p300/CREB-Binding Protein Enhances the Prolactin-Mediated Transcriptional Induction through Direct Interaction with the Transactivation Domain of Stat5, but Does Not Participate in the Stat5-Mediated Suppression of the Glucocorticoid Response

Edith Pfitzner, Ruth Jähne, Manuela Wissler, Elisabeth Stoecklin and Bernd Groner1

Institute for Experimental Cancer Research Tumor Biology Center D-79106 Freiburg, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stat5 was discovered as a PRL-induced member of the Stat (signal transducer and activator of transcription) family. Its induction by many other cytokines and interleukins suggests that Stat5 plays a crucial role not only in mammary epithelial, but also in hematopoietic cells. Cell type- and promoter- specific functions of Stat5 are most likely modulated by the interaction with other transcription factors. We recently showed cross-talk between Stat5 and the glucocorticoid receptor. The activated glucocorticoid receptor forms a complex with Stat5 and enhances Stat5-mediated transcriptional induction. Conversely, Stat5 diminishes the induction of glucocorticoid-responsive genes. Here, we investigated the role of p300/CBP(CREB-binding protein), a transcriptional coactivator of several groups of transcription factors, in Stat5-mediated transactivation and in the cross-talk between Stat5 and the glucocorticoid receptor. p300/CBP acts as a coactivator of Stat5. Its ectopic expression enhances PRL-induced Stat5-mediated transcriptional activation. Consistent with this observation, we find that the adenovirus E1A protein, which binds to p300/CBP, suppresses Stat5-induced transcriptional activation. This inhibition requires the Stat5 transactivation domain and the p300/CBP binding site of E1A. Coimmunoprecipitation and mammalian two-hybrid assays demonstrate a direct interaction between the carboxyl-terminal transactivation domain of Stat5 and p300/CBP. p300/CBP also positively interacts with the glucocorticoid receptor and enhances glucocorticoid receptor-dependent transcriptional activation of the mouse mammary tumor virus-long terminal repeat promoter. Overexpression of p300/CBP does not counteract the Stat5-mediated inhibition of glucocorticoid receptor-dependent transactivation, i.e. the repression of the glucocorticoid response by Stat5 is not a consequence of competition for limiting amounts of p300/CBP.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Studies on mammary epithelial cells have provided important insights on the coordinate action of steroid and peptide hormones (1, 2, 3). Milk protein gene expression in mammary epithelial cells is regulated by the lactogenic hormones, PRL, insulin, and glucocorticoids. The signal transduction pathways used by these hormones and their interactions at the level of gene transcription have been investigated (4, 5). Binding of PRL to its receptor at the cell surface induces receptor dimerization and activation of the cytoplasmic Janus kinase 2 (Jak2) (6, 7, 8). Jak2 phosphorylates and activates Stat5, a transcription factor that belongs to the family of signal transducers and activators of transcription (Stat) (8, 9, 10, 11). Phosphorylation of tyrosine residue 694 causes Stat5 dimerization and confers specific DNA-binding ability to promoter sequences in target genes. GAS-sites ({gamma}-interferon activated sites) in the promoter or upstream enhancer regions of several milk protein genes (ß-casein, ß-lactoglobulin, and whey acidic protein) are required for maximal transcription during lactation and PRL induction (12, 13, 14, 15).

Stat5, originally called mammary gland factor (MGF), was discovered as a regulator of the ß-casein gene promoter that binds to the ß-casein gene promoter GAS site (15) and was cloned from sheep tissue as MGF/Stat5 (16). Two highly related Stat5 genes, Stat5a and Stat5b, have been found in mouse and human DNA (17, 18, 19). The encoded proteins are greater than 95% identical, form heterodimers after phosphorylation and activation, and are both able to confer the PRL response to the ß-casein gene promoter (18).

Glucocorticoids, in concert with PRL and insulin, induce the transcription of the ß-casein gene in vivo and in HC11 mammary epithelial cells in vitro (20, 21). Glucocorticoids act through the glucocorticoid receptor (GR), a member of the nuclear receptor family of ligand-inducible transcription factors. Binding of glucocorticoid hormone leads to activation of the latent receptor, dimerization, nuclear translocation, specific DNA-binding to glucocorticoid-response elements, and transcriptional activation (22, 23). GR also acts as a negative transcriptional regulator and cross-couples to other transcription factors like AP-1 (Jun/Fos) or nuclear factor (NF)-{kappa}B (24, 25). Nuclear receptors must interact with additional factors such as SRC-1, TRIP-1, RIP-140/160, TIF-1, SMART, and N-CoR to mediate both activation and repression of gene expression (26, 27).

The p300/CBP (CAAT-binding protein) proteins represent a family of transcription coactivators that potentiate the activity of several groups of transcription factors by interaction with their activated forms (28, 29). This was originally observed for the protein kinase A-activated form of CREB (cAMP response element-binding protein) (30, 31, 32) and the adenovirus E1A protein (33) and has been extended to other transcription factors (e.g. ATF, c-Jun, c-Fos, c-Myb, Sap-1, tax, Myo D, P/CAF, SRC-1, NF-{kappa}B (28, 29). Most important for the studies described here, p300/CBP has been shown to be a coactivator of the GR and also of the interferon-{gamma}- and interferon-{alpha}-induced members of the Stat family, Stat1 and Stat2 (34, 35, 36, 37). The different Stat proteins (Stat1, 2, 3, 4, 5a, 5b, 6) are mainly related due to their similar mode of action by various cytokines (10). Their sequence homologies and functional relatedness are rather limited. Knock-out mice, in which Stat1, Stat5a, or Stat5b have been inactivated, indicate absolutely specialized functions for the different members of the Stat family (38, 39, 40, 41). Sequence comparison showed that the carboxyl-terminal transactivation domains are the most dissimilar parts of the different Stat molecules (42). Various Stat proteins might therefore contact distinct coactivators or members of the transcription machinery for transcriptional activation. p300 and CBP are similar in sequence and functionally interchangeable in most cases. p300 exhibits histone acetyltransferase activity and associates with a protein with the same enzymatic activity (P/CAF) (43, 44, 45). Accumulation of histone acetyltransferase at specific genomic sites has been implicated in the induction of gene expression through the remodeling of chromatin structure (46, 47). CBP also plays a role in the negative cross-talk between the GR and AP-1. Nuclear receptors, including the GR, interact with CBP and inhibition of GR-mediated AP-1 activity results from competition for limiting amounts of p300/CBP (35, 36).

The analysis of the mechanism by which PRL and glucocorticoid hormones synergize in their transcriptional control has revealed a direct interaction between Stat5 and the GR (48, 49). This interaction results in an enhancement of Stat5-mediated transcriptional activation and an inhibition of GR-dependent transcription. We further investigated the molecular mechanisms of this transcription factor interaction and the potential role of p300/CBP. Our results show that p300 directly interacts with the transactivation domain of Stat5 and enhances PRL-induced transcriptional activation. This can be observed with Stat5, mStat5a, and mStat5b. Cytokine-activated, but not latent, Stat5 is able to interact with p300/CBP in vitro. The adenoviral p300-binding protein E1A inhibits Stat5- mediated transcriptional induction. The inhibition can be overcome by overexpression of p300. In contrast, Stat5 inhibition of glucocorticoid-mediated transactivation cannot be restored by overexpression of p300. These results indicate that repression of the glucocorticoid response by Stat5 is not the result of competition for limiting amounts of p300, but is most likely due to the direct complex formation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
p300 Enhances PRL-Induced Transcription by MGF/Stat5, Mouse (m)Stat5a, and mStat5b
p300, a protein with intrinsic histone acetylase activity, has been found to be a crucial contributor to the activation of transcription by diverse classes of exogenously regulated transcription factors (29). Stat5 is a member of a gene family that regulates the response to cytokines and was initially identified in the study of PRL action. We have investigated the functional and physical interactions between Stat5 and p300.

We have previously shown that heterologous cell systems, supplied with the genes encoding the PRL receptor, Stat5, and a ß-casein promoter-luciferase construct, can be used to study PRL-induced signaling and transcription, normally restricted to mammary epithelial cells. To investigate whether the p300 coactivator is involved in the transcriptional regulation exerted by Stat5, HeLa cells were transfected with expression vectors for the PRL receptor, MGF/Stat5, and the ß-casein gene promoter-luciferase construct as a reporter. The cells were treated with PRL, and luciferase activities were measured. PRL treatment of the cells caused an approximately 5-fold stimulation of the reporter gene (Fig. 1AGo, lanes 1 and 2). Cotransfection of increasing amounts of p300 increased the PRL-dependent transactivation (lanes 3 to 8). p300 specifically enhanced the Stat5-induced transcription; basal activity of the ß-casein promoter was not affected.



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Figure 1. p300 Enhances PRL-Induced, Stat5-Mediated Transactivation

HeLa cells (A, C, and D) or 293 cells (B) were transfected with expression vectors encoding MGF/Stat5 (Stat5) (A and B), mSTAT5a (C), mStat5b (D), the PRL receptor (Prl R), and the ß-casein gene promoter-luciferase reporter (ß-Cas-luc). The indicated amounts (µg) of p300 were cotransfected. Transfected cells were either not treated (solid bars) or treated for 14 h with 1 µg/ml PRL (hatched bars) as indicated. Cellular extracts were prepared and luciferase activities were determined.

 
The induction-enhancing effect of p300 is not dependent on a specific cell line. Consistent results were obtained in the adenovirus-transformed human embryonic kidney cell line 293 (Fig. 1BGo) and COS7 cells (data not shown). Stat5 induction resulted in about an 18-fold stimulation of the reporter gene in 293 cells (Fig. 1BGo, lanes 1 and 2). Increasing amounts of p300 further enhanced the PRL-induced activity of the ß- casein reporter gene up to 120-fold (lanes 3–8).

Two closely related variants of Stat5 are encoded by the mouse genome (mStat5a and mStat5b) (18). These proteins are greater than 95% identical and differ mainly in their carboxyl-terminal transactivation region. They can form homo- and heterodimers, and both variants are able to confer the PRL response to the ß-casein gene promoter, underlining their functional similarity (13, 18). MGF/Stat5, used in the former experiments, is the sheep homolog of Stat5 and very similar in sequence to mStat5a. To investigate whether p300 is involved in the transcriptional regulation exerted by both variants of Stat5, HeLa cells were transfected with ß-casein reporter plasmid and expression vectors for the PRL receptor, Stat5a (Fig. 1CGo), and Stat5b (Fig. 1DGo). Both Stat5 homologs stimulated the activity of the reporter gene about 6-fold (lanes 1 and 2). A p300 expression vector was included in the transfection protocol (lanes 3 and 4). In the absence of PRL stimulation of the cells, p300 expression did not affect basal luciferase activity (lanes 3). Treatment with PRL caused a luciferase induction. This induction was 2-fold higher in the p300-transfected cells (lanes 4) when compared with the the controls (lanes 2). The enhancement of PRL induction by p300 was observed for Stat5a as well as for Stat5b.

E1A Inhibits PRL-Induced Transactivation by Stat5
The adenovirus E1A protein binds to p300 and CBP and inactivates their function as coactivators of CREB-mediated transcription (50, 51). The selective inhibition of p300 function allowed us to investigate whether p300 is necessarily required for the PRL-induced transactivation by Stat5. HeLa cells were transfected with expression vectors for the PRL receptor, Stat5, and the ß-casein promoter reporter construct. Treatment of the cells with PRL resulted in the induction of luciferase activity (Fig. 2AGo, lanes 1 and 2). Inclusion of an expression vector for E1A in the transfection scheme resulted in the suppression of PRL induction (lanes 3 and 4). This suppression was overcome by the augmentation of p300 expression. Enhanced expression of p300 not only restored PRL-induced transactivation, but resulted in higher induction levels than in the control cells (compare lanes 2 and 8).



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Figure 2. E1A Suppresses PRL-Induced Transactivation by Stat5 and p300 Restores Induction

HeLa cells were transfected with expression vectors encoding MGF/Stat5 (A and B), Stat5{Delta}750VP16 (C), the PRL receptor, and the ß-casein-luciferase reporter gene. Expression plasmid (50 ng) encoding E1A or the mutants thereof, {Delta}CR1 and {Delta}CR1, or the indicated amount of p300 expression plasmid were cotransfected as indicated. Transfected cells were either not treated (solid bars) or treated for 14 h with 1 µg/ml PRL (hatched bars). The schematic structures of E1A, {Delta}CR1, {Delta}CR1, Stat5, and Stat5{Delta}750VP16 are shown. A, PRL-induced transcriptional activation is repressed by E1A and restored by exogenous expression of p300. B, The p300 but not the Rb binding function of E1A is necessary for repression of Stat5-mediated transcription. C, E1A inhibits transcriptional activation by Stat5, but not by Stat5{Delta}750VP16.

 
We characterized the domains of E1A involved in the suppression of Stat5- induced transcription. The deletion mutant {Delta}CR1 of E1A is lacking the p300 and the Rb-binding functions; the mutant {Delta}CR2 lacks the Rb-binding function, but retains its p300-binding function (52). The structure of these molecules is schematically shown in Fig. 2Go. {Delta}CR1 expression did not interfere with Stat5 induction of transcription (Fig. 2BGo, lanes 5 and 6), and {Delta}CR2 inhibited PRL-induced transcription (lanes 7 and 8) to a similar extent as the E1A wild-type molecule (lanes 1 and 2). A third variant of E1A, lacking amino acids 64–68 and unable to bind p300 but still able to bind Rb, was tested. This variant did not affect PRL inducibility of the reporter construct (data not shown). This indicates that the p300-binding site within the E1A molecule is required for suppression of Stat5-mediated transcription.

The Carboxyl-Terminal Transactivation Domain of Stat5 Is Required for E1A-Dependent Suppression of Induction
We have previously reported that the transactivation domain of Stat5 is located in the very carboxyl-terminal region. This transactivation domain is autonomously active when fused to a heterologous DNA-binding domain. The strength of the transactivation domain was found to be weaker than those of Stat6 and VP16 (53). Exchange of the Stat5 transactivation domain with that of VP16 resulted in a molecule that retained its cytokine regulation, but which had a stronger transactivation potential (53, 54). We investigated whether the transactivation domain of Stat5 participates in the inhibitory effect of E1A on the transcriptional activation. A molecule lacking the endogenous transactivation domain of Stat5, but comprising the VP16 transactivation domain (Stat5{Delta}750VP16), was used for this purpose.

PRL induction of cells transfected with Stat5{Delta}750VP16 leads to a much stronger transcriptional activation of the ß-casein reporter (Fig. 2CGo, lanes 1 and 2) compared with the wild-type Stat5 (Fig. 2BGo, lanes 1 and 2). Coexpression of E1A with Stat5{Delta}750VP16 (Fig. 2CGo, lanes 3 and 4), {Delta}CR1 (lanes 5 and 6), or {Delta}CR2 (lanes 7 and 8) did not interfere with the transcriptional activation by Stat5{Delta}750VP16. Even a 10-fold increase of E1A vector in the cotransfection scheme did not cause inhibition (data not shown). These experiments show that E1A inhibition of transcriptional activation is dependent upon the presence of the Stat5 transactivation domain and can be circumvented by the fusion of a truncated Stat5 to the VP16 transactivation domain. These experiments indicate that p300 acts as a coactivator of Stat5 through the transactivation domain.

p300 Interacts with the Carboxyl-Terminal Transactivation Domain of Stat5
Coactivators are thought to be transcription factors that stabilize complexes between regulated activator proteins and the basal transcription factors through direct protein-protein interactions (26). Since p300 acts as a Stat5 coactivator, we investigated their potential interaction in coimmunoprecipitation experiments. COS7 cells were transfected with expression vectors encoding PRL receptor, Stat5, and p300. Transfected cells were treated with PRL, nuclear extracts were prepared, and proteins were immunoprecipitated with p300-specific antibodies. The immunoprecipitates were analyzed by Western blotting and developed with an antiserum specific to the carboxyl terminus (lanes 1–3) or an antiserum specific for a more amino-terminal region of Stat5 (lanes 4–7). The band in Fig. 3Go, lane 2, indicates that the p300-specific antiserum coimmunoprecipitates Stat5, most likely due to a direct protein-protein interaction. When control antibodies, with specificity for the yeast protein GAL4, were used in the immunoprecipitation reaction, no Stat5 was detected (lane 3).



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Figure 3. p300 Interacts with the Carboxyl-Terminal Transactivation Domain of MGF/Stat5

A, Stat5 coimmunoprecipitates with p300. COS7 cells were transfected with expression vectors encoding the PRL receptor, p300, and Stat5 (lanes 1–5) or with expression vectors encoding the PRL receptor, p300, and Stat5{Delta}750. Cells were treated for 1 h with 1 µg/ml PRL, and nuclear extracts were prepared. One-tenth of the nuclear extracts were loaded directly onto a SDS/polyacrylamide gel (lanes 1, 4, and 6; input). The remainder was immunoprecipitated with p300-specific antibodies (lanes 2, 5, and 7; {alpha}-p300) or with unrelated antibodies (lane 3; unrel.). All samples were analyzed by Western blotting with an antiserum raised against the carboxyl terminus of Stat5a (lanes 1–3; {alpha}-Stat5C) or an antiserum raised amino acids 661–677 of Stat5a (lanes 4 to 7; {alpha}-Stat5N).

 
The suppression experiments shown in Fig. 2CGo indicate that the carboxyl-terminal region of Stat5 is involved in the p300 interaction. To corroborate this conclusion, we performed immunoprecipitations from nuclear extracts of cells transfected with Stat5{Delta}750, a variant in which the carboxyl-terminal transactivation domain (amino acids 751–794) of Stat5 has been deleted (55). Only a very weak band can be detected in Fig. 3Go (lane 7), indicating that the carboxyl-terminal transactivation domain of Stat5 is necessary for the interaction with p300.

A second, independent method was employed to confirm this conclusion, a mammalian two-hybrid assay. For this purpose we fused the GAL4 DNA-binding domain (amino acids 1–147) to the carboxyl-terminal transactivation domain of Stat5 (amino acids 722–794) (55). This fusion construct (GAL4-Stat5TA) was cotransfected with a Galp3TK-Luciferase into COS7 cells. The reporter construct is regulated by three palindromic GAL4-binding sites and a minimal thymidine kinase (TK) promoter. A 4-fold induction of transcription was observed (Fig. 4Go, lanes 1 and 2). Addition of increasing amounts of a p300-VP16 expression vector, encoding a p300 fused to the VP16 transactivation domain (56) significantly enhanced the activity of the reporter gene (lanes 2- 5). Transfection of p300-VP16 in the presence of a control vector, encoding only the GAL4 DNA-binding domain (GAL4-DBD), had no affect on the activity of the reporter construct (lanes 6–9). These results indicate that the transactivation domain of Stat5 directly interacts with p300 and that the transactivation domain of Stat5 is necessary and sufficient for this interaction.



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Figure 4. p300 Interacts with the Carboxyl-Terminal Transactivation Domain of Stat5 in the Mammalian Two-Hybrid Assay

The reporter plasmid Galp3TK-luciferase (2.5 µg) was transfected in HeLa cells with plasmids expressing GAL4-Stat5TA (1 µg) or GAL4-DBD (1 µg) alone or in combination with increasing amounts (0.5 to 6 µg) of expression vectors encoding p300-VP16. Cellular extracts were prepared and luciferase activities were determined.

 
To determine the domains of p300/CBP responsible for binding to Stat5, individual fragments of CBP were expressed as glutathione-S-transferase (GST) fusion proteins and tested for their interaction with Stat5. We compared the binding capacity of latent nonactivated Stat5 with that of the tyrosine-phosphorylated activated form. The GST-CBP fusion proteins were expressed in bacteria, bound to glutathione-Sepharose beads, and reacted with cell extracts containing Stat5. Bound Stat5 was visualized upon gel electrophoresis and Western blotting. The latent form of Stat5 was derived from whole-cell extracts of HeLa cells, stably transfected with the PRL receptor and Stat5a. The presence of Stat5 in this cellular extract is shown in Fig. 5Go (lane 1, upper panel). No binding of latent Stat5 was observed to the GST-CBP fusion molecules (lanes 2–7, upper panel). To investigate whether phosphorylation and dimerization of Stat5 is necessary for the interaction with p300/CBP, binding experiments were also performed with whole-cell extracts from cells treated with PRL and containing the activated form of Stat5. The fusion protein comprising the region of CBP between amino acids 451 and 721 was found to interact with the activated form of Stat5 (lane 3, lower panel). This indicates that dimerization of Stat5 is required for the interaction with the distinct part of p300/CBP and is consistent with our coimmunoprecipitation experiments shown in Fig. 3Go in which activated Stat5 was present.



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Figure 5. Activated Stat5a Interacts with the CREB-Binding Domain of p300/CBP in Vitro

GST-fusion proteins of p300/CBP bound on glutathione-Sepharose beads were incubated with whole-cell extracts from untreated (upper panel) or PRL-treated (lower panel) HeLa cells stably expressing the PRL receptor and Stat5a. Specifically bound Stat5a was visualized by Western blotting using an anti-Stat5a antibody.

 
Stat5 Represses Glucocorticoid Induction of the MMTV-LTR Promotor in a Dose-Dependent Manner
Stat5, as shown above, and the GR both utilize p300 as a coactivator (35, 36). We have investigated the effect of Stat5 activation on the induction of the GR-mediated transactivation of the MMTV-LTR. MMTV-LTR contains a promoter region with several GR-binding sites and responds to induction by glucocorticoid hormones (57). COS7 cells were transfected with expression vectors encoding the PRL receptor, the GR, Stat5, and a MMTV-LTR luciferase reporter gene (Fig. 6Go). In the absence of transfected GR, no luciferase activity was detected, whether or not the cells were treated with dexamethasone (lanes 1–3). A strong induction was observed when the GR, but no Stat5, was transfected and the cells were induced with dexamethasone (lanes 4 and 5) or dexamethasone plus PRL (lane 6). Increasing amounts of Stat5 in the transfection scheme did not affect dexamethasone inducibility of the MMTV-LTR as long as Stat5 was not activated by PRL (lanes 8, 10, 12, 14, and 16). PRL addition to the cells, i.e. Stat5 activation, suppressed the dexamethasone inducibility of the MMTV-LTR in a Stat5 dose-dependent manner (lanes 9, 11, 13, 15, and 17). These results could be explained as the consequence of competition of the GR and Stat5 for limiting amounts of p300/CBP or by the interference of GR function through complex formation with Stat5.



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Figure 6. Stat5 Represses GR-Mediated Transcriptional Activation in a Dose-Dependent Manner

COS7 cells were transfected with MMTV-LTR-luciferase reporter plasmid (4 µg) and expression vectors encoding for the glucocorticoid receptor (2 µg), the PRL receptor (250 ng), and increasing amounts of Stat5 expression vector as indicated. Transfected cells were either not treated or treated as indicated with 10-7 M dexamethasone (open bars) and/or 5 µg/ml ovine PRL (solid bars) for 16 h before harvesting.

 
Increased Expression of p300 Does Not Counteract Stat5-Mediated Inhibition of GR Function
The simultaneous activation of Stat5 and the GR causes an enhancement of the Stat5-dependent transcription of the ß-casein gene promoter luciferase construct and a repression of the GR-mediated induction of the MMTV-LTR luciferase construct (48). Since p300 is a coactivator for both factors, we determined whether it is involved in the repression process. We investigated the possibility that the Stat5-mediated repression of the GR-induced transcription can be compensated by the augmented expression of p300.

Introduction of the GR into COS7 cells resulted in the strong inducibility of a MMTV-LTR luciferase reporter gene by dexamethasone (Fig. 7Go, lanes 1 and 2). Expression and induction of Stat5 suppresses the MMTV-LTR luciferase induction (lanes 3 and 4). Addition of increasing amounts of p300 enhanced the glucocorticoid-dependent transactivation in a dose- dependent manner (lanes 5, 7, and 9). This indicates that endogenous p300 expression is limiting the extent of GR induction. The suppression of dexamethasone induction of the MMTV-LTR by the activated Stat5 was not relieved, however, by the increase in p300. Although the absolute values of luciferase activity increased slightly in the presence of 5 µg and 10 µg of p300 vector in the transfection, the repression of induction by activated Stat5 persisted (lanes 4, 6, 8, and 10). We conclude that repression of GR-mediated induction by activated Stat 5 does not result from competition for limiting amounts of p300, a mechanism suggested in the functional interaction between nuclear receptors and AP-1 (35, 36). We propose that repression of GR function by Stat5 is a consequence of their complex formation.



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Figure 7. Increased Expression of p300 Does Not Counteract Stat5-Mediated Repression of Glucocorticoid Induction of the MMTV-LTR Promoter

COS7 cells were transfected with MMTV-LTR-luciferase reporter plasmid (4 µg) and expression vectors encoding for the glucocorticoid receptor (2 µg), the PRL receptor (250 ng), Stat5 (50 ng), and the indicated amounts (µg) of p300 expression plasmids. Cells were either untreated or treated as indicated with 10-7 M dexamethasone (open bars) and/or 5 µg/ml ovine PRL (solid bars).

 
Phosphorylation of Stat5 on Tyrosine 694, but Not the Carboxyl-Terminal Transactivation Domain, Is Required for Inhibition of GR Function
Specific phosphorylation of tyrosine 694 by Jak2 is essential for the activation of Stat5 (13). It causes dimerization and nuclear translocation of Stat5. We used a mutant of Stat5, in which tyrosine 694 was replaced by a phenylalanine residue and tested its ability to repress transactivation of the MMTV-LTR by the GR. This mutant was not able to repress GR transactivation (Fig. 8Go, lanes 6 and 7). Activation of Stat5 through tyrosine phosphorylation is a necessary prerequisite for the inhibitory effect. We also investigated whether the transactivation domain of Stat5 participates in the inhibitory effect. A mutant lacking the carboxyl-terminal transactivation domain, Stat5{Delta}750, inhibited the glucocorticoid-induced transactivation of the MMTV-LTR reporter plasmid as well as wild-type Stat5 (Fig. 8Go; compare lanes 4 and 5 with 8 and 9). This result supports our finding that interaction of Stat5 with p300 is not involved in the repression process.



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Figure 8. Stat5 Y 694 F, a Tyrosine Phosphorylation-Deficient Variant of Stat5, Cannot Repress GR Function; Stat5{Delta}750, Lacking the Transactivation Function, Represses GR-Mediated Transcription Activation

COS 7 cells were transfected with MMTV-LTR luciferase reporter plasmids (4 µg) and expression vectors encoding the GR (2 µg), the PRL receptor (250 ng), Stat5, Stat5 Y694F, or Stat5{Delta}750 plasmids (50 ng each). Cells were treated as indicated with 10-7 M dexamethasone (open bars) and/or 5 µg/ml ovine PRL (solid bars).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid and peptide hormones regulate specific gene transcription through the activation of latent transcription factors. The glucocorticoids are lipophilic hormones that enter the cell and bind to their specific receptor in the cytoplasm. This leads to the activation, dimerization, and DNA binding of the receptor and induction of target genes (22). PRL binds to a trans-membrane receptor and induces its dimerization and the activation of the associated kinase Jak2. Jak2 phosphorylates the receptor as well as signaling components, e.g. Stat5. Tyrosine phosphorylation of Stat5 induces its dimerization, translocation to the nucleus, specific DNA binding, and induction of gene transcription (5).

A direct connection between the glucocorticoid- and PRL-induced signal transduction pathways was recently found that involves complex formation between the GR and Stat5 (48, 58, 59). This interaction enhances PRL-induced Stat5-activated transcription, but suppresses glucocorticoid-responsive transcriptional activation. We have further investigated this interaction and examined the role of the coactivator p300/CBP in the induction process. Members of the p300/CBP protein family were originally detected through their ability to bind to E1A and CREB (31, 33) and subsequently identified as coactivators for nuclear hormone receptors (35, 36). p300/CBP act as integrators for several signal transduction pathways (28, 29), e.g. they may mediate the cross-talk between nuclear receptors and AP-1 (35, 36) and connect IFN-{gamma} and Ras/AP-1 signaling (60).

We found that p300 enhances PRL-induced transcriptional acitvation by Stat5a and Stat5b. These two variants of the Stat5 protein are encoded by highly related genes and show a sequence identity of greater than 95% at the amino acid level. The most pronounced sequence diversity is located in their carboxyl- terminal transactivation region (18, 55), the region required for p300-mediated coactivation. Both proteins can form homo- and heterodimers and are able to confer the PRL response to the ß-casein gene promoter, i.e. they are functionally very similar (13, 18). This is supported by our finding that Stat5a and Stat5b, despite their sequence divergence in the carboxyl-terminal region, can cooperate with p300.

The involvement of p300 in Stat5-induced transcription was confirmed in experiments in which we observed that E1A inhibits Stat5-induced transcriptional activation. E1A is thought to bind directly to p300 and thereby prevents it from functioning as a coactivator of other transcription factors (61), but E1A has also been shown to be able to interact with other crucial cellular regulators such as the transcriptional repressor Dr1, YY1, the general transcription factor TBP, and Rb (62, 63, 64, 65). It also participates in the control of transcription, DNA synthesis, and cell cycle regulation and differentiation. Mutational analysis of the E1A protein has shown that some activities of E1A are distinct from others and are carried out independently by different regions of the protein (66). Our observations that E1A-mediated inhibition of Stat5-induced transcriptional activation can be overcome by overexpression of p300 and that inhibition by E1A requires the presence of an intact p300-binding site of E1A indicate that the effect of E1A on Stat5 action is based on its physical interaction with p300.

293 cells are adenovirus transformed and express endogenous E1A. It might have been anticipated that E1A expression would not allow Stat5-mediated transactivation. In fact, we observed an 18-fold induction of the ß-casein reporter gene. We conclude that the residual, functional amounts of p300 are sufficient for transcriptional activation by Stat5 (Fig. 1BGo, lanes 1 and 2). Increasing amounts of p300 further enhanced the PRL-induced activity of the ß-casein reporter gene up to 120 fold (lanes 3–8). The higher induction in 293 cells as compared with HeLa cells might result from a modification of p300 by E1A. An E1A-induced phosphorylation of p300 has been shown to influence the interaction with transcription factors (DRF) and enhance the induction of responsive promoters (67).

Our data might have interesting implications for the pathology of adenovirus infections. If E1A expression, upon viral infection, interferes with Stat5-induced transcription, processes dependent upon crucial cytokines and growth factors, such as interleukin (IL)-2, IL-3, IL-5, IL-7, IL-9, IL-10, IL-15, PRL, granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), GH, TPO, and epidermal growth factor (EGF) might be affected. These cytokines have been shown to exert at least part of their signaling potential through the activation of Stat5 (17, 19, 68, 69, 70, 71, 72, 73, 74, 75, 76).

The inhibition of Stat5-induced transcription by E1A requires the Stat5 transactivation domain. Coimmunoprecipitation experiments and mammalian two-hybrid assays revealed a direct interaction between p300 and Stat5. Interactions between p300 and two other members of the STAT family, Stat1 and Stat2, have recently been described, and the carboxyl-terminal regions of Stat1 and Stat2 have been found to be involved in those interactions (34, 37). In Stat1 there is an additional contact region in the amino terminus (37). The carboxyl-terminal regions of Stat1 and Stat2 both function as transactivation domains, but are distinct in character. The carboxyl terminus of Stat2, a highly acidic region, does not undergo serine phosphorylation. The carboxyl- terminal transactivation domain of Stat5 could form an amphipathic {alpha}-helix with clusters of acidic residues on one face and hydrophobic residues on the other (55). Despite these differences, p300 acts as a coactivator for all three Stat factors.

Our observation that p300 is a coactivator of Stat5 reveals at least three interesting protein-protein interactions in the lactogenic hormone scheme: p300 interacts with Stat5 and the GR; Stat5 and the GR interact with each other. Since the GR enhances Stat5 action at the level of the ß-casein gene promoter and Stat5 antagonizes the GR at the level of the MMTV-LTR, we analyzed the effect of p300 on Stat5-mediated repression on glucocorticoid response. p300 enhances the GR-dependent transcriptional activation of the MMTV-LTR in the absence of activated Stat5, but does not counteract Stat5-mediated repression of induction. Analysis of Stat5 variants in transfection assays indicate that the activation of Stat5 through tyrosine phosphorylation is required for the repression of Stat5 on GR function. However, the transactivation domain of Stat5, which is also the domain responsible for the interaction with p300/CBP, is not required. Competition for p300 has been invoked in the interaction between Stat1 and AP-1 (60) and is probably also responsible for the E1A inhibition of Stat5 transactivation described here. Repression of GR transactivation by Stat 5, however, is not the consequence of competition for limiting amounts of p300, but most likely results from the complex formation between the two transcription factors.

Complex formation between Stat5 and the GR has been observed in COS7 cells overexpressing GR and Stat5 (48) but also in HC11 mammary epithelial cells expressing physiological levels of GR and Stat5 (59). Decreased binding of the GR to glucocorticoid- response elements in the presence of activated Stat5 might be a possible mechanism for repression. This, however, could not be observed in in vitro bandshift experiments with extracts from transfected cells (data not shown). A further possible mechanism for the Stat5-mediated inhibition of the GR transactivation function is that a complex of Stat5 and GR might cause sterical hindrance and interfere with contacts between the GR and coactivators or the basal transcription machinery. It will be of interest to investigate whether complex formation between GR and Stat5 also influences the transrepression function of the GR on AP-1 and NF-{kappa}B-responsive genes. p300/CBP seems to be an important integrator in these different signaling pathways involved in inflammation reaction and immune response. The observed negative cross-talk of Stat5 on GR function might also have implications for T lymphocyte development and differentiation as well as in erythropoiesis. GR is thought to control thymocyte apoptosis and proliferation of erythroblasts (77).

GR activation has a synergistic effect on the transcription of the ß-casein gene promoter activated by Stat5 (48). When p300 was added, a further increase in transcription, above the GR-mediated effect, was observed (not shown). These results indicate that the GR/Stat5 complex recruits high levels of p300. The ß-casein reporter construct used in our experiments contains, in addition to the Stat5-binding sites, several binding sites for regulator proteins such as C/EBP and YY1 (15). C/EBPß has been shown to interact and collaborate with p300, and relief of YY1 transcriptional repression has been described to be mediated by p300 (78, 79). This indicates that there are several contact sites for p300 in the ß-casein promoter and that p300 might coordinate the cooperation between different regulatory factors that bind to the ß-casein promoter. The recruitment of this coactivator and its associated histone acetyl transferase activity may result in a chromatin configuration that allows the efficient assembly and enhanced stabilization of the preinitiation complex, and its local concentration may eventually determine the extent of transcriptional induction.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The ß-casein gene promoter (-344 to -1) luciferase construct and the expression vectors for sheep MGF/Stat5 (pXM-MGF), mStat5a (pXM-Stat5a), mStat5b (pXM-Stat5b), the deletion mutant Stat5{Delta}750 (pXM-MGF{Delta}750), the point mutant Stat5 Y694F (pXM-MGF Y694F), and the long form of the PRL receptor (pcDNAI-PrlR) were described previously (13, 16, 55). To construct Stat5{Delta}750VP16, the transactivation domain of VP16 (amino acids 411–489) was amplified by PCR and fused in frame to the 3'-end of pXM-MGF{Delta}750 (53). The human GR expression vector pRSVhGRa (GR) (80), the MMTV-LTR-luciferase reporter (81), and the expression vectors for pCMV (cytomegalovirus promoter enhancer)-p300 and p300-VP16 have been described (33, 56). The same is true for the RSV LTR (Rous sarcoma virus long terminal repeat)-E1A 12S constructs and the mutants E1A{Delta}CR1 and E1A{Delta}CR2 (52), and the reporter plasmid Galp3TK-luciferase, which contains three copies of the UASG cloned upstream of the TK promoter (82). The plasmids encoding the GAL4-Stat5TA fusion comprise the DNA-binding and dimerization domains of yeast GAL4 protein (amino acids 1–147) and the carboxyl-terminal transactivation domain of Stat5 (amino acids 722–794). GAL4-DBD and GAL4-Stat5TA have been described previously (55).

Cell Culture and Transfection
HeLa cells, 293 cells, and COS7 cells were maintained in DMEM containing 10% FCS, 2 mM glutamine, and 50 µg/ml gentamycin. Transient transfection experiments were performed using the calcium phosphate precipitation technique (13). Half-confluent cells in 10-cm dishes were cotransfected with the ß-casein luciferase reporter constructs (2 µg) and expression vectors for Stat5 (2 µg) or its variants, the PRL receptor (250 ng), and p300 or E1A expression plasmids, as indicated in the figure legends. To monitor the GR-dependent transcriptional activation, the MMTV-LTR-luciferase reporter construct (4 µg) was cotransfected with an expression vector encoding the GR (2 µg), the PRL receptor (250 ng), and the indicated amounts of Stat5 and p300. Plasmid (0.5 µg) encoding the ß-galactosidase gene driven by the CMV promoter was included to monitor transfection efficiency. The DNA was adjusted to 10 µg with empty vector-DNA. One day after transfection, the cells were treated with 5 µg/ml ovine PRL and/or 10-7 M dexamethasone for 16 h before harvesting.

Luciferase and ß-Galactosidase Assays
Two days after transfection cells were harvested, washed twice in cold PBS, and lysed in 25 mM glycylglycine, pH 7.8, 1 mM dithiothreitol (DTT), 15 mM MgSO4, 4 mM EGTA, and 1% Triton for 10 min at 4 C. Samples were centrifuged for 5 min at 14,000 rpm. Cleared supernatants were used for luciferase and ß-galactosidase assays. For ß-galactosidase determination, 20 µl of extracts were added to 200 µl of reaction buffer containing 100 mM Na-phosphate, pH 8.0, 1 mM MgCl2, and 1 x Galacton (Tropix, Bedford, MA) and incubated for 30 min at room temperature. Measurements were made by injecting 300 µl of accelerator solution (10% Emerald luminescent amplifier and 0.2 N NaOH), and the samples were counted for 20 sec in the Luminometer 953 (Berthold, Pforzheim, Germany). Luciferase activities (100 µl extracts and 300 µl accelerator solution) were quantified in the same apparatus and normalized to the ß-galactosidase activities. At least three independent experiments were performed.

Coimmunoprecipitation Assay
COS7 cells were transfected with expression plasmids encoding Stat5 or Stat5{Delta}750, p300, and the PRL receptor. One day after transfection, the cells were treated with 5 µg/ml ovine PRL for 1 h before harvesting. Nuclear extracts from the induced cells were prepared. Protein (200 µg) was incubated with 4 µg of p300-specific monoclonal antibodies (Upstate Biotechnology, Lake Placid, NY) or as a control, with antibodies against the GAL4 DNA-binding domain (Santa Cruz Biotechnology, Santa Cruz) overnight at 4 C at constant agitation. Protein A/G-Sepharose-coupled beads (Pierce, Rockford, IL) were added for 1 h. The beads were pelleted and washed five times with incubation buffer (20 mM HEPES,pH 7.9, 100 mM NaCl, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM DTT). The immunoprecipitates were separated by SDS-PAGE, and the Western blots were developed with antiserum specific against the carboxyl terminus of Stat5a ({alpha} Stat5C) or an antiserum against amino acids 661–677 of Stat5a ({alpha} Stat5N).

Mammalian Two-Hybrid Assay
Half-confluent HeLa cells in 10-cm dishes were cotransfected with the reporter plasmid Galp3TK-luciferase (2.5 µg) and expression vectors for GAL4-Stat5TA (1 µg), the GAL4-DBD (1 µg), and p300-VP16. Plasmid (0.5 µg) encoding the ß-galactosidase gene driven by the CMV promoter was included to monitor transfection efficiency. Luciferase assays were performed as described.

GST-Fusion Protein Interaction Assay and Western Blot Analysis
The expression vectors for the GST-CBP fusion proteins have been described previously (83). GST-fusion proteins were purified from bacterial extracts using glutathione-Sepharose beads as specified by the manufacturer (Pharmacia, Freiburg, Germany). Whole-cell extracts from HeLa cells stably expressing the PRL receptor and Stat5a were prepared as described previously (13). The cells were either untreated, to obtain the latent form of Stat5a, or treated with 5 µg/ml ovine PRL for 1 h before harvesting, to obtain the activated form of Stat5a. For binding assays, 150 µg protein from whole-cell extracts were incubated with 10–20 µg of GST-fusion proteins bound on beads at 4 C for 1 h under constant agitation in 500 µl 50 mM Tris-HCl (pH 8), 100 mM NaCl, 0,3 mM DTT, 10 mM MgCl2, 10% glycerol and 0,1% Nonidet P-40 (NP40). The protein complexes were washed five times in binding buffer. The bound proteins were eluted from the beads with SDS sample buffer and analyzed by SDS-PAGE. Western blot analyses were done using the antiserum specific against the carboxyl terminus of Stat5a.


    ACKNOWLEDGMENTS
 
We thank R. Eckner (Zürich, Switzerland) and T. Kouzarides (Cambridge, U.K.) for plasmids and reagents; C. Beisenherz, M. Frische, R. Moriggl, B. Schnierle, and C. Shemanko (Freiburg, Germany) for discussions and critical reading of the manuscript; and I. Fernandez for editorial assistance.


    FOOTNOTES
 
1 Address requests for reprints to: Dr. Bernd Groner, Institute for Experimental Cancer Research, Tumor Biology Center Freiburg, Breisacher Strasse 117, D-79106 Freiburg, Germany. Back

Received for publication January 30, 1998. Revision received June 4, 1998. Accepted for publication July 2, 1998.


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 ABSTRACT
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 DISCUSSION
 MATERIALS AND METHODS
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