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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo) 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. 2Go, 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).

 
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. 1AGo). 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. 1AGo). The distal E-box with a sequence similar to the proximal E-box is positioned 3' of the SRE core sequence (Fig. 1BGo). 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. 1AGo. 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. 3Go; P < 0.001). A stimulation in CRE-CAT was also observed in response to PModS S300 but was significantly lower than FSH stimulation (Fig. 3Go). 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.

 
As shown in Fig. 4Go, 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. 2Go). 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. 4Go). 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. 5AGo). In contrast to the Sertoli cell data (Fig. 5AGo), control stromal cells were not effected by a mutation in the E-box of the SRE (Fig. 5BGo).



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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).

 
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. 4Go). 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. 1Go.) and TRE-tk-CAT (downstream of SRE) reporter plasmids were cotransfected with the Id expression plasmids (Fig. 6Go). The Id sense had no effect on the SIE or TRE CAT activity (Fig. 6Go). This observation suggests that the regulation of the c-fos promoter by bHLH proteins is at the level of the SRE (Fig. 4Go).



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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.

 
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. 7AGo). As shown in Fig. 7BGo, 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. 7BGo). 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. 7BGo). 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. 7BGo (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.

 
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. 8AGo). In contrast, the addition of the E12/E47 antibody resulted in a single major supershifted band labeled 5 (Fig. 8AGo). 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. 7BGo.



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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.

 
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. 8BGo). This is in contrast to the Sertoli cell gel shift data shown in Fig. 7BGo. The single shifted band was displaced when excess unlabeled SRE was included in the binding reaction (Fig. 8BGo), 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. 8BGo). 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. 5BGo). This is in contrast to Sertoli cells in which the activity of mutSRE-EB reporter plasmid was significantly lower as compared with wtSRE (Fig. 5AGo). 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. 9Go). This gel shift was displaced by adding either excess SRE or E-box oligoneucleotides. The shifted band in Fig. 9Go may therefore correspond to band 1 observed in Fig. 7BGo. Gel shift data from Figs. 7BGo (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. 9Go). 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.

 
To further characterize the proteins binding to the SRE oligo, the gel shift (Fig. 9Go) 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. 10Go). 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. 10Go). This observation suggests that a multimeric complex involving bHLH proteins and SRF appears to cause the gel shift with SRE (Fig. 10Go). Control gel shifts with an Oct-1 oligonucleotide, mutSRE-EBoligo, or nonimmune serum showed no bands being detected in the blot (Fig. 10Go). 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. 11Go).



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Figure 10. Immunoblot of SRE Gel Mobility Shift Assay (Similar to Fig. 9Go) 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).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 7BGo 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. 8Go). 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. 7BGo). 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. 11Go). 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. 11Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Ham’s 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 (Ham’s 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 20–30% 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 70–100 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{gamma}-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 Student’s 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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