Basic Helix-Loop-Helix Proteins Can Act at the E-Box within the Serum Response Element of the c-fos Promoter to Influence Hormone-Induced Promoter Activation in Sertoli Cells
Jaideep Chaudhary and
Michael K. Skinner
Center for Reproductive Biology Department of Genetics and Cell
Biology Washington State University Pullman, Washington
99164-4231
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ABSTRACT
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The Sertoli cell is a terminally differentiated
testicular cell in the adult required to maintain the process of
spermatogenesis. Previously basic helix-loop-helix (bHLH) factors and
c-fos have been shown to influence Sertoli
cell-differentiated functions. The induction of Sertoli cell
differentiation appears to involve the serum response element (SRE) of
the c-fos promoter to activate c-fos and
intermediate bHLH factor(s) that regulate downstream Sertoli
cell-differentiated genes (e.g. transferrin expression).
The SRE of the c-fos promoter is influenced through the
serum response factor (SRF). Interestingly, an E-box nucleotide
sequence is present within the SRE. bHLH proteins act through E-box
elements, and the current study investigates the possibility that bHLH
proteins may directly influence the SRE of the c-fos
promoter. The activation of the c-fos promoter in Sertoli
cells was found to be inhibited with the overexpression of the
inhibitory HLH protein Id. Analysis of major response elements within
the c-fos promoter demonstrated that the expression of Id
specifically inhibited the activation of SRE in Sertoli cells and no
other elements tested. Mutations in the E-box of the SRE also inhibited
the activation of SRE, suggesting the direct role of bHLH proteins in
regulating SRE activity in Sertoli cells. In contrast, the activation
of SRE containing a mutated E-box was comparable to wild-type SRE in
control stromal cells. Analysis of SRE oligonucleotide gel mobility
shift assays with nuclear extracts from Sertoli cells demonstrated the
presence of both the SRF and the ubiquitously expressed bHLH protein
E12/E47. In contrast, no E12/E47 was detected in the SRE
oligonucleotide gel shift using control stromal cell nuclear extracts.
Observations suggest the binding of E12/E47 to SRE may be a
cell-specific event. The SRF and bHLH proteins appear to bind to the
SRE and activate the c-fos promoter in Sertoli cells.
Observations provide evidence that a bHLH protein can interact with the
SRE of the c-fos promoter to influence hormone-induced
promoter activation. Cross-talk between these nuclear transcription
factors appears to be instrumental in the control of Sertoli
cell-differentiated functions.
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INTRODUCTION
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Sertoli cells in the testis are epithelial cells that form the
seminiferous tubule and provide the cytoarchitectural arrangements and
microenvironment required for developing germinal cells (1). Sertoli
cell fate is established in the embryonic gonad at the time of testis
and male sex determination. The majority of Sertoli cell-differentiated
functions appear at the onset of puberty and increase to optimal levels
in the adult animal. The expression of large numbers of specific gene
products (2) establishes the differentiated Sertoli cell. An example of
a Sertoli cell gene product is the iron-binding protein transferrin
(3), which transports iron to the developing germ cells. The induction
and increase in the expression of Sertoli cell genes during puberty
reflect the progression of Sertoli cell differentiation.
Sertoli cell-differentiated functions are modulated by a number of
regulatory agents including FSH and PModS. FSH is an endocrine hormone
that directly influences Sertoli cell function (4). PModS is a
testicular paracrine factor produced by peritubular myoid cells
surrounding the seminiferous tubule that acts on Sertoli cells (1). The
actions of both FSH and PModS on Sertoli cells involve the activation
of immediate early genes such as c-fos (5, 6), and the
c-fos, in turn, appears to activate intermediate factors
responsible for transcription of Sertoli cell-specific genes (6). The
actions of FSH on Sertoli cell gene expression can also be mediated
through the cAMP response element (CRE) present on the promoters of a
number of genes (7). The protooncogene c-fos is a gene whose
transcription is rapidly activated by a number of factors (8) and
stimuli in a variety of cell types. The rapid and transient activation
of this gene has been correlated with entry of the cell into the cell
cycle for cell proliferation (9) and growth arrest followed by terminal
cell differentiation (10). The expression of c-fos alone is
not likely to be sufficient to induce cellular differentiation (11).
Recently PModS and FSH were shown to regulate the c-fos
promoter at the level of the cis-acting regulatory element
termed the serum response element (SRE) (6). The SRE is a 29-bp region
within the c-fos promoter that binds serum response factor
(SRF) (12). Along with the SRF-binding domain, the SRE also contains an
E-box sequence (CATCTG, 3' to the SRF core) that can bind basic
helix-loop-helix (bHLH) transcription factors (13).
bHLH proteins are a class of transcription factors involved in the
control of cell-specific differentiation of a number of tissues
including muscle and brain (14, 15). An example in muscle is a family
of bHLH transcription factors (i.e. the MyoD family), which
when expressed are sufficient to orchestrate the expression of
muscle-differentiated functions (16). We have recently shown that bHLH
proteins are present in Sertoli cells and are involved in regulating
Sertoli cell-differentiated functions such as transferrin expression
(17). The bHLH proteins have a conserved helix-loop-helix domain
essential for dimerization and a basic domain. The paired basic region
mediates DNA binding to a consensus hexanucleotide sequence (CANNTG)
termed an E-box element (18). The presence of an E-box in the SRE of
the c-fos promoter suggests that bHLH proteins are potential
candidates in regulating c-fos activity in Sertoli cells.
Previous observations suggest that bHLH proteins like E12 and MyoD can
physically interact with SRF (19). However, implications for such an
interaction in regulating cellular function at the transcriptional
level are not known. The interactions of bHLH proteins with the SRE of
the c-fos may be required to promote growth arrest and
cellular differentiation. In the present study we provide evidence that
the interactions between SRF and Sertoli cell bHLH proteins may be
instrumental in maintaining Sertoli cell-differentiated functions in
response to regulatory agents such as FSH.
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RESULTS
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The hypothesis was tested that bHLH proteins are involved in
regulating the transcription of the protooncogene c-fos in
Sertoli cells. A chloramphenicol acetyl transferase (CAT) reporter
construct containing the proximal 400 bp of the human c-fos
promoter (Fig. 1A
) was transiently
transfected into cultured Sertoli cells. Sertoli cells were isolated
from 20-day-old rat testis and cultured under serum-free conditions.
The cells were also cotransfected with an Id (inhibitor of
differentiation) expression plasmid driven by the retroviral Rous
sarcoma virus promoter to overexpress Id. As a control, both
sense Id [Id(S)], which generates a functional protein, and
antisense Id [Id(A)], which generates a nonfunctional protein, were
used. After transfections, the cells were left untreated (control) or
were treated with either FSH or partially purified PModS preparation
from a size exclusion column termed PModS(S300) (6) for 48 h
before harvesting the cells for CAT activity. As shown in Fig. 2
, both FSH and PModS(S300) stimulated
c-fos CAT activity (9- and 12-fold, respectively;
P < 0.001). In the presence of Id(S), the ability of
FSH and PModS(S300) to stimulate c-fos CAT activity
decreased significantly (P < 0.001). In contrast, the
antisense Id had no effect on either FSH- or PModS(S300)-stimulated
Sertoli cells. The presence of Id had no effect on the viability of
Sertoli cells (data not shown). Dose-response curves with increasing
amounts of sense Id had no further inhibitory effects (data not shown),
which may be due, in part, to the small (2.5-fold increase) effects of
FSH and S300 on the basal plasmid. Id is a natural dominant negative
HLH protein and acts as a negative regulator of bHLH action. Id lacks
the DNA-binding domain (the basic region) but has an intact
helix-loop-helix domain, thus making it available for dimerization with
Sertoli cell E-box binding proteins (i.e. bHLH proteins) but
rendering the dimer ineffective for binding to E-box sequence(s). This
negative regulation of the c-fos promoter by Id suggests
that bHLH proteins are involved in up-regulating the c-fos
promoter activity.

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Figure 1. The c-fos Promoter
A, Schematic representation of the proximal (-400 bp)
c-fos promoter and the location of the major response
elements. B, Sequence of the SRE (-300 bp) in the c-fos
promoter. The E-box sequence in the SRE is in bold
letters. SRF binds to most of the 5'-end of the SRE. E12 is a
bHLH protein that can bind to the E-box sequence at the 3'-end of the
SRE. The asterisks indicate the region of dyad symmetry
within the SRE.
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Figure 2. Regulation of c-fos Promoter in
Sertoli Cells by the Dominant Negative HLH Protein Id
The Sertoli cells were cultured in serum-free conditions and
transfected with a CAT reporter plasmid driven by 400 bp of the
proximal c-fos promoter. The cells were also
cotransfected with Id(S) or Id(A) expression plasmids. After
transfections the cells were left untreated (Control) or were treated
with either FSH (FSH, 100 ng/ml) or PModS(S300) (S300, 50 µg/ml).
Data are expressed in terms of relative CAT activity (fold stimulation)
with control (transfected with c-fos reporter construct
only) set at 1. Data are presented as the mean ± SEM
from at least three different experiments done in triplicate. The
statistical analysis (ANOVA) is shown (with different
superscript letters indicating a significant difference
(P < 0.001).
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Several cis-acting elements have been characterized in the
proximal (-400 bp) 5'-flanking region of the c-fos promoter
as targets for various stimuli (Fig. 1A
). Central to these is the SRE,
which mediates the action of a number of stimuli including growth
factors and cAMP. cAMP has been shown to be a potent activator of the
c-fos gene. Multiple elements are known to contribute to the
induction of c-fos by cAMP, but the presence of a consensus
CRE site at -60 is the most active element (8). Induction of the
c-fos promoter by FSH is believed to be mediated through the
SRE and CRE (-60 bp) (6). Analysis of the c-fos promoter
reveals the presence of two E-box sequences. The proximal E-box
(CATCTG) is located immediately 3' to the CRE (Fig. 1A
). The distal
E-box with a sequence similar to the proximal E-box is positioned 3' of
the SRE core sequence (Fig. 1B
). The presence of two E-boxes suggests
that the activation of the proximal c-fos promoter may be
mediated through the binding of bHLH proteins to these E-boxes. To
confirm this possibility and to understand the mechanism by which the
bHLH proteins regulate c-fos expression, CAT reporter gene
constructs containing the CRE and SRE of the c-fos promoter
were generated. A schematic representation of the locations of these
elements within the c-fos promoter is shown in Fig. 1A
.
These constructs were transiently transfected into cultured Sertoli
cells. In addition to the reporter plasmids, the cells were also
cotransfected with Id(S) or Id(A) expression plasmids. After the
transfections, the cells were incubated in the absence (control) or
presence of either FSH or PModS(S300) for 48 h followed by CAT
assays.
FSH stimulation of Sertoli cells transfected with CRE-CAT resulted in
significantly higher CAT activity (Fig. 3
; P < 0.001). A
stimulation in CRE-CAT was also observed in response to PModS S300 but
was significantly lower than FSH stimulation (Fig. 3
). The presence or
absence of the dominant negative HLH protein Id had no effect on
FSH-stimulated CRE CAT activity. The CRE-CAT construct used in this
study consisted of the proximal 120 bp of c-fos promoter,
which, along with the CRE, also contains the proximal E-box. The
absence of any effect of Id on this construct suggests that this
proximal E-box is either not involved in regulation of FSH-stimulated
CRE-CAT activity or the proximal E-box-mediated control of
c-fos gene is independent of Id. Previously, this E-box was
shown to bind bHLH-Zip proteins like USF (upstream stimulatory factor)
(20). Gel shift data with Sertoli cell nuclear extracts indicate that
this E-box does bind bHLH proteins (data not shown). Under the
experimental conditions used in the present study, the bHLH complex
binding to the proximal E-box (-55 bp) does not appear to be regulated
by FSH.

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Figure 3. Relative CAT Activity of CRE-CAT Reporter Construct
in the Presence or Absence of Id
Cultured Sertoli cells were transfected with the reporter plasmid
containing the proximal 120 bp of the proximal c-fos
promoter along with Id(S) or Id(A) expression plasmids. The primary
response element in the proximal 120 bp of the c-fos
promoter is the CRE. The cells were subsequently treated with FSH (FSH,
100 ng/ml), PModS(S300) (S300, 50 µg/ml), or were left untreated
(control). The data are expressed as fold stimulation and relative CAT
activity with control (without expression plasmids) set as 1. Data are
presented as mean ± SEM from four different
experiments carried out in triplicate. As determined by ANOVA, presence
of Id(S) or Id(A) had no effect on FSH-stimulated CRE-CAT activity.
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As shown in Fig. 4
, the SRE of the
c-fos promoter is activated by both FSH and PModS(S300)
(P < 0.001). The magnitude of the SRE-tk-CAT reporter
construct stimulation was similar to the whole c-fos
promoter (Fig. 2
). Therefore, the SRE appears to play a significant
role in regulating c-fos promoter activity in Sertoli cells.
Cotransfection with an Id sense expression plasmid significantly
reduced the SRE-tk-CAT activity in response to both FSH and PModS(S300)
(Fig. 4
). No reduction in CAT activity was observed when Sertoli cells
were cotransfected with the Id antisense expression plasmid. The
observed decrease in SRE-tk-CAT activity in the presence of Id suggests
the binding of bHLH dimers to the E-box in the SRE is required for
optimal activity. This hypothesis was confirmed by transfecting the
Sertoli cells with an SRE-tk-CAT plasmid in which the E-box was mutated
(mutSRE-EB). The activity of mutSRE-EB was significantly lower as
compared with the wild-type SRE-tk-CAT plasmid (wtSRE) (Fig. 5A
). In contrast to the Sertoli cell data
(Fig. 5A
), control stromal cells were not effected by a mutation in the
E-box of the SRE (Fig. 5B
).

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Figure 4. Regulation of the SRE-tk-CAT Activity in Sertoli
Cells Cultured in the Absence (Control) or Presence of FSH (FSH, 100
ng/ml) or PModS (S300), 50 µg/ml (S300)
The cells were transfected with SRE-tk-CAT alone or in the presence of
Id(S) or Id(A) expression plasmids. To determine the effect of FSH,
S300 and the presence of Id(S) and Id(A) on the tk-promoter, the
Sertoli cells were also transfected with the tk-CAT plasmid only. The
data are expressed as relative CAT activity (fold stimulation) with
control (transfected with tk-CAT plasmid) set at 1.0. Data is presented
are mean ± SEM from four different experiments
carried out in triplicate. The statistical analysis (ANOVA) is shown
(with different superscript letters indicating a
significant difference (P < 0.001).
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Figure 5. Effect of E-box Mutations on the Activation of
SRE-tk-CAT plasmid in Cultured Sertoli Cells (A) and Stromal Cells (B)
The E-box in the wild-type SRE (CATCTG, wt-SRE-tk-CAT) was mutated
(CAAAATG, mut-SRE-EB-tk-CAT) to disrupt the binding of potential bHLH
proteins. These reporter constructs were transfected in cultured cells,
and the cells were either left untreated (control) or treated with FSH
or PModS S300 (Sertoli cells) or 10% bovine calf serum (stromal cells)
before harvesting for CAT assay. The data are expressed as mean ±
SEM relative CAT activity (fold stimulation, with
wt-SRE-tk-CAT activity = 1) is representative of three different
experiments done in triplicate. The different superscript
letters represent a significant difference
(P < 0.001).
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As a control for the SRE-tk-CAT, the thymidine kinase minimal promoter
CAT construct (tk-CAT) was transfected into Sertoli cells in the
absence or presence of the Id(S) or Id(A) constructs. Both FSH and S300
were found to stimulate the tk-CAT construct 2- to 3-fold above control
levels (Fig. 4
). The Id overexpression had no effect on the basal
tk-CAT plasmid activity. To determine whether bHLH proteins
specifically act at the SRE of the c-fos promoter, the other
major c-fos promoter response elements were examined.
SIE-Tk-CAT (sis-inducible element, upstream of SRE; Fig. 1
.) and
TRE-tk-CAT (downstream of SRE) reporter plasmids were cotransfected
with the Id expression plasmids (Fig. 6
).
The Id sense had no effect on the SIE or TRE CAT activity (Fig. 6
).
This observation suggests that the regulation of the c-fos
promoter by bHLH proteins is at the level of the SRE (Fig. 4
).

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Figure 6. Relative CAT Activity of SIE and TRE-CAT Reporter
Constructs in the Presence or Absence of Id
The cells were subsequently treated with FSH (FSH, 100 ng/ml),
PModS(S300) (S300, 50 µg/ml), or were left untreated (control). The
data are expressed as fold stimulation and relative CAT activity with
control (without expression plasmids) set as 1. Data are presented as
mean ± SEM from three different experiments carried
out in triplicate.
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Gel shift experiments were performed to address the question of whether
SRF and bHLH proteins bind to the SRE. Nuclear extracts were obtained
from freshly isolated Sertoli cells (Fr) or cultured in the presence of
FSH (F) or PModS (S300) (P). A gel shift with an Oct-1 oligonucleotide
was used to check the integrity and quality of the nuclear extracts.
All the extracts resulted in a similar amount of Oct-1 gel shift (Fig. 7A
). As shown in Fig. 7B
, three major
migrating bands were observed when a radiolabeled SRE oligonucleotide
(lacking the 5'-ets domain) was used as a probe in the low-stringency
binding reaction with Sertoli cell nuclear extracts. The presence of
excess unlabeled SRE in the binding reaction abolished both the bands
(band 1 and band 2, Fig. 7B
). To determine whether the transcription
factors binding to the SRE are similar to those that can bind to the
E-box sequence, excess E-box oligo was added in the low-stringency
binding reaction. Addition of excess E-box oligo only displaced the
slower migrating band 1. These displacement data suggest that the
protein(s) present in band 1 consist of either E-box binding proteins
only or both E-box and SRE-binding protein(s). In contrast, the faster
migrating band 2 may be primarily due to the binding of transcription
factors to SRE. Band 3 was not completely displaced by either excess
SRE or E-box, suggesting that it may be due to nonspecific binding
(Fig. 7B
). Addition of excess unlabeled Oct-1 in the binding reaction
had no effect on the SRE gel shift (negative control, data not shown),
indicating that the displacement observed with excess unlabeled SRE and
E-box was specific. The excess unlabeled oligonucleotides were titered,
and optimal doses were used in the gel shift reactions shown in Fig. 7B
(data not shown).

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Figure 7. Gel Mobility Shift Assay
A, Gel mobility shift assay with 32P-labeled Oct 1 (panel
A) or SRE (panel B) oligonucleotides and nuclear extracts from Sertoli
cells. The nuclear extracts were prepared from cells that were freshly
isolated (Fr) or cultured and treated with FSH, 100 ng/ml (F); PModS
(S300), 50 µg/ml (P), or control (untreated, C). Excess unlabeled SRE
or E-box oligonucleotide is indicated. The gel is representative of
three different experiments. Multiple bands (1 2 ) indicated with
arrows were observed when the binding reaction was
carried out under low-stringency conditions.
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Gel supershift experiments were performed to identify the proteins
binding to the SRE oligo under low-stringency binding conditions.
Inclusion of an SRF antibody in the binding reaction between SRE oligo
and nuclear extracts from FSH-treated Sertoli cells resulted in two
supershifted bands labeled 3 and 4 (Fig. 8A
). In contrast, the addition of the
E12/E47 antibody resulted in a single major supershifted band labeled 5
(Fig. 8A
). As a control, nonimmune serum was not found to cause a
supershift. Observations indicate that both SRF and E12/E47
appear to be involved in the SRE gel shift observed. This supports the
gel shift displacement data in Fig. 7B
.

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Figure 8. Gel Mobility Shift Assay
A, Gel mobility supershift experiments with 32P-labeled SRE
oligonucleotide and nuclear extracts from FSH-treated Sertoli cells.
Supershifts were observed when antibody to either SRF (bands 4 and 3)
or E12/E47 (band 5) were included in the binding reaction. No
supershift was observed when nonimmune serum (NIS) was present in the
binding reaction. B, Gel mobility shift assay with
32P-labeled SRE oligonucleotide and nuclear extracts from
cultured stromal cells. No supershift was detected when E12/E47
antibody was present in the binding reactions. Excess unlabeled SRE and
mutated SRE*, but not E-box, oligonucleotide displaced the shifted
band. The gel is representative of three different experiments.
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Whether the binding of bHLH proteins to the SRE is a cell-specific
event was addressed by using bovine ovarian stromal cells. The gel
shift experiment using nuclear extracts from cultured bovine stromal
cells and SRE oligonucleotide resulted in a single shifted band (Fig. 8B
). This is in contrast to the Sertoli cell gel shift data shown in
Fig. 7B
. The single shifted band was displaced when excess unlabeled
SRE was included in the binding reaction (Fig. 8B
), but was only
partially displaced in the presence of excess SRE with mutated E-box.
Presence of E12/E47 antibody did not result in a super shift. The SRE
gel shift was also not competed in the presence of excess unlabeled
E-box oligonucleotide (Fig. 8B
). Observations suggest that the binding
of SRF to SRE does not require the presence of bHLH proteins in bovine
stromal cells. This prompted the investigation of the activation of
wtSRE-tk-CAT and mutSRE-EB-tk-CAT in bovine stromal cells. As
previously presented, the wtSRE and mutSRE-EB reporter plasmids in
bovine stromal cells were activated to similar levels when the cells
were stimulated with 10% bovine calf serum (Fig. 5B
). This is in
contrast to Sertoli cells in which the activity of mutSRE-EB reporter
plasmid was significantly lower as compared with wtSRE (Fig. 5A
). The
difference in the gel shift profiles and transcriptional activation
data between Sertoli cells and stromal cells suggests that the binding
of bHLH proteins to SRE and its activation may be a cell-specific
event.
Interestingly, when the binding reaction between the SRE
oligonucleotide and the nuclear extracts from FSH- and
PModS(S300)-treated Sertoli cells was performed at higher stringency
(i.e. increased KCl concentrations), a single gel shift band
was observed (Fig. 9
). This gel shift was
displaced by adding either excess SRE or E-box oligoneucleotides. The
shifted band in Fig. 9
may therefore correspond to band 1 observed in
Fig. 7B
. Gel shift data from Figs. 7B
(band 1), 8A, and 9 suggest that
SRF and bHLH proteins are present in the retarded band. To further
confirm this observation, an SRE oligonucleotide with mutations in the
E-box (mutSRE-EBoligo) was used as a probe in the gel shift assay.
Using mutSRE-EBoligo as a probe, a faster migrating band was expected
due to the binding of transcription factor(s) to the intact SRE core
sequence. In contrast, no retarded band was observed with the
mutSRE-EBoligo probe (Fig. 9
). This observation suggests that either
the E-box is part of the core SRE and is required for the binding of
transcription factor(s), such as SRF to SRE, or that the binding of
bHLH proteins to the E-box is necessary for the stable in
vitro binding of SRF to the SRE core sequence.

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Figure 9. Gel Mobility Shift Assay Carried Out under
High-Stringency Conditions with 32P-Labeled SRE or Mutated
SRE* Oligonucleotides and Nuclear Extracts from Sertoli Cells
The nuclear extracts from cells treated with FSH, 100 ng/ml (F), PModS
(S300), 50 µg/ml (P), or untreated (control, C). Excess unlabeled SRE
or E-box oligonucleotide is indicated and displaced the gel shift. No
gel shift band was detected when mutated SRE* oligonucleotide was used
as a probe. The gel is representative of three different experiments.
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To further characterize the proteins binding to the SRE oligo, the gel
shift (Fig. 9
) was blotted onto a nitrocellulose membrane and probed
with specific antibodies to various bHLH proteins such as USF and the
E2A gene products, E12 and E47. A positive band was detected with an
antibody to E12/E47, suggesting that this ubiquitously expressed bHLH
protein is present in the complex binding to the SRE (Fig. 10
). In contrast, USF was not found in
the complex (data not shown). E47 forms heterodimers with cell-
specific bHLH proteins. Another transcription factor that binds to the
SRE is a 67-kDa protein SRF. Interestingly, when the same SRE gel shift
blot was probed with an SRF antibody, a band similar in migration to
the E12/E47 was observed (Fig. 10
). This observation suggests that a
multimeric complex involving bHLH proteins and SRF appears to cause the
gel shift with SRE (Fig. 10
). Control gel shifts with an Oct-1
oligonucleotide, mutSRE-EBoligo, or nonimmune serum showed no bands
being detected in the blot (Fig. 10
). Due to the native gel conditions
most of the proteins were seen at the top of the gel, and no proteins
migrated into the gel without binding DNA as determined with a silver
stain of the gel (data not shown). The immunoblot data support the gel
shift and transfection data, which suggests that activation of the
c-fos promoter at the level of the SRE involves the binding
of SRF and bHLH proteins (Fig. 11
).

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Figure 10. Immunoblot of SRE Gel Mobility Shift Assay
(Similar to Fig. 9 ) with E12/E47 and SRF Antibody
Nuclear extracts from Sertoli cells cultured in the absence (C) or
presence of FSH, 100 ng/ml (F) or PModS (S300), 50 µg/ml (P) were
used. The DNA protein complexes were electophoretically separated on
5% polyacrylamide gels. The gels were then either dried and
autoradiographed (gel shift) or electrophoretically transferred to
nitrocellulose membrane and immunoblotted with antibodies to SRF or
E12/E47 (blot). The data are representative of three different
experiments. As a control, the gel shift immunoblot with Oct 1 and
nuclear extracts from FSH-treated Sertoli cells was probed with E12/E47
and SRF antibody.
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Figure 11. Schematic of Potential bHLH Proteins and SRF
Interactions on the SRE of the c-fos Promoter to
Activate c-fos Expression and Influence Sertoli Cell
Differentiation
The E-box and SRE cooperatively bind a multiprotein complex consisting
of SRF and bHLH proteins. This complex may be involved in
tissue-specific activation of c-fos in response to FSH
or PModS (S300). It is possible that the interactions between SRF and
bHLH may be mediated by another protein (coactivator).
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DISCUSSION
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It has been proposed that a small number of transcription factors
may be capable of initiating the entire process of differentiation. One
such group of transcription factors are the bHLH proteins. Data
accumulating over the last several years have established that bHLH
proteins are sufficient to initiate differentiation programs in a
number of tissues and cell types (14, 15). The examples include the
MyoD family and NeuroD, which are involved in muscle cell and neuronal
cell differentiation (15), respectively. Another example relevant to
the current model is the bHLH protein daughterless involved
in Drosophila sex determination (21). Recently, a knockout
of the E2A gene containing the ubiquitously expressed E12/E47 bHLH
factors demonstrated effects on a number of tissues (22);
however, effects on reproductive tissues were not investigated. Another
group of transcription factors induced during differentiation are the
immediate early genes such as c-fos (8). The
c-fos oncoprotein is a component of the AP-1 transcription
factor complex and is a member of a multigene family that includes jun,
jun-related genes, and other fos-related genes (23). The
c-fos is also considered a master switch necessary for both
cell division and differentiation.
Sertoli cell expression of both c-fos and bHLH proteins is
required for the activation of differentiated functions such as
transferrin gene expression (6). Regulatory agents such as the
endocrine hormone FSH and testicular paracrine factor PModS(S300)
promote Sertoli cell differentiation through an increase in the
expression of c-fos and bHLH transcription factors (6, 17).
Therefore, the importance of bHLH proteins as key differentiation
factors and the observation that the E-box sequences are present in the
c-fos promoter led to the hypothesis that a cascade of
events leading to Sertoli cell differentiation may involve the bHLH
proteins acting on immediate early genes such as c-fos. To
investigate this hypothesis, transient transfection experiments were
performed.
In transient transfection experiments, the overexpression of Id
down-regulated the activity of the c-fos promoter. Id is a
natural dominant negative HLH protein that lacks the DNA-binding basic
domain. It can dimerize with Sertoli cell bHLH proteins, but the dimers
fail to bind to an E-box. Therefore, Id can titer out the endogenous
bHLH proteins by forming a nonfunctional dimer resulting in the loss of
transcriptional activation (24). The ability of the dominant negative
HLH protein Id to inhibit the c-fos promoter is the first
direct evidence that the activity of the c-fos promoter is
dependent on Sertoli cell bHLH proteins. As previously shown, the
stimulation of the c-fos promoter is essential for the
stimulation of Sertoli cell-differentiated functions (6). This is in
contrast to the myoblasts, in which bHLH proteins such as MyoD/myogenin
negatively regulate the c-fos promoter (25). Unlike Sertoli
cells, the myoblasts do not express c-fos once they initiate
differentiation (25).
The 5'-flanking region of the c-fos promoter consists of a
number of response elements that can act as targets for various stimuli
(26). The induction of the c-fos promoter in Sertoli cells
by PModS(S300) is primarily through the SRE (6). FSH can also activate
c-fos expression through multiple transduction pathways
integrating at the level of the SRE and the CRE. Therefore, reporter
constructs consisting of SRE and the proximal region of the
c-fos promoter containing CRE were both used in transient
transfection experiments to understand the mechanism by which bHLH
proteins regulate the expression of the c-fos promoter in
Sertoli cells.
CRE represents a binding site for CRE-binding protein
(CREB)-related transcription factors (27). CREB is a target for protein
kinase A that is itself activated by the second messenger cAMP
(27). Actions of FSH on Sertoli cells are mediated through cAMP (4).
The CRE-CAT construct used in the present study had CRE as the primary
response element. A similar region of the c-fos promoter
(-70 to -48), which contains this CRE sequence, was shown to confer
cAMP inducibility to a heterologous promoter (28). The activation of
CRE-CAT reporter construct by FSH is therefore most likely due to the
cAMP/protein kinase A pathway. Apart from the CRE, this promoter
fragment also contains an E-box (CATCTG) sequence just 3' to the CRE
(proximal E-box). bHLH-ZIP proteins such as USF bind to this proximal
E-box (20). The absence of any effect of Id overexpression on the
CRE-CAT construct suggests that FSH activation of CRE by CREB-related
proteins does not involve bHLH proteins. Although evidence from gel
shift experiments suggests bHLH proteins bind to the proximal E-box,
their expression does not appear to be regulated by FSH.
The SRE is a highly conserved region of imperfect dyad symmetry located
300 bp upstream of the cap site (12). The central element
CC(A/T)6G (also known as the CArG box) is the specific
binding site for the nuclear factor termed the SRF (30). In addition to
the SRF-binding domain, SRE also consists of multiple overlapping
enhancers that can act independently when appended to a reporter gene
(8). This complex structure is a target for an interplay of multiple
positive and negative interactions. It appears that stimulation of
c-fos transcription by regulatory agents is mediated through
accessory nuclear factors that act cooperatively with or independently
of SRF at the SRE. Experimental evidence suggests the important role
for one of the SRE-binding proteins, p62 ternary complex factor.
Ternary complex factor is encoded by a family of ets-related genes such
as elk-1 and SAP-1 that do not bind directly to the SRE, but bind to a
purine-rich motif 5' to the SRE when SRF is bound (31). The ternary
complex formation may not be necessary for induction of
c-fos (31). At the 3'-end of the SRE palindrome
protein complexes such as SRE-binding protein (32) and E12/E47 have
been shown to bind and effect c-fos expression in certain
cell types (13, 25). Negative regulation of the SRE through mutations
in the E-box and overexpression of Id suggest that Sertoli bHLH
proteins directly act at the level of the SRE. Another transcription
factor complex that binds SRE is SRE-binding protein and is composed,
in part, of the dimer of C/EBPß, a member of the basic
leucine-zipper family of transcription factors (32). cAMP is known to
activate C/EBPß by causing its translocation to the nucleus (33).
Metz and Ziff (33) have reported an increase in the binding of C/EBPß
at the C/E site of the SRE in the c-fos promoter upon
treatment of PC12 cells with forskolin, a cell line known to be
sensitive to cAMP stimulation. The induction of SRE-CAT reporter
construct by FSH may be due to the cAMP-mediated translocation and
binding of C/EBPß to the C/E site. Our gel shift and blot data are in
contrast to this assumption because the binding of C/EBPß and SRF to
the SRE is mutually exclusive (37). The presence of SRF in the complex
binding to the SRE oligonucleotide excludes the possibility that
C/EBPß is also present in the complex.
Interestingly, the ubiquitous bHLH protein E12/E47 is also present in
the complex binding to the SRE and is consistent with the reported
observations that the binding of E12/E47 and SRF may not be mutually
exclusive (13). The binding of the bHLH proteins to the SRE is due to
the presence of an E-box immediately 3' to the SRE core sequence. The
bHLH proteins bind as heterodimers to consensus E-box sequences. The
heterodimers consist of ubiquitously expressed proteins such as E12 or
E47 and more cell-specific bHLH proteins such as MyoD (18). The E47-E47
homodimer has been shown to bind E-box only in the context of Ig
enhancer in B cells. The antibody used to detect E47 cross-reacts with
another splice variant of the E2A gene, E12. Unlike E47, E12 does not
efficiently homodimerize but can heterodimerize with cell-specific bHLH
proteins. It remains to be investigated whether a heterodimer between
either of the E2A gene products and a yet unknown Sertoli cell-specific
bHLH protein or an E47 homodimer may be binding to the SRE E-box. The
E-box in the SRE is part of the dyad symmetry and is required for
efficient binding of the SRF (34, 35). In NIH 3T3 cells, the
transcription of the c-fos gene is severely impaired when
the E-box of SRE is mutated (34). Similar down-regulation of SRE with
mutations in the E-box were also observed in Sertoli cells. This
observation suggests that the formation of a multiprotein complex may
be required for efficient transcription of the c-fos gene
driven by the SRE (34). The proposal is made that the SRE may be
constitutively occupied by SRF in vivo and its activation
possibly requires phosphorylation of SRF and its association with
accessory nuclear factors. In the current transient transfection
experiments, SRF may be constitutively bound to the SRE, but activation
of the reporter gene may require phosphorylation of SRF and binding of
bHLH proteins to the E-box. Mutations in the E-box or overexpression of
Id can titer out the bHLH proteins, making them unavailable for binding
to the E-box of the SRE followed by down-regulation of the
promoter.
The gel shift data presented in Fig. 7B
showing the presence of two
bands are in contrast to that reported by Greenberg et al.
(34). They reported the presence of a single shifted band using HeLa
nuclear extracts and an SRE probe similar to the one used in the
present study. A gel shift with an alternate stromal cell extract,
however, did provide a single gel-shifted band (Fig. 8
). Observations
presented in the current study suggest that SRF, together with the
E12/E47/bHLH dimer, bind the SRE as a multiprotein complex resulting in
the appearance of a second gel shift band (band 1, Fig. 7B
). The
displacement of the SRE-retarded band with excess E-box and supershift
with E12/E47 and SRF antibody support this observation. In Sertoli
cells, E-box-binding proteins appear to be required for a stable
complex formation between SRF and SRE. Formation of such a complex
in vitro between SRF/bHLH/E12 was recently proposed (19).
In vitro this complex required the intact dimerization
domain of SRF and the bHLH domain of MyoD and E12, but the functional
implications for such a complex were not reported. The formation of
such a multiprotein complex between SRF and bHLH proteins is likely to
be a cell-specific event. In the myoblasts, binding of MyoD or
Myogenin/E12 dimer and SRF to SRE oligo was shown to be mutually
exclusive, and such an interaction was believed to cause a
down-regulation of the c-fos promoter (25). To confirm
in vivo that the SRF/E-box complex is required for the
induction of c-fos in Sertoli cells, the overexpression of
SRF and E12/E47 in the presence of wild-type SRE were considered.
However, previous reports suggest that overexpression of SRF alone has
no effect on the c-fos reporter construct (36).
Overexpression of E12/E47 would be expected to provide analogous data
to Id overexpression and support the gel shift data.
The mechanisms by which bHLH proteins regulate the activity of the
c-fos promoter appear to be different in different cell
types. Sertoli cells appear to require activation of c-fos
to sustain differentiated function. The bHLH proteins may together with
SRF up-regulate c-fos. In myoblasts, c-fos plays
a major role in proliferation, but not in differentiation (25). The
bHLH proteins, which are expressed only during differentiation, can
alone inhibit c-fos activity (25). These mechanisms of
repression vs. stimulation of differentiation may be
required to diversify the pathways of c-fos promoter
induction. SRF, by its ability to interact with other members of
distinct transcription factor families, may modify c-fos
promoter activity.
The cooperative interaction of bHLH proteins with other transcription
factors such as SRF may be a mechanism by which bHLH transcription
factors acquire more specificity for transcriptional activation. This
is supported by the observation that a proximal E-box near the CRE has
a sequence similar to the E-box in the SRE, but does not play any role
in regulating promoter activity. The SRE E-box, on the other hand, by
virtue of being close to the SRF-binding domain, allows interaction
between bHLH proteins and SRF. This interaction may modify the
activities of interacting transcription factors. Another possibility,
which cannot be ruled out, is the presence of an adapter protein that
may mediate the interaction between SRF and bHLH transcription factors
(Fig. 11
). In summary, this study demonstrates that in Sertoli cells,
bHLH proteins can regulate the c-fos promoter activity
specifically at the level of the SRE. The gel shift data support the
hypothesis that the regulation of the c-fos promoter by bHLH
proteins possibly involves the formation of a multiprotein complex at
the SRE consisting of SRF and E47 (Fig. 11
).
 |
MATERIALS AND METHODS
|
---|
Cell Preparation and Culture
Sertoli cells were isolated from the testis of 20-day-old rats
by sequential enzymatic digestion with a modified procedure described
by Tung et al. (37). Decapsulated testis fragments were
digested first with trypsin (1.5 mg/ml; Gibco BRL,
Gaithersburg, MD) to remove the interstitial cells and then with
collagenase (1 mg/ml type I; Sigma Chemical Co., St.
Louis, MO) and hyaluronidase (1 mg/ml; Sigma Chemical Co.). Sertoli cells were then plated under serum-free conditions
in 24-well Falcon plates at 1 x 106 cells per well.
Cells were maintained in a 5% CO2 atmosphere in Hams
F-12 medium (Gibco BRL) with 0.01% BSA at 32 C. Sertoli
cells were treated with either FSH (100 ng/ml; o-FSH-16, National
Pituitary Agency) PModS (S300) (50 µg/ml), or vehicle alone (Hams
F-12, Control). These optimal concentrations of FSH and PModS (S300)
have previously been shown to dramatically stimulate cultured Sertoli
cell-differentiated functions (38, 39). Due to changes in bioactivity
of these substances, the presentation of exact concentrations
(i.e. molarities) is not informative. The cells were
cultured under serum-free conditions for a maximum of 5 days with a
media change and treatment after 48 h of culture. Cell number and
viability did not change during the culture in the absence or presence
of treatment (38, 39). Bovine ovaries were obtained from young
nonpregnant cycling heifers less that 10 min after death from the local
slaughterhouse. The stromal cells from bovine ovaries were prepared and
cultured as previously described (40).
Plasmids
The CAT reporter plasmid (PBL-CAT2) with thymidine kinase
minimal promoter (41) was used to make the
SRE:5'-CAGGATGTCCAAATTAGGACATCTGC-3'-CAT reporter plasmids. The
SRE-tk-CAT plasmid was constructed by cloning synthetic annealed SRE
oligonucleotide into PBL-CAT2 plasmid upstream of thymidine kinase
minimal promoter. The site-specific mutations in the E-box of SRE
(GCAAAATG) mSRE-EBCAT were introduced into the SRE-tk-CAT plasmid by
using the Quick Change site-directed mutagenesis kit
(Stratagene, La Jolla, CA). The CRE-CAT plasmid had 120
bp of the 5'-flanking sequence from the transcriptional start
site of the human c-fos promoter with 5'-TGACGTTT-3'
sequence at -60 bp (42). The entire 400 bp of the c-fos
promoter CAT construct were generously provided by Dr. Jeff Holt
(Vanderbilt University, Nashville, TN). The pREP Id-1S and pREP Id-1AS
were provided by Dr. Jay Cross (McGill University, Montreal, Quebec,
Canada) (43).
Transfections and CAT Assays
Sertoli cells or stromal cells, cultured for 48 h, were
transfected with a reporter gene construct by the calcium phosphate
method coupled with hyperosmotic shock (10% glycerol) as previously
described (6, 17). Briefly, 1.5 µg reporter plasmid with or without
1.5 µg of expression plasmid in 150 µl of transfection buffer [250
mM CaCl2 mixed 1:1 (vol/vol) with 2x Hebes (28
mM NaCl, 50 mM HEPES and 1.47 mM
Na2HPO4, pH 7.05)] were added to each well of
a 24-well plate containing 1 x 106 Sertoli cells in 1
ml of Ham F12 with 0.01% BSA and incubated at 32 C for 4 h. After
incubation, the cells were subjected to a hyperosmotic shock. The media
were aspirated, and 1 ml of 10% glycerol in HBSS was added. The cells
were incubated for 3 min, and the wells were washed twice before adding
fresh Ham F-12 media. Various treatments were subsequently added and
cells incubated for an additional 48 h before harvesting for CAT
assays. Assay of CAT activity was performed with the
[14C]chloramphenicol conversion, as previously described
(6). The average conversion of CAT substrate for treated cells ranged
between 2030% conversion. This assay was found to be linear with the
protein concentration used.
Gel Mobility Shift Assay
Gel shift assays were performed with nuclear extracts of
cultured Sertoli and stromal cells. The cells were isolated as
described above and cultured in 150 mm x 20-mm tissue culture
dishes (Nunclon, Naperville, IL). The cells were treated after
48 h in culture with either FSH, PModS(S300), 10% bovine calf
serum (stromal cells only), or not treated for controls. After 72
h, the cells were scraped off the tissue culture dishes and washed once
with PBS. The nuclear extracts of these cells were then prepared as
described by Guillou (44). Typically 70100 µg of protein were
obtained from 108 plated cells. The double-stranded DNA
probes used in gel retardation assays were: the muscle creatine kinase
(mck) E-box (CACCTG) containing flanking sequence (5'-GAT CCC CCC
AAC ACC TGC TGC CTG A-3') and the SRE oligonucleotide
(5'-GAT GTC CAT ATT AGG ACA TCT GC-3'), SRE-Ebox (5'-ATT AGG ACA TCT
GC-3'), and mutated SRE (5'-GAT GTC CAT ATT AGG ACA AAA
TGC-3'). The single-stranded oligonucleotides were 5'-32P
end labeled with 32P
-ATP (150 µCi/µl, New England
Nuclear, Boston, MA) and polynucleotide kinase (10 U/µl,
Boehringer Mannheim, Indianapolis, IN). The complementary
oligonucleotides were annealed, electrophoretically purified, and were
then used as probes in gel shift assays.
The gel retardation assay used was a modification of the protocol
described by Garner and Rezvin (45). The final reaction volume (high
stringency) of 20 µl contained 0.5 ng (
50,000 cpm) of
5'-32P-end-labeled double-stranded probe, 100 ng sonicated
salmon sperm DNA, 2 µg Poly dI-dC (United States Biochemical Corp., Cleveland, OH), 20 µg BSA, 20 mM
HEPES (pH 8.0), 4 mM Tris (pH 7.9), 50 mM KCl,
600 µM EDTA and EGTA, 500 µM dithiothreitol, and 5 µg
nuclear proteins. The low- stringency binding reaction was carried out
using 10 ng salmon sperm DNA, 1.5 µg poly dI-dC, and 25
mM KCl. After incubation at room temperature for 20 min, 5
µl of the binding reaction were electrophoretically separated on a
5% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA.
The gel was subsequently dried and autoradiographed. For the
competition experiments, excess unlabeled oligonucleotide (x250 molar
excess) was also included in the binding reaction. The gel supershift
experiments were performed by incubating the nuclear extracts with 2
µg of either SRF antibody (Santa Cruz Biotechnology, Inc., SC-335x) or E12/E47 antibody (SC-36a) at room temperature
for 45 min before adding the radiolabeled probe.
Immunoblot Procedure
A gel shift assay was performed in duplicate on the same gel
using radioactive and nonradioactive SRE oligonucleotide. The gel shift
using radioactive SRE was dried and autoradiographed. A gel shift assay
using nonradioactive SRE probe on the polyacrylamide gel was
electrophoretically transferred to a nitrocellulose membrane (BA85,
Schleicher & Schuell, Inc., Keene, NH) by electrophoresis
in Tris-glycine buffer containing 12% methanol. The blot was then
blocked in 5% nonfat milk in TBSN [50 mM Tris (pH 7.4),
150 mM NaCl, and 0.05% Nonidt P-40] and incubated with a
1:3000 dilution of antibodies to E12/E47, SRF, USF, and
c-fos (Santa Cruz Biotechnology, Inc.) for
3 h. After three washes of 15 min each, the blot was hybridized
with a secondary antibody (1:3000 dilution; directed against rabbit
IgG) conjugated to horseradish peroxidase for 1 h at room
temperature. After five washes in TBSN, the immune complex was detected
using the enhanced chemiluminescent (ECL) detection kit
(Amersham). The c-fos antibody was included to
determine the specificity of the blot. As an internal control, a gel
shift was carried out using AP-1 oligo and blotted with
c-fos, E12, USF, and SRF antibody. When AP-1 was used as a
probe, a band in the blot was observed only with c-fos
antibody (11).
Statistical Analysis
All the data were obtained from a minimum of three
different experiments unless otherwise stated. Each data point was
converted to a relative CAT activity with the mean and SEM
from multiple experiments determined as indicated in the figure
legends. Data were analyzed by a Students t test or ANOVA
as indicated in the figure legends. Different superscript letters
denote a statistical difference as stated in the figure
legends.
 |
ACKNOWLEDGMENTS
|
---|
We thank Rachel Mosher and Gene Herrington for technical
assistance. We thank Dr. Andrea Cupp and Susan Cobb for assistance in
preparation of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Michael K. Skinner, Center for Reproductive Biology, Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99164-4231. E-mail:
Skinner{at}mail.wsu.edu
This work was supported by NIH Grants.
Received for publication March 17, 1998.
Revision received January 29, 1999.
Accepted for publication February 2, 1999.
 |
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