Attenuation of Glucocorticoid Signaling through Targeted Degradation of p300 via the 26S Proteasome Pathway

Qiao Li, Anna Su, Jihong Chen, Yvonne A. Lefebvre and Robert J. G. Haché

Department of Medicine (Q.L., Y.A.L., R.J.G.H.) and Department of Biochemistry, Microbiology and Immunology (Y.A.L., R.J.G.H.), University of Ottawa, and the Ottawa Health Research Institute (Q.L., A.S., J.C., Y.A.L., R.J.G.H.), Ottawa, Ontario, Canada K1Y 4E9

Address all correspondence and requests for reprints to: Robert J. G. Haché or Qiao Li, The Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, Ontario, Canada, K1Y 4E9. E-mail: rhache@ohri.ca or qli{at}ohri.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The effects of acetylation on gene expression are complex, with changes in chromatin accessibility intermingled with direct effects on transcriptional regulators. For the nuclear receptors, both positive and negative effects of acetylation on specific gene transcription have been observed. We report that p300 and steroid receptor coactivator 1 interact transiently with the glucocorticoid receptor and that the acetyltransferase activity of p300 makes an important contribution to glucocorticoid receptor-mediated transcription. Treatment of cells with the deacetylase inhibitor, sodium butyrate, inhibited steroid-induced transcription and altered the transient association of glucocorticoid receptor with p300 and steroid receptor coactivator 1. Additionally, sustained sodium butyrate treatment induced the degradation of p300 through the 26S proteasome pathway. Treatment with the proteasome inhibitor MG132 restored both the level of p300 protein and the transcriptional response to steroid over 20 h of treatment. These results reveal new levels for the regulatory control of gene expression by acetylation and suggest feedback control on p300 activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE SPECIFIC ACETYLATION of histones leads to a relaxation of DNA-histone interactions within chromatin that generally facilitates specific gene transcription (1). Histone acetylation appears to be mediated by transcriptional coactivators such as P/CAF, p300/CREB-binding protein (CBP), and TAFII250 that contain intrinsic histone acetyltransferase activity (HAT) activities (2, 3, 4, 5).

Additionally, no-histone proteins, including transcription factors and the coregulatory proteins themselves, also may be regulated through acetylation. In several instances, the HAT activities of p300/CBP have been shown to be responsible for transcription factor acetylation (6, 7, 8). Acetylation of p53 and GATA1 by p300/CBP potentiates their transcriptional effects by enhancing their DNA-binding activities (9, 10), while acetylation of the p160 coactivator ACTR leads to the dissociation of ACTR from estrogen receptor (ER) to down-regulate transcriptional activation (11). Thus the effects of acetylation on gene expression through no-histone proteins are complex and can alternatively facilitate or attenuate transcription by altering the interactions of transcription factors with DNA and coregulatory proteins.

Glucocorticoid receptor (GR) is a nuclear hormone receptor that is released from a transcriptionally inert chaperone complex in the cytoplasm to directly activate specific gene transcription in response to glucocorticoids (12). A number of coactivators, such as SRC-1 and p300/CBP, have been implicated in nuclear receptor signaling (13, 14, 15). However, the specifics of their involvement in the activation of transcription remains to be clarified. In particular, while p300 is thought to be generally important for transcriptional regulation by nuclear receptors, the relevance of the HAT activity of p300 for the function of individual receptors is unclear. It has been suggested that the HAT domain of p300 is dispensable for the activation of transcription mediated by retinoic acid receptor (16). However, the HAT activity of p300 appears to be critical for estrogen-induced histone hyperacetylation and transcriptional activation (11). These results suggest that the requirement for p300 and its HAT activity vary between transcription factors.

The complex nature of the effects of acetylation on specific gene transcription is further illustrated by the effects of histone deacetylase inhibitors on gene expression. The histone deacetylase inhibitors, sodium butyrate and trichostatin A (TSA), enhance gene expression from a number of promoters, such as heat shock protein 70, TRßA, HIV-1, and G{alpha}i2 gene promoter (6, 7, 17, 18). By contrast, butyrate prevents the induction of ovalbumin and transferrin genes by estrogen (19). It also impedes the activation of the tyrosine aminotransferase gene and episomal or chromosomally integrated mouse mammary tumor virus (MMTV) promoter by glucocorticoids (20, 21, 22, 23) and progestins (24).

However, for MMTV it has recently been shown that histone acetylation was maximal before transactivation of transcription and that deacetylation correlated with the attenuation of MMTV transcription (25). This is consistent with the general observation that histone acetylation correlates with transcriptional activation, and histone deacetylation correlates with repression. Furthermore, the result suggests that the repressive effects of butyrate and TSA treatment on glucocorticoid-induced MMTV transcription are mediated through targets other than the histones within the transcriptional regulatory region.

The ubiquitin-dependent protein degradation pathway is involved in the regulation of many basic cellular processes such as cell cycle and division, differentiation and development, signal transduction, and apoptosis (26, 27, 28, 29, 30, 31, 32). Ubiquitin-dependent mechanisms are also responsible for the selective degradation of transcriptional activators (33, 34, 35), nuclear hormone receptors (36, 37, 38), and coactivators (39, 40). One recent study has suggested that ubiquitination regulates activator function by serving as a dual signal for transcriptional activation and activator destruction (41).

In the present work we have determined that sodium butyrate treatment induces the degradation of p300 through the 26S proteasome pathway. The activation of transcription by GR was strongly potentiated by p300 in a manner that was dependent on its HAT activity, and the degradation of p300 correlated with the long-term repressive effects of sodium butyrate on glucocorticoid-mediated transcription. By contrast, repression of GR-mediated transcription at early times after inhibition of cellular deacetylases correlated with a temporal shift in the association of GR with SRC-1 and abrogated binding of GR to p300 before its degradation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Potentiation of GR-Induced Transcription by p300 Is Dependent upon Its HAT Activity
To initiate our study of the effects of acetylation on the induction of transcription by steroid hormone receptors, we examined transcriptional activation by GR from a transiently transfected MMTV construct with the promoter proximal steroid hormone-dependent regulatory region driving transcription of a chloramphenicol acetyltransferase (CAT) reporter gene (Fig. 1AGo). Transfections were performed in HeLa cells in which rat GR was stably expressed at moderate levels to ensure proper folding and modification of the receptor. The transfected cells were treated with the synthetic steroid dexamethasone (dex) for 20 h before harvest.



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Figure 1. MMTV Promoter and the Transcriptional Augmentation by SRC-1

A, Schema of the MMTV-CAT reporter employed. Numbers denote the base pair distance relative to transcription initiation site of the MMTV promoter. B, Increasing amounts of an SRC-1 expression vector, 50 ng (+), 100 ng (++), and 200 ng (+++), respectively, were cotransfected with a constant amount of MMTV-CAT reporter (100 ng) into HeLa-GR cells. Dex treatment (100 nM) was for 20 h. Transfections were repeated a minimum of four times in triplicate with CAT activity expressed as the fold induction (normalized to protein concentration) relative to the untreated controls. Error bars represent the SEMs of triplicate samples within representative experiments.

 
GR-mediated transcription from a simple promoter has been shown to be dependent upon SRC-1 (13, 42), but little other information on specific coregulatory function in the activation of GR-mediated transcription is available. Expression of exogenous SRC-1 in the HeLa-GR cells resulted in an up to 3-fold increase in glucocorticoid-induced MMTV expression, indicating that SRC-1 is also likely important for the induction of complex promoters by GR (Fig. 1BGo).

Coexpression of p300 also resulted in an increase in glucocorticoid-induced MMTV transcription (Fig. 2AGo). The magnitude of induction correlated with the amount of p300 coexpressed. By contrast, transfections with a vector expressing a version of p300 (p300-hm) containing a 50-amino-acid deletion in the HAT domain that abrogates acetyltransferase activity had no effect on GR-induced transcription (Fig. 2BGo). Western analysis confirmed that p300-hm was expressed at the same levels as wild-type p300 (Fig. 2CGo). Together, these results indicated that the function of p300 in GR action depended on its HAT activity.



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Figure 2. The Effects of p300 and E1A on Transcriptional Activation of the MMTV Promoter

A, CAT activities of the pHCWT MMTV-CAT reporter were assessed after 20 h of hormone induction (dex, 100 nM) in the presence of increasing amounts of p300 expressed by cotransfection of 50 ng (+), 100 ng (++), and 200 ng (+++) of p300 expression vector. B, Transfections were performed identically to those in panel A except that the p300 cDNA used (p300-hm) contained a deletion comprising 50 amino acids of the HAT domain (aa 1472–1522) resulting in the production of a p300 mutant lacking HAT activity. C, Western analysis of the level p300 construct expression in panels A and B. Equal amounts of whole-cell extracts (100 µg) were resolved by SDS-PAGE and probed with antibody against p300 (N-15, Santa Cruz Biotechnology, Inc.). The first lane of each series shows the signal obtained in the absence of cotransfected p300 followed by the signals obtained upon transfection of 50, 100, and 200 ng of p300 or p300-hm expression vector, respectively, as indicated by the triangles above the lanes, with the breadth of the triangle reflecting the increasing amounts of transfected DNAs in the lanes from left to right. A nonspecific band representing endogenous protein recognized by the p300 antibody on Western blots is shown below as a control for sample loading (Con). D, The hormone-induced transcription from the MMTV promoter was measured in HeLa-GR cells in which either wild-type 12S E1A protein or mutant 12S E1A (E1A/E1A-C, 100 ng of each expression plasmid transfected) were expressed. E1A-C contains a deletion of aa 2–36 that specifically abrogates the interaction of E1A with p300. Treatment with 100 nM dex was for 20 h. Transfections were repeated a minimum of four times in triplicate with CAT activity expressed as the fold induction (normalized to protein concentration) relative to the untreated controls. Error bars represent the SEMs of triplicate samples within representative experiments.

 
To confirm that endogenous p300 played an important role in the response to steroid, we employed the adenovirus E1A oncoprotein. E1A interferes with p300-dependent transcription by directly repressing the acetylation activity of p300, possibly through its physical interaction with p300 (43). Introducing wild-type 12S E1A into the HeLa-GR cells almost completely abolished GR-mediated transcription from the MMTV promoter, whereas a truncated form of E1A containing a 34-amino-acid deletion that makes it specifically deficient in binding to p300 (44) had little, if any, effect on glucocorticoid-induced transcription (Fig. 2DGo).

Repression of GR-Mediated Transcription by Sodium Butyrate
To assess the broader role of acetylation in the activation of MMTV transcription, we compared the effects of sodium butyrate and TSA treatment on glucocorticoid-induced transcription (Fig. 3Go). TSA is a potent and specific inhibitor of histone deacetylases while sodium butyrate is an inhibitor of deacetylation that is effective at higher concentrations (45, 46, 47).



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Figure 3. Butyrate and TSA Inhibit GR-Mediated Transcription from the MMTV Promoter

After transfection of MMTV-CAT reporter (100 ng), the HeLa-GR cells were incubated in the absence (-) or the presence (+) of dex (100 nM), sodium butyrate (10 mM), or TSA (160 nM) for 20 h. CAT activity was expressed as the fold induction (normalized to protein concentration) relative to the untreated controls. Error bars represent the SEMs from four experiments performed in duplicate.

 
Addition of 16–50 nM TSA, concentrations sufficient to completely block cellular HAT activities and induce the accumulation of acetylated histones (47), had no effect on steroid induction (data not shown), consistent with a previous report that histone acetylation was without effect on the response of transiently transfected MMTV templates to steroid (22). By contrast, addition of 10 mM sodium butyrate to the culture medium with dex strongly repressed the accumulation of CAT activity at 20 h of treatment (Fig. 3Go). As the levels of TSA added to incubations were raised further, effects independent of the blockage of histone acetylation become apparent. Indeed, whereas TSA concentrations as high as 100 nM had no effect on steroid induction (data not shown), we observed a modest decrease in CAT activity when the TSA concentration was increased to 160 mM (Fig. 3Go). Together, these results indicated that sodium butyrate repressed GR- mediated transcription through a process distinct from the inhibition of histone deacetylation.

Butyrate Repression of GR-Mediated Transcription Correlates with the Induction of p300 Degradation Through the 26S Proteasome Pathway
To begin to probe for the molecular basis of the repressive effects of butyrate on glucocorticoid-induced transcription, we examined the effects of butyrate treatment on SRC-1 and p300 protein. Whole-cell extracts were prepared from HeLa cells treated with hormone and/or sodium butyrate for 4 and 20 h, respectively. Antibodies against p300 and SRC-1 were used to track the levels of endogenous p300 and SRC-1 (Fig. 4AGo).



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Figure 4. The 26S Proteasome Selectively Regulates the Level of p300 Protein

A, Butyrate treatment causes p300 proteolysis and MG132 blocks the p300 degradation. Whole-cell extracts of Hela-GR cells were used for Western analysis with antibodies against p300 (N-15, Santa Cruz Biotechnology, Inc.) and SRC (Affinity BioReagents, M341) to assess the protein levels of p300 and SRC-1 after 4 h (lanes 1–8) and 20 h (lanes 9–16) of Dex (100 nM), sodium butyrate (10 mM), and/or MG132 (1 µM) treatment. B, Experimental setup as in panel A except that the TSA treatment was at 16 nM for lane 2, 50 nM for lane 3, and 160 nM for lane 4.

 
Four hours of butyrate/dex treatment had no discernible effect on the levels of p300 and SRC-1 in the cells (Fig. 4AGo, lanes 1–8). However, by 20 h of butyrate treatment the levels of p300 had dropped precipitously. This decrease was independent of steroid treatment and was reversed by the addition of MG132, a specific inhibitor of protein degradation through the 26S proteasome pathway (Fig. 4AGo, lanes 9–16). Interestingly, MG132 treatment alone did not appear to significantly stabilize the endogenous p300, suggesting that proteasome-mediated degradation was a specific response to butyrate.

By contrast to the induced degradation of p300, SRC-1 levels were unaffected by either treatment at both 4 and 20 h. Moreover, the levels of GR were only moderately affected by treatment of the cells with MG132, although GR levels were down-regulated upon steroid treatment in a manner that was insensitive to butyrate treatment (data not shown).

We also tested the effect of 20 h of TSA treatment on p300 levels (Fig. 4BGo). At the 16–50 nM concentrations that failed to affect GR-mediated MMTV transcription, TSA treatment also had no discernible affect on p300 levels (lanes 2 and 3). However, when the concentration of TSA was raised to 160 nM, a level that moderately repressed glucocorticoid-induced transcription (Fig. 3Go), p300 levels were affected (Fig. 4BGo, lane 4). Indeed, the reduction in p300 levels in response to butyrate and TSA was, in each instance, proportional to the reduction observed in GR- activated MMTV transcription.

To determine whether the stabilization of p300 by MG132 treatment rescued the inhibition of MMTV transcription by butyrate, we measured the response of the MMTV promoter after treatment with MG132 (Fig. 5Go). Previously, it has been established that MG132 treatment can reversibly block proteolytic activity of the 26S proteasome without affecting cell viability and growth for as long as 20 h of treatment (48). Treatment with MG 132 alone had no affect on the induction of MMTV transcription by dex. However, addition of MG132 to the tissue culture medium completely reversed the inhibitory effect of butyrate on glucocorticoid-induced MMTV transcription, returning transcription to the levels induced by dex alone.



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Figure 5. MG132 Relieves the Down-Regulation of MMTV Promoter by Sodium Butyrate

The activity of the MMTV-CAT reporter (100 ng) in the HeLa-GR cells was monitored after 20 h of treatment with dex (100 nM), sodium butyrate (10 mM), and/or MG132 (1 µM) as indicated. Transfections were repeated a minimum of four times in triplicate with CAT activity expressed as the fold induction (normalized to protein concentration) relative to the untreated controls. Error bars represent the SEMs of triplicate samples within representative experiments.

 
Therefore, our results indicate that p300, but not SRC-1 or GR, is degraded through the 26S proteasome as a result of the inhibition of the deacetylation of no-histone proteins in the cell. Further, this degradation correlated directly with the repressive effects of butyrate on GR-mediated transcription subsequent to 20 h of dex treatment.

Butyrate Alters the Association of GR with SRC-1/p300 in Response to Hormone Treatment
The relatively normal levels of p300 that were maintained over the first 4 h of butyrate treatment suggested that the effects of butyrate on glucocorticoid-induced MMTV transcription also might only take effect after at least 4 h into the response. To test this hypothesis, we examined the effects of sodium butyrate and TSA treatment on the induction of MMTV transcription over the first 4 h of dex treatment (Fig. 6Go.) In contrast to expectations, butyrate also significantly reduced dex-induced transcription at 4 h, although the fold reduction was somewhat less than that at 20 h. Furthermore, MG132 treatment was unable to abrogate the butyrate effect on GR-activated transcription at 4 h (data not shown). Interestingly, 160 nM TSA was a slightly more affective inhibitor at 4 h than 20 h (Fig. 6Go), whereas 50 nM TSA continued to have no effect on steroid-induced transcription (data not shown).



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Figure 6. Sodium Butyrate and TSA Inhibit GR-Mediated Transcription at 4 h of Treatment

After transfection of MMTV-CAT reporter (100 ng), the HeLa-GR cells were incubated in the absence (-) or the presence (+) of dex (100 nM), sodium butyrate (10 mM), or TSA (160 nM) for 4 h before harvesting for CAT assay. CAT activity was expressed as the fold induction (normalized to protein concentration) relative to the untreated controls. Error bars represent the SEMs from four experiments performed in duplicate.

 
These results indicated an early effect of butyrate on MMTV transcription that was mediated through a mechanism other than the induction of p300 degradation. To initiate study of this mechanism, we examined the effect of butyrate treatment on the interaction between GR and SRC-1/p300 (Fig. 7Go). Cells were treated with hormone and butyrate as indicated, and coimmunoprecipitations were performed from nuclear extracts. Analysis of the immunoprecipitates showed that glucocorticoid treatment led to association of GR with both coactivators over at least the first 2 h after steroid administration (Fig. 7AGo, lane 1; Fig. 7BGo, lane 2). However, by 4 h after steroid treatment, association between GR and SRC-1/p300 declined (Fig. 7AGo, lane 2; Fig. 7BGo, lane 3), which is consistent with the profile of transcriptional activation of MMTV that has been described for GR, with a rapid initial transcriptional response followed by a decreased steady state response (49). Whereas a cyclic association between ER and coactivators has been observed at specific promoters in chromatin immunoprecipitation experiments (50), our results show that coactivator association with a steroid receptor in solution is also temporally regulated in response to steroid treatment.



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Figure 7. Butyrate Alters the Transient Interaction of GR with SRC and p300

A, Nuclear extracts of HeLa-GR cells were first immunoprecipitated with an antibody against GR (Affinity BioReagents, MA1–510). Then antibody against SRC (Affinity BioReagents, MA1–840) was used for Western blotting (lanes 1–5). Lanes 6–10 were Western blots of 20% of nuclear extract (NE) input that had been used for the immunoprecipitation. Nuclear extracts of HeLa wild-type cells were included as negative control (lanes 5 and 10). The duration of dex (100 nM) and/or sodium butyrate (10 mM) treatment is indicated in hours. B, The experimental set up was the same as in panel A except that p300 antibody (N-15, Santa Cruz Biotechnology, Inc.) was used for immunoprecipitation and GR antibody (M-20, Santa Cruz Biotechnology, Inc.) was used for the Western blot (lanes 1–6). The result shown is representative of the results obtained in four independent experiments.

 
Butyrate treatment altered the interaction of GR with SRC-1 and p300 in two ways. First, addition of butyrate to the culture media with dex resulted in a temporal shift in the association of SRC-1 with GR (Fig. 7AGo, lanes 3 and 4). Thus, SRC-1 failed to form a complex with GR 2 h after treatment but was coimmunoprecipitated with GR 2 h later, at 4 h post treatment. The change of association did not appear to reflect a barrier to the nuclear uptake of GR, as translocation of GR to the nucleus upon steroid treatment was unaffected by butyrate treatment (data not shown). In the second instance, butyrate treatment did not shift the time course of p300 interaction with GR, but essentially abolished the association between the two proteins (Fig. 7BGo, lanes 5 and 6).

Together, these results indicate that the repression of GR-mediated transcription at early times after administration of steroid results from multiple effects on GR-coactivator interactions.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our results demonstrate that p300 makes an important contribution to the activation of transcription by GR and that the effect of p300 is dependent upon its HAT activity. Furthermore, we show that inhibition of cellular deacetylase activities by sodium butyrate represses GR-activated transcription by altering the interaction of p300 and SRC-1 with GR and inducing the degradation of p300 through the 26S proteasome pathway. Thus, in addition to revealing a new pathway for the modulation of p300 function, we provide molecular evidence for means through which several of the complex actions of acetylation on the activation of transcription by GR are mediated.

Degradation of a protein through the 26S proteasome pathway is a tightly regulated ATP-dependent process and has important functions in cellular regulation and signal transduction (27). Proteins are generally targeted to the proteasome pathway through polyubiquitination. The addition of p300 to the list of proteins degraded through the proteasome suggests that the function of p300 may be dynamically regulated by ubiquitin ligases. Whether targeting of p300 to the 26S proteasome is triggered directly by specific acetylation of p300, or indirectly through the activation of effector proteins, remains to be determined.

We note that while treatment with the proteasome inhibitor MG132 abrogated the degradation of p300 induced by butyrate treatment, it had no effect on the levels of p300 detected in the absence of butyrate. This suggests that p300 degradation may be mediated through multiple pathways. Thus, targeting p300 to the 26S proteasome pathway may be a response to events that occur when histone deacetylases are inhibited, or upon specific hyperacetylation of components of transcription-regulatory complexes. However, this mode of degradation may differ from the means through which steady state levels p300 are maintained. Alternatively, the inability of MG132 to affect p300 levels in the absence of deacetylase inhibitors may simply reflect the long-lived stability of p300 when functional deacetylases are present to efficiently reverse acetylation.

It was proposed previously that ER{alpha}, PR, CBP, and SRC-1 are all degraded through the 26-proteasome pathway (36, 37, 39, 51). By contrast, in the present work, neither SRC-1 nor GR appeared to be strongly sensitive to treatment with the same proteasome pathway inhibitors employed in the previous studies. In this study we monitored the effects of proteasome pathway inhibition on the levels of SRC-1 and p300 endogenous to Hela cells and moderate levels of stably expressed GR, whereas in previous work, the SRC-1, ER{alpha}, and CBP were overexpressed in Hela cells by transient transfection. One possibility for the difference in results is that forced protein overexpression activates stress response pathways that attempt to rebalance the levels of critical transcription factors. More simply, it may be that overproduction via transient transfection leads to the generation of misfolded or improperly modified proteins that become specifically targeted to the 26S proteasome pathway in ways that do not happen for the correctly folded and modified endogenous factors. Interestingly, a recent report indicates that pretreatment of cells with MG132 results in the stabilization of the steady state levels of naïve GR (52).

Whereas p300 has previously been shown to be important for the induction of transcription by ER{alpha}, a specific requirement for p300 HAT activity has not been demonstrated (53). Indeed, other work has indicated that the HAT domain p300 is not required for the potentiation of transcriptional activation by retinoic acid receptors (16). Our result showing a dependence of GR-mediated transcription on the HAT activity of p300 suggests a selective requirement for p300 HAT activity by individual nuclear receptors. Whether the requirement for p300 HAT activity in GR-activated transcription is influenced by the cellular milieu, or displays aspects of promoter specificity, remains to be determined.

The early repressive effects of butyrate treatment on glucocorticoid-mediated transcription resulted from a mechanism distinct from the degradation of p300, as p300 levels had not declined significantly by 4 h into the treatment regimen. While temporal cycles of transcription factor/coactivator association with specific promoters have been shown previously for transcriptional responses to estrogen (50), our results reveal two broad effects of butyrate on GR-coactivator interactions in solution. While migration of GR to the nucleus upon steroid treatment appeared normal, association of GR with SRC-1 was delayed by several hours, and p300 association was essentially abrogated by butyrate treatment. These results suggest that inhibition of deacetylase activities led to specific modifications of GR or the coactivators that interfered with their association. Interestingly, the inability to detect SRC-1-GR interactions at 4 h post treatment in the absence of butyrate appears consistent with our newly obtained results that indicate that the bulk of liganded GR reassociates with the chaperone complex by 4 h after steroid treatment (data not shown). Whether butyrate affects interactions of other coregulatory factors with GR beyond those tested here remains to be determined.

One important question for further study is to delimit the relative contribution of effects of alterations in coactivator/GR interactions and p300 stability for the effects of butyrate on the activation of transcription by GR at the MMTV promoter. Results at present suggest that both pathways contribute to the butyrate effect and are supported by additional data that MG132 is unable to reverse the effect of butyrate on GR-activated transcription at the 4-h time point (data not shown). To resolve the relative contribution of the two pathways will likely require the development of HAT-competent p300 mutants that escape butyrate- induced proteasome degradation as well as characterization of the early effects of butyrate that alter GR-p300 and GR-SRC-1 interactions.

Thus, the control of p300 protein levels through the 26S proteasome pathway plays a central role in regulating long-term cellular responses to the inhibition of deacetylases while rapid effects of sodium butyrate treatment result from a direct interference in the normal interactions with transcription factors. Determining whether these complex changes result from a single signaling event or multiple distinct events will be the next challenge.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The plasmids for wild-type human p300 (pCI-p300) and mutant p300 with a 50-amino-acid deletion (aa 1472–1522) in the HAT domain (p300-hm) that abrogates HAT activity have been described previously (54). Expression vectors for wild-type 12S E1A, pcDNA1-E1A, and a mutated 12S E1A containing a deletion of amino acids 2–36, pcDNA3-E1A, that abrogates the interaction of E1A with p300 without affecting the association with retino-blastoma protein have also been described (44). pCR3-hSRC-1A expressing full-length human SRC-1a was described by Onate et al. (13). The MMTV-CAT reporter plasmid, pHC-WT, which contains MMTV sequences from -237 to +127 including the complex GRE between -178 and -80 is the same construct employed previously (55).

Cell Culture and Transfection
HeLa-GR cell line was created by selecting HeLa cells for the expression of rat GR from stably integrated p6R-GR expression plasmid (56). The cells were maintained in DMEM supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT). Transient transfections were performed in 35-mm dishes using 1.2 µl of the polyethylenimine agent ExGen 500 (Fermentas, Hanover, MD) mixed with 100 ng of reporter DNA, the amounts of cDNA expression vectors indicated in individual experiments. Carrier DNA was added to a total of 200 ng DNA per transfection. After transfection, the cells were cultured in DMEM supplemented with 10% charcoal and dextran-treated fetal bovine serum (HyClone Laboratories, Inc.) for 24–40 h, and then treated with dex (100 nM), butyrate (10 mM), and TSA (16–160 nM as indicated) for 4–20 h. The cells were washed twice with PBS, harvested in FT buffer (10 mM Tris-HCl, pH 7.4; 10 mM EDTA; 0.25 M sucrose). Cytosol was prepared by subjecting the cells to three cycles of freeze-thaw in dry-ice-ethanol followed by centrifugation at 16,000 x g for 10 min and 4 C. CAT assays were performed according to the standard scintillation counting protocol (Promega Corp., Madison, WI). CAT activity was expressed as the fold induction (normalized to protein concentration) relative to the untreated controls.

Assays for Protein Expression and Stability
Cells were treated with steroid, deacetylase inhibitors, and MG132 as indicated in individual experiments. After harvesting, the cells were washed twice in PBS, and whole-cell extracts were prepared by incubation of the cells in buffer W [50 mM Tris-HCl, pH 7.6; 400 mM NaCl; 10% glycerol; 0.5 mM dithiothreitol (DTT); 0.5 mM phenylmethylsulfonyl fluoride; and 1% Nonidet P-40 (NP-40)] at 4 C for 30 min, followed by centrifugation at 16,000 x g at 4 C for 10 min. The protein concentrations of the extracts were quantified by Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA) in duplicate. Extracts were separated by SDS-PAGE for Western analysis with antibodies against p300 (N-15, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and SRC-1 (Affinity BioReagents, Golden, CO; MA1–840). All experiments were repeated a minimum of three times.

Analysis of Protein-Protein Interactions
Protein-protein interactions were assessed by coimmunoprecipitation of factors from nuclear extracts. Nuclei were isolated by lysing cells treated as indicated in individual experiments in buffer L (10 mM Tris-HCl, pH 7.4; 10 mM KCl; 1.5 mM MgCl; 0.5 mM DTT; and 0.5% NP-40) for 10 min at 4 C followed by centrifugation at 16,000 x g for 1 min at 4 C. Nuclear extracts were prepared by incubation of the isolated nuclei in buffer N (50 mM Tris-HCl, pH 7.4; 400 mM KCl; 20% glycerol; 1.5 mM MgCl; 0.2 mM EDTA; and 0.5 mM DTT) at 4 C for 30 min followed by centrifugation at 16,000 x g for 10 min. For coimmunoprecipitation, the KCl concentration of nuclear extracts was adjusted to 150 mM and the NP-40 concentration was adjusted to 0.5%. Immunoprecipitation was carried out by incubating 300 µg of nuclear extracts with antibodies specific against GR (Affinity BioReagents, MA1–510) or p300 (Santa Cruz Biotechnology, Inc., N-15) at 4 C for 1 h, followed by incubation with 30 µl of 50% protein A slurry for an additional hour. Immunoprecipitates were washed three times in buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% glycerol, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5% NP-40 and then were separated on SDS-PAGE and subjected to Western analysis with antibodies specific for SRC (Affinity BioReagents, MA1–840) or GR (Santa Cruz Biotechnology, Inc., M-20) as indicated. All binding assays shown are representative of the results of a minimum of three independent experiments.


    ACKNOWLEDGMENTS
 
We are grateful to Drs. Ogryzko, Nakatani, O’Malley, and Yammamoto for plasmids. We thank J. Kochan, L. Pope, and C. Bayer for excellent technical assistance.


    FOOTNOTES
 
This work was funded by grants from Canadian Institutes of Health Research (CIHR) and Cancer Research Society to R.J.G.H and Y.A.L., respectively. Q.L. was supported by Junior and Senior Research Fellowship from CIHR, and R.J.G.H. is a CIHR Investigator.

Abbreviations: CAT, Chloramphenicol acetyltransferase; CBP, CREB binding protein; dex, dexamethasone; DTT, dithiothreitol; ER, estrogen receptor; GR, glucocorticoid receptor; HAT, histone acetyltransferase; MMTV, mouse mammary tumor virus; NP-40, Nonidet P-40; SRC-1, steroid receptor coactivator 1; TSA, trichostatin A.

Received for publication April 25, 2002. Accepted for publication August 12, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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