Cooperative Effects of STAT5 (Signal Transducer and Activator of Transcription 5) and C/EBP ß (CCAAT/Enhancer-Binding Protein-ß) on ß-Casein Gene Transcription Are Mediated by the Glucocorticoid Receptor

Shannon L. Wyszomierski and Jeffrey M. Rosen

Department of Molecular and Cellular Biology Baylor College of Medicine Houston, Texas 77030-3498


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ß-Casein gene transcription is controlled primarily by a composite response element (CoRE) that integrates signaling from the lactogenic hormones, PRL, insulin, and hydrocortisone, in mammary epithelial cells. This CoRE contains binding sites for STAT5 (signal transducer and activator of transcription 5) and C/EBPß (CCAAT/enhancer-binding protein-ß) and several half-sites for glucocorticoid receptor (GR). To examine how interactions among these three transcription factors might regulate ß-casein gene transcription, a COS cell reconstitution system was employed. Cooperative transactivation was observed when all three factors were expressed, but unexpectedly was not seen between STAT5 and C/EBPß in the absence of full-length, transcriptionally active GR. Cooperativity required the amino-terminal transactivation domain of C/EBPß, and neither C/EBP{alpha} nor C/EBP{delta} was able to substitute for C/EBPß when cotransfected with STAT5 and GR. Different GR determinants were needed for transcriptional cooperation between STAT5 and GR as compared with those required for all three transcription factors. These studies provide some new insights into the mechanisms responsible for high level, tissue-specific expression conferred by the ß-casein CoRE.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Composite response elements (CoREs) are found in the promoters of most genes and control spatially and developmentally regulated patterns of gene transcription (1, 2). CoREs are composed of groups of transcription factor-binding sites for both positively and negatively acting transcription factors that integrate signal transduction pathways. Interestingly, the signal transduction pathways and downstream transcription factors integrated by CoREs are generally not individually spatially or developmentally specific. Moreover, the level of transcription from a CoRE is typically greater than the transcriptional activity of each transcription factor alone (2). Although these defining properties, specificity of expression and enhanced level of transactivation, have been observed repeatedly from the CoREs in different genes, the mechanisms by which they are accomplished remain largely undetermined.

The regulation of ß-casein gene transcription is controlled primarily by a CoRE that integrates signaling from the lactogenic hormones, PRL, insulin, and hydrocortisone, in mammary epithelial cells (reviewed in Ref. 3). The individual transcription factors, which bind to and activate the ß-casein CoRE, have been well characterized in vitro, and in several cases in vivo analysis has been performed as well. Signal transducer and activator of transcription (STAT) 5, glucocorticoid receptor (GR), and CCAATT/enhancer binding protein-ß (C/EBPß) have been identified as important activators of transcription (reviewed in Ref. 4 ; schematic representations of the ß-casein proximal promoter are shown in Refs. 4, 5). The CoRE in the proximal promoter of the ß-casein gene contains a consensus and a nonconsensus binding site for STAT5, at least three binding sites for C/EBP family members, and several half-palindromic binding sites for GR (half- GREs) (6, 7, 8, 9, 10, 11). These half-GREs are closely interspersed with the binding sites for the other transcription factors. Although half-GREs are not the canonical elements known for eliciting GR responses, the importance of these elements in the ß-casein promoter has been unequivocally demonstrated by site-directed mutagenesis (7). Understanding how STATs, GR, and C/EBPs interact with each other and act in a concerted manner should provide a clearer understanding of how the ß-casein CoRE conveys high level, mammary-specific gene expression.

Efforts to understand the mechanism by which transcriptional activity is enhanced by the ß-casein gene CoRE were initiated by the analysis of STAT5 and GR interactions using a COS-7 cell reconstitution system. Direct protein-protein interactions of STAT5 and GR were demonstrated, resulting in transcriptional synergy at the ß-casein promoter (12). Two STAT5 proteins, STAT5a and STAT5b, which are encoded by different genes (13), are both capable of transcriptional synergy with GR (14) and are associated with GR in the mammary epithelium and HC11 mammary epithelial cells (15).

The C/EBPs are a family of transcription factors that contain an amino-terminal transactivation domain, which differs among family members, and a carboxy-terminal basic leucine zipper domain (bZIP) responsible for dimerization and DNA binding, which is more highly conserved among family members (16, 17). Multiple C/EBP isoforms can be generated from the intronless genes, which encode several different C/EBPs by either differential translation start site utilization (18) or selective proteolysis (19). For example, from a single C/EBPß mRNA, at least three transcripts can be translated, two activating isoforms called LAPs (originally identified as liver-enriched activating proteins) and one dominant negative isoform called LIP (originally identified as liver-enriched inhibitory protein) (20). ß-Casein gene expression is reduced 85% to 100% in mammary epithelial cells derived from C/EBP ß knockout (KO) mice (21, 22).

Interaction and transcriptional cooperation between C/EBPß and GR have been studied in transactivation of several genes, including {alpha}-1 acid glycoprotein, phosphoenolpyruvate carboxykinase (PEPCK), and herpes simplex virus thymidine kinase (HSV) (23, 24, 25, 26). Although GR has been shown to interact with C/EBPß, C/EBP{alpha}, and C/EBP{delta} (24, 25), transcriptional cooperation with GR was demonstrated to be specific for C/EBPß for at least two of these genes (PEPCK and HSV) (24, 26).

Transcriptional cooperation between C/EBPß and GR had not been studied previously in ß-casein transactivation. Additionally, transcriptional cooperation between STAT5 and C/EBP family members had not been examined in transactivation of any other gene to our knowledge. Studies were initiated to analyze the potential cooperative effects between STAT5, C/EBPß, and GR on ß-casein gene transcription. Transcriptional cooperation of STAT5, C/EBP family members, and GR on the ß-casein transactivation was shown to be specific to the LAP C/EBPß isoform. Unexpectedly, STAT5 and C/EBPß did not exhibit cooperative effects in the absence of GR. Transcriptional cooperation between the three proteins required full-length GR in a transcriptionally active state. The determinants for STAT5 and GR transcriptional cooperativity were also found to be different from those required for cooperation among all three proteins. These studies have helped elucidate some of the mechanisms involved in transcription factor cooperation in ß-casein transactivation and have provided a better understanding of how the ß-casein CoRE facilitates interactions and increased transcription not observed with the individual transcription factors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cooperative Transactivation Regulates ß-Casein Gene Expression
COS-1 cells express little or no endogenous STAT5, GR, and C/EBPß (S. L. Wyszomierski, unpublished observations). They, therefore, can be used as a versatile reconstitution system to study the combinatorial effects of these transcription factors on ß-casein reporter constructs without the complications of variable endogenous levels of these factors. A PRL receptor expression construct and reporter construct containing a luciferase gene driven by the –2,300/+490 sequences of the rat ß-casein gene were transiently cotransfected with different combinations of transcription factor expression constructs or the corresponding empty vectors. Reporter gene activity was measured after a desired hormone treatment was administered for 24 h. As shown previously, transfection of STAT5a or STAT5b into COS cells followed by PRL treatment leads to a 4- to 5-fold induction of ß-casein promoter activity that is not seen in the absence of STAT5 (Fig. 1CGo, lane 2, and Ref. 13). Under these conditions, cotransfection of STAT5a and GR followed by treatment with PRL and hydrocortisone (HC) gave a 55% ± 6% SEM increase in ß-casein promoter activity as compared with STAT5a alone (Fig. 1AGo, lane 5 and Fig. 1CGo, lane 8). Because of the critical role of C/EBPß on ß-casein gene expression observed in C/EBPß-deficient mammary epithelial cells (21, 22), studies of the potential cooperative effects between C/EBPß and STAT5 and between C/EBPß and GR at the ß-casein promoter were initiated. For these studies, an expression construct that expressed only the LAP isoforms of C/EBPß was used.



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Figure 1. Cooperative Regulation of ß-Casein Gene Transcription by STAT5, GR, and C/EBPß

A, Each transcription factor (50 ng), indicated by a "+", or 50 ng of the appropriate empty vector as a control, indicated by a "–", was transiently transfected into COS-1 cells in 35- mm wells. Treatment with either HC alone (black bars) or HC + PRL (striped bars) was performed for 24 h. A representative experiment is shown (lanes 1–8). Each treatment group was performed in triplicate. Error bars denote the SEM. All differences reported were statistically significant (P < 0.05). Fold induction (gray bars) was calculated vs. the level of transactivation seen with STAT5a alone without PRL treatment. Lanes 9–11 show the average fold induction from 40 experiments. Error bars denote the SEM. B, Increasing amounts of LAP were added in the presence of 50 ng of STAT5 and GR or empty vector plasmids. Amounts of LAP expression vectors were as follows: lanes 1, 5, and 11, 5 ng; lanes 2, 7, and 12, 10 ng; lanes 3, 8, and 13, 25 ng; lanes 4, 9, and 14, 100 ng; data not shown, 200 ng. The experiment was repeated three times. A representative experiment is shown. Each treatment group was performed in triplicate. Error bars denote the SEM. C, Different combinations of transcription factors were transfected and treated with different combinations of HC and PRL. Hormone treatments are indicated as follows: –HC –PRL, black bars; –HC +PRL, white bars; +HC –PRL, dark striped bars, +HC +PRL, light striped bars. This experiment was repeated nine times. A representative experiment is shown. Each treatment group was performed in triplicate. Error bars denote the SEM. All differences reported were statistically significant (P < 0.05).

 
Cotransfection of LAP alone into COS-1 cells resulted in no significant change in the level of basal transcription from the ß-casein reporter construct (Fig. 1AGo, lane 4). Surprisingly, cotransfection of STAT5a and LAP also did not lead to a further stimulation of ß- casein promoter activity over that found with STAT5a alone (Fig. 1AGo, lane 6). When all three transcription factors are cotransfected, however, an overall increase in the transcriptional activity was observed reproducibly (Fig. 1AGo, lane 8). With LAP present, an increase of 38% ± 6% SEM was seen over the level of transactivation imparted by STAT5a and GR in the absence of LAP, or 107% ± 10% SEM compared with STAT5 alone. In terms of fold induction, reporter gene transcription is increased 4.2-fold by STAT5a alone, 6.5-fold by STAT5a + GR, and 8.5-fold by STAT5a + GR + LAP after hormone treatment over the uninduced level (Fig. 1AGo, lanes 9–11). Therefore, the ability of LAP to cooperate with STAT5a was dependent on the presence of GR. The same response was observed if STAT5b was used in place of STAT5a (data not shown). Since no difference was seen between the two highly related STAT5 transcription factors for this response, all subsequent analyses were performed only with STAT5a.

To determine whether the lack of a cooperative effect between STAT5 and LAP was due to a limiting amount of LAP expression, titration experiments were performed. Varying amounts of LAP ranging from 5 ng to 200 ng were cotransfected alone, with STAT5a or with STAT5a + GR. At all concentrations of LAP tested, GR was necessary for the cooperative effects with STAT5a (Fig. 1BGo, lanes 11–14). Cooperativity between STAT5a and LAP was not seen in the absence of GR at any concentration of LAP (Fig. 1BGo, lanes 6–9). Additionally, LAP did not affect the basal level of transcription from the ß-casein promoter at any concentration (Fig. 1BGo, lanes 1–4).

Even before the specific transcription factors responsible for conveying these effects were identified, it was known that PRL and glucocorticoids are both essential for ß-casein gene expression (27, 28). Accordingly, the hormonal dependence of the cooperativity between STAT5, GR, and LAP was examined. In the absence of PRL, no induction of the ß-casein promoter was seen, regardless of HC treatment (Fig. 1CGo, odd numbered lanes). Consistent with previous observations, transcriptional cooperation between STAT5a and GR was dependent upon both HC and PRL (Fig. 1CGo, lanes 5–8). The cooperative transcriptional effects of STAT5a, GR, and LAP were also dependent on both HC and PRL (Fig. 1CGo, lanes 13–16). These results confirm that LAP addition to this reconstitution system mimics the in vivo hormonal requirements for ß-casein gene transcription.

The Role of the C/EBPß Transactivation Domain
The bZIP domain of C/EBPß is required for interaction of C/EBPß with GR (25), but the amino-terminal portions of C/EBPß are crucial for transcriptional cooperation with GR in PEPCK transactivation (26). In the COS cell reconstitution system, the transactivation domain of STAT5 is not required for the transcriptional cooperation between STAT5 and GR (7, 14). Accordingly, the role of the transactivation domain of C/EBPß in regulating transcriptional cooperativity in ß-casein transactivation by these factors was examined. LIP is a naturally occurring, dominant-negative isoform of C/EBPß (20). When LIP was cotransfected with STAT5a, an inhibition of transcription was observed (Fig. 2Go, lane 7). Inhibition by LIP also was observed when LIP was cotransfected with STAT5a and GR (Fig. 2Go, lane 10). Therefore, addition of GR does not circumvent the need for the C/EBPß transactivation domain in regulating ß-casein gene transcription. Furthermore, LIP inhibited cooperative transactivation by STAT5, GR, and LAP (Fig. 2Go, lane 11). These data are consistent with the observation that LIP markedly inhibited ß-casein gene expression in CHOk1 cells (our unpublished results). CHOk1 cells contain endogenous C/EBPß, STAT5, and GR and are one of the few nonmammary cell lines that can activate milk protein gene transcription without the addition of exogenous transcription factors (our unpublished results and Ref. 29).



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Figure 2. LIP Does Not Cooperate with STAT5 and GR

Each transcription factor (50 ng), indicated by a "+", or 50 ng of the appropriate empty vector as a control, indicated by a "–", was transiently transfected into COS-1 cells in 35- mm wells. All samples are +HC +PRL. The experiment was repeated three times. A representative experiment is shown. Each treatment group was performed in triplicate. Error bars denote the SEM. All differences reported were statistically significant (P < 0.05).

 
Next, to determine whether cooperative transactivation of the ß-casein promoter fragment was specific for C/EBPß as compared with other C/EBP family members, cotransfection experiments were performed with C/EBP{alpha} and C/EBP{delta} expression constructs. Neither C/EBP{alpha} nor C/EBP{delta} exhibited cooperative transactivation with STAT5a (Fig. 3AGo, lanes 2 and 4) or with STAT5a and GR (Fig. 3AGo, lanes 6 and 8). The same cooperative effect was seen with C/EBPß as compared with the C/EBPß/LAP- only expression construct (Fig. 3AGo, lane 7). This lack of activity of C/EBP{alpha} and C/EBP{delta} on the ß-casein promoter reporter construct was particularly interesting given that both C/EBPs were considerably more active than C/EBPß when their activity was compared using a multimerized C/EBP binding site [D9-CAT (chloramphenicol acetyltransferase)] reporter construct in COS-1 cells (data not shown). This finding of C/EBPß specificity is consistent with previous reports of the selective roles of different C/EBPs in mammary gland development (see Discussion).



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Figure 3. Transcriptional Cooperation Is Specific for C/EBPß and Requires Its Amino Terminus

Transfection was performed as described in Fig. 1Go. All samples are +HC +PRL. The experiment in panel A was repeated six times. The experiment in panel B was repeated three times. Representative experiments are shown. Each treatment group was performed in triplicate. Error bars denote the SEM. All differences reported were statistically significant (P < 0.05).

 
To further confirm that the N-terminal transactivation domain of C/EBPß was required for transcriptional cooperativity, expression constructs for C/EBP chimeras were obtained (30) containing the activation domain of C/EBP{alpha} fused to the b-ZIP domain of C/EBPß (C/EBP{alpha}ß) or the activation domain of C/EBPß fused to the b-ZIP domain of C/EBP{alpha} (C/EBPß{alpha}). Only C/EBPß{alpha} elicited a cooperative effect on ß-casein transactivation with STAT5a and GR (Fig. 3BGo, lane 6). Like C/EBP{alpha}, C/EBP{alpha}ß did not cooperate with STAT5a and GR (Fig. 3BGo, lane 5), and neither protein cooperated with STAT5a alone (Fig. 3BGo, lanes 1–3). Taken together, these results indicate that cooperative transcriptional regulation of ß-casein promoter activity with STAT5 and GR requires, and is specific to, the N-terminal transactivation domain of C/EBPß.

Regions of GR Needed for Transcriptional Cooperation
The DNA binding domain (DBD) of GR is required for the protein-protein interaction with C/EBPß (24), and the transactivation function, TAF-2, in the ligand binding domain of GR is required for transcriptional cooperation with C/EBPß (24, 25). Transcriptional cooperation with STAT5 requires the N-terminal portions of GR (14). A protein-protein interaction domain in GR for STAT5 interaction has not been mapped, but is thought to reside in TAF-1 (see Fig. 4Go). Given these observations, experiments were undertaken to determine whether both portions of the GR molecule would, therefore, be required for cooperative activation of the ß-casein reporter construct. Expression constructs for N- and C-terminal truncations of GR (31) were expressed at comparable levels to the full-length GR in COS-1 cells (Fig. 4Go, lane 3 for GR 407–795). The immunoblot shown in Fig. 4Go was probed with an anti-GR antibody recognizing a C-terminal epitope. This antibody (Fig. 4Go, lane 2), therefore, did not detect GR 3–556. An antibody to an epitope in the DBD of GR was used to verify GR 3–556 production (data not shown).



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Figure 4. Wild-Type and Mutant GRs Employed in Transient Transfection Assays

Wild-type and mutant GR proteins are represented schematically. The following abbreviations are used: TAF, transactivation domain/function; LBD, ligand binding domain. An X indicates a point mutation. Twenty five micrograms of protein extract were used per lane. Immunoblotting was performed using the anti-GR (P-20) antibody from Santa Cruz Biotechnology, Inc. Black arrows indicate GR proteins. Open arrowheads indicate nonspecific cross-reactive material. The proteins examined are as follows: Lane 1, wild-type GR in pSTC vector; lane 2, GR 3–556; lane 3, GR 407–795; lane 4, GR C482S; lane 5, wild-type GR in pCR3.1 vector, lane 6, GR 108–317{Delta}; lane 7, G30IIB; lane 8, nontransfected COS cell extract.

 
Consistent with previously published results (14), GR 3–556 was capable of transcriptional cooperativity with STAT5a alone, while GR 407–795 was not (Fig. 5AGo, lanes 5 and 7). Because GR 3–556 is constitutively active, it gave a higher level of transactivation than wild-type GR when cotransfected with STAT5a (Fig. 5AGo, lane 2 vs. lane 5); however, further transactivation was not seen upon addition of LAP (Fig. 5AGo, lane 6). Addition of LAP also did not influence the lack of transcriptional cooperation between STAT5a and GR 407–795 (Fig. 5AGo, lane 8). These data indicate that the regions of GR that are necessary for transcriptional cooperation between STAT5 and GR and between C/EBPß and GR in pairs are also necessary for transcriptional cooperation between all three transcription factors on the ß-casein promoter.



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Figure 5. Regions of GR Required for Transcriptional Cooperation

Transfection was performed as described for Fig. 1Go. The type of GR, wild-type (wt) or mutant (mutants are explained in Fig. 4Go) is indicated below each graph. All samples are +HC +PRL. The experiments were repeated three times. A representative experiment is shown for each. Treatment groups were performed in triplicate. Error bars denote the SEM. All differences reported were statistically significant (P < 0.05).

 
To further examine the importance of the protein-protein interactions between these transcription factors, a GR mutant, GR C482S, which is not capable of binding to DNA due to a point mutation in the second zinc finger of the DBD (Fig. 4Go, lane 4), was used. This point mutation diminished the level of transcriptional cooperativity between STAT5a and GR (Fig. 5BGo, lane 3; in agreement with Refs. 7, 14). Abolishing DNA binding of GR did not, however, effect transcriptional cooperation between STAT5a, GR, and LAP (Fig. 5BGo, lane 5). Although the independence of GR transcriptional effects from the DNA binding activity of GR may be a result of the COS cell overexpression system (W. Doppler, personal communication), the fact that the transcriptional cooperativity still occurs emphasizes the importance of the protein- protein interactions of GR with STAT5 and C/EBPß in regulating ß-casein gene expression.

The Role of Transactivation by GR
The data thus far suggested several possible mechanisms for the observed GR- dependent transcriptional cooperation. One possibility was that GR was playing a structural role. For example, GR might act as a bridging molecule between STAT5 and C/EBPß, thereby helping to provide a favorable conformation for cooperative transactivation. Alternatively, GR could play a transactivational role dependent on the presence of C/EBPß. To differentiate between these possibilities, the GR antagonist RU486 and transactivation-deficient GR mutants were used.

RU486 allows DNA binding of GR but blocks transactivation by keeping the C-terminal TAF-2 domain of GR in a conformation unable to interact with the rest of the transcriptional machinery through coactivators (32). Surprisingly, when STAT5a and GR were cotransfected and RU486 treatment was performed, the same level of transactivation was seen as with HC treatment (Fig. 6Go, lanes 3 and 4). However, when STAT5a and GR were cotransfected with LAP and treated with RU486, no additional transactivation occurred (Fig. 6Go, lane 6). GR was cotransfected with a mouse mammary tumor virus (MMTV)-reporter gene to verify that RU486 did not activate consensus GREs in this cell system. As expected, RU486 did not activate the MMTV-reporter construct and inhibited HC activation as well (Fig. 6Go, gray bars, lanes 7–10). These data suggest that transactivation by GR is an important aspect of the transcriptional cooperativity between STAT5, GR, and C/EBPß.



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Figure 6. RU486 Permits Transcriptional Cooperation between STAT5 and GR but Not STAT5, GR, and C/EBPß

Transfection was performed as described for Fig. 1Go. Black bars indicate ß-casein promoter activity (lanes 1–6). These samples are all +PRL. Gray bars indicate MMTV promoter activity (lanes 7–10). These samples are all –PRL. Treatment with HC or RU486 is indicated below the graph. H, HC; R, RU486; HR, HC + RU486. The ß-casein portion of the experiment was repeated seven times. The MMTV portion of the experiment was repeated twice. A representative experiment is shown. Each treatment group was performed in triplicate. Error bars denote the SEM. All differences reported were statistically significant (P < 0.05). Note: The small induction seen with RU486 on MMTV (lane 9) was not statistically significant over the basal (lane 7).

 
To complement these studies using RU486, TAF-1 mutants of GR deficient in transactivation were obtained. The TAF-1 domain of GR has been shown not only to be essential for activation of transcription by GR on many promoters, but is also necessary for repression of AP-1 transcriptional activity by GR (33). The two GR mutants used are shown schematically in Fig. 4Go. GR 108–317{Delta} contains a deletion that eliminates the core of the TAF-1 domain. It is not transcriptionally active and cannot repress AP-1-dependent transcription. GR30IIB contains three point mutations, which were identified using a large-scale mutagenesis and screening strategy in yeast. Mutation of these three amino acids severely compromises transactivation by GR but does not affect GR repression of AP-1-dependent transcription (34). The fact that the repressive ability of GR is maintained suggests that the overall structure of the protein is not severely altered by the point mutations. Both proteins were expressed at levels comparable to the wild-type GR in COS-1 cells (Fig. 4Go, lanes 6 and 7). In the absence of LAP, GR30IIB was able to cooperate transcriptionally with STAT5a similar to wild-type GR, while GR 108–317{Delta} could not (Fig. 7Go, lanes 4 and 6). Neither GR mutant was capable of transcriptional cooperativity with STAT5a and LAP (Fig. 7Go, lanes 5 and 7). Thus, the use of the GR antagonist RU486 and GR mutants containing TAF-1 mutations revealed that the transcriptional activity of GR is not necessary for cooperative transactivation between STAT5 and GR. However, transactivation by GR is an essential component of the transcriptional cooperativity between STAT5, GR, and C/EBPß at the ß-casein promoter.



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Figure 7. The TAF-1 Domain of GR Is Required for Transcriptional Cooperation with STAT5 and C/EBPß

Transfection was performed as described for Fig. 3Go. All samples are +HC +PRL. The experiment was repeated twice. A representative experiments is shown. Each treatment group was performed in triplicate. Error bars denote the SEM. All differences reported were statistically significant (P < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Despite the fact that STAT and C/EBP sites are found in close proximity in a number of promoters in addition to the ß-casein promoter, including the oncostatin M and serine protease inhibitor-3 promoters (35, 36), before these studies, transcriptional cooperativity between STAT5 and C/EBPß had not been reported. Transcriptional cooperativity between C/EBPß and GR had also not been studied with respect to ß-casein transactivation. These analyses revealed that transcriptional cooperativity between STAT5, GR, and C/EBPß is dependent on transcriptionally active GR. In addition, this enhancement of ß-casein transcription was specific for C/EBPß, since cooperative transactivation was not observed with either C/EBP{alpha} or C/EBP{delta}. The results indicate that the domains of GR necessary for interaction and transcriptional cooperativity with STAT5 and C/EBPß individually were also required for cooperativity between all three proteins. Finally, different determinants are required for cooperativity between STAT5 and GR alone as compared with STAT5, GR, and C/EBPß together.

The experiments in this study were performed using a COS-1 cell reconstitution system. The advantage of this system is its versatility. Endogenous STAT5, GR, and C/EBPß were not detected by Western blotting before transfection even when 100 µg of total cellular protein were analyzed. After transfection, the protein levels of each transcription factor were easily visualized by Western blotting from as little as 5 µg of total cellular protein. Additionally, no increase in reporter gene activity was detected after PRL or hydrocortisone treatment in the absence of exogenously added transcription factors (demonstrated in Fig. 1AGo, lane 1, and Fig. 2Go, lane 1, and in data not shown). Any effects observed on ß-casein transactivation were entirely dependent on the transfection of exogenous transcription factors. This allowed the comparison of combinatorial effects of intact transcription factors with the analysis of the transcription factors containing deletions and point mutations, as well as with chimeric proteins. One limitation of using this reconstitution system is that a lower level of hormonal induction of ß-casein promoter activity was observed than has been reported in other cell systems (10, 27, 29). A second limitation is that the overexpression of these transcription factors may force interactions not necessarily sufficient at lower endogenous levels of these same transcription factors. High expression may help stabilize protein-protein interactions and obviate the need for weaker protein-DNA interactions (W. Doppler, personal communication). Although it may not be possible for a reconstitution system to mimic all aspects of an in vivo process, the results reported herein correlate well with observations obtained from studies performed using mammary epithelial cells, mammary gland extracts, and knockout mice (4).

This finding that C/EBPß transactivation of ß-casein is GR dependent agrees with previous studies. In a cytotoxic T cell line, glucocorticoid induction of ß- casein promoter activity was dependent on the region of the CoRE containing the C/EBP binding sites (37). Cooperation with STAT5 and GR was specific to C/EBPß. C/EBP{alpha} and C/EBP{delta} could not substitute for C/EBPß. This is consistent with results obtained in mouse models and in experiments using mammary epithelial cell lines. Four C/EBP binding sites were found in the ß-casein proximal promoter. Mutation of these binding sites severely decreased ß-casein promoter activity in stably transfected HC11 mammary epithelial cells. C/EBPß was the predominant protein in extracts from HC11 cells that bound to the C/EBP sites (6). In mice, deletion of C/EBPß severely decreased ß-casein gene expression, while deletion of C/EBP{alpha} exhibited no effect on ß-casein gene expression (22). Although some binding of C/EBP{delta} to the ß-casein CoRE was detected using extracts from HC11 cells, this interaction was minor compared with C/EBPß (6). Additionally, C/EBP{delta} levels are highest in the mammary gland during involution, a stage when ß-casein expression is down-regulated (38, 39). The ß-casein promoter is one of only a few promoters on which specificity for a C/EBP family member has been reported. Transcriptional cooperation with GR is specific for C/EBPß in transactivation of the PEPCK and HSV promoters as well (24, 26). Synergy with the Sp-1 transcription factor on the CYP2D5 promoter is also specific for C/EBPß (40). These data suggest that one way of conferring specificity for individual C/EBP family members is obligate interaction and cooperation with other proteins at the promoter.

LIP, the dominant negative isoform of C/EBPß, inhibited ß-casein transactivation both in the presence and absence of LAP. The mechanisms of the ß-casein gene repression by LIP are particularly interesting because LIP expression in the mammary gland is high during pregnancy and is severely decreased during lactation (8). These data strongly suggest physiological relevance for repression of the ß-casein gene by LIP. Because LIP has a greater DNA binding affinity than LAP (20), it is likely that LIP inhibits ß-casein transactivation by binding to the C/EBP binding sites in the CoRE, preventing LAP from binding. Elucidation of the mechanism of ß-casein repression by LIP in the absence of LAP requires further experimentation. Several possibilities exist. In the CoRE of the rat ß-casein proximal promoter, one of the C/EBP binding sites overlaps with a nonconsensus STAT5 binding site. This site alone does not bind STAT5 with high affinity (6, 8) but may bind STAT5 as a tetramer in cooperation with the consensus STAT5 binding site. Tetramerization of STAT5 on suboptimal DNA binding sites has been demonstrated for many other promoters (41, 42, 43). Therefore, LIP binding to the overlapping C/EBP site may inhibit transactivation by STAT5 by preventing formation of a tetrameric STAT5 complex. Additionally, the ß-casein proximal promoter contains a Yin Yang-1 (YY1) binding site known to be important for repression of ß-casein gene transcription (44, 45). LIP interacts directly with YY1 (46) and YY1 interacts with several histone deacetylases (47, 48). Therefore, LIP may contribute to the recruitment of histone deacetylases and active repression of ß-casein transactivation.

One surprising result of the experiments described herein was the finding that transcriptional cooperation between STAT5 and GR in the absence of C/EBPß did not require transactivation by GR. RU486-bound wild-type GR and GR with point mutations in TAF-1 did not eliminate the increase in transactivation observed when GR was cotransfected with STAT5 as compared with activation by STAT5 alone. Deletion of amino acids (a.a.) 108–317 of GR did, however, eliminate the cooperative effect. Although coimmunoprecipitation experiments are required for confirmation, this further maps the interaction domain between STAT5 and GR within the region previously shown to be essential (a.a. 1–407) (14). These results suggest that cooperative transcription between STAT5 and GR in this system is the result of a structural effect rather than a transactivational effect of GR. Binding of STAT5 and GR to their adjacent elements on this promoter (half-GREs for GR) may strengthen the STAT5 interaction with the ß-casein promoter, allowing it to exert increased transcriptional effects. This may either result in or be a result of prolonged tyrosine phosphorylation of STAT5 (49). GR also may be mediating chromatin remodeling events, such as those that have been shown to take place on the MMTV promoter even if GR is bound to RU486 (50). However, this seems less likely since the reporter genes in these experiments were transiently introduced into the cells rather than stably integrated into the chromatin.

There are several nonexclusive, testable models that may explain the effects of GR on transcriptional cooperativity between STAT5 and C/EBPß. One model predicts that a component of GR-dependent C/EBPß activation is a required interaction of C/EBPß with GR to help relieve an inhibitory conformation of C/EBPß. Experiments using the D9-CAT reporter gene construct, driven by multimerized C/EBP binding sites (data not shown), support this hypothesis. Using this reporter construct, C/EBPß also exhibited very little activity in COS-1 cells in the absence of GR, but exhibited increased activity when GR was present. The amino-terminal transactivation domain of C/EBPß contains two repression domains, which, through an intramolecular interaction, inhibit transactivation and may decrease the DNA binding of C/EBPß (51, 52). This repression can be relieved by phosphorylation of C/EBPß via several kinase-mediated cascades including ras- activated MAPK cascades (51, 52, 53). This inhibitory conformation can also be relieved by interaction with other proteins (proposed in Refs. 51, 52). The relief of C/EBPß repression by protein-protein interactions has been convincingly demonstrated with the myb protein on the mim-1 promoter. C/EBPß and myb interact (54) and exhibit transcriptional synergy on the mim-1 promoter (55, 56). In CV-1 cells, C/EBPß bound to its cis-regulatory element but was inactive on the mim-1 promoter in the absence of myb. In the presence of myb, transcriptional synergy was seen (57).

The N terminus of C/EBPß interacts with the E1A region of p300/CBP (58). It is likely that this interaction requires the open, non repressed conformation of C/EBPß. In CV-1 cells, p300/CBP exerted minimal effects on the mim-1 promoter with C/EBPß alone. When constitutively activated ras was cotransfected, p300/CBP enhanced transcription by C/EBPß on the mim-1 promoter. Cotransfection of myb without ras allowed the same enhancement by p300/CBP to occur, and the transcriptional synergy previously observed by C/EBPß and myb was greatly enhanced (59). Therefore, a common theme emerges that may explain some of the specificity of expression from a CoRE. Interaction of another protein acting on the CoRE with C/EBPß may relieve the inhibitory conformation of C/EBPß and allow recruitment of transcriptional activators such as p300/CBP.

Another model predicts that the transactivation function of GR contributes a second component to the GR dependence of C/EBPß activation of ß-casein. It appears unlikely that changing the C-terminal conformation of GR by binding RU486 and mutating the N-terminal transactivation domain (TAF-1) would both disrupt the interaction between GR and C/EBPß. Nevertheless, both methods of eliminating GR transactivation abolished transcriptional cooperativity with C/EBPß and STAT5. Additionally, using the D9-CAT reporter, the GR30IIB mutant activated C/EBPß to a similar extent as wild-type GR (data not shown). One possibility is that C/EBPß and GR recruit a coactivator complex together that neither can effectively recruit alone. Boruk et al. (24) have proposed another mechanism to explain the transcriptional cooperativity. They theorized that C/EBPß recruits an activation complex to the HSV promoter after which the activity of this activation complex is enhanced by TAF-2 of GR. This enhancement could be independent of GR binding to DNA or interaction with C/EBPß. It should be noted that this mechanism and the coactivator mechanism postulated above are not mutually exclusive.

Because C/EBPß and GR cannot activate ß-casein gene transcription in the absence of STAT5, cooperativity between a STAT5-recruited activation complex and an activation complex recruited jointly by C/EBPß and GR seems likely. It has been demonstrated that multiple coactivator activities are required for transactivation by retinoic acid receptor, hepatic nuclear factor-1, and NF-{kappa}B (60, 61, 62). It is very likely, therefore, that the recruitment of multiple coactivators is necessary for high level transactivation of many genes. CoREs may accomplish this by using multiple transcription factors as a way to impart specificity of gene expression. Analysis of the coactivators recruited by STAT5, GR, and C/EBPß to the ß-casein promoter and their contribution to transactivation is an important area of future investigation.

There are still some aspects of regulation of the ß-casein CoRE observed in vivo that cannot be readily explained by these data. In STAT5a-deficient mice, the level of activated STAT5b is severely reduced, yet ß-casein expression is only marginally affected (63). Analysis of ß-casein expression in STAT5a- and STAT5b-deficient mammary gland transplants has not yet been reported, so it is still not known whether a small amount of STAT5 is sufficient for transactivation in vivo. In contrast, transactivation in cell culture systems is highly dependent on STAT5 (9, 13). Decreasing the level of STAT5 in the COS-1 reconstitution system severely decreased ß-casein transactivation even in the presence of GR and C/EBPß (data not shown). In the reconstitution system, transactivation also is observed in the absence of C/EBPß, while in C/EBPß-deficient mammary epithelial cells, ß-casein expression is severely reduced (by 85–100%) (21, 22). One possibility is that C/EBPß may play an additional role in ß-casein transactivation before formation of the hypothesized activation complexes. C/EBPß was recently found to interact with the SWI/SNF complex, an ATP-dependent chromatin remodeling complex (64), and this may be one possible explanation for the discrepancies between the in vivo and cell culture observations. Modification of the reconstitution system to examine the effects of STAT5, C/EBPß, and GR on the ß-casein transactivation with the reporter construct stably integrated into the chromatin may provide further information on how these transcription factors act on the ß-casein CoRE. Additionally, analysis of GR-deficient mammary epithelial cells may help confirm the importance of GR in transactivation from the ß- casein CoRE.

In summary, several unique roles for GR at the CoRE located in the ß-casein proximal promoter have been observed. GR appears to promote the formation of an activated conformation of C/EBPß as well as prolong the activated state of STAT5 (49). Additionally, it is likely that GR, C/EBPß, and STAT5 together recruit an activation complex to the ß-casein CoRE that cannot be efficiently recruited by any of the transcription factors individually. Reconciliation of the differences between the in vivo and cell culture observations and analysis of the composition and assembly of the proposed activation complexes are important avenues for future investigation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
For the majority of the studies reported herein, the –2,300/+490 ß-casein promoter subcloned into pGL2 luciferase vector (Promega Corp., Madison, WI) was used as the reporter construct. The responsiveness of the –2,300/+490 ß-casein promoter has been reported previously (27, 65). Two other reporter genes were used as well: D9-CAT, which contains multiple copies of the C/EBP binding site from the albumin promoter, which was kindly provided by Dr. Ueli Schibler (University of Geneva, Geneva, Switzerland) and MMTVLUC. Both have been previously described (20, 66, 67). PCMVßGAL was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). The PRL receptor expression vector, pECE-PRL-R-L, which was kindly provided by Dr. Paul Kelly (INSERM Unité 344 Paris, France) and the expression vectors for STAT5a and STAT5b, pRcCMVSTAT5a and pcDNA3STAT5b, have been previously described (49, 68). Expression vectors for C/EBPß-LAP and C/EBPß-LIP, pSCTLAP{Delta}3, and pSCTLIP, were kindly provided by Dr. Ueli Schibler and have been previously described (20). For the comparative experiments, we used expression vectors for C/EBP{alpha}, ß, and {delta} , which were potentially capable of generating multiple isoforms if the alternative downstream translation start sites were employed, because expression vectors capable of producing only activating isoforms of C/EBP{alpha} and C/EBP{delta} were not readily available. Expression vectors for C/EBP{alpha}, C/EBPß, and C/EBP{delta} in the pSV2sport expression vector as well as expression vectors for chimeric fusion proteins C/EBP {alpha}ß and C/EBP ß{alpha} were kindly provided by Drs. Gerald Elberg and Sophia Tsai (Baylor College of Medicine) and have been previously described (30). These cDNAs were subcloned into the pSCT expression vector using restriction enzyme sites in the multiple cloning sites of both vectors. The original cDNAs of C/EBP {alpha}, C/E 66 Pß, and C/EBP{delta} were kindly provided to Drs. Elberg and Tsai from Dr. Steven McKnight (University of Texas Southwestern Medical Center, Dallas, TX). Dr. Rainer Lanz (Baylor College of Medicine) kindly provided the pSTC and pSCT vectors. Wild-type GR in the pSTC vector and the following GR mutant constructs, pSTC GR 3–556, pSTC GR 407–795 (X-795), and pSTC GR C482S, were kindly provided by Dr. Rainer Lanz and Dr. Sandro Rusconi (University of Fribourg, Fribourg, Switzerland) and have been previously described (69). Wild-type GR in the p6R vector and GR 108–317{Delta} and GR30IIB in the same vector were kindly provided by Drs. Jorge Iniguez-Lluhi and Keith Yamamoto (University of California San Francisco) and have been previously described (34). Because the Rous sarcoma virus (RSV) promoter drove these vectors and RSV is C/EBPß responsive, these cDNAs were subcloned into the pCR3.1 expression vector (Invitrogen, Carlsbad, CA), which has a CMV promoter like the vectors used for all the other transcription factors. All plasmids were purified using QIAGEN DNA maxi-prep kits (QIAGEN, Valencia, CA).

Cell Culture, Transient Transfection, and Reporter Gene Assays
DMEM, trypsin-EDTA, donor horse serum, and glutamine were purchased from JRH Biosciences (Lenexa, KS). FBS was purchased from Summit Biotechnologies (Fort Collins, CO). Gentamicin, insulin, and hydrocortisone were purchased from Sigma (St. Louis, MO). RU486 was obtained from Roussel/UCLAF (Romainville, France). Ovine PRL (lot AFP10692C) was kindly provided by the National Hormone and Pituitary program (Bethesda, MD). COS-1 cells were obtained from the ATCC (Manassas, VA). COS-1 cells were routinely passaged in DMEM + 10% FBS in the presence of gentamicin. COS-1 cell transfections were performed 1 day after passaging the cells into the 35-mm wells of six-well tissue culture plates. Transfection was performed using Superfect Reagent (QIAGEN). In each well, 50 ng of pCMVßgal, 50 ng of pECEPRL-R-L, and 200 ng of -2,300/+490 ß- casein LUC were transiently cotransfected with different combinations of transcription factor expression constructs or the corresponding empty vectors as controls. Usually, 50 ng of each transcription factor were used; 2 µg total DNA and 10 µl of Superfect were used per 35-mm well. Transfections were performed according to the manufacturer’s instructions. During transfection and thereafter, the cells were maintained in DMEM + 10% charcoal-stripped horse serum with gentamicin and 5 µg/ml insulin. Twenty-four hours after transfection, treatment with hydrocortisone (1 µg/ml), RU486 (1 x 10-7 M) and/or ovine PRL (1 µg/ml) was performed for 24 h as indicated. Luciferase assays were performed by standard methods on a MLX microtiter plate luminometer (Dynex Technologies, Chantilly, VA). Luciferin was purchased from Molecular Probes, Inc. (Eugene, OR) and used to make substrate containing 1 mM luciferin, 0.1 M Trizma phosphate, 12 mM MgCl2, and 2.4 mM ATP. ß-Galactosidase assays were performed by standard protocols (70). O- Nitrophenyl ß-D-galactopyranoside was purchased from Sigma. CAT assays were performed using a CAT enyzme-linked immunosorbent assay (ELISA) kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s instructions.

Statistical Analysis
Univariate ANOVA was used to test for equality of mean relative light units (RLU)/ßGAL values across treatment groups. The null hypothesis was that all treatments had the same mean values of RLU/ßGAL. ANOVA runs were performed using data from triplicate samples in treatments from a single experiment with necessary Bonferroni corrections to P values based on the number of multiple tests. ANOVA runs were also performed using data from all treatments in all experiments combined. For these analyses, fitted values of marginal means and their SEs were used in hypothesis tests for equal means, with necessary Bonferroni corrections to P values based on the number of multiple tests. Analysis was performed using the SPSS statistical software package (SPSS Version 10, SPSS, Inc., Chicago, IL).

Antibodies and Western Blot Analysis
SDS-PAGE and Western blot analysis was performed by standard protocols that have been previously described (49). STAT5a and GR were separated on 7.5% running gels and the C/EBPs were separated on 12% running gels. Affinity purified rabbit polyclonal anti- STAT5a antibody has been previously described (68). The following anti-GR antibodies were used: rabbit polyclonal anti-GR (P-20) TransCruz antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:2,000 dilution and the monoclonal anti-GR antibody, BuGR2 (Affinity BioReagents, Inc. Golden, CO) at a 1:600 dilution. Rabbit polyclonal antibodies anti-C/EBP{alpha} (14AA), C/EBPß (C-19), and C/EBP{delta} (C-22) from Santa Cruz Biotechnology, Inc. were used at a 1:1,000 dilution to detect those proteins.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Leif Peterson for assistance and advice in statistical analysis of the data presented herein. The authors thank Drs. Li-yuan Yu-Lee, Rainer Lanz, and Michelle Kallesen for critical reading of the manuscript and Ms. Alvenia Daniels for secretarial assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Jeffrey M. Rosen, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030-3498. E-mail: jrosen{at}bcm tmc.edu.

This work was supported by NIH Grant CA-16303.

Received for publication September 15, 2000. Revision received November 6, 2000. Accepted for publication November 9, 2000.


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