protected region.
Mutagenesis of sequences within each of these DNaseI-protected regions
diminished CSEn activity (6), indicating that CSEn activity is governed
by the interactions of multiple elements. However, the identity of
factors binding to these elements has not been established, nor has the
function of isolated GT-IIC- or SphI/SphII-related enhansons been
tested.
In the present study, TEF-1 and CSEF-1 binding and functional
activities were examined for each of the GT-IIC- and SphI/SphII-like
regions. Each of the enhansons were shown to bind to TEF-1 and CSEF-1
with comparable relative binding affinities that varied considerably
(
2 orders of magnitude) (
Figs. 26



and Table 2
). When the
individual enhansons were concatenated (3- to 9-mers), virtually all of
the enhansons stimulated hCS promoter activity in an
orientation-independent manner when cloned upstream of the
hCSp.LUC gene and transfected into BeWo cells (Fig. 7
),
indicating that each of these structures can contribute to CSEn
function. Moreover, there was a linear relationship between the
relative binding affinity of each enhanson for TEF-1 and CSEF-1 and the
relative enhancer activity (Fig. 8
). This is consistent with the
concept that CSEn function is governed by the binding of multiple
copies of TEF-1 and CSEF-1 to the enhansons, Enh1-Enh5. Although Enh2
displays the weakest binding and functional activity and exists within
a CSEn domain that is not protected by DNaseI footprinting (6), it may
contribute, nevertheless, toward mediating enhancer function. The fact
that it resides within a region not protected by DNaseI may reflect a
lower bound of affinity for which DNaseI footprints may be observed.
Interestingly, mutation of either Enh3 or Enh4 results in 70100%
loss of enhancer function in the context of the intact enhancer (6),
although there is a
20-fold difference in the TEF-1 or CSEF-1
binding affinity for the individual enhansons (Fig. 6
), and Enh4 is a
more potent enhancer when multimerized (Fig. 7
). This result may
reflect the importance of the spatial organization of the modular
enhancer and illustrates that a relatively weaker enhanson may possess
inordinate functional significance in the context of the intact
enhancer, which is likely the result of cooperative interactions. In
this regard it is of interest that the Enh3 and Enh4 enhansons are
centrally located, suggesting that they may comprise part of a core
enhancer structure. Taken together these data support the concept that
the cooperative interaction of TEF-1 and CSEF-1 to these multiple,
low-affinity binding sites plays an essential role in mediating CSEn
functional activity; however, they do not exclude the possibility that
other factors may be involved in enhancer function. For example,
recent evidence indicates that M-CAT (GT-IIC-like) elements
involved in both muscle-specific and non-muscle-specific
transcription may be modulated by additional factors that bind to
flanking sequences adjacent to the M-CAT motifs (12).
Although many enhancers function through the interactions of multiple,
unique proteins, several enhancers and regulatory elements appear to
operate by the binding of single transcription factors to multiple,
low-affinity binding sites. For example, ovalbumin gene regulation by
estrogen occurs by estrogen receptor binding to several
half-palindromic TGACC motifs instead of the typical palindromic
estrogen response element (ERE) (13). Cooperative binding of the
estrogen receptor to these weak, relatively widely spaced half-sites
provides for synergistic activation of the ovalbumin gene by estrogen.
In a very similar manner, estrogen regulation of the progesterone
receptor gene is governed by weak, nearly palindromic EREs that are
coupled to estrogen receptor half-sites (14). Regulation of the
immunoglobulin heavy chain µ-enhancer is modulated by the binding of
the enhancer-binding regulatory protein, NF-µNR, to four adjacent
binding sites that flank the enhancer core. Like the GT-IIC- and
SphI/II-like elements of CSEn, the individual NF-µNR binding sites
display a range of binding affinities spanning 2 orders of magnitude
(15). Nevertheless, when low- and high-affinity NF-µNR binding sites
are present on the same molecule, both sites are occupied at
concentrations of NF-µNR that would only be expected to occupy the
high-affinity site by itself. Accordingly, the juxtaposition of low-
and high-affinity binding sites within the µ-enhancer results in
cooperative binding of NF-µNR to the enhancer (15). Finally, the SV40
enhancer functions through the binding of TEF-1 to multiple GT-IIC and
SphI/SphII enhansons (16, 17, 18) that differ by 4- to 10-fold in their
ability to bind TEF-1 (GT-IIC
SphI 
SphII) (16). These
differences in binding affinity closely mirror those observed with the
GT-IIC- and SphI/SphII-like CSEn enhansons (
Figs. 26



and Table 2
).
Because mutation of the low affinity TEF-1/CSEF-1 binding sites results
in an enhancer with lower functional activity (6), these sites are
important for enhancer function. Consequently, it is likely that, as is
the case with the SV40 enhancer, cooperative binding of TEF-1/CSEF-1 to
the multiple low-affinity sites is important for full CSEn functional
activity.
TEF-1 is a member of a highly conserved family of regulatory proteins
that includes the yeast factor TEC1 (19), the Aspergillus
nidulans factor AbaA (20), and the Drosophila scalloped
(sd) gene product (21). In addition to TEF-1, a number of
distinct TEF-1 homologs from humans (22), mice (22), and chicken (23, 24) have been cloned. TEFs are involved in the regulation of a diverse
set of processes, including skeletal and cardiac muscle gene expression
(12, 23, 24, 25, 26), SV40 (16, 17, 18), CSEn (3, 4, 5, 6), and human papillomavirus type
16 E6 and E7 (27) enhancer control. The mouse TEF-1, TEF-3, and TEF-4
homologs display a complex pattern of expression during development
that implicate these homologs in myogenesis and cardiogenesis, as well
as central nervous system development and organogenesis (22). Despite
the diversity of these different products, all the members of this
family contain a very highly conserved TEA/ATTS DNA-binding domain that
recognizes the DNA sequences related to the prototypic GT-IIC and
SphI/SphII enhansons (28).
Although the exact structure of the TEA/ATTS domain is not yet known,
it has been proposed that this 80-amino acid-containing domain contains
either three
-helices or one
-helix and two ß-sheet structures
(20, 21, 28). Mutational analysis of the putative
-helical and/or
-helical/ß-sheet structures demonstrates that the first
-helical and third
-helical/ß-sheet are critical for DNA
binding; however, the carboxyl terminus of TEF-1 can also modulate
DNA-binding affinity (18). Nevertheless, expression of a synthetic
DNA-binding domain containing all three of the
-helical and/or
-helical/ß-sheet motifs established that these structures are
sufficient to determine the binding specificity to the unrelated GT-IIC
and SphI/SphII enhansons (18). Thus DNA-binding specificity of these
family members resides within the TEA/ATTS domain. Given the striking
similarity in the relative binding affinities of CSEF-1 and TEF-1 for
the unrelated GT-IIC and SphI/SphII enhansons (
Figs. 26



and Table 2
),
it seems likely that CSEF-1 is an as yet unidentified member of this
family. It is noteworthy that the TEA/ATTS domain is sufficient for
cooperative binding to tandemly repeated GT-IIC and Sph enhansons and
that the cooperativity is required for binding to low-affinity
enhansons (22). This lends further support to the concept that the
multiple, low-affinity GT-IIC- and SphI/SphII-like sites within CSEn
may act via cooperative binding of TEF-1 and/or CSEF-1.
It is possible that multiple low-affinity binding sites with or without
interspersed high-affinity sites provide a mechanism for generating a
transcriptional rheostat that senses different levels of physiological
signals and generates fine-tuned control of gene expression. Such a
mechanism may help to explain hCS gene expression during
pregnancy, which is gradually up-regulated from nearly silent
expression during early pregnancy to maximal expression in the third
trimester. It should be emphasized that the mechanism by which TEF-1
and/or CSEF-1 mediate enhancer function is unknown. Overexpression of
intact TEF-1 in several cell lines (11, 17) results in a dominant
negative inhibition of reporter gene activity, suggesting that limiting
cofactors are required for its transactivating functions (17). Using
GAL4-TEF-1 chimeras, Hwang et al. (18) have been able to
demonstrate that three distinct, but interdependent, domains were
required for both transactivation and squelching functions, providing
additional support for the limiting cofactor model. In contrast, we
previously demonstrated that TEF-1-mediated transrepression may be
accounted for by interactions with TBP that inhibit TBP from binding
the TATA element. Interestingly, the same three TEF-1
activation/squelching domains identified earlier (18) were required for
TBP binding (11). These latter results suggest an alternate model in
which TEF-1 is a repressor, whose binding to the enhancer may allow it
to interact with TBP and negatively regulate transcription initiation.
Further evidence for such a repressor model is presented in the
accompanying article in which it is shown that TEF-1 binding to
multiple enhancers (CSEn2 and CSEn1 or CSEn2 and CSEn5) is associated
with a composite silencer activity in pituitary GC cells (30).
Accordingly, a cofactor might be required as part of a switch mechanism
in cell types in which TEF-1 acts as a transactivator, but not
presumably in BeWo cells in which CSEF-1 appears to mediate
transactivation. Further studies will be required to elucidate these
mechanisms.
 |
MATERIALS AND METHODS
|
---|
Cell Transfection
Placental trophoblast BeWo cells (ATCC, Rockville, MD) were
grown in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD)
supplemented with 10% FBS (BioWhittaker, Walkersville, MD), 100 U/ml
penicillin (Life Technologies, Inc.), 100 µg/ml streptomycin (Life
Technologies, Inc.) and 2 mM glutamine (Life Technologies,
Inc.). Cells were maintained at 37 C in an atmosphere containing 5%
CO2 and 100% humidity. Transfections were performed with
double CsCl2-purified plasmids (15 µg) as described in
detail (6). Because of the large number of constructs that had to be
evaluated, multiple transfections with different batches of cells grown
at different times were required. Consequently, reporter gene activity
was normalized to ß-galactosidase activity instead of protein per our
normal protocol (6, 7, 11, 30, 32). This was accomplished by
cotransfection of a CMV ßGal plasmid (5 µg) and
subsequent analysis of ß-galactosidase activity.
Plasmid Construction
All reporter plasmids were cloned into
pA3LUC (29). Construction of
CSp.LUC, which contains the hCS 496-bp promoter,
was described previously (6). Complementary oligonucleotides harboring
CSEn sequences protected in footprinting experiments (6) were
synthesized at the Molecular Biology Core Facility, Mayo Clinic (Table 1
). We devised a simple and efficient PCR-based method to subclone all
the synthetic oligonucleotides in tandem repeats with defined spacing,
orientation, and repeat number (31). Briefly, phosphorylated individual
enhanson monomers that contained GG and CC overhangs at the 3'-ends
(Table 1
) were mixed with a synthetic linker containing a
BglII site and similarly affixed 3'-GG and 3'-CC overhangs.
Enhanson monomers and synthetic linker (10:1) were mixed and ligated
with T4 DNA ligase, and 200- to 300-bp DNA fragments containing
tandemly repeated monomers and linkers were selected on a 2% agarose
gel. Oligomers recovered after the gel had been subjected to several
freeze/thaw cycles were amplified by PCR using the synthetic linker
oligonucleotides as primers. Self-priming, due to the repetitive nature
of the oligomers, generated large DNA PCR products. After
BglII restriction digestion, clear bands corresponding to
DNA fragments containing different numbers of repeated monomers bounded
by the linker oligonucleotide were visible on the gel. These oligomers
were ligated to the BglII-treated
pA3LUC to generate constructs
containing Enh1-Enh5 sequences in different numbers and orientations.
Positive clones were screened by BglII digestion, and the
sequence of the insert was confirmed by dideoxy-nucleotide sequencing
(Molecular Biology Core Facility, Mayo Clinic).
Data Analysis
Data were subjected to multivariate ANOVA using post
hoc Bonferroni t tests to assess individual differences
among the multiple comparisons.
Gel Shift Assays
The pXJ40-TEF-1A plasmid (17), generously provided by
Dr. Pierre Chambon and Irwin Davidson (University of Strasbourg,
Strasbourg, France), was used to generate TEF-1 protein by in
vitro translation (TNT, Promega, Madison, WI) using pBluescript in
a mock translation reaction for negative control as described
previously (7). Large-scale nuclear extracts were isolated from
cultured BeWo cells according to the method of Dignam et al.
(32).
Gel shift probes, which contain the same sequences as the
oligonucleotides listed in Table 1
, except for the absence of
protruding 5'-GG and 3'-CC ends, were 5'-end labeled with
[
-32P]ATP (Amersham Corp., Arlington Heights, IL) and
with polynucleotide kinase to a specific activity 2 x
106 cpm/pmol. The labeled probe was purified through a
Bio-Gel P-60 (Bio-Rad, Richmond, CA) column. Probe (30,000 cpm) and 2.5
µl of in vitro-generated TEF or 20 µg of BeWo nuclear
extract were used for gel shift analyses under conditions described in
detail previously (33).
The TEF-1 and CSEF-1 affinities to Enh1-Enh5 were measured by gel shift
competition experiments. Increasing concentrations (0.05 to 500
nM) of unlabeled double-stranded oligonucleotides were
mixed with labeled GT-IICSV probe (30,000 cpm) and BeWo
cell nuclear extracts (20 µg). After autoradiography, the TEF-1-DNA
and CSEF-1-DNA complexes were analyzed by densitometry (NIH Image).
Rabbit anti-chicken TEF-1 antibody was generously provided by Drs.
Charles Ordahl and Iain Farrance (University of California San
Francisco) and used for gel supershift experiments. Compared with
normal gel shift analyses, less nuclear extract (10 µg) and more DNA
probe (80,000 cpm) was used to enhance the sensitivity. After a 30-min
incubation, 3 µl TEF-1 antibody were added to the binding reaction
and the incubation was continued for an additional 15 min. After
electrophoresis, the gel was dried and exposed to Kodak x-ray film for
2 days.
UV Cross-Linking
For cross-linking studies, 120,000 cpm of DNA probe and 80 µg
BeWo nuclear extracts were incubated and subsequently resolved by
electrophoresis. The wet nondenatured gel was placed on ice and
irradiated with UV light (Stratalinker, Stratagene, La Jolla, CA) for
1 h. Autoradiography was performed overnight at 4 C with an
intensifying screen on top of the gel. Gel slices containing the CSEF-1
complexes were excised and soaked in 200 µl 2x SDS-PAGE loading
buffer (100 mM Tris·HCl (pH 7.6), 300 mM KCl,
1 mM EDTA, 10 mM dithiothreitol) at 4 C for 30
min. The cross-linked CSEF-1-DNA complex was resolved by 10% SDS-PAGE.
The gel was dried and exposed to Kodak x-ray film with intensifying
screens at -20 C for 2 days.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to express their appreciation to Drs. Pierre
Chambon and Irwin Davidson for the pXJ140 TEF-1 expression plasmid and
to Drs. Charles Ordahl and Iain Farrance for the generous gift of the
chicken TEF-1 antibody. We thank Ruth Kiefer for preparing the
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Norman L. Eberhardt, Endocrine Research Unit, 4407 Alfred, Mayo Clinic, Rochester, Minnesota 55905.
This work was supported by NIH Grants DK-41206 and DK-51492 (to
N.L.E.).
Received for publication November 12, 1996.
Revision received March 18, 1997.
Accepted for publication May 22, 1997.
 |
REFERENCES
|
---|
-
Miller WL, Eberhardt NL 1983 Structure and evolution of
the growth hormone gene family. Endocr Rev 4:97130[Medline]
-
Walker WH, Fitzpatrick SL, Barrera-Saldana HA,
Resendez-Perez D 1991 The human placental lactogen genes:
structure, function, evolution and transcriptional regulation. Endocr
Rev 12:316328[Abstract]
-
Walker WH, Fitzpatrick SL, Saunders GF 1990 Human placental
lactogen transcriptional enhancer. Tissue specificity and binding with
specific proteins. J Biol Chem 265:1294012948[Abstract/Free Full Text]
-
Fitzpatrick SL, Walker WH, Saunders GF 1990 DNA sequences
involved in the transcriptional activation of a human placental
lactogen gene. Mol Endocrinol 4:18151826[Abstract]
-
Jacquemin P, Oury C, Peers B, Morin A, Belayew A, Martial JA 1994 Characterization of a single strong tissue-specific enhancer
downstream from the three human genes encoding placental lactogen. Mol
Cell Biol 14:93103[Abstract]
-
Jiang SW, Eberhardt NL 1994 The human chorionic
somatomammotropin gene enhancer is composed of multiple DNA elements
that are homologous to several SV40 enhansons. J Biol Chem 269:1038410392[Abstract/Free Full Text]
-
Jiang SW, Eberhardt NL 1995 Involvement of a protein distinct
from transcription enhancer factor-1 (TEF-1) in mediating human
chorionic somatomammotropin gene enhancer function through the GT-IIC
enhanson in choriocarcinoma and COS cells. J Biol Chem 270:1390613915[Abstract/Free Full Text]
-
Nickel BE, Bock ME, Nachtigal MW, Cattini PA 1993 Differential expression of human placental growth hormone variant and
chorionic somatomammotropin genes in choriocarcinoma cells treated with
methotrexate. Mol Cell Endocrinol 91:159166[CrossRef][Medline]
-
Nickel BE, Cattini PA 1991 Tissue-specific expression and
thyroid hormone regulation of the endogenous placental growth hormone
variant and chorionic somatomammotropin genes in a human
choriocarcinoma cell line. Endocrinology 128:23532359[Abstract]
-
Lytras A, Cattini PA 1994 Human chorionic somatomammotropin
gene enhancer activity is dependent on the blockade of a repressor
mechanism. Mol Endocrinol 8:478489[Abstract]
-
Jiang SW, Eberhardt NL 1996 TEF-1 transrepression in BeWo
cells is mediated through interactions with the TATA-binding protein,
TBP. J Biol Chem 271:95109518[Abstract/Free Full Text]
-
Larkin SB, Farrance IK, Ordahl CP 1996 Flanking sequences
modulate the cell specificity of M-CAT elements. Mol Cell Biol 16:37423755[Abstract]
-
Kato S, Tora L, Yamauchi J, Masushige S, Bellard M, Chambon P 1992 A far upstream estrogen response element of the ovalbumin gene
contains several half-palindromic 5'-TGACC-3' motifs acting
synergistically. Cell 68:731742[Medline]
-
Kraus WL, Montano MM, Katzenellenbogen BS 1994 Identification
of multiple, widely spaced estrogen-responsive regions in the rat
progesterone receptor gene. Mol Endocrinol 8:952969[Abstract]
-
Scheuermann RH 1992 The tetrameric structure of NF-mu NR
provides a mechanism for cooperative binding to the immunoglobulin
heavy chain mu enhancer. J Biol Chem 267:624634[Abstract/Free Full Text]
-
Davidson I, Xiao JH, Rosales R, Staub A, Chambon P 1988 The
HeLa cell protein TEF-1 binds specifically and cooperatively to two
SV40 enhancer motifs of unrelated sequence. Cell 54:931942[Medline]
-
Xiao JH, Davidson I, Matthes H, Garnier JM, Chambon P 1991 Cloning, expression, and transcriptional properties of the human
enhancer factor TEF-1. Cell 65:551568[Medline]
-
Hwang JJ, Chambon P, Davidson I 1993 Characterization of the
transcription activation function and the DNA binding domain of
transcriptional enhancer factor-1. EMBO J 12:23372348[Abstract]
-
Laloux I, Jacobs E, Dubois E 1994 Involvement of SRE element
of Ty1 transposon in TEC1-dependent transcriptional activation. Nucleic
Acids Res 22:9991005[Abstract]
-
Andrianopoulos A, Timberlake WE 1994 The Aspergillus nidulans
abaA gene encodes a transcriptional activator that acts as a genetic
switch to control development. Mol Cell Biol 14:25032515[Abstract]
-
Campbell S, Inamdar M, Rodrigues V, Raghavan V, Palazzolo M,
Chovnick A 1992 The scalloped gene encodes a novel, evolutionarily
conserved transcription factor required for sensory organ
differentiation in Drosophila. Genes Dev 6:367379[Abstract]
-
Jacquemin P, Hwang JJ, Martial JA, Dolle P, Davidson I 1996 A
novel family of developmentally regulated mammalian transcription
factors containing the TEA/ATTS DNA binding domain. J Biol Chem 271:2177521785[Abstract/Free Full Text]
-
Stewart AF, Larkin SB, Farrance IK, Mar JH, Hall DE, Ordahl CP 1994 Muscle-enriched TEF-1 isoforms bind M-CAT elements from
muscle-specific promoters and differentially activate transcription.
J Biol Chem 269:31473150[Abstract/Free Full Text]
-
Azakie A, Larkin SB, Farrance IK, Grenningloh G, Ordahl CP 1996 DTEF-1, a novel member of the transcription enhancer factor-1
(TEF-1) multigene family. J Biol Chem 271:82608265[Abstract/Free Full Text]
-
Farrance IK, Mar JH, Ordahl CP 1992 M-CAT binding factor is
related to the SV40 enhancer binding factor, TEF-1. J Biol Chem 267:1723417240[Abstract/Free Full Text]
-
Farrance IK, Ordahl CP 1996 The role of transcription enhancer
factor-1 (TEF-1) related proteins in the formation of M-CAT binding
complexes in muscle and non-muscle tissues. J Biol Chem 271:82668274[Abstract/Free Full Text]
-
Ishiji T, Lace MJ, Parkkinen S, Anderson RD, Haugen TH, Cripe
TP, Davidson I, Chambon P, Turek LP 1992 Transcriptional enhancer
factor (TEF)-1 and its cell-specific co-activator activate human
papillomavirus-16 E6 and E7 oncogene transcription in keratinocytes and
cervical carcinoma cells. EMBO J 11:22712281[Abstract]
-
Burglin TR 1991 The TEA domain: a novel, highly conserved
DNA-binding motif [letter]. Cell 66:1112[Medline]
-
Maxwell IH, Harrison GS, Wood WM, Maxwell F 1989 A DNA
cassette containing a trimerized SV40 polyadenylation signal which
efficiently blocks spurious plasmid-initiated transcription.
Biotechniques 7:276280[Medline]
-
Jiang SW, Trujillo MA, Eberhardt NL 1997 The placental human
chorionic somatomammotropin enhancers form a composite silencer in
pituitary cells in vitro. Mol Endocrinol 11:12331244[Abstract/Free Full Text]
-
Jiang SW, Trujillo MA, Eberhardt NL 1996 An efficient method
for generation and subcloning of tandemly repeated DNA sequences with
defined length, orientation and spacing. Nucleic Acids Res 24:32783279[Abstract/Free Full Text]
-
Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res 11:14751489[Abstract]
-
Jiang SW, Shepard AR, Eberhardt NL 1995 An initiator element
is required for maximal human chorionic somatomammotropin gene promoter
and enhancer function. J Biol Chem 270:36833692[Abstract/Free Full Text]