The Human Chorionic Somatomammotropin Enhancers Form a Composite Silencer in Pituitary Cells in Vitro
Shi-Wen Jiang and
Norman L. Eberhardt
Endocrine Research Unit Departments of Medicine and
Biochemistry/Molecular Biology Mayo Clinic, Rochester, Minnesota
55905
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ABSTRACT
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The human GH (GH) gene family includes
the pituitary-specific hGH-1, placental-specific chorionic
somatomammotropin (hCS-5, hCS-2, and
hCS-1), and hGH-2 genes. These duplicated,
nearly identical genes are localized on approximately 50 kb of DNA on
chromosome 17q23-q24.
An enhancer (CSEn2), located downstream of the hCS-2 gene,
participates in mediating placental-specific hCS gene
expression. In the preceding paper we demonstrated that CSEn2 activity
derives from the cooperative binding of transcription factor-1, TEF-1,
and a placental-specific factor CSEF-1 to multiple enhansons,
Enh1-Enh5, that are related to the SV40 GT-IIC and SphI/SphII
enhansons. Here we demonstrate that two copies of CSEn2 or a single
copy of CSEn2 linked to either of the other two enhancers in the
hGH/hCS locus, CSEn1 and CSEn5, act
cooperatively to enhance hCS promoter activity in
choriocarcinoma (BeWo) cells, but silence the promoter in pituitary GC
cells. Mutation of Enh4, an essential GT-IIC-like enhanson in the
context of the intact enhancer, abolishes silencer activity, and
multimerized GT-IIC enhansons mimic the intact CSEn enhancer/silencer
activities in BeWo and GC cells, respectively. By antibody-mediated
supershift, Western, and far Western analyses, we identified TEF-1 as
the GT-IIC-binding factor in pituitary cells. The data suggest that
TEF-1 may be involved in pituitary-specific repression of placental
GH/CS gene transcription through long-range interactions
between the multiple CS enhancers present on the
GH/CS gene locus.
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INTRODUCTION
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Human chorionic somatomammotropin (hCS) is a member of
the GH gene family (1, 2). The gene locus contains five
genes, including the pituitary hGH-1 and the placental
hGH variant, hGH-2, and three hCS
genes, hCS-5, hCS-1, and hCS-2 (see
Fig. 1A
). Although the multiple hCS and
hGH genes share approximately 9396% sequence identity,
their differential cell-specific expression indicates that these genes
are regulated by unique mechanisms. The mechanism for regulating
pituitary-specific expression of the hGH gene by the
POU-homeodomain containing transcription factor GHF1/Pit1 has been well
established (3). A central enigma associated with this expression
pattern is the fact that the GHF1/Pit1-binding sites in the
hCS promoters are conserved. Morever, these promoters are as
active as the hGH promoter in pituitary GC cells and
function via a GHF1/Pit1-dependent mechanism (4), indicating that
unique repressor mechanisms must operate to suppress hCS
gene expression in the pituitary. Evidence for such repressor
mechanisms has been provided by Nachtigal et al. (5), who
showed that unique elements associated with hCS genes can
repress their expression. Recent studies of the control of
hCS placental-specific expression have focused on the
characterization of an enhancer, CSEn2 (6, 7, 8, 9, 10), which is localized
downstream of the hCS-2 gene at the 3'-end of the
hGH/hCS gene locus (11). In the preceding paper,
we demonstrated that CSEn2 is composed of multiple enhansons,
Enh1-Enh5, which are homologous to the GT-IIC, and SphI
enhansons that comprise the Simian Virus 40 enhancer (SVEn)(12). The
studies also demonstrated that CSEn activity is dependent on the
cooperative binding of transcription factor-1 (TEF-1) and a
placental-specific factor, CSEF-1, to Enh1-Enh5 (12). In the current
paper we demonstrate that CSEn2 in conjunction with either of two other
homologous enhancers present on the hGH/hCS
locus, CSEn1 or CSEn5, form a composite silencer in pituitary cells
(see Fig. 1
).

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Figure 1. Schematic Diagram of the
hGH/hCS Chromosomal Locus (A) and
Nucleotide Sequence Comparison of the Three CS
Enhancers, CSEn1, CSEn2, and CSEn5 (B)
The localization of the individual hGH and
hCS genes and the three copies of the CS
enhancer are depicted. The enhancer sequence comparison (GCG PILEUP,
Genetics Computer Group, Madison, WI) also shows the locations of the
DNaseI-resistant footprints (FP1-FP5, open boxes), the
location and orientation (arrows) of the GT-IIC-related
enhansons (Enh1 and Enh4), SphI/SphII-related enhansons (Enh2, Enh3 and
Enh5), and direct repeat structures (DR) as defined in previous studies
(6 12 ), and the structures and location of the block mutants (EM1-EM8)
that were each constructed in the context of an otherwise intact
enhancer (6 ).
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CSEn2 function was originally thought to be mediated by transcription
enhancing factor-1 (TEF-1), a transactivator involved in SVEn function.
Transactivation by TEF-1 was proposed to require a limiting coactivator
because its overexpression led to squelching in a number of systems (7, 13, 14, 15). Nevertheless, in BeWo cells strong evidence that TEF-1 was not
responsible for CSEn transactivation was obtained (7). First,
transactivation was correlated with the presence of a 30-kDa protein,
designated CSEF-1, that binds to the GT-IIC enhanson and competes for
TEF-1 binding to that DNA element. Second, CSEn was found to be very
active in COS-1 cells, which express high levels of CSEF-1 and very low
levels of TEF-1. Third, down-regulation of TEF-1 levels by antisense
oligonucleotides led to an increase of CSEn activity in BeWo cells.
Finally, TEF-1 inhibited hCS promoter activity in BeWo cells
independently of the GT-IIC binding site, suggesting that it interacted
with components of the basal transcription apparatus. Subsequently, we
demonstrated that TEF-1 specifically interacts with the TATA box
binding protein, TBP, and that it inhibits TBP binding to the TATA box
(8). Overexpression of TBP in BeWo cells alleviated the squelching
effect of TEF-1, suggesting that in BeWo cells TEF-1 function is
limited to transrepression (8).
In the current study we provide additional evidence that TEF-1 plays a
major role in transrepression and that it may function as part of a
molecular enhancer/silencer switch. We discovered that multiple copies
of CSEn2 acted cooperatively in BeWo cells to synergistically activate
the hCS promoter, but repressed the activity of the same
expression construct in pituitary GC cells. The DNA element mediating
GC cell-specific repression was localized to the GT-IIC motif by
mutation analysis and confirmed by demonstrating enhancer/silencer
function of constructs containing multiple GT-IIC motifs. A single
protein-GT-IIC complex was detected in GC cells, and TEF-1 was
identified as the GT-IIC binding factor by gel supershift and Western
blot analyses using TEF-1 antibodies. The CSEn-mediated transrepression
did not depend on specific hCS promoter elements and may be
mediated by direct interaction with basal transcription factors,
consistent with the TEF-1-TBP interaction identified in BeWo cells (8).
Interestingly, combinations of CSEn2 and the additional copies of the
CSEn1 or CSEn5, which are associated with the placental
GH/CS gene locus, acted cooperatively to repress
hCS promoter activity in GC cells, suggesting that silencing
might be an in vivo function of the multiple enhancers on
the GH/CS locus. Accordingly, CSEn
pituitary-specific silencing functions may participate with the
pituitary-specific repressor elements (PSF) associated with the
placental hGH/hCS members (5) to ensure absence
of hCS promoter activity in the pituitary.
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RESULTS
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Multiple CSEn and SVEn Repress hCS Promoter Activity in Pituitary
Cells
In previous studies we and others established that a single copy
of CSEn was able to stimulate the hCS promoter in BeWo
cells, but lacked any activity in pituitary GC cells or HeLa cells (6, 9). Curiously, in footprinting studies, the identical protection
patterns were observed in both BeWo, HeLa, and GC cells (see Fig. 1B
for footprint locations and associated enhansons). Mutation of these
protected regions resulted in loss of enhancer activities in BeWo cells
(6), and we demonstrated that these regions contain multiple enhansons
that cooperatively bind TEF-1 and CSEF-1 (12). The presence of
identical footprinting patterns over functional enhancer regions raised
the question of why CSEn lacked activity in HeLa and GC cells. We had
previously shown that multiple copies of CSEn2 acted synergistically to
stimulate hCS promoter activity in BeWo cells (6) and COS-1
cells (7). We therefore decided to reassess the activities of promoter
constructs containing one and two copies of CSEn2 in COS-1, HeLa, and
GC cells (Fig. 2
).

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Figure 2. Multiple Copies of the CSEn2 and SV40 Enhancers
Exhibit Selective Repressor Activity in Rat Pituitary GC Cells
GC (A), BeWo (B), COS-1 (C), and HeLa (D) cells were transfected with
15 µg of the designated plasmids, CSp.LUC,
CSEn2 CSp.LUC,
(CSEn2)2_CSp.LUC,
SVEn CSp.LUC,
(SVEn)2 CSp.LUC,
CMVEn CSp.LUC, and
(CMVEn)2 CSp.LUC as
described in Materials and Methods. Luciferase reporter
activity was normalized to protein concentration and expressed as light
units/µg protein ± SE. ANOVA analyses for each of
the experiments A-D were significant (P < 0.0001).
Individual comparisons were made by posthoc Dunnetts
t tests (P < 0.05) for values less
than the mean (§) or greater than the mean (¶) of the control,
CSp.LUC.
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Confirming previous results (6, 7), CSEn2 produced strong synergistic
stimulation in BeWo and COS-1 cells. Remarkably, when the same set of
plasmids were introduced into pituitary GC cells, the constructs
containing two copies of CSEn2 were repressed significantly (Fig. 2A
).
To exclude any possibility that this represents a nonspecific
phenomenon resulting from insertional alteration of the plasmid
structure, we subcloned two copies of the cytomegalovirus (CMV)
enhancer and SV40 enhancer at the same positions as the CSEn-containing
constructs. Unlike CSEn, one or two copies of CMVEn produced strong and
cooperative stimulation of hCS promoter in GC cells (Fig. 2A
), indicating that some sequences specific to CSEn were involved in
repression. Interestingly, like CSEn, two copies of the SV40 enhancer
significantly repressed hCS promoter activity in GC cells
(Fig. 2A
). These data indicate that CSEn and SVEn are functionally
related and suggest that some common feature accounts for this
pituitary cell-specific negative regulation.
CSEn Repressor Action Is Mediated through GT-IIC Enhansons
As the first step to understand the mechanism of the negative
regulation, we determined which CSEn DNA motifs were required for this
silencing function. Site-specific CSEn mutants of the individual
enhansons in the context of the intact enhancer (EM1-EM8, see Fig. 1B
)
were subcloned at BglII and PstI sites of
hCSp.LUC to generate plasmids containing two copies of the
individual mutants. These constructs were then compared with wild type
CSEn in transfected BeWo and GC cells (Fig. 3
).
Consistent with our previous results obtained with a single copy of
CSEn mutants (6), mutations EM3, EM4, EM5, EM6, and EM7 cause
significant reduction in enhancer activity in BeWo cells. However, the
previously observed loss of enhancer activity with single copies of EM1
and EM8 are largely restored after duplication, a phenomenon observed
previously with SV40 enhancers (16, 17). In GC cells, while duplicate
copies of the wild type CSEn mediate 3- to 4-fold repression,
significant derepression is observed only with the duplicate EM5 mutant
(Fig. 3A
). In contrast, mutations of several of the enhansons
significantly affect enhancer function in placental cells (Fig. 3B
).
These results indicate that Enh4, the GT-IIC-related enhanson (Fig. 1B
), is the only essential element for silencer function in pituitary
cells. The data suggest that GT-IIC binding factors present in GC cells
are crucial for silencer function.

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Figure 3. The GT-IIC Enhanson of CSEn2 Is Essential for
Pituitary Silencer and Placental Enhancer Activity
GC (A) and BeWo (B) cells were transfected with 15 µg of the
designated plasmids, CSp.LUC,
(EM1)2 CSp.LUC through
(EM8)2 CSp.LUC, which
contain block mutations in individual CSEn enhansons (6 ) as described
in Materials and Methods. The differences in luciferase
reporter activities (light units/µg protein ± SE)
were significant by ANOVA analyses (P < 0.0001 for
data in A and B). Individual comparisons were made by
posthoc Dunnetts t tests
(P < 0.05) for values less than the mean (§) of
the control, CSp.LUC (A), or
(CSEn2)2_CSp.LUC
(B).
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Since EM5 specifically disrupts Enh4, the highest affinity binding site
for TEF-1 (12), we tested whether multiple copies of this
GT-IIC-like enhanson exhibited independent repressor activity. The
artificial enhancers containing GT-IIC 12-mers and 16-mers stimulate
hCS promoter activity in BeWo cells (Fig. 4B
). The GT-IIC 8-mer stimulated hCS promoter
activity 2.7-fold in BeWo cells, a statistically insignificant effect.
In GC cells, the GT-IIC 8-mer repressed hCS promoter
activity as much as the 12-mers and 16-mers in GC cells (Fig. 4A
). Thus
the multimerized GT-IIC enhansons are efficient at mimicking silencer
activity in GC cells, indicating that this enhanson is an essential
silencer element. These experiments indicate that the isolated GT-IIC
enhansons can independently mediate both enhancer and silencer
activity, suggesting that GT-IIC binding factors may be responsible
for the switch mechanism between placental and pituitary cells.

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Figure 4. Multiple Copies of the GT-IIC Enhansons Are
Sufficient to Generate Pituitary Silencer and Placental Enhancer
Activity
GC (A) and BeWo (B) cells were transfected with 15 µg of the
designated plasmids, CSp.LUC,
(GT-IIC)8 CSp.LUC,
(GT-IIC)12 CSp.LUC,
and (GT-IIC)16 CSp.LUC
as described in Materials and Methods. Basal expression
of the CSp.LUC gene was 5.9 ± 0.3 (SE)
x 104 and 18.1 ± 3.1 (SE) x
104 light units/µg protein in GC and BeWo cells,
respectively. Differences in luciferase reporter activities (light
units/µg protein ± SE) were significant by ANOVA
analyses (P < 0.0001 for data in A and B).
Individual comparisons were made by posthoc Dunnetts
t tests (P < 0.05) for values less
than the mean (§) or greater than the mean (¶) of the control,
CSp.LUC.
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Specific hCS Promoter Elements Are Not Required for
Enhancer-Silencer Switching
Functional elements mediating hCS promoter
activity in GC and BeWo cells have been well established in previous
studies. In transfected GC cells the GHF1, Sp1, and TATA box elements
are important for optimal hCS promoter activity (4, 18).
Despite the conservation of the pituitary-specific GHF1 sites, the
hCS genes are not expressed in the pituitary, indicating
that negative regulatory mechanisms suppress their expression.
Accordingly, it is possible that CSEn silencer activity in pituitary
cells functions through specific CS promoter elements.
Consequently, mutated hCS promoters were coupled to
luciferase expression vectors containing two copies of CSEn, and their
functional activities were examined in transfected BeWo and GC cells.
Like the results with a single copy of CSEn in BeWo cells (18), the
proximal, distal, or both GHF1 mutations had no impact on either basal
(data not shown) or CSEn-stimulated hCS promoter activity
(Fig. 5B
). In contrast, mutation of the TATA and Sp1
elements significantly reduced basal (data not shown) and
CSEn-stimulated activity (Fig. 5B
). In GC cells, mutations of the
proximal, distal, or both GHF1 sites caused a 2- to 3-fold reduction in
promoter basal activity (data not shown), but had no effect on
CSEn-mediated silencer activity (Fig. 5A
). Mutation of the TATA and Sp1
elements also decreased the basal promoter activity 3- and 8-fold,
respectively (data not shown). While CSEn repressor activity was not
affected by the TATA box mutation, it was significantly increased with
the Sp1 site mutation (Fig. 5A
). These data indicate that Sp1, while
not an essential element, exerts a reciprocal modulatory effect on
enhancer/silencer function, serving to increase enhancer activity in
placental cells and diminish silencer activity in pituitary cells.

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Figure 5. Sp1 Elements Present on the CS
Promoter Contribute to CSEn2 Cooperative Interactions that Result in
Synergistic Enhancer Action, but Antagonize the Silencer Activity
GC (A) and BeWo (B) cells were transfected with 15 µg of the
designated plasmids, CSp.LUC, pGHF1p.LUC,
dGHF1p.LUC, pdGHF1p.LUC,
SP1p.LUC, and TATAp.LUC and their cognate
constructs that contain two copies of CSEn2 as described in
Materials and Methods. The measured light units for the
least active (EN2)2_Sp1p.LUC gene in GC cells was 1929 ± 267 (SE)
light units with a signal-noise ratio of 11.0 ± 1.5
(SE), a value within the linear range of the instrument as
determined by limiting dilutions of partially purified luciferase
(2505 x 106 light units). Basal expression of the
CSp.LUC gene was 7.6 ± 0.7 (SE) x
104 and 11.5 ± 1.5 (SE) x
104 light units/µg protein in GC and BeWo cells,
respectively. The fold differences in luciferase reporter activities
(light units/µg protein ± SE) for each cognate pair
were calculated and the differences were significant by ANOVA analyses
(P < 0.0001 and < 0.0006 for GC (A) and BeWo
(B) cell data, respectively). Individual comparisons were made by
posthoc Dunnetts t tests
(P < 0.05) for values less than themean (§) or
greater than the mean (¶) of the control,
(En2)2 CSp.LUC.
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CSEn1 and CSEn5 Cooperate with CSEn2 to Generate Both Enhancer and
Silencer Function
The human hGH/hCS locus contains two other
copies of CSEn, designated CSEn1 and CSEn5, that are located downstream
of the hCS-1 and hCS-5 genes, respectively (Fig. 1A
). The CSEn1 and CSEn5 enhancers contain substitutions within or near
individual enhansons that have been reported to render these enhancers
nonfunctional (see Fig. 1A
)(9). Nevertheless, since these enhancers are
composed of multiple elements, it is possible that these duplicated
sequences could interact with each other. To study the potential
cooperativity among these native enhancers, single copies of CSEn1 and
CSEn5 were inserted into expression vectors containing either one or no
copy of CSEn2. In BeWo cells, CSEn2, CSEn1, and CSEn5 alone stimulate
hCS promoter activity by 8-, 5-, and 3-fold, respectively.
All of these effects were statistically significant compared with the
activity of the hCSp.LUC gene (multivariate ANOVA
P < 0.0001; posthoc Dunnetts t
test P < 0.05), suggesting they are competent
enhancers, albeit less effective than CSEn2 (Fig. 6B
, compare with CSEn2 CSp.LUC in Fig. 1
) This is
contrary to the results of Jacquemin et al. (9) that
indicated that CSEn1 and CSEn5 lack enhancer activity. This discrepancy
may be due to the different sensitivity of the chloramphenical acetyl
transferase and luciferase reporter systems used in the two studies.
Importantly, constructs containing the multiple enhancer combinations,
CSEn1-CSEn2 and CSEn5-CSEn2, produced strong cooperative stimulatory
activity (
15-fold) in BeWo cells. In fact, constructs containing
CSEn1-CSEn2 or CSEn5-CSEn2 were nearly as strong as constructs
containing two copies of CSEn2 (Fig. 6B
). These results point to the
possibility that the multiple CSEn enhancers present on the
hGH/hCS locus might be able to interact in
vivo to synergistically stimulate placental hCS gene
expression.

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Figure 6. The Additional Enhancers, CSEn5 and CSEn1, Present
on the hGH/hCS Genomic Locus Can Participate in
Cooperative Enhancer and Silencer Function with CSEn2
GC (A) and BeWo (B) cells were transfected with 15 µg of the
designated plasmids,
(CSEn2)2_CSp.LUC,
CSEn1 CSp.LUC, CSEn5 CSp.LUC,
CSEn1-CSEn2 CSp.LUC, and
CSEn5-CSEn2 CSp.LUC as described in
Materials and Methods. Basal expression of the
CSp.LUC gene was 4.5 ± 0.8 (SE) x
104 and 14.9 ± 2.0 (SE) x
104 light units/µg protein in GC and BeWo cells,
respectively. Differences in luciferase reporter activities (light
units/µg protein ± SE) were significant by ANOVA
analyses (P < 0.0001 for data in A and B). Individual comparisons
were made by posthoc Dunnetts t tests
(P < 0.05) for values less than the mean (§) of
the control,
(En2)2 CSp.LUC.
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In GC cells, like CSEn2, neither a single copy of CSEn1 or CSEn5
exhibited significant repressor activity on its own (Fig. 6A
, compare
CSEn2 CSp.LUC activity in Fig. 1A
;
CSEn1 CSp.LUC and CSEn5 CSp.LUC
activities not significantly different than hCSp.LUC, ANOVA
P > 0.05). However, when combined with an additional
copy of CSEn2, both CSEn1 and CSEn5 exhibited strong cooperative
repressor function. Indeed, the
CSEn1-CSEn2 CSp.LUC and
CSEn5-CSEn2 CSp.LUC genes were about as active as the
(CSEn2)2 CSp.LUC gene and were
not statistically different from two copies of CSEn2 (Fig. 6A
). These
results are consistent with the observation that in CSEn1 and CSEn5 the
essential GT-IIC motif required for silencer function (Fig. 3A
) is
perfectly conserved (Fig. 1B
). These data suggest that CSEn1, CSEn2,
and CSEn5 comprise a composite placental-specific enhancer and
pituitary-specific silencer whose functions are mediated by cooperative
interactions.
GC Cell TEF-1 Binds the CSEn GT-IIC Enhanson
As demonstrated above, the GT-IIC enhanson was essential for
CSEn2-mediated silencer activity in GC cells (Fig. 3A
), suggesting that
TEF-1 binding to this enhanson might mediate repressor activity. In gel
shift experiments using GC cell nuclear extracts, a single complex was
detected with GT-IICSV and GT-IICCS
oligonucleotides (Fig. 7
), and this complex comigrated
with complexes formed by BeWo and HeLa nuclear extracts that were
previously shown to contain TEF-1 (7). Since the GT-IICS
and GT-IICSV probes contain divergent flanking sequences,
the factor must recognize the TGGAATG core sequences present in both of
these enhansons. This view is supported by the fact that mutation of
the TGTGGAATGTGT sequences blocks formation of this complex (Fig. 7
).
Also, the complex is competed away by an 80-fold excess of unlabeled
GT-IICSV oligonucleotide and to a lesser extent by a
320-fold excess of an SphISV oligonucleotide, but not a
320-fold excess of the mutated GT-IICSV oligonucleotide
(data not shown). Since the complex was competed by SphISV
oligonucleotide, albeit with relatively lower affinity, the GC factor
also recognizes SphI sequences, a property that is shared by TEF-1
(19).

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Figure 7. GC cell TEF-1 Is the Major Factor Binding to the
GT-IIC Enhanson
GC cell nuclear proteins (15 µg) that interacted with the GT-IIC
enhanson were analyzed by gel shift analysis and compared with the same
amount of protein from BeWo, COS-1, and HeLa nuclear extracts in the
gel shift assay as described in Materials and Methods.
Probe GT-IICSVMUT contains mutations in the core consensus,
but identical flanking sequences as the wild type GT-IICSV
probe (7 ). Specific TEF-1 and CSEF-1 (COS-1 and BeWo cells) complexes
are marked.
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To establish that TEF-1 was contained in the GT-IIC complex from GC
cells, the complex was further characterized using a TEF-1-specific
antibody. In gel shift experiments, addition of the antibody generated
a new complex with slower migration and a correspondingly decreased
density of the original complex (Fig. 8A
). No supershift
band was observed in either nonimmunized serum or nonextract controls.
Presence of TEF-1 in GC cells was established by Western blot analysis
which showed the presence of a 53 kDa protein that reacted with the
antibody (Fig. 8B
) and by Northern blot analysis with the TEF-1 cDNA
probe demonstrating the characteristic 12- and 3.5-kb TEF-1 transcripts
(data not shown). Also, mild heat treatment (55 C for 2 min) of GC
nuclear extracts abolished specific complex formation (data not shown),
another property characteristic of TEF-1 (7). To further establish that
TEF-1 was present in the complex, the protein-DNA complex was isolated
from a gel shift experiment (Fig. 8C
) and transferred onto
nitrocellulose membrane, and far Western analysis using the TEF-1
antibody indicated that a band at the same position as TEF-1-GT-IIC
complex was visualized in the GT-IIC probe lane, but not the GT-IIC
mutant lane (Fig. 8D
). Taken together, these results confirm that TEF-1
is the major factor present in GC cells that binds to the GT-IIC
enhanson. These data provide strong support for the concept that TEF-1
may be involved in mediating the CSEn repressor function in pituitary
cells.

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Figure 8. TEF-1 Is Expressed in GC Cells and Specifically
Binds to the GT-IIC Enhanson
A, Gel supershift experiment with wild type GT-IICSV (W)
and mutant GT-IICSVMUT (M) enhanson probes and 7.0 µg GC
cell nuclear extract. Different amounts of the TEF-1 antibody were
included in each binding reaction along with a nonimmune serum control
(NI). TEF-1- and TEF-1-antibody-GT-IICSV complexes are
marked. B, Western blot analysis. GC cell nuclear extract (70 µg) was
resolved by SDS-PAGE, transferred to nitrocellulose, and detected by
TEF-1 antibody as indicated in Materials and Methods. C
and D, Far Western blot analysis. The protein-GT-IIC complex from a
large scale gel shift experiment (C) was excised, transferred to
nitrocellulose, and incubated with TEF-1 antibody as described in
Materials and Methods (D). A positively reacting protein
was detected by the antibody in the complex containing the wild type
GT-IICSV (W), but not the mutant GT-IICSVMUT
(M) probe.
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DISCUSSION
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In previous studies we demonstrated that the CS
enhancer, CSEn2, was composed of multiple enhansons that were related
to those comprising the SV40 enhancer (6, 12). The finding of SVEn
GT-IIC enhanson-like sequences in CSEn2 suggested that TEF-1 might be
involved in its function. We and others subsequently provided evidence
that a protein distinct from TEF-1, CSEF-1, or "f" bound to the
GT-IIC enhanson and was responsible for CSEn2 transactivation in BeWo
(6, 9) and COS-1 cells (6). Since TEF-1 has been proposed to be a
principal transactivator for the SV40 enhancer and it is ubiquitously
expressed, we could not understand why CSEn2 lacked significant
enhancer activity in certain cell types, including pituitary GC cells
and HeLa cells. Since we had demonstrated that multiple copies of CSEn2
produced a synergistic response in BeWo (6) and COS-1 cells (7), we
reasoned that reexamination of these reporter constructs with their
increased sensitivity might reveal additional information about
TEF-1-mediated enhancer function.
Surprisingly, we discovered that multiple copies of CSEn2 produced a
significant silencing activity in GC cells (Fig. 2
). The observed
repression is not caused by insertional alteration of the reporter gene
structure because subcloning of two copies of CMVEn at the same
positions resulted in high level stimulation of hCS promoter
activity. In addition, multiple copies of the SV40 enhancer exhibited
similar repressor activity on the hCS promoter (Fig. 2
),
suggesting that the repressor activity was caused by a structural
feature common to these two related enhancers. CSEn mutation analysis
indicated that the GT-IIC enhansons were essential for repressor
activity, a finding confirmed by demonstrating that multiple GT-IIC
enhansons also exhibited silencer activity (Figs. 3
and 4
). A single
GT-IIC complex with mobility identical to that produced by authentic
TEF-1 was detected from GC cell nuclear extracts. Gel supershift,
Western, and far Western blot analyses of this complex using TEF-1
antibodies confirmed that TEF-1 from GC cells specifically bound to the
GT-IIC motif of CSEn (Figs. 7
and 8
). These results, together with our
earlier observation that TEF-1 negatively regulates CSEn activity in
BeWo cells (7, 8), support the concept that TEF-1 may be a dominant
transrepressor in selected cell types.
TEF-1 was identified initially as a transactivator, by virtue of its
binding to the essential GT-IIC and SphI enhansons for the SV40
enhancer and its ability to activate in vitro transcription
(19). Nevertheless, overexpression of TEF-1 in transfected cells
negatively regulated wild type SV40 and artificial GT-IIC-containing
enhancers, a phenomenon attributed to squelching of a specific cofactor
(13, 14, 15). We observed that overexpression of TEF-1 in BeWo cells only
led to decreased CSEn2 enhancer activity while down-regulation of TEF-1
levels resulted in increased enhancer activity, suggesting that TEF-1
was a transrepressor in BeWo cells (7). Consistent with these
observations, Takahashi et al. (20) reported that
overexpression of TEF-1 in human SV40 transformed keratinocytes
repressed involucrin gene expression. Immunohistochemistry demonstrated
high levels of TEF-1 in basal cells, which do not express involucrin,
whereas in the suprabasal keratinocytes, TEF-1 expression is repressed
and involucrin expression is abundant.
In follow-up studies, we demonstrated that TEF-1 interacted with the
TATA box binding protein, TBP, and this interaction interfered with
in vitro TBP binding to the TATA element (8). Three lines of
evidence supported the concept that TBP is involved in TEF-1-mediated
transrepression in vivo. First, the TEF-1 proline-rich and
zinc finger domains that were shown to be involved in TEF-1 squelching
effects (15) were required for TBP binding. Second, overexpression of
TBP alleviated the TEF-1-mediated squelching in transfected BeWo cells.
Finally, the TATA and initiator (InrE) elements (18) play an essential
role in mediating TEF-1 function (8). Although Sp1 is clearly capable
of augmenting enhancer (18, 21) (Fig. 4
) and antagonizing silencer
function (Fig. 4
), its absence does not abrogate enhancer function.
Several factors, including p53 (22, 23), E1A (24), Dr1 (25),
even-skipped (26, 27) and Tax1 (28), have been found to exert their
negative function through interaction with TBP, suggesting that TBP may
be a common target for negative regulation. It is not clear how the
same set of factors can also serve as transactivators in certain cells.
It is possible that participation of additional factor(s), including
cell-specific TAFs or other coactivators, may change their mode of
interaction with TBP.
Since both CSEn and SVEn contain multiple GT-IIC and SphI motifs, it is
perhaps not surprising to observe a similar pattern of function of
these two enhancers in different cells, including the silencer activity
in GC cells (Fig. 1
). Several previous studies have implicated the
GT-IIC enhanson in negative regulation in plants, yeast, and mammalian
systems, suggesting that this might represent a primordial regulatory
mechanism. Evidence for an activator-repressor switch mechanism
mediated by GT-IIC-related sequences emerged from studies of the small
subunit of pea ribulose-1,5-bisphosphate carboxylase gene expression in
transgenic tobacco plants (29). This gene contains a light-inducible
transcriptional enhancer with four SV40 core enhansons that represses
transcription in the dark. The SV40 core enhancer also functions as a
repressor in yeast. Zhang-Keck et al. (30) demonstrated that
when positioned adjacent to a yeast upstream activator sequence), the
SV40 core sequence inhibited CYC1 promoter activity.
Southwestern analysis revealed a protein of 68 kDa that bound to the
SV40 core enhanson; however, the identity of this factor(s) or its
relationship to TEF-1, if any, has not been established. Interestingly,
SVEn repressor activity has been observed previously in pituitary
cells. Dana and Karin (31) used SV40 promoters as controls in early
studies of cAMP regulation of the hGH gene and observed that
reporter constructs containing SVEn were expressed with 2030 fold
lower activity than constructs containing only the SV40 promoter. In
our current study, the factor involved in GT-IIC-mediated repression
was identified, and the data provide a possible physiological role for
this negative regulatory pathway.
We propose that the multiple CS enhancers act collectively
as a switchable enhancer/silencer in placental syncytiotrophoblast and
pituitary, respectively. The molecular control of the enhancer/silencer
switch is due to factors that bind to the essential GT-IIC enhanson,
since it is required for both enhancer and silencer activity (Fig. 3
).
The simplest model would be that TEF-1 and CSEF-1 bind to the GT-IIC
enhanson in a mutually exclusive manner and the overall CSEn activity
would be determined by competition between these factors. In placental
and COS-1 cells, which contain relatively low levels of TEF-1, the
GT-IIC enhansons will be dominantly occupied by CSEF-1, resulting in
strong CSEn stimulation. In GC cells, which contain abundant TEF-1, but
no detectable CSEF-1 binding activity, CSEn is switched to a repressor.
This model assumes that TEF-1 acts dominantly as a transrepressor;
however, it cannot be excluded that TEF-1 functions as a transactivator
via the interaction with other coactivators in certain cell types. For
example, this mechanism might explain why CSEn and SVEn act positively,
albeit weakly, in HeLa cells (Fig. 2
), which appear to lack or contain
very low CSEF-1-like binding activity (Fig. 7
).
Clearly, other factors may be involved in controlling GT-IIC-containing
enhancers. Recently, additional GT-IIC binding factors that are
distinct from TEF-1, but share the TEA/ATTS DNA binding domain, have
been identified from different tissues (32). Unlike TEF-1, which
appears to be expressed ubiquitously, these factors are expressed in
restricted tissues and are involved in control of a variety of genes.
GT-IIC binding factors distinct from TEF-1 participate in activation of
muscle-specific genes through binding to the M-CAT motif, a GT-IIC
variant found in myosin heavy chain genes (33, 34, 35). Inamdar et
al. (36) found that a Drosophila factor,
sd, is essential for taste development. Yasunami et
al. (37) cloned several embryonic factor cDNAs from neural
precursor cells. Preliminary studies suggest that the expression of
these genes is restricted to certain stages of brain development.
Therefore, it is probable that multiple GT-IIC binding factors exist in
different tissues, although their transactivation and/or
transrepression functions remain to be determined.
Our results have important implications for the tissue-specific control
of the linked, nearly identical hGH and hCS
genes, which has long been considered enigmatic. The hCS
promoter retains the two essential GHF-1 elements, required for
pituitary-specific hGH expression. Indeed, these sites
mediate high level hCS promoter activity in transfected GC
cells (4). Therefore why arent the closely linked hCS
genes expressed in the pituitary? Nachtigal et al. (5) have
found that PSF elements associated with the placental members of
hGH/hCS gene family can repress hCS
promoter activity in transfected pituitary cells. Two proteins, PSF-A
and PSF-B, present in pituitary but not in placental nuclear extracts,
were found to bind to the PSF elements. Thus it is possible that
cooperative interactions between CSEn1, CSEn2, and CSEn5 operating in
conjunction with the PSF-A and PSF-B repressors could ensure the
absence of hCS gene expression in the pituitary.
While previous studies have shown that CSEn1 and CSEn5 exhibit minor
activity in placental cells and may not play significant roles in
hCS expression, our findings suggest this may not be the
case. Although single copies of CSEn1 and CSEn5 are weak enhancers in
choriocarcinoma cells (Fig. 6
), in cooperation with CSEn2, they can
work almost as efficiently as CSEn2. This is especially true in terms
of their repressor activity in GC cells. Neither CSEn1, CSEn2, or CSEn5
exhibit silencer activity on their own, but in combination they
cooperate to repress hCS promoter activity (Fig. 6
).
Clearly, for such a mechanism to operate in vivo, long
distance interactions between the CSEn5 [locus position: 16,689 bp
(Genebank Accession No. J03071)], CSEn1 (locus position: 31,422 bp)
and CSEn2 (locus position: 54, 237 bp) would be required. Comparing
DNAse I sensitivity of the hGH-1 and hCS genes,
Nickel and Cattini (38) have shown that all the genes on the
hGH/hCS locus are maintained in a relatively open
configuration in pituitary tissue. Thus such long range interactions
among the multiple CS enhancer/silencers in pituitary are
not precluded. Further studies will be required to establish the
physiological significance of this mechanism.
 |
MATERIALS AND METHODS
|
---|
Cell Transfection and Luciferase Assays
BeWo cells were maintained in RPMI 1640 (GIBCO, Grand Island,
NY). Rat anterior pituitary tumor (GC), COS-1 (ATCC, Rockville, MD) and
HeLa (S3, ATCC) cells were maintained in DMEM (GIBCO). All media were
supplemented with 10% FBS (BioWhittaker, Walkersville, MD), 100 U/ml
penicillin (GIBCO), 100 µg/ml streptomycin (GIBCO), and 2
mM L-glutamine (GIBCO). Cells were grown at 37 C in an
atmosphere containing 5% CO2 and 100% humidity. Cell
transfections were performed by electroporation at optimal capacitance
and voltage using 15 µg double CsCl gradient purified plasmid DNA and
5 x 106 cells. In all cases the data reflect a
minimum of three independent transfections, and luciferase activities
were normalized to cell protein content. Cell plating, harvesting, and
luciferase and protein assays have been described in detail (6).
Data Analysis
Data were subjected to multivariate ANOVA with
posthoc Dunnetts one-tailed t tests for values
greater than or less than the mean of the control to assess individual
comparisons.
Plasmid Constructions
All reporter plasmids were constructed with the firefly
luciferase expression vector
pA3LUC (39). The parent plasmid
493CSp.LUC contains the hCS-1 5'-flanking region
(493 bp) preceding the reporter gene. En2 CSp.LUC and
(En2)2_CSp.LUC contain one and
two copies of CSEn2, respectively, subcloned at the BglII
(nucleotide -993 relative to the transcription initiation site) and
PstI sites (nucleotide -285) and were constructed as
described previously (6). CSEn1 and CSEn5 sequences were PCR amplified
from bacteriophage
clones
EB-11 and
EB-4 (40) using primers
CSEn p31 and CSEN p32 (Table 1
). After
BglII digestion, CSEn1 and CSEn5 were ligated to
BglII-digested 493CSp.LUC to generate
En1 CSp.LUC and En5 CSp.LUC.
Similarly, ligation of the CSEn1 and CSEn5 BglII
fragments with BglII-treated
(En2)2 CSp.LUC results in the
replacement of the upstream En2 to yield
En1-En2 CSp.LUC and
En5-En2 CSp.LUC.
The CSEn2 mutants (EM1-EM8) were created by mutagenesis and subcloned
at the BglII site as described previously (6). Except for
EM1 and EM7, the mutant enhancer fragments were PCR amplified by
PA3_p11 and PA3_p12 primers (Table 1
), digested with
PstI, and ligated to their corresponding parental plasmid
(EM1-EM8), which were digested with PstI to give
(EM2)2_CSp.LUC,
(EM3)2_CSp.LUC,(EM4)2 CSp.LUC,
(EM5)2_ CSp.LUC,
(EM6)2 CSp.LUC, and
(EM8)2 CSp.LUC. Since
PstI sites had been introduced into the EM1 and EM7
mutations for the convenience of screening, the PstI site of
the 493CSp.LUC vector was first converted to KpnI
and the EM1 and EM7 enhancer fragments were subcloned at the
KpnI site. An additional copy of EM1 and EM7 were inserted
at the BglII site to obtain
(EM1)2 CSp.LUC and
(EM7)2 CSp.LUC.
Reporter plasmids carrying mutations at the hCS GHF-1, Sp1,
and TATA elements, either containing or lacking CSEn2 at the
BglII site, were described previously (18). Insertion
of an additional copy of CSEn2 at the PstI site of
these promoter mutants generated
(En2)2 pGHF1p.LUC,
(En2)2_dGHF1p.LUC
(En2)2 pdGHF1p.LUC,
(En2)2 SP1p.LUC, and
(En2)2 TATAp.LUC.
A fragment containing the SVEn 72-bp repeats was isolated by GeneClean
(BIO 101, Vista, CA) from BamHI/HindIII-digested
pED4Neo (41). The CMVEn was PCR amplified from the pUHD 151 plasmid
(42) using CMV1 and CMV2 primers (Table 1
).
After T4 DNA polymerase treatment and BglII linker addition,
these fragments were subcloned into the BglII site of
493CSp.LUC to generate SVEn CSp.LUC and
CMVEn CSp.LUC. Like CSEn, these viral enhancers
were also inserted at the PstI site through linker addition,
to yield (SVEn)2 CSp.LUC and
(CMVEn)2 CSp.LUC.
Complementary oligonucleotides containing two GT-IIC motifs
(OGT256)(13) were synthesized (Molecular Biology Core Facility, Mayo
Clinic) to construct reporter plasmids containing multiple GT-IIC
binding sites. After phosphorylation, annealing, and ligation of
monomers, 4-mers containing eight copies of the GT-IIC motif were
selected by eluting the 120-bp DNA fragment from a 2% agarose gel
using GeneClean. BglII linkers were added and the DNA
fragments were cloned into 493CSp.LUC to yield
(GT-IIC)8 CSp.LUC.
Subsequently, additional 2-mers and 4-mers containing four and eight
GT-IIC motifs, respectively, were cloned into the PstI sites
of these plasmids to generate
(GT-IIC)12 CSp.LUC and
(GT-IIC)16 CSp.LUC. Sequences
of all plasmid constructions were confirmed by dideoxy DNA sequencing
(Molecular Biology Core Facility, Mayo Clinic).
Gel Shift and Gel Supershift Assays
The sequences, labeling, and purification of double-stranded
oligonucleotide probes, GT-IICCS, GT-IICSV,
GT-IICSVMUT, were described by Jiang and Eberhardt (7).
Crude nuclear extracts from BeWo, GC, HeLa, and COS-1 cells were
isolated from cultured cells according to Dignam et al.
(43). After DNA-protein binding using 15,000 cpm of probe and 15 µg
nuclear extract, samples were resolved on 4.0% polyacrylamide gels and
the DNA-protein complexes were visualized by exposure to Kodak x-ray
film at -70 C for 12 days. To increase the sensitivity of the gel
supershift experiment, 60,000 cpm probe and 7 µg GC cell nuclear
extracts were used. After the initial incubation, TEF-1 antibodies were
added and incubation continued for 15 min longer before
electrophoresis. The rabbit anti-chicken TEF-1 antibody was generously
provided by Drs. Charles Ordahl and Iain Farrance (University of
California, San Francisco).
Western and Far Western Blot Analyses
For direct detection of TEF-1, 70 µg GC cell nuclear extracts
were resolved by 9% SDS-PAGE and transferred onto 0.2 µm
nitrocellulose membrane for antibody detection (7). To obtain
sufficient DNA-protein complexes for far Western analysis, gel shift
assays with GC cell extracts were scaled up 3-fold. After
electrophoresis, the wet gel was exposed to Kodak x-ray film at 4 C
overnight to localize the position of the DNA-protein complex. The gel
band containing the complex was excised, electrotransferred onto
0.2-µm nitrocellulose membrane, and subjected to primary antibody
binding (1:1500 dilution), secondary alkaline phosphatase-conjugated
antibody binding, and color development as described previously
(7).
 |
ACKNOWLEDGMENTS
|
---|
The authors express their appreciation to Drs. Charles Ordahl
and Iain K. G. Farrance for the anti-chicken TEF-1 antibodies, Dr.
Randall Kaufman for the pED4neo vector, Dr. Hermann Bujard for the pUHD
151 plasmid, and Nicole Henry and Ruth Kiefer for secretarial and
editorial help.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Norman L. Eberhardt, Ph.D., 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 23, 1997.
 |
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