Repression of Hepatocyte Nuclear Factor 4{alpha} by Tumor Suppressor p53: Involvement of the Ligand-Binding Domain and Histone Deacetylase Activity

Yutaka Maeda, Shawn D. Seidel, Gang Wei, Xuan Liu and Frances M. Sladek

Environmental Toxicology Graduate Program (Y.M.), Departments of Cell Biology and Neuroscience (S.D.S., F.M.S.) and Biochemistry (G.W., X.L.), University of California, Riverside, California 92521

Address all correspondence and requests for reprints to: Frances M. Sladek, Ph.D., Department of Cell Biology and Neuroscience, 5429 Boyce Hall, University of California, Riverside, Riverside, California 92521.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tumor suppressor p53 is known to inhibit transactivation by certain nuclear receptors, and overexpressed p53 is known to correlate with poor differentiation in liver cancer. Therefore, we investigated whether wild-type p53 might also affect the function of hepatocyte nuclear factor 4{alpha}1 (HNF4{alpha}1), an orphan receptor required for liver differentiation. Our results show that HNF4{alpha}1-mediated transactivation is repressed by p53 but that the mechanism of repression is not due to inhibition of HNF4{alpha}1 DNA binding. Rather, transfections with Gal4 fusion constructs indicate that the repression is via the ligand-binding domain of HNF4{alpha}1. Furthermore, we found that p53 in human embryonic kidney whole-cell extracts preferentially bound the ligand-binding domain of HNF4{alpha}1 and that the activation function 2 region was required for the binding. Competition for coactivator CREB binding protein could not entirely account for the repression but trichostatin A, an inhibitor of histone deacetylase activity, could reverse p53-mediated repression of HNF4{alpha}1. In contrast, p53-mediated repression of transcriptional activation of the same promoter by another transcriptional activator, CCAAT/enhancer-binding protein-{alpha}, could not be reversed by the addition of trichostatin A. These results suggest that p53, like other transcriptional repressors, inhibits transcription by multiple mechanisms, one of which involves interaction with the ligand-binding domain and recruitment of histone deacetylase activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
p53 is a well characterized tumor suppressor gene involved in cell cycle control, DNA repair, and apoptosis. p53 carries out these diverse functions by binding DNA response elements in the promoter regions of genes critical to those cellular processes and activating those genes (e.g., p21, GADD45, bax). p53 is also known to repress the transcription of other genes that lack p53 response elements, including the c-fos, c-jun, retinoblastoma, IL-6, and bcl-2 genes (reviewed in Ref. 1). Whereas, by and large, the mechanism of transcriptional repression by p53 is much less well understood than that of transcriptional activation, several mechanisms have recently been proposed: binding to TATA-binding protein (2); squelching of transcription factors IIB and IID (3); and sequestration of coactivator p300/CREB binding protein (CBP) (4, 5). p53 has also been found to repress transcription of genes containing p53-binding elements in their promoter regions by recruitment of mSin3A and histone deacetylase (HDAC) activity (6, 7). Histone deacetylation is thought to keep chromatin in a closed conformation, thereby inhibiting transcription (reviewed in Ref. 8).

There are several reports of p53 blocking transcription by nuclear receptors. Nuclear receptors are a superfamily of ligand-dependent transcription factors that play a critical role in growth, development, and differentiation of nearly every cell type in the body. Members of the superfamily include steroid receptors, vitamins A and D receptors, TR, and a large number of orphan receptors, some of which have recently been found to respond to fatty acids (PPARs), bile acids (farnesoid X receptor), cholesterol derivatives (liver X receptor), and xenobiotics (steroid and xenobiotic receptor, benzoate X receptor, and pregnane X receptor) (reviewed in Refs. 9, 10, 11, 12, 13). Like other transcriptional activators, nuclear receptors are known to activate transcription by binding specific DNA response elements in promoter/enhancer regions of genes and subsequently recruiting a variety of different coactivator complexes which, by some poorly defined mechanism, activate transcription by RNA polymerase II (reviewed in Ref. 14). p53 has been reported to block the activation of several different nuclear receptors by binding the DNA binding domain (DBD) of the receptor and, in at least some cases, subsequently inhibiting DNA binding [TR (15), GR (16), ER (17)].

Since overexpressed p53 is known to correlate with poor liver differentiation in liver cancer (18, 19, 20), we wanted to determine whether p53 might also affect hepatocyte nuclear factor 4{alpha}1 (HNF4{alpha}1), an orphan nuclear receptor (NR2A1) known to be important for liver differentiation. In addition to liver, HNF4{alpha}1 is also expressed in adult kidney, intestine, and pancreas but not other tissues (reviewed in Ref. 21). HNF4{alpha}1 is considered to be an orphan receptor in that a ligand has not yet been definitively identified for it (22, 23). However, HNF4{alpha}1 is known to be essential for development in organisms ranging from insects to mammals and to regulate many essential genes related to nutrient transport and metabolism. For example, HNF4{alpha}1 positively regulates genes involved in the transport of lipids and vitamins as well as genes involved in lipid, amino acid, and glucose metabolism. It also regulates genes involved in the regulation of several serum proteins such as blood coagulation factors, erythropoietin, and antithrombin III. HNF4{alpha}1 is directly linked to several human diseases: the HNF4{alpha} gene has been found to be mutated in maturity-onset diabetes of the young type 1, and HNF4{alpha}1 DNA binding sites have been found to be mutated in patients with hemophilia B Leyden and maturity onset diabetes of the young type 1 (21). Therefore, since a tissue-specific knockout indicates that HNF4{alpha}1 is essential for proper functioning of the adult liver (24), repression of HNF4{alpha}1-mediated transcription is anticipated to impede the function of the liver and possibly other organs as well.

Like other nuclear receptors, HNF4{alpha}1 recruits coactivators of the p160 and p300/CBP families via an interaction that depends primarily on the activation function 2 (AF-2) region (putative helix 12) in the ligand-binding domain (LBD) (25, 26, 27, 28, 29). These coactivator complexes contain or are associated with histone acetylase activity, which serves to decondense the chromatin, thereby facilitating transcription initiation (8, 14, 30). However, unlike most other receptors, HNF4{alpha}1 interacts with coactivators and activates transcription in a constitutive fashion, i.e. in the absence of an exogenously added ligand.

In the current work, using transient transfection analysis, we show that p53 represses HNF4{alpha}1-mediated activation on a HNF4{alpha}1-responsive promoter (derived from the apolipoprotein B (apoB) gene) in several different cell lines. We also provide evidence indicating that the repression is via a mechanism not previously described for p53 but one that is reminiscent of corepressors. Namely, repression occurs via interaction of p53 with the LBD of HNF4{alpha}1. Furthermore, the interaction appears to be dependent on the presence of the AF-2 and to result in recruitment of HDAC activity. Finally, we present evidence suggesting that this mechanism of repression may not be applicable to basic leucine zipper protein CCAAT/enhancer-binding protein-{alpha} (C/EBP{alpha}), indicating that p53, like other corepressors, inhibits transcription by multiple mechanisms.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
p53 Represses HNF4{alpha}1-Mediated Transactivation in Vivo
To determine whether p53 represses HNF4{alpha}1-mediated transactivation, transient transfection assays were performed in three different cell lines: Saos-2, a human osteosarcoma cell line that is deficient in both endogenous p53 and endogenous HNF4{alpha}1 protein (our unpublished results and this study); HepG2, a human hepatocellular carcinoma cell line that is at least partially differentiated for the liver phenotype and expresses endogenous HNF4{alpha}1 (Ref. 31 and our unpublished data) and low levels of wild-type p53 protein (32); and 293T, a human embryonic kidney cell line that transfects very well and displays no endogenous HNF4{alpha}1 protein but contains high levels of wild-type p53 protein (see Fig. 1BGo). The results indicate that in all three cell lines, wild-type p53 repressed transactivation by transfected HNF4{alpha}1 from a reporter construct containing four HNF4{alpha}1 sites from the human apoB promoter (Fig. 1AGo, compare lanes 4, 8, and 11 to lanes 3, 7, and 10, respectively). In HepG2 cells, p53 also repressed transcription of the reporter construct alone, presumably by interference with the endogenous HNF4{alpha}1 protein (compare lane 6 to lane 5). The repression was relatively greater in the Saos-2 cells than in the HepG2 and 293T cells, which may or may not be related to the fact that the Saos-2 cells do not express endogenous p53 protein. To verify that repression was not due to a loss of expression of HNF4{alpha}1 protein, immunoblot analysis was performed on the same whole-cell extracts from 293T cells as those used to determine the luciferase activity in Fig. 1AGo. The results indicate that the level of HNF4{alpha}1 protein was not affected by cotransfection of the p53 vector (Fig. 1BGo, top panel; compare lane 3 to lane 2), indicating that the repression is via blocking of HNF4{alpha}1 function.



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Figure 1. p53 Represses HNF4{alpha}1-Mediated Transactivation in Several Different Cell Lines

A, Transient transfection assays were performed into the indicated cell lines with the pMT7. HNF4{alpha} (HNF4{alpha}) (+, 0.1 µg for Saos-2 and HepG2 cells, 1.0 µg for 293T cells) and pcDNA-p53 (p53wt) (+, 0.1 µg for Saos-2 cells, 0.05 µg for HepG2 cells, and 1.0 µg for 293T cells) expression vectors (-, appropriate amount of salmon sperm DNA or pcDNA3.1 empty vector) and the ApoB.-85-47. E4.Luc reporter construct (2 µg) (diagrammed at top, not drawn to scale) as indicated and as described in Materials and Methods. Shown is the mean luciferase activity in RLU from one representative experiment of several experiments ± SD of triplicate samples. Luc, Luciferase. B, Immunoblot analysis (IB) of the same 293T whole-cell extracts used to determine the luciferase activity in panel A with the indicated transfected vectors (bottom) and the primary antisera (right) as described in Materials and Methods. {alpha}-HNF4{alpha}, {alpha}445; {alpha}-p53wt, DO-1. Material from approximately one tenth of a well was loaded in each lane. Low transfection efficiencies of Saos-2 and HepG2 cells and endogenous HNF4{alpha}1 protein in HepG2 cells prevented a similar experiment from being done in those cell lines.

 
p53 Does Not Inhibit HNF4{alpha}1 DNA Binding
To determine whether p53 might repress HNF4{alpha}1-mediated transactivation via inhibition of DNA binding, as has been reported for other nuclear receptors (15, 16, 17), EMSA was performed with a 32P-labeled oligonucleotide containing the HNF4{alpha}1 element from the human apoB promoter and HNF4{alpha}1 and p53 proteins expressed in mammalian cells (Fig. 2AGo). We used two different immunoaffinity methods to purify p53: one was via an antisera (12CA5) that recognizes the hemagglutinin (HA) tag on the N terminus, and the other was via an antisera (421) that recognizes the C terminus of p53 (Fig. 2BGo). (The 421 antisera has been shown previously to activate p53 DNA binding, suggesting that it may change the conformation of the protein (33).) The results indicate that, even in the presence of excess amounts of p53 protein (up to 200 ng vs. approximately 20 ng of HNF4{alpha}1 protein, as determined by immunoblot analysis, data not shown), regardless of the p53 purification method, the ability of HNF4{alpha}1 to bind DNA was not altered (Fig. 2AGo, compare lanes 2, 3, and 4 to lane 1 and lanes 6, 7, and 8 to lane 5). This suggests that the repression observed in vivo was not due to an inhibition of HNF4{alpha}1 DNA binding but to an alternative mechanism.



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Figure 2. p53 Does Not Inhibit HNF4{alpha}1 DNA Binding in Vitro

A, EMSA with COS-7 nuclear extracts containing overexpressed HNF4{alpha}1 protein and increasing amounts (0, 50, 100, and 200 ng) of wild-type HA-tagged p53 protein (p53wt) purified by two different antisera (421 and 12CA5) (see panel B) as indicated and as described in Materials and Methods. Shown is an autoradiograph of the shift gel with the free probe (32P-ApoB-85-47), the HNF4{alpha}1:DNA complex (arrow), and an endogenous band from the COS extracts (*) indicated, and verified as described previously (55 ). B, Silver-stained 10% SDS gel of purified p53 protein used in shift reactions in panel A.

 
p53 Represses Activation by and Interacts with the HNF4{alpha} LBD
Since the HNF4{alpha} LBD is known to be essential for transactivation in vivo, we hypothesized that p53 might interfere with the function of the LBD. We therefore constructed a Gal4 fusion with the HNF4{alpha} LBD (Gal4.HNF4.LBD) (Fig. 3AGo) and cotransfected it into 293T cells along with the p53 expression vector. The results indicate that the Gal4.HNF4.LBD fusion protein activated transcription well (Fig. 3BGo, compare lane 4 to lane 3), as expected from previous results (34). However, cotransfection with the p53 expression vector repressed the Gal4.HNF4.LBD-mediated transcription to approximately 50% (compare lane 6 to lane 4) but had no significant effect on the basal activation by the Gal4 DBD (compare lane 5 to lane 3), which, in any case, was only slightly above the reporter alone (compare lane 3 to lane 1). Similar p53-mediated repression of activation by Gal4.HNF4.LBD was observed in Saos-2, HepG2, and 293 cells (data not shown). These results suggest that p53 inhibits transactivation by HNF4{alpha} by interfering with the function of the LBD and are consistent with the notion that p53 does not inhibit HNF4{alpha}1 DNA binding.



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Figure 3. p53 Represses Transactivation by the HNF4{alpha} LBD

A, Schematic representation of Gal4. DBD and GST HNF4{alpha} fusion constructs used in the assays in panels B, C, and D. Full-length rat HNF4{alpha}1 protein is shown at the top with the traditional nuclear receptor domains indicated, A–F. Zn++, Zinc finger region (C domain). Numbers indicate amino acids. B, Transient transfection assay in 293T cells with the Gal4 responsive reporter (2 µg) and the indicated expression vectors (0.1 µg Gal4 vectors, 1.0 µg pcDNA-p53) (diagrammed at the top) as described in Fig. 1AGo. C, Immunoblot analysis (IB) with {alpha}-p53 antisera (DO-1) of GST pull-down assay with the indicated GST proteins (~10–20 µg) and whole-cell extracts from 293T and 293 cells (1 mg) as described in Materials and Methods. 10% input represents 10% of extract (100 µg) used in the assay. Shown is one representative experiment of several that were performed. D, Immunoblot analysis as in panel C with 293T extracts but with less GST protein (~2 µg) and including the HNF4{alpha} LBD construct lacking the AF-2 (GST.HNF4.C360).

 
To determine whether p53 interacts with the HNF4{alpha} LBD, a glutathione-S-transferase (GST) pull-down assay was performed with whole-cell extracts from 293T cells, and the presence of p53 protein was verified by immunoblot analysis. The results indicate that the endogenous p53 protein bound very well (well over 10%) to the GST.HNF4.LBD but only marginally to the GST.HNF4.DBD and not at all to the GST control (Fig. 3CGo, top panel; compare lane 4 to lanes 3 and 2). Since 293T cells express the SV40 large T antigen, and since large T antigen is known to interact with p53 (33), we performed a similar experiment with whole-cell extracts from 293 cells that lack the large T antigen and observed the same result, namely that the endogenous p53 protein bound the HNF4.LBD but not the GST control or the HNF4.DBD (Fig. 3CGo, bottom panel; compare lane 4 to lanes 2 and 3). Since p53 also repressed transactivation by Gal4.HNF4.LBD in 293 cells (data not shown), these results suggest that repression of and interaction with the HNF4{alpha} LBD by p53 is not mediated via the SV40 large T antigen.

Since the AF-2 region of the LBD is essential for interaction of nuclear receptors with coactivators and corepressors (14), we next determined whether the AF-2 of HNF4{alpha}1 was also required for interaction with p53. The results indicate that the LBD construct lacking the AF-2 (GST.HNF4.127.360) (see Fig. 3AGo) did not bind p53 protein in 293T whole-cell extracts (Fig. 3DGo, compare lane 4 to lane 5), indicating that the AF-2 is required for interaction with p53 and suggesting that inhibition is via the AF-2 of HNF4{alpha}1. (The construct lacking the AF-2 did, however, bind in vitro translated HNF4{alpha}1, indicating that the lack of binding to p53 was not due to a gross malfolding of the bacterially expressed GST protein; data not shown).

Repression by p53 Is Only Partially Recovered by Overexpression of CBP
The AF-2 of nuclear receptors is known to interact with coactivators CBP/p300 (reviewed in Ref. 35), and p53 is known to interact with p300 (36) and repress activation by other transcriptional activators via competition for p300 (4, 5). Therefore, to determine whether repression of HNF4{alpha}1-mediated transactivation is due to sequestration of limiting amounts of an endogenous coactivator such as CBP/p300, we overexpressed CBP in the transient transfection assay. The results indicate that CBP enhanced the ability of HNF4{alpha}1 to activate its reporter construct both in the absence and presence of cotransfected p53 (Fig. 4Go, compare lane 6 to lane 4 and lane 7 to lane 5). However, they also indicate that even in the presence of the exogenously added CBP, the cotransfected p53 still reduced the luciferase activity to approximately 60% compared with 46% in the absence of added CBP (lanes 7 and 5). These results indicate that there is only limited reversal of the p53-mediated repression by cotransfection with CBP, indicating that a mechanism of repression other than sequestration of CBP by p53 is involved.



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Figure 4. p53 Repression Is Only Partially Recovered by Overexpression of CBP

Transient transfection assay in 293T cells with the ApoB.-85-47 Luc reporter construct (2 µg) and the indicated expression vectors and 5 µg of coactivator CBP (pRC/RSV-mCBP.HA.RK) as described in Fig. 1AGo. Percentages show RLU relative to samples lacking the p53wt vector.

 
p53-Mediated Repression of HNF4{alpha}1 Is Reversed by HDAC Inhibitor Trichostatin A (TSA)
The AF-2 of nuclear receptors has also been found to be involved in the interaction of corepressors that recruit HDAC activity to promoters (14). Since p53 has been shown to directly repress transcription of target genes by recruiting HDAC1 to a promoter containing a p53 binding site (6, 7), we reasoned that p53 might also repress HNF4{alpha}1-mediated transcription by recruiting HDAC activity. To test this hypothesis, transient transfection assays into 293T cells with the Gal4 fusion constructs were performed in the presence of TSA, a specific inhibitor of HDAC activity (37, 38). The results indicate that in the presence of TSA, the repression by p53 of the Gal4.HNF4.LBD-mediated transactivation was completely reversed (Fig. 5AGo; compare lane 8 to lane 6 vs. lane 4 to lane 2), indicating that recruitment of HDAC activity to the HNF4{alpha} LBD may be involved in p53-mediated repression.



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Figure 5. TSA Reverses p53-Mediated Repression of HNF4{alpha}

A, Transient transfection assay in 293T cells with the Gal4 responsive reporter and the indicated expression vectors in the absence (-) or presence (+) of HDAC inhibitor TSA as described in Fig. 3BGo and Materials and Methods. B, Transient transfection assay in 293T cells with the ApoB.-85-47 Luc reporter construct (2 µg) and the indicated expression vectors (C/EBP{alpha}, 0.1 µg pMT2. C/EBP{alpha}; HNF4{alpha}, 0.1 µg pMT7. HNF4{alpha}1; p53wt, 1.0 µg pcDNA-p53) in the absence (-) or presence (+) of TSA as described in Fig. 1AGo and Materials and Methods. Shown are the relative luciferase activities given in percentages based on the samples lacking the p53wt vector arbitrarily put at 100%.

 
We next determined whether TSA could also reverse the p53-mediated repression of full-length HNF4{alpha}1 by using the HNF4{alpha}1 reporter construct ApoB.-85 -47.E4.Luc. The results indicate that TSA partially reversed the repression of HNF4{alpha}1 by p53 (Fig. 5BGo; compare lane 4 to lane 2). The significance, if any, of the fact that 100% reversal was achieved with the Gal4.HNF4.LBD, but not the full-length HNF4{alpha}1, is not known but could be due to the fact that other domains, such as the DBD, the AF-1, and the F domain, are present in the full-length HNF4{alpha}1 construct but not the Gal4.HNF4.LBD construct.

Since we had previously reported that the -85 to -47 region of the apoB promoter also responds to the basic leucine zipper protein C/EBP{alpha} (39), and since C/EBP{alpha} has been shown previously to be repressed by p53 (40), we tested whether that repression might also be reversed by TSA. The results indicate that treatment with TSA did not reverse the repression of C/EBP{alpha} by p53 (Fig. 5BGo; compare lane 8 to lane 6), suggesting that repression of C/EBP{alpha} by p53 may act via a mechanism other than recruitment of HDAC activity. These and the previous results suggest that the effect of the reversal of p53-mediated repression by TSA is a specific one and might be related to interaction with the LBD.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the current study we show for the first time that tumor suppressor protein p53 can repress the constitutive transactivation activity of orphan receptor HNF4{alpha}1 in vivo (Fig. 1Go). We also show that the repression is not due to an inhibition of HNF4{alpha}1 DNA binding by p53 but rather to an inhibition of LBD function (Figs. 2Go and 3Go). We show that endogenous p53 in 293T and 293 whole-cell extracts can interact in vitro with the HNF4{alpha} LBD in an AF-2-dependent fashion (Fig. 3Go). Repression could not be completely accounted for by competition for limiting amounts of coactivators such as CBP (Fig. 4Go). Rather, it appeared to be due, at least partially, to recruitment of HDAC activity, as it was reversed by addition of TSA, a specific inhibitor of HDAC activity (Fig. 5Go). In contrast, repression of basic leucine zipper protein C/EBP{alpha} by p53 could not be reversed by TSA (Fig. 5Go), suggesting that there may be multiple mechanisms of repression. Taken together, our results support the notion that p53 represses transactivation of at least certain nuclear receptors by a mechanism other than inhibition of DNA binding and one that involves the LBD, the AF-2, and recruitment of HDAC activity.

New Mechanism of p53-Mediated Repression
Previous work by others showed that p53 inhibited transactivation of certain nuclear receptors by interaction with the DBD and inhibition of DNA binding (15, 16, 17, 41). Our results indicate that p53 also inhibits transactivation by HNF4{alpha}1 albeit via a different mechanism, one that is more reminiscent of a corepressor than an inhibitor of DNA binding. Hallmarks of corepressors include repression of nuclear receptors only in the absence of ligand, interaction with the receptor LBD, and recruitment of HDAC activity (14), conditions all found in this study. What is not known at this point is whether binding of the endogenous p53 in the cell extracts to the HNF4{alpha} LBD is direct or indirect. Whereas we ruled out involvement of the SV40 T antigen (Fig. 3Go), it is still possible that some cellular protein or another viral protein is required for the interaction. However, it should be noted that p53 repressed activation by HNF4{alpha}1 in cell lines lacking known viral genes (Fig. 1Go; Saos-2 and HepG2) and that repression was via the LBD in those cell lines as well (data not shown). Finally, it appears that repression via interaction with the LBD may be applicable to other nuclear receptors as we have evidence that repression of RAR/RXR activity by p53 is reversed by the addition of ligand, suggesting involvement of the LBD (data not shown).

Multiple Mechanisms of p53-Mediated Repression
In addition to inhibition of DNA binding and recruitment of HDAC activity, there appears to be additional mechanisms of repression mediated by p53 since TSA did not completely reverse the repression of full-length HNF4{alpha}1 (Fig. 5BGo). One such mechanism could be competition for coactivators such as CBP (Fig. 4Go). p53 also repressed C/EBP{alpha} by a mechanism other than recruitment of HDAC activity (Fig. 5BGo), which may or may not involve inhibition of C/EBP{alpha} DNA binding (40, 42). Others have also found multiple mechanisms of repression by p53 of another liver-specific gene, although both of those mechanisms required binding of p53 to a p53 response element (43). This mechanism, however, is apparently not involved here as we saw no evidence of p53 binding the ApoB element in the gel shift assay (Fig. 2AGo), and p53 did not repress the ApoB reporter construct in the absence of HNF4{alpha}1 in Saos-2 cells (Fig. 1A). The existence of multiple mechanisms of repression is evidently not uncommon for corepressors. Two well characterized transcriptional repressors, silencing mediator of retinoid and thyroid hormone receptors (SMRT) and retinoblastoma protein (Rb), have also been found to repress transcription by multiple mechanisms, one of which involves recruitment of HDAC activity under certain conditions and another that involves interaction with the basal transcription machinery (44, 45, 46).

Consequences of p53-Mediated Repression
HNF4{alpha} and C/EBP{alpha} are both critical for liver function and differentiation (24, 47, 48). Therefore, repression of their transcriptional activity by p53 could explain why overexpression of p53 has been shown to correlate with poor liver differentiation in liver cancer (18, 19, 20). Furthermore, p53-mediated repression of C/EBP{alpha} and HNF4{alpha}1 could play a role in liver toxicity of adenoviral vectors, such as those used in gene therapy, as an increase in p53 protein levels due to adenovirus infection correlated with a decrease in albumin gene expression, presumably via inhibition of C/EBP{alpha}/ß transcriptional activity (40). Under normal conditions in vivo, however, wild-type p53 protein levels are very low and are increased only in response to cellular stress such as DNA damage, withdrawal of growth factors, and hypoxia (reviewed in Ref. 49). It has been assumed, with good reason, that tight regulation of p53 protein levels is required to prevent blockage of the cell cycle and induction of apoptosis. Perhaps another reason to keep p53 protein levels low is to avoid interference with normal cellular processes such as differentiation. Finally, an increase in p53 protein levels may also serve as a mechanism to prevent commitment of cellular resources for processes such as differentiation during times of stress.

In conclusion, this report of p53-mediated repression of an additional nuclear receptor suggests that, in addition to cell cycle control, DNA repair, and apoptosis, repression of transcriptional activators involved in cellular differentiation might be a general property of p53. And, like those other processes, it appears that there are multiple mechanisms by which p53 carries out this repression, one of which is to interact with the LBD and recruit HDAC activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The expression vectors containing rat HNF4{alpha}1 (pMT7.HNF4{alpha}1) (50), wild-type human p53 in pcDNA1 (pcDNA-p53) (51), and rat basic-leucine zipper protein C/EBP{alpha} (pMT2.C/EBP{alpha}) (52) have been described previously. The HNF4{alpha} GST fusion constructs were made by inserting PCR products from the original rat HNF4{alpha}1 cDNA (53) corresponding to fragments containing amino acids (aa) 127–374 (GST.HNF4.LBD), aa 45–125 (GST.HNF4.DBD), and aa 127–360 (GST.HNF4.127.360) into the EcoRI/XhoI sites of the pGEX6P-1 vector (Pharmacia Biotech, Uppsala, Sweden), using appropriate primers containing EcoRI (sense) or XhoI (antisense) sites. Gal4.HNF4.LBD (pFA-HNF4{alpha}.LBD) was constructed by first inserting the appropriate BamHI/NotI fragment from the GST.HNF4.LBD vector containing aa 127–374 of rat HNF4{alpha}1 into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA). Subsequently, the appropriate BamHI/XbaI fragment from pcDNA3.1 was inserted in frame with Gal4.DBD in the pFA-CMV vector (Stratagene, La Jolla, CA). The reporter construct ApoB.-85-47.E4.Luc was constructed by ligating four tandem copies of a double-stranded oligonucleotide (ApoB.-85-47) corresponding to nucleotides -85 to -47 relative to the transcriptional start site of the human apoB gene and containing a four-base overhang on the 5'-end (underlined) (5'-GATCCGGGAGGCGCCCTTTGGACCTTTTGCAATCCTGGCGCTC-3', top strand) into the BamHI site of pZLuc.E4, which contains the adenovirus E4 TATA box driving the firefly luciferase gene. pZLuc.E4 was generated by removal of an approximately 100-bp BamHI fragment containing five RGC response elements from 5RGC.E4.Luc (54). The reporter construct pFR-luc contains a synthetic promoter with five Gal4 binding elements driving the expression of the luciferase gene (Stratagene). All PCR constructs were verified by dideoxy sequencing. pRc/RSV-mCBP.HA.RK containing full-length mouse CBP was kindly provided by R. Goodman.

Transient Transfection Assays
Cell lines—human embryonic kidney cells (293 and 293T), human hepatocarcinoma cells (HepG2), and human osteogenic sarcoma cells (Saos-2)—were routinely maintained in DMEM supplemented with penicillin-streptomycin and 10% FBS (5% for 293 and 293T cells) at 37 C under 5% CO2. One day before transfection, cells were seeded in six-well plates (Falcon 3046, Becton Dickinson and Co., Franklin Lakes, NJ) at 0.5 x 106 cells per well. DNA mixtures containing 2 µg luciferase reporter construct, 0.1–1.0 µg of HNF4{alpha} expression vector, and 0.1–1.0 µg p53 expression vector (pcDNA-p53), as indicated, were added to each well using Lipofectin according to the manufacturer’s protocol (Life Technologies, Inc., Gaithersburg, MD). Total DNA was brought up to 10 µg with either the appropriate amount of empty vector (e.g. pcDNA1 for samples lacking p53) or salmon sperm DNA. After approximately 24 h, transfected cells were harvested and luciferase activity was determined as previously described (55). For cell treatment, cells were treated with 100 ng/ml TSA (Sigma, St. Louis, MO) for 8 h before harvest. All assays were performed in triplicate in at least two independent experiments, unless indicated otherwise. Results are given in relative light units (RLU, an arbitrary unit) or fold induction (RLU relative to transfections lacking expression vectors) as indicated.

GST Pull-Down Assay
In vitro protein-protein interaction assays were performed using GST fusion proteins expressed in E. coli strain BL21(DE3)(pLysS) and bound to glutathione-agarose (Sigma) using the manufacturer’s protocol (Pharmacia Biotech). In general, 20 µl of a 1:1 GST-protein/glutathione agarose bead slurry (i.e. 10 µl packed beads, ~10–20 µg protein) in 100 µl NETN equilibration buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.01% Nonidet P-40) were incubated with 100 µl of 10 mg/ml whole-cell extract (see below) for 12 h at 4 C with gentle agitation. Protein complexes were isolated by pelleting the beads at 3,000 rpm for 30 sec in a Sorvall MC 12-V Microfuge (E.I. Dupont de Nemours & Co., Inc., Newtow, CT) and washing three times with 400 µl of NETN equilibration buffer followed by resuspension in SDS-sample loading buffer. After fractionation by 10% SDS-PAGE, proteins were transferred to polyvinylidene difluoride membrane (Immobilon, Millipore Corp., Bedford, MA) for immunoblot analysis (see below). GST.HNF4.DBD beads were prewashed in buffer containing 1 M NaCl to remove contaminating DNA. Whole-cell extracts were prepared by harvesting approximately 107 293 or 293T cells by gentle scraping in 1 ml ice-cold PBS and resuspending in 0.2 ml ice-cold lysis buffer [50 mM Tris-HCl (pH.7.4), 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol]. The lysis mixture was incubated for 1 h at 4 C and then cleared by centrifugation at 12,000 rpm for 15 min at 4 C.

Immunoblot Analysis
Immunoblot analysis was performed as previously described (55) except that the blot was preincubated in blocking buffer [5% nonfat dried milk, 0.01% sodium azide, 1x Tris-buffered saline with Tween 20 (TBST)] and then incubated for 16 h with a 1:5,000 dilution of {alpha}445 antiserum for HNF4{alpha}1 (53) or 0.5 µg/ml final concentration of DO-1 antibody for p53 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in blotting buffer (1% nonfat dried milk, 0.002% sodium azide, 1x TBST). The blot was washed twice with 1x TBST and incubated for 2 h in the same blotting buffer with a 1:5,000 dilution of goat antirabbit (for {alpha}445) or goat antimouse (for DO-1) IgG Fab' conjugated to alkaline phosphatase (The Jackson Laboratory, West Grove, PA). The blot was washed three times in 1x TBST and developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate toluidinium using a standard protocol (56).

EMSA
Shift reactions and gel analysis were carried out as previously described, with minor modifications (55). A standard mobility shift reaction mixture (15 µl) contained 400 ng of COS-7 cell crude nuclear extract with overexpressed HNF4{alpha}1 incubated at 4 C for 1 h with purified p53. The incubations were continued for another 30 min at room temperature with 3 ng of 32P-labeled double-stranded oligonucleotide probe (ApoB.-85-47) and nonspecific DNA [1 µg of poly(dIdC), 1 µg of sonicated denatured salmon sperm DNA] before 5 µl were loaded onto a 6% native polyacrylamide gel in 0.25x Tris-borate-EDTA. After electrophoresis, gels were subjected to autoradiography for 16 h at room temperature. Crude nuclear extracts of pMT7. HNF4{alpha}1-transfected COS-7 cells were prepared as previously described (55). HA epitope-tagged wild-type human p53 was immunoaffinity purified with either the 421 antibody (recognizes the C terminus of p53) or the 12CA5 antibody (recognizes the HA tag at the N terminus) antibody from HeLa cells infected with a recombinant vaccinia virus as previously described (54, 57).


    ACKNOWLEDGMENTS
 
We thank H. Brar, M. Mittal, J. Xun, S. Corneillie, S. Desai, and L. Nepomuceno for technical assistance.


    FOOTNOTES
 
This work was supported by NIH Grants DK-53892 to F.M.S. and CA-75180 to X.L. and fellowships to Y.M. from the Japan Society for the Promotion of Science and University of California Toxic Substances Research and Teaching Program.

Abbreviations: aa, Amino acid; AF-2, activation function 2; apoB, apolipoprotein B; CBP, CREB binding protein; C/EBP{alpha}, CCAAT/enhancer-binding protein-{alpha}; CREB, cAMP response element binding protein; DBD, DNA-binding domain; GST, glutathione-S-transferase; HA, hemagglutinin; HDAC, histone deacetylase; HNF, hepatocyte nuclear factor; LBD, ligand-binding domain; RLU, relative light units; TBST, Tris-buffered saline with Tween 20; TSA, trichostatin A.

Received for publication June 1, 2001. Accepted for publication October 1, 2001.


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