The Coactivator p300 Directly Acetylates the Forkhead Transcription Factor Foxo1 and Stimulates Foxo1-Induced Transcription

Valérie Perrot and Matthew M. Rechler

Growth and Development Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Matthew M. Rechler, Growth and Development Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Building 10, Room 8D12, Bethesda, Maryland 20892. E-mail: mrechler{at}helix.nih.gov.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The FOXO (Forkhead box class O) subgroup of forkhead transcription factors controls the expression of many genes involved in fundamental cellular processes. Until recently, studies conducted on posttranslational modifications of Forkhead proteins were restricted to their phosphorylation. In this report, we show that the coactivator p300 directly acetylates lysines in the carboxyl-terminal region of Foxo1 in vivo and in vitro, and potently stimulates Foxo1-induced transcription of IGF-binding protein-1 in transient transfection experiments. The intrinsic acetyltransferase activity of p300 is required for both activities. Our results suggest that acetylation of Foxo1 by p300 is responsible, at least in part, for its increased transactivation potency, although acetylation of histones cannot be excluded. Insulin, the major negative regulator of Foxo1-stimulated transcription, potently enhances p300 acetylation of Foxo1. Three consensus protein kinase B/Akt phosphorylation sites whose phosphorylation is stimulated by insulin are required for insulin-induced acetylation of Foxo1. In contrast to its importance in regulating the transcriptional activity of Foxo1 in the absence of insulin, acetylation plays only a minor role compared with phosphorylation in insulin inhibition of Foxo1 transcriptional activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HIGHLY CONSERVED subfamily of Forkhead box class O (FOXO) transcription factors (1, 2) was initially identified at chromosomal translocations in several human tumors (3, 4, 5, 6). Members of the FOXO subfamily include FOXO1, FOXO3a (7), FOXO4 (4, 5), and FOXO6 (8). A closely related ortholog of FOXO1, Daf-16, is expressed in Caenorhabditis elegans (9, 10). The FOXO proteins regulate the transcription of genes involved in key cellular processes such as cell cycle progression, apoptosis, DNA repair, response to oxidative stress, differentiation, and glucose metabolism (reviewed in Refs.11, 12, 13, 14, 15, 16). They contain a highly conserved central DNA binding domain (the Forkhead Box) that binds to a FOXO response element (5'-TTGTTTAC-3') located in the promoter of their target genes (17, 18) and a carboxyl-terminal transactivation domain (3, 19, 20). They also share three consensus phosphorylation sites for serine/threonine protein kinase B (PKB)/Akt (21). Insulin (22), as well as cytokines and growth factors (23, 24, 25, 26), inhibit FOXO-stimulated transcription by activating a phosphatidylinositol 3-kinase-PKB/Akt pathway (27, 28). PKB/Akt-mediated phosphorylation of Foxo1 at consensus sites located at Thr24, Ser253, and Ser316 (29) greatly reduces insulin inhibition of target gene transcription and triggers the export of Foxo1 from the nucleus (23, 30, 31, 32). In addition to PKB/Akt, serum and glucocorticoid-inducible kinase (33) and inhibitor of nuclear factor-{kappa}B kinase (34) also induce cytoplasmic redistribution and transcription inhibition by phosphorylating FOXO proteins.

Whereas the role of phosphorylation in the regulation of FOXO activity has been extensively characterized, the importance of other posttranslational modifications of the FOXO proteins such as ubiquination (34, 35, 36) and acetylation (37, 38, 39, 40, 41) has only recently emerged. Ubiquitination of phosphorylated FOXO proteins targets them for proteosomal degradation, whereas the effect of acetylation of FOXO proteins on their transcriptional activity remains to be resolved.

The possibility that FOXO1 might be acetylated was first suggested by the observation of Nasrin et al. (42), showing that the adenoviral oncoprotein E1A inhibited transcription stimulated by Daf-16 or FOXO1. E1A might act by inhibiting binding of the FOXO proteins to the closely related coactivators, p300 and CBP [cAMP-responsive element-binding protein (CREB)-binding protein] (43, 44, 45). p300 and CBP possess intrinsic acetyltransferase activity (46, 47), which enables them to regulate transcription by acetylating histones and a growing list of transcription factors (48, 49). Indeed, while this study was in progress, several reports have appeared showing that p300/CBP can acetylate members of the FOXO subfamily (37, 38, 40, 41).

The functional implications of FOXO acetylation by p300/CBP have not been studied extensively, and the results obtained have been conflicting. Acetylation by CBP inhibited the transcriptional activity of FOXO4 (38, 41), whereas acetylation by p300 stimulated the transcriptional activity of FOXO3a (40). The present study was undertaken to determine directly the effect of p300 on Foxo1-stimulated transcription of the IGF-binding protein-1 (IGFBP-1) promoter in the absence and presence of insulin, and to determine whether acetylation of Foxo1 by p300 was involved in its regulation of transcription. We demonstrate that the coactivator p300 directly acetylates Foxo1 in vivo and in vitro, and that p300 markedly increases Foxo1-stimulated IGFBP-1 transcription when its acetyltransferase activity is intact, suggesting that acetylation of Foxo1 by p300 may contribute to the enhancement of Foxo1-stimulated transactivation. We also observed that insulin potently enhances Foxo1 acetylation by p300 and that phosphorylation of the PKB/Akt sites is necessary for insulin-dependent acetylation. Acetylation of Foxo1 is an important regulator of Foxo1 transcriptional activity in the absence of insulin but plays a much less important role than phosphorylation in insulin inhibition of Foxo1 transcriptional activity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
p300 Acetyltransferase Activity Is Required for Acetylation of Foxo1 in Vivo
In light of the previous observation that CBP binds to FOXO1 and Daf-16 (42), we first examined whether the related coactivator p300 could acetylate Foxo1 when the two proteins were coexpressed. Immunoblot analysis using an antibody specific for acetyl-lysine demonstrated p300-dependent acetylation of Foxo1 in human embryonic kidney (HEK)-293T cells (Fig. 1AGo). Foxo1 proteins remained unacetylated in the absence of p300. Foxo1 acetylation also was observed in H4IIE rat hepatoma cells (Fig. 1BGo), indicating that this posttranslational modification is not cell type specific. Moreover, acetylation of Foxo1 by p300 depends on the acetyltransferase activity of the coactivator protein because an acetylase-deficient p300 point mutant (p300-DY) (50) failed to induce Foxo1 acetylation (Fig. 1CGo). Our results corroborate the recent reports showing that FOXO proteins can be acetylated (37, 38, 39, 40, 41) and further show that p300 acetyltransferase activity is required for Foxo1 acetylation.



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Fig. 1. Foxo1 Is Acetylated in Vivo by Wild-Type p300 But Not by an Acetylase-Deficient p300 Mutant

HEK-293T (A) and H4IIE (B) cells were cotransfected with expression vector for myc-tagged Foxo1 together with FLAG-tagged p300 or the empty expression vector as indicated. Foxo1 proteins were immunoprecipitated (IP) with anti-myc antibody followed by immunoblotting (WB) with antibody to acetyl-lysine. The expression levels of Foxo1 protein and p300 were examined by immunoblotting total cellular lysates with anti-Foxo1 and anti-FLAG antibodies, respectively. Representative examples of three to five independent experiments are shown. The acetylation signal is weaker in H4IIE cells than in HEK-293T cells because of a lower efficiency of transfection. C, HEK-293T cells were cotransfected with myc-tagged Foxo1 and FLAG-tagged wild-type p300 or the acetylase-deficient myc-tagged p300 mutant (p300-DY). Immunoprecipitation with anti-myc antibody was followed by immunoblotting with anti-acetyl-lysine antibody as indicated. The level of Foxo1 protein in cells cotransfected with empty vector control in this experiment was too high to be able to appreciate a further increase in abundance in the presence of p300.

 
p300 Stabilizes Foxo1 Protein in an Acetyltransferase-Independent Manner
In addition to acetylating Foxo1, p300 appeared to increase its abundance in comparison with cells expressing the vector control (Fig. 1AGo). The fact that Foxo1 protein levels were increased to a comparable extent in cells expressing either wild-type p300 or the p300-DY mutant (Fig. 1CGo) suggested that the up-regulation of Foxo1 by p300 was unrelated to its acetylation. This was demonstrated by monitoring the disappearance of the forkhead protein in cells overexpressing wild-type (Fig. 2AGo) or mutant (Fig. 2BGo) p300 when de novo protein synthesis was inhibited with cycloheximide. As seen in Fig. 2Go, coexpression of either wild-type or mutant p300 prolonged the half-life of Foxo1 protein. Because Foxo1 is not acetylated by p300-DY, these results indicate that p300 stabilizes Foxo1 protein in an acetyltransferase-independent manner.



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Fig. 2. p300 Stabilizes Foxo1 Protein Levels in an Acetyltransferase-Independent Manner

HEK-293T cells were transfected with Foxo1 together with empty vector and either wild-type p300 (A) or the acetylase-deficient mutant p300-DY (B). Twenty-four hours after transfection, cycloheximide (10 µg/ml) was added and the abundance of Foxo1 in cell lysates was determined at the indicated times by Western blotting. The intensity of the Foxo1 protein band was quantified using NIH imaging software. The percent of Foxo1 proteins remaining is plotted; levels at 0 h were taken as 100%. The graphs shown are representative of three independent experiments. Although our results indicate that overexpression of either p300 or p300-DY stabilizes expressed-Foxo1, the higher abundance of the exogenously expressed proteins may have contributed to the slower clearance.

 
p300 Interacts with and Directly Acetylates Foxo1 Protein
The previous results suggested that p300 might directly acetylate Foxo1. To test this hypothesis, we first examined whether the two proteins interacted physically using in vitro glutathione-S-transferase (GST) pull-down assays (Fig. 3AGo). Lysates prepared from HEK-293T cells overexpressing p300 were tested for interactions with bacterially expressed GST control or GST-fused FOXO1. The results shown in Fig. 3AGo indicated that FLAG-p300 was specifically pulled down by interaction with GST-FOXO1 but not by GST alone, demonstrating that the two proteins can interact in vitro. Endogenous Foxo1 also binds to p300. It was detected in lysates of HEK-293T cells after immunoprecipitation with antibody to p300 (Fig. 3BGo).



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Fig. 3. Foxo1 Interacts with p300 in Vitro and in Vivo

A, GST pull-down assay. FLAG-tagged wild-type p300 was transiently expressed in HEK-293T cells. Cellular lysates were mixed with GST-FOXO1 or GST bound beads, and a GST pull-down assay was performed. Reaction products were resolved on SDS-PAGE and bound p300 was visualized by immunoblotting with anti-FLAG antibody. B, Immunoprecipitation. Nuclear extracts from HEK-293T cells were immunoprecipitated with anti-Foxo1 or anti-p300 antibodies. The precipitated proteins were analyzed by Western blotting using anti-Foxo1 antibody. C and D, Immunoprecipitation. HEK-293T cells were transfected with FLAG-tagged wild-type p300 (C) or p300-DY (D), together with myc-tagged full-length Foxo1 or GFP-tagged Foxo1 208–652 expression vectors as indicated. Nuclear extracts were prepared and immunoprecipitated with anti-myc or anti-GFP antibodies, or control IgG from nonimmune mouse serum (IgG). The precipitated proteins were analyzed by Western blotting using anti-FLAG or anti-myc antibody. A representative experiment is shown.

 
To confirm that p300 interacts with Foxo1 in vivo and to delineate the region in Foxo1 involved in this interaction, HEK-293T cells were transfected with FLAG-p300 and full-length Foxo1 protein or its carboxyl-terminal region (Foxo1 208–652). Cell lysates immunoprecipitated with antibodies against tagged-Foxo1 proteins and subjected to Western blotting using antibody against FLAG-tagged p300 confirmed the results observed in Fig. 3AGo and further demonstrated that p300 was able to interact with the carboxyl-terminal region of Foxo1 (Fig. 3CGo). Similar results were obtained with p300-DY (Fig. 3DGo), indicating that the acetyltransferase-deficient mutant can bind to Foxo1 and stabilize it but cannot acetylate it.

We next established that p300 can directly acetylate Foxo1 by performing in vitro acetylation assays. Recombinant His6-tagged full-length p300 protein (graciously purified and provided by T. P. Yao and C. H. Lai, Duke University, Durham, NC) was incubated with GST-fused Foxo1 proteins in the presence of [1-14C]acetyl-coenzyme A (CoA) (Fig. 4Go). As expected, histones were strongly acetylated by p300. His6-tagged p300 protein directly acetylated GST-Foxo1 1–652 but not its amino-terminal region encompassing amino acids 1–207 or the GST protein alone. These data indicate that Foxo1 itself is a specific target of p300 acetyltransferase activity and that the acetylation site(s) is (are) located exclusively in the carboxyl-terminal region of Foxo1.



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Fig. 4. Foxo1 Is a Target of Acetylation by p300

A, Autoradiogram of the in vitro acetylation assay. Recombinant GST-Foxo1 1–207 (lane 3), GST-Foxo1 1–652 (lanes 4–5) or GST protein alone (lane 2) were incubated with 0.8 µg of baculovirus-expressed His6-tagged full-length p300 in the presence of [1-14C]acetyl-CoA for 1 h at 30 C. Core histones (Histones) (lane 1) were used as a positive control. Reaction products were separated by SDS-PAGE and processed for autoradiography. The labeled band of acetylated GST-Foxo1 1–652 protein after a 12-h exposure is indicated. A representative experiment is shown. B, Coomassie blue staining of SDS-PAGE separation of purified recombinant proteins used in the in vitro acetylation assay. The top part of the gel with the autoacetylated full-length p300 was cut off. The numbers on the left indicate the migration positions of molecular mass markers (in kilodaltons). The asterisks indicate the positions of intact GST-Foxo1 1–652 in the right two lanes and histones in lane 1.

 
p300 Increases Foxo1-Stimulated Transcription in an Acetyltransferase-Dependent Manner
To examine the effect of p300 on the transcriptional responses induced by Foxo1, transient transfection assays were performed using an IGFBP-1 promoter-luciferase reporter bearing two copies of a FOXO response element. In agreement with the observation of Nasrin et al. (42), overexpression of the adenovirus E1A protein, which can competitively inhibit the binding of transcription factors to p300/CBP and thereby inhibit transcriptional coactivation (43, 51), strongly reduced basal expression of the IGFBP-1 promoter (data not shown). E1A inhibition was overcome by overexpression of p300 (data not shown). These results suggest that p300/CBP or an endogenous coactivator homolog may be involved in the stimulation of IGFBP-1 promoter activity by Foxo1.

Indeed, transcription of the IGFBP-1 promoter induced by Foxo1 was markedly increased when p300 was overexpressed (12.5 ± 0.9-fold, mean ± SE, n = 7) (Fig. 5AGo). By contrast, overexpression of p300-DY failed to enhance transactivation of the IGFBP-1 promoter by Foxo1 (Fig. 5BGo). These results suggest that the acetyltransferase activity of p300 is required not only for acetylation of Foxo1 but also for the stimulation of Foxo1-induced transcription, further supporting the hypothesis that Foxo1 acetylation may play a critical role in the stimulation of Foxo1 transcriptional activity by p300.



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Fig. 5. p300 Stimulates the Transcriptional Activity of Full-Length Foxo1 in an Acetyltransferase-Dependent Manner

A, H4IIE cells were transfected with the expression vector for p300, pCMV5/Foxo1, and the IGFBP-1 luciferase reporter plasmid. The relative luciferase activity of cells transfected with Foxo1 proteins in the absence of p300 was taken as 100%. Results are displayed as the mean ± SE of seven independent experiments. B, H4IIE cells were transiently transfected with the IGFBP-1 luciferase reporter plasmid and pCMV5/Foxo1 together with either wild-type p300 (wt), mutant p300-DY (DY) or the empty expression vector (Control). The mean ± SE of three independent experiments is shown. For unexplained reasons, the stimulation of Foxo1-stimulated transcription by wild-type p300 was lower in these experiments than in the experiments shown in panel (A).

 
Insulin Potently Enhances the Acetylation of Foxo1 by p300
Insulin is the major negative regulator of Foxo1-stimulated transcription. This is thought to occur predominantly through phosphorylation of its three PKB/Akt consensus sites (Thr24, Ser253, and Ser316) (22, 23, 52). To begin exploring whether insulin might also regulate Foxo1 transcriptional activity through another posttranslational modification such as acetylation, we examined the effect of insulin on p300-induced acetylation of Foxo1 (Fig. 6Go). Surprisingly, addition of insulin to p300-expressing cells led to a rapid and transient increase of acetylated Foxo1 (Fig. 6AGo). Although the acetyl-lysine signal was maximal at 5 min in the experiment shown, the peak of acetylation varied from 1–10 min in different experiments. As seen in Fig. 6BGo, the maximal enhancement of p300-dependent acetylation of Foxo1 by insulin was 5.9 ± 1.1 (mean ± SE, n = 3). Foxo1 acetylation was not observed with the acetylase-deficient p300-DY even in the presence of insulin (Fig. 6CGo), excluding the possibility that after insulin treatment p300-DY might have recruited another acetyltransferase protein that could acetylate the forkhead protein.



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Fig. 6. Stimulation of p300-Dependent Foxo1 Acetylation by Insulin

A, Myc-tagged Foxo1 was expressed in HEK-293T cells together with FLAG-tagged p300 or empty vector. After 24 h, cells were switched to serum-free medium overnight before incubation with recombinant human insulin (0.25 µg/ml) for the indicated times. Foxo1 proteins were immunoprecipitated with anti-myc antibody followed by Western blotting with antibody to acetyl-lysine. A representative experiment is shown. B, Quantification of the maximal enhancement of p300-dependent acetylation of Foxo1 by insulin. Foxo1 acetylation in the absence of insulin in the same experiment was taken as 1. Results are displayed as the mean ± SE (n = 3). C, HEK-293T cells were transiently transfected with myc-tagged Foxo1 and FLAG-tagged p300 or myc-p300-DY mutant and treated with insulin as described in panel (A). D, HEK-293T cells overexpressing myc-tagged Foxo1 and FLAG-tagged p300 were either untreated or treated with insulin (0.25 µg/ml) in the presence (+TSA) or absence of TSA (0.4 µM), and harvested at the indicated time points. The immunoprecipitates were subjected to Western blotting with anti-acetyl-lysine antibody. The levels of the different tagged proteins were examined in total cell lysates by immunoblot analysis with the antibodies to myc and FLAG.

 
The observation that Foxo1 acetylation is a transient event after insulin treatment suggested that after an initial increase, the abundance of acetylated Foxo1 might decrease due to the induction of a deacetylase. In fact, treatment with the histone deacetylase inhibitor trichostatin A (TSA) dramatically increased the levels of acetylated Foxo1 in HEK-293T cells (Fig. 6DGo), although the Foxo1 protein levels were identical in untreated and treated cells. Identical results were obtained when another deacetylase inhibitor, sodium butyrate, was used (data not shown). The increased levels of acetylated Foxo1 after TSA treatment also were seen in cells that had not been treated with insulin, suggesting that the deacetylase activity may be constitutive and that the enhancement of Foxo1 acetylation by insulin most likely results from increased acetylase activity.

Phosphorylation of the PKB/Akt Sites Is Required for Insulin-Enhanced Acetylation of Foxo1
We next determined whether phosphorylation of the PKB/Akt sites in Foxo1 might be required for its acetylation. Wild-type Foxo1 1–652 and a Foxo1 mutant in which the three PKB/Akt phosphorylation sites were mutated to alanine [Foxo1/Thr24Ala/Ser253Ala/Ser316Ala (Foxo1/AAA)] were transfected into HEK-293T cells together with p300. As shown in Fig. 7Go, in the absence of insulin, Foxo1/AAA was acetylated to a greater extent than wild-type Foxo1, suggesting that basal phosphorylation of the three PKB/Akt sites might have inhibited acetylation as proposed for FOXO3a (37). Moreover, in contrast to wild-type Foxo1, acetylation of Foxo1/AAA decreased during incubation with insulin, indicating that phosphorylation of the PKB/Akt sites may be required for insulin-induced acetylation.



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Fig. 7. Phosphorylation of the PKB/Akt Sites Is Required for Insulin-Induced Acetylation of Foxo1 by p300

A, HEK-293T cells were transfected with FLAG-tagged p300 along with myc-tagged Foxo1 wild-type (wt) or Foxo1/Thr24Ala/Ser253Ala/Ser316Ala (AAA). Cells were treated with insulin (0.25 µg/ml) for the indicated times. Foxo1 proteins were immunoprecipitated with anti-myc antibody followed by immunoblotting with anti-acetyl-lysine antibody. The position of Foxo1 and the heavy chain of the immunoprecipitating antibody are indicated by an arrowhead and arrow, respectively. The membrane was stripped and reprobed with the anti-FOXO1-pThr24 and anti-FOXO1-pSer253 antibodies. The expression levels of Foxo1 proteins and p300 were assessed by immunoblotting total cellular lysates with anti-Foxo1 and anti-FLAG antibodies, respectively. The weaker signal with Foxo1/AAA compared with wild-type Foxo1 could represent lower transfection efficiency or decreased immunoreactivity. B, Quantification of the p300-dependent acetylation of wild-type Foxo1 and Foxo1/AAA by insulin. Wild-type Foxo1 acetylation in the absence of insulin was taken as 100%. Results are displayed as the mean ± SE (n = 3).

 
Effect of p300 and Acetylation on Insulin Inhibition of Foxo1-Mediated Transcription
Because p300 overexpression markedly stimulated Foxo1-induced transcription in the absence of insulin, we next examined the effect of the coactivator on insulin inhibition of transactivation stimulated by Foxo1 (Fig. 8Go). As we previously reported (19, 20), insulin potently inhibited transactivation by wild-type Foxo1 (Fig. 8Go). Even though cotransfection with p300 stimulated Foxo1 acetylation, it did not affect insulin inhibition of Foxo1-stimulated transactivation of the IGFBP-1 promoter. Overexpression of p300 caused a smaller increase in Foxo1/AAA transcriptional activity in the absence of insulin than is seen with wild-type Foxo1 even though its acetylation was increased. Mutation of the PKB/Akt sites or acetylation of additional sites in Foxo1/AAA may have limited the ability of p300 to enhance Foxo1/AAA-stimulated transcription. Insulin inhibited Foxo1/AAA transcriptional activity to only a small extent in the presence or absence of p300, confirming that phosphorylation was the predominant determinant of insulin inhibition of Foxo1 transcriptional activity.



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Fig. 8. Insulin Inhibition of Foxo1-Mediated Transcription Is Decreased by Mutation of the Three PKB/Akt Sites But Not by Cotransfection with p300

H4IIE cells were transiently transfected with the IGFBP-1 luciferase reporter plasmid and p300 or the corresponding empty vector control, together with expression vectors encoding wild-type Foxo1 or Foxo1/Thr24Ala/Ser253Ala/Ser316Ala (Foxo1/AAA). Two independent Foxo1/AAA clones were used. After 48 h, half of the cells were treated with recombinant human insulin (0.25 µg/ml). Relative luciferase activity is plotted. The activity with Foxo1 in the absence of p300 and insulin is taken as 100%. The mean ± SD of two experiments is plotted.

 
Effect of p300 on the Subcellular Localization of Foxo1 in the Presence and Absence of Insulin
Nuclear exclusion of Foxo1 is thought to play a major role in insulin-induced inhibition of transcription (23, 32). We examined whether alterations in the subcellular localization of Foxo1 proteins in H4IIE cells could account for the enhancement of basal Foxo1-stimulated transcription by p300. As previously reported (20, 23, 32), indirect immunofluorescence demonstrated the predominant nuclear localization of Foxo1 under basal conditions, and its subsequent redistribution to the cytoplasm after insulin stimulation (Fig. 9Go). Overexpression of p300 led to an even greater nuclear localization of Foxo1 but did not prevent its translocation to the cytoplasm in response to insulin. An identical result was obtained when the p300-DY mutant was overexpressed (data not shown), indicating that acetylation is not required for Foxo1 redistribution to the cytoplasm. Altogether, these results indicate that the small increase in nuclear localization of Foxo1 after p300 overexpression in the absence of insulin is not sufficient to account for the marked stimulation of Foxo1-induced transcription by p300 under basal conditions.



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Fig. 9. Subcellular Localization of Foxo1 in Hepatocytes Transfected with p300

Myc-tagged Foxo1 was expressed in subconfluent H4IIE cells, together with FLAG-tagged p300 or the empty vector control. Twenty-four hours after transfection, cells were transferred to serum-free medium for an additional 16 h before the addition of insulin (1 µg/ml), fixed, and analyzed by immunofluorescence for nuclear staining (DAPI, bottom) and myc expression using specific monoclonal antibody and FITC (fluorescein isothiocyanate)-conjugated antimouse IgG (top). Shown are representative images of six independent experiments with similar results.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Posttranslational modification of transcription factors is a prominent mechanism of transcription regulation (53, 54, 55). Although the role of phosphorylation in the regulation of Foxo1 transcriptional activity has been extensively documented, the possible contribution of other posttranslational modifications has only recently been considered. In this report, we show that overexpression of the coactivator p300 potently stimulates Foxo1-induced IGFBP-1 transcription and directly acetylates Foxo1 protein in vivo and in vitro. The acetyltransferase activity of p300 is required for both effects. Although the precise role of Foxo1 acetylation remains to be elucidated, our results suggest that acetylation of Foxo1 by p300 may be responsible, at least in part, for its increased transactivating activity.

Nasrin et al. (42) were the first to provide suggestive evidence that CBP might regulate FOXO1-induced IGFBP-1 transcription. They reported that the adenoviral protein E1a inhibited transcription stimulated by Daf-16 or FOXO1, possibly by competitively inhibiting the binding of the transcription factors to the C/H3 domain of p300/CBP (44). The argument was not conclusive, however, because E1A also can inhibit transcription by mechanisms that do not involve the two coactivators (51). We now demonstrate that overexpression of p300 potently increases Foxo1-stimulated IGFBP-1 transcription approximately 12-fold.

The acetyltransferase activity of p300 is required for p300 enhancement of Foxo1-mediated IGFBP-1 transcription. No stimulation of IGFBP-1 transcription was seen when a p300 mutant deficient in acetyltransferase activity was used, consistent with what has been reported for other transcription factors: erythroid Kruppel-like factor (56), HNF-4 (57), c-myc (58), and sterol regulatory element-binding protein 1 (59). Our results, however, contrast with the increased stimulation of Foxo4 transcriptional activity observed after cotransfection of an acetylase-deficient mutant of CBP recently reported by Fukuoka et al. (38).

As we demonstrate in this report, Foxo1 is acetylated by p300 in vivo and by recombinant p300 in vitro. Although p300 might acetylate Foxo1 indirectly, by recruiting other acetyltransferases such as p300/CBP-associated factor (P/CAF) (60) or steroid receptor coactivator-1 (SRC-1) (61), which have been shown to bind to FOXO1 (42), our in vitro results demonstrate that Foxo1 can be directly acetylated by p300. We show that p300 specifically binds to and acetylates the carboxyl-terminal region of Foxo1 that contains the transactivation domain (Foxo1 208–652) but does not acetylate the amino-terminal protein fragment (Foxo1 1–207). Consistent with our results, Fukuoka et al. (38) identified three lysine residues in the same region of Foxo4 that were acetylated by CBP, and Brunet et al. (39) demonstrated that five carboxyl-terminal lysines in FOXO3a were acetylated in response to oxidative stress. All five of the FOXO3a acetylation sites and two of the three sites in Foxo4 are conserved in Foxo1. In fact, the residue corresponding to Lys242 of Foxo1 is acetylated in both FOXO3a and Foxo4.

The fact that the acetyltransferase activity of p300 is required for both the enhancement of Foxo1-stimulated transcription and for the acetylation of Foxo1 suggests that acetylation of Foxo1 by p300 per se may be responsible for its potentiation of Foxo1-stimulated transcription, although acetylation of histones cannot be excluded. Indeed, a direct relationship between acetylation of specific lysines on transcription factors and increased transcriptional activity (48) has been documented for several transcription factors, including p53 (62), GATA-1 (63, 64), Drosophila T-cell factor-1 (65), erythroid Kruppel-like factor (56, 66), HIV-1 Tat protein (67), c-Myb (68), MyoD (69), the estrogen receptor (70), the androgen receptor (71), and HNF-4 (57).

Acetylation of other members of the FOXO subfamily by p300/CBP has had variable effects on their transcriptional activity. Motta et al. (40) showed that overexpression of p300 increased FOXO3a transcriptional activity toward the Bim promoter, and that the increased activity was inhibited by the NAD-dependent deacetylase SIRT1. Because SIRT1 can deacetylate both p300 and FOXO3a, however, the inhibition of p300-enhanced transcription could result from deacetylation of either p300 or Foxo3a. By contrast, acetylation of Foxo4 by CBP appears to inhibit its transcriptional activity (38). Van der Horst et al. (41) confirmed that CBP overexpression decreased FOXO4 induction of p27. These differences are undoubtedly functionally significant. Although many actions of the FOXO transcription factors are interchangeable, it is now appreciated that they exhibit specific nonredundant functions during development (72). Foxo1 null mice are embryonic lethals due to defective development of the vascular system, whereas Foxo4 null mice are viable and have no gross defects. Structural heterogeneity of the FOXO proteins may lead to acetylation of different sites by p300/CBP with different functional consequences. Our results demonstrating that p300 binds to the carboxyl-terminal region of Foxo1 are in contrast to a recent study showing that p300 interacts with the first 52 amino acids of FOXO3a (37), suggesting that p300 may recognize multiple sites in FOXO proteins. It remains to be determined whether the divergent effects of p300/CBP on the transcriptional activity of acetylated Foxo1 and Foxo4 result from the acetylation of different sites or other structural differences that might affect promoter selectivity.

Acetylation of transcription factors can regulate their transcriptional activity by altering their DNA binding activity and interaction with transcription-regulatory proteins, as well as their stability and subcellular localization. Our analysis reveals that acetylation of Foxo1 by p300 does not alter its stability or localization. Exogenous expression of wild-type p300 but also the acetyltransferase-defective p300-DY mutant increased the half-life of Foxo1 protein in cells, indicating that acetylation itself is not critical for stabilization of the forkhead protein. Furthermore, stabilization of Foxo1 by p300 is unique in that it does not require its acetylation, in contrast to other transcription factors such as p53 (73, 74, 75), HNF-4 (57), Smad7 (76), and sterol regulatory element-binding protein 1 (59), for which stabilization has been demonstrated to be associated with their acetylation. Similarly, overexpression of p300 has only limited effects on the subcellular distribution of Foxo1 that are unlikely to account for the marked stimulation of Foxo1-induced transcription. This differs from the marked effects of acetylation of HNF-4 preventing its nuclear export (57) or acetylation of the RelA subunit of the nuclear factor-{kappa}B heterodimer, preventing it from being sequestered in the cytoplasm (77). The enhancement of Foxo1-induced transcription by p300 might occur because acetylation increases DNA binding as reported for the transcription factors p53 (62, 78), GATA-1 (63), E2F-1 (79), HNF-4 (57), c-Myb (68), RelA (77), and E2F-1,2,3 (80), or by facilitating the recruitment of coactivators to the promoter in vivo (81).

Insulin is the major negative regulator of Foxo1-stimulated transcription. We examined whether insulin regulated Foxo1 acetylation and, if so, how this might affect its transcriptional activity. We observed that insulin potently enhances Foxo1 acetylation by p300. The increase was transient, however, suggesting the existence of negative regulators of Foxo1 acetylation. In support of this hypothesis, we showed that the abundance of acetylated Foxo1 is considerably increased when deacetylases are inhibited whether or not insulin is present, suggesting that deacetylase activity is constitutive and that insulin acts instead by stimulating p300 acetyltransferase activity. Moreover, as in the absence of insulin, acetylation was not observed with p300-DY, indicating that insulin treatment did not lead to the recruitment of another acetyltransferase that could acetylate the Forkhead protein.

Although insulin and the cytokine erythropoietin both activate PKB/Akt and stimulate phosphorylation of FOXO proteins, insulin enhances p300-stimulated acetylation of Foxo1, whereas erythropoietin inhibited p300 binding to and acetylation of FOXO3a (37). Mahmud et al. (37) concluded that dephosphorylation of FOXO3a was a prerequisite for acetylation. By contrast, we observed that insulin stimulated the phosphorylation and acetylation of wild-type Foxo1, and that insulin-induced acetylation was reduced in Foxo1/AAA in which the PKB/Akt sites were mutated, indicating that insulin-stimulated phosphorylation of the PKB/Akt sites did not inhibit and probably promoted Foxo1 acetylation. Phosphorylation was not required for basal acetylation, however, because acetylation was increased in Foxo1/AAA in the absence of insulin.

Although insulin increases the acetylation of Foxo1 by p300, insulin’s ability to inhibit Foxo1-stimulated transcription is unchanged by p300 overexpression and acetylation (Fig. 10Go). Insulin-induced phosphorylation of the three PKB/Akt sites in Foxo1 leading to its export from the nucleus remains the predominant determinant of Foxo1 transcriptional activity and is unaffected by increased acetylation. A similar dissociation of insulin’s effect on p300-induced acetylation and its effects on Foxo1 transcriptional activity is seen in Foxo1/AAA in which the three PKB/Akt sites have been mutated. Insulin’s ability to inhibit Foxo1-stimulated transcription is greatly reduced irrespective of whether p300 is overexpressed or whether insulin decreases p300-induced acetylation in the mutant. Thus, even though insulin regulates the level of acetylation of Foxo1 and Foxo1/AAA by p300, the effects on Foxo1 acetylation do not significantly affect its ability to inhibit Foxo1 transcriptional activity. Insulin inhibition of Foxo1-stimulated transcription is predominantly determined by insulin-induced phosphorylation and the resulting export of the transcription factor from the nucleus and inhibition of Foxo1-stimulated transcription, which are unaffected by p300 overexpression and increased acetylation of Foxo1.



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Fig. 10. Schematic Diagram Summarizing the Effect of Acetylation and Phosphorylation on Foxo1-Stimulated IGFBP-1 Promoter Activity

Wild-type Foxo1 (lines 1 and 2) and Foxo1/AAA (lines 3 and 4) were cotransfected with empty vector (lines 1 and 3) or p300 (lines 2 and 4). The effects of p300 and insulin on posttranslational modifications of Foxo1 (lysine acetylation and phosphorylation of the three PKB/Akt sites) are indicated. Foxo1 transcriptional activity in the absence of insulin (+p300/–p300) is indicated by solid arrows to the left. The effect of insulin on Foxo1 transcriptional activity (+Insulin/–Insulin) is indicated by solid arrows to the right. In the absence of insulin, p300 stimulates acetylation of wild-type Foxo1 and, to a greater extent, Foxo1/AAA, suggesting that basal phosphorylation of the PKB/Akt sites might inhibit acetylation. p300 markedly stimulates transcription of wild-type Foxo1 and, to a lesser extent, Foxo1/AAA. Insulin stimulates the phosphorylation of the PKB/Akt sites in wild-type Foxo1 and inhibits Foxo1 transcriptional activity. Although insulin stimulates the acetylation of wild-type Foxo1 by p300, it inhibits Foxo1 transcriptional activity to the same extent as in the absence of p300. The inability of insulin to inhibit Foxo1/AAA-stimulated transcriptional activity is not affected by p300, even though insulin inhibits p300-induced acetylation of Foxo1/AAA. These results indicate that insulin inhibition of Foxo1-stimulated transcription is predominantly determined by phosphorylation of the PKB/Akt sites and is not significantly affected by the additional acetylation. Ac, Acetyl; S, serine; T, threonine; pS, phosphoserine; pT, phosphothreonine.

 
In summary, we have shown that Foxo1 and its carboxyl-terminal protein fragment are targets of the p300 acetyltransferase. p300 Binds to and acetylates lysines located in the carboxyl-terminal region of Foxo1. p300 Also functions as a coactivator and potentiates Foxo1-induced transactivation in the absence of insulin. Our results suggest that direct acetylation of Foxo1 by p300 may be responsible for p300 enhancement of Foxo1-stimulated transcription, although acetylation of histones cannot be excluded. The enhanced transcription may reflect increased binding of acetylated Foxo1 to DNA motifs in the promoter of target genes or interactions with coactivators but does not appear to involve stabilization or enhanced nuclear localization of Foxo1. Acetylation of Foxo1 by p300 is increased by insulin but, paradoxically, insulin inhibition of transcription still occurs in the presence of p300. This most likely reflects the preeminent role of phosphorylation in the inhibition of Foxo1 transcriptional activity by insulin, although secondary effects of acetylation cannot be excluded.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Sodium butyrate and cycloheximide were purchased from Sigma (St. Louis, MO). Trichostatin A was obtained from Cell Signaling Technology (Beverly, MA). Restriction enzymes were purchased from Invitrogen (Life Technologies Inc., Carlsbad, CA). Taq polymerase was obtained from PerkinElmer Life Sciences-Applied Biosystems (Foster City, CA), and human recombinant insulin (Humulin U-100 regular) was from Eli Lilly & Co. (Indianapolis, IN).

Cell Lines
H4IIE rat hepatoma cells and HEK-293T cells were grown as monolayer cultures in low-glucose and high-glucose DMEM (Life Technologies Inc.), respectively. Cells were grown at 37 C in the presence of 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and antibiotics (50 U/ml of penicillin and 50 µg/ml of streptomycin) in a humidified atmosphere of 5% CO2.

Plasmid Constructs
The expression vector encoding pcdef3/FLAG-p300 was kindly provided by Dr. D. M. Livingston (Dana-Farber Cancer Institutes, Boston, MA). The pcDNA/myc-p300 acetylase-deficient mutant, p300-DY (Lysine 1399 converted to tyrosine), was a generous gift of Dr. T. P. Yao (Duke University) (50). The expression vectors encoding pcdef3/E1A and mutant pcdef3/E1a{Delta}2–36 were graciously obtained from Dr. A. B. Roberts [National Cancer Institute, National Institutes of Health (NIH)]. The rat IGFBP-1 (p925GL3) and the pG5E1b promoter-luciferase reporter plasmids used for luciferase assays were described previously (19, 20). pCMV5/c-Myc-Foxo1 1–652 and pCEF/GFP-Foxo1 208–652 were previously reported (20).

Site-Directed Mutagenesis
Mutations were introduced using a PCR-based method (QuikChange site-directed mutagenesis kit; Stratagene, La Jolla, CA) following the manufacturer’s instructions. The three PKB/Akt consensus phosphorylation sites Thr24, Ser253, and Ser316 were mutated to alanine in pCMV5/c-Myc-Foxo1 1–652. Mutated oligonucleotides used for DNA amplification are available on request. The sequence of all constructs was confirmed by automated DNA sequencing using a rhodamine fluorescent terminator sequencing kit (PerkinElmer Life Sciences-Applied Biosystems).

Transient Transfection and Luciferase Assays
H4IIE cells were transfected as previously described (20) using diethylaminoethyl-dextran (Amersham Pharmacia Biotech, Piscataway, NJ). The luciferase and ß-galactosidase assays were performed as described (20), cotransfecting pRSV-ß-galactosidase to allow for normalization of transfection efficiency. The total amount of transfected DNA was standardized by addition of empty control vector as required. Assays were performed in duplicate and are represented as mean ± SEM of at least three independent experiments.

Expression and Purification of Foxo1 Proteins
The pGEX/Foxo1 1–207 construct was obtained by PCR using the sense primer, 5'-ATAGAATTCATGGCCGAGGCGCCCCAG-3' and the antisense primer 5'-ATACTCGAGTTACTTCCAGCCCGCCGAGCT-3'. The PCR fragment was subcloned into the EcoRI-XhoI sites of pGEX-4T-1 vector (Amersham Pharmacia Biotech). The pGEX/Foxo1 1–652 construct was generated according to the same procedure, using the sense primer 5'-ATAGGATCCATGGCCGAGGCGCCCCAG-3' and the antisense primer 5'-ATACTCGAGTTAGCCTGACACCCAGCTGTG-3', and insertion of the PCR fragment into the BamHI-XhoI sites of pGEX-4T-1 vector. GST fusion proteins were purified using glutathione-Sepharose beads (Amersham Pharmacia Biotech) following the manufacturer’s instructions.

In Vitro Acetylation Assay
Recombinant Foxo1 proteins were acetylated in vitro by His6-tagged full-length p300 purified from baculovirus-infected cells and generously provided by T. P. Yao and C. H. Lai (Duke University). The acetylation reactions were performed in a buffer containing 50 mM Tris base, pH 8.0; 0.1 mM EDTA; 1 mM dithiothreitol and 10% (vol/vol) glycerol, supplemented with 0.8 µg of His6-tagged full-length p300 and substrate in the presence of 0.05 µCi of [1-14C]acetyl-CoA (Amersham Pharmacia Biotech). Reactions were performed at 30 C for 1 h and quenched with Laemmli buffer. Reaction products were then subjected to 8–16% SDS-PAGE and analyzed by autoradiography, followed by Coomassie blue staining. In addition, the acetylated proteins were spotted onto P81 phosphocellulose filter paper and counted for 14C activity by liquid scintillation counting.

GST Pull-Down Assay
Lysates from HEK-293T cells transiently expressing FLAG-tagged p300 were incubated with 3 µg of GST-FOXO1 (Upstate Biotechnology, Lake Placid, NY) or GST. Binding reactions mixtures were precipitated using glutathione-Sepharose beads (Amersham Pharmacia Biotech). Bound proteins were recovered from the beads by boiling the beads in sample buffer and then resolved on SDS-PAGE. Proteins were transferred to nitrocellulose membrane and subjected to Western blot analysis with anti-FLAG antibody (Covance, Princeton, NJ).

Immunoprecipitation and Western Blot Analysis
HEK-293T and H4IIE cells were transfected using the LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer’s instructions. Cells were switched to serum-free medium overnight before incubation with recombinant human insulin (0.25 µg/ml) for the indicated times. Lysis was performed in the presence of 5 mM sodium butyrate, an inhibitor of protein deacetylases, using radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.2), 500 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate] containing 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride. Lysates were cleared by centrifugation and then either resolved directly by SDS-PAGE and transferred onto nitrocellulose membrane, and/or first immunoprecipitated with anti-myc (9E10) or anti-GFP (green fluorescent protein) antibodies (Covance). Western blots were detected using anti-acetyl-lysine (4G12) (Upstate Biotechnology) antibody. The expression level of Foxo1 and p300 constructs was examined by immunoblotting analysis using anti-Foxo1 and anti-p300 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. Proteins were visualized with an enhanced chemiluminescence detection system (Pierce, Rockford, IL). Autoradiographs were scanned and the images analyzed using the software program NIH image 1.62 (NIH, Bethesda, MD). In addition, nuclear extracts from HEK-293T cells were obtained using the nuclei EZ prep isolation buffer (Sigma) and processed as described in Ref.20 .

Immunofluorescence
Immunofluorescence experiments were performed as described previously (20). Briefly, H4IIE and HEK-293T cells were transiently transfected with the LipofectAMINE Plus reagent (Life Technologies). After 24 h, cells were serum-starved overnight, before incubation with recombinant human insulin (1 µg/ml) for 1 h at 37 C. Then, cells were fixed, permeabilized and mounted with medium containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Vectashield, Vector Labs, Burlingame, CA). Cells were visualized using a fluorescence microscope.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. T. P. Yao and Ms. C. H. Lai for generously purifying and providing His6-p300 full-length protein and for the p300-DY mutant expression vector. We thank Drs. D. M. Livingston and A. B. Roberts for the kind gifts of DNA plasmids. We are grateful to Drs. D. LeRoith, L. Scavo, and K. Ge [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH)] for critical reading of the manuscript. We also thank G. Poy (NIDDK, NIH) for technical assistance with the sequencing.


    FOOTNOTES
 
Present address for V.P.: Unité Mixte de Recherche, Institut National de la Santé et de la Recherche Médicale, Unité 449/Institut National de la Recherche Agronomique 1235, Laënnec Medical School, University Claude Bernard of Lyon 1, 69372 Lyon Cedex 08, France.

First Published Online May 12, 2005

Abbreviations: CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; CoA, coenzyme A; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; FOX, Forkhead Box; FOXO, human FOX subfamily O [previously known as FKHR (Forkhead in rhabdomyosarcoma)]; Foxo, mouse FOXO; FOXO1 (Foxo1), previously known as human (mouse) FKHR; FOXO3a, previously known as human FKHRL1 (FKHR-like-1); FOXO4, previously known as AFX (acute-lymphocytic-leukemia-1 fused gene from chromosome X); GFP, green fluorescent protein; GST, glutathione-S-transferase; HEK, human embryonic kidney; HNF-4, hepatocyte nuclear factor-4; IGFBP-1, IGF binding protein-1; PKB, protein kinase B; TSA, trichostatin A.

Received for publication July 20, 2004. Accepted for publication May 3, 2005.


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