Polyamines Modulate the Interaction between Nuclear Receptors and Vitamin D Receptor-Interacting Protein 205
Yutaka Maeda,
Christophe Rachez1,
Leo Hawel, III,
Craig V. Byus,
Leonard P. Freedman and
Frances M. Sladek
Environmental Toxicology Graduate Program (Y.M.), Departments of Cell Biology and Neuroscience (F.M.S.) and Biomedical Sciences (L.H., C.V.B.), University of California, Riverside, California 92521; and Cell Biology Program (C.R., L.P.F.), Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Address all correspondence and requests for reprints to: Frances M. Sladek, Ph.D., Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, California 92521. E-mail: frances.sladek{at}ucr.edu.
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ABSTRACT
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Nuclear receptors (NR) activate transcription by interacting with several different coactivator complexes, primarily via LXXLL motifs (NR boxes) of the coactivator that bind a common region in the ligand binding domain of nuclear receptors (activation function-2, AF2) in a ligand-dependent fashion. However, how nuclear receptors distinguish between different sets of coactivators remains a mystery, as does the mechanism by which orphan receptors such as hepatocyte nuclear factor 4
(HNF4
) activate transcription. In this study, we show that HNF4
interacts with a complex containing vitamin D receptor (VDR)-interacting proteins (DRIPs) in the absence of exogenously added ligand. However, whereas a full-length DRIP205 construct enhanced the activation by HNF4
in vivo, it did not interact well with the HNF4
ligand binding domain in vitro. In investigating this discrepancy, we found that the polyamine spermine significantly enhanced the interaction between HNF4
and full-length DRIP205 in an AF-2, NR-box-dependent manner. Spermine also enhanced the interaction of DRIP205 with the VDR even in the presence of its ligand, but decreased the interaction of both HNF4
and VDR with the p160 coactivator glucocorticoid receptor interacting protein 1 (GR1P1). We also found that GR1P1 and DRIP205 synergistically activated HNF4
-mediated transcription and that a specific inhibitor of polyamine biosynthesis,
-difluoromethylornithine (DFMO), decreased the ability of HNF4
to activate transcription in vivo. These results lead us to propose a model in which polyamines may facilitate the switch between different coactivator complexes binding to NRs.
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INTRODUCTION
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NUCLEAR RECEPTORS (NRs) comprise a family of transcription factors that play a role in nearly every aspect of growth, development, and disease processes (reviewed in Refs. 1 and 2). The best characterized members of the family, such as the steroid and thyroid hormone and vitamins A and D receptors, are known to regulate transcription by binding their cognate lipophilic ligands and subsequently undergoing a conformational change that alters their ability to interact with coregulatory proteins. These coregulatory proteins typically consist of complexes that either repress (corepressors) or activate (coactivators) transcription. The coactivator complexes can be crudely divided into complexes that either contain histone acetyl transferase (HAT) activity [e.g. the p160-p300/CBP-PCAF complexes] or ones that do not [e.g. the vitamin D receptor (VDR)-interating protein (DRIP)/thyroid hormone receptor associated protein (TRAP)/ARC/mediator complexes]. We and others have proposed previously that nuclear receptors interact first with the coactivator complexes that contain HAT activity, allowing for acetylation and hence decondensation of the chromatin, and that subsequently the other coactivator complexes bind the receptors and interact with the basal transcription machinery to initiate transcription (reviewed in Refs. 3, 4, 5, 6).
An increasing amount of evidence indicates that both types of coactivators interact with the activation function-2 (AF-2) region of the ligand binding domain (LBD) of the NRs via LXXLL motifs (NR interaction regions or NR boxes). p160 family members glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator-1 (SRC1) bind the AF-2 and help recruit p300/CBP, which has also been found to bind via the AF-2. DRIP205 (also known as TRAP220, RB18A, PBP, TRIP2), a component of the DRIP/TRAP complex, also interacts with the AF-2 via LXXLL motifs (6, 7, 8). However, what is not clear is how a given nuclear receptor makes the switch from the p160/p300 complex to the DRIP complex. Whereas the switch from corepressor to coactivator binding is apparently made by a conformational change in the receptor upon ligand binding, both types of coactivators bind liganded receptor and bind principally via the AF-2. This suggests that perhaps a change not in the receptor but in the coactivators is required to make the switch from one complex to another. However, what might cause that switch is completely unknown at this point.
In an attempt to answer some of these questions, we examined the interaction between an orphan member of the nuclear receptor superfamily, hepatocyte nuclear factor 4
(HNF4
, NR2A1) and the DRIP complex. HNF4
is a somewhat unusual receptor in that it activates transcription in a constitutive fashion (i.e. in the absence of exogenously added ligand). HNF4
, a highly conserved member of the superfamily, is critical to early development and is directly linked to several human diseases including diabetes and hemophilia (reviewed in Ref. 9). Whereas an endogenous ligand has not yet been definitively identified for HNF4
(10, 11), we and others (12, 13, 14, 15, 16) have shown that HNF4
interacts with coactivators GRIP1 and p300/CBP in the absence of added ligand and that transactivation by HNF4
is completely dependent upon the AF-2.
In this study, we show that the HNF4
LBD also interacts with a complex containing DRIP proteins in the absence of exogenously added ligand. In investigating this interaction, we found that the polyamine spermine enhanced the interaction between DRIP205 and not only the HNF4
but also the VDR LBD. In contrast, spermine decreased the interaction of both receptors with p160 family member GRIP1. These and other results lead us to propose a model in which polyamines such as spermine help promote the switch from the p160/p300 complex to the DRIP complex.
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RESULTS
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HNF4
Interacts with the DRIP Complex in the Absence of an Exogenously Added Ligand
To determine whether HNF4
could interact with the DRIP complex in the absence of exogenously added ligand, a pull-down assay using Namalwa B cell nuclear extracts and glutathione S-transferase (GST).HNF4.LBD (see Fig. 1
) was performed in a fashion similar to that originally used to identify the DRIP complex (17, 18). The results indicate that the HNF4
LBD pulled down a complex that was similar but not identical with that pulled down by the VDR LBD in the presence of its ligand, 1,25-dihydroxy vitamin D3 [1,25-(OH)2D3; Fig. 2A
, top, compare lane 4 with lane 2]. Immunoblot analysis indicated that DRIP205, DRIP130, and, to a lesser extent, DRIP150 were present in the HNF4
complex (Fig. 2A
, bottom panels, lane 4). Transient cotransfection analyses verified that the interaction with the DRIP complex is a functional one. Transfected DRIP205 wild type (DRIP205.wt) stimulated the ability of HNF4
1 to activate a reporter gene in the absence of added ligand 4.7-fold (Fig. 2B
, compare lane 4 with lane 3). Removal of the region C terminal to the LBD, the F domain, resulted in a greater degree of stimulation by DRIP205 (6.1-fold; lanes 7 and 8), just as was found previously for GRIP1 and CBP (12). The AF-2 was required for the stimulation by DRIP205.wt, as it was for all other transactivation activity (lanes 5 and 6).

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Figure 1. Constructs Used in this Study
Top, wt rat HNF4 1, from which all HNF4 constructs were derived. Numbering indicates aa residues; AF, conventional nomenclature for NR domains; DBD, DBD consisting of the zinc fingers (Zn2+) and hinge region (domain D). DRIP205.wt is the full-length, wt human DRIP205 sequence. DRIP205.mut contains double mutations in the two NR boxes. DRIP205.box encompasses aa 527714 and contains the two wt NR boxes.
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Figure 2. HNF4 Interacts with the DRIP Complex in the Absence of Exogenously Added Ligand
A, Pull-down assay with 2.5 mg of Namalwa B cell nuclear extracts in the presence or absence of 1 µM 1,25-(OH)2D3 (+D3) and the indicated GST fusion proteins (VDR LBD, HNF4 DBD and LBD, GST control; see Fig. 1 ) as described in Materials and Methods. Top, Silver stain of SDS-PAGE. Bottom, Immunoblot analysis (Western blots) of a gel done in parallel with antisera to DRIP205 (1:100), DRIP150 (1:500), or DRIP130 (1:500) as indicated (37 ). B, Transient transfection assay as described in Materials and Methods into Saos2 cells with 2.0 µg reporter construct ApoB.-8547.Luc, 0.1 µg of the indicated HNF4 expression vector (see Fig. 1 ), and 5.0 µg of pcDNA3.DRIP205.wt. Shown is the mean luciferase activity in relative light units (RLU) ±SD of triplicates from one representative experiment of three. DRIP205.wt also stimulated HNF4 -mediated transcription in the absence of added ligand in 293T and CV-1 cells (data not shown). Stimulation was also observed when normalized to a ß-galactosidase control, although it was not as great because DRIP205.wt also enhanced the expression from the ß-galactosidase vector (data not shown).
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Polyamines Enhance the Interaction between HNF4
LBD and DRIP205 in Vitro
To determine whether DRIP205 interacts directly with the LBD of HNF4
, as it does with other nuclear receptors (17, 19, 20, 21, 22), GST pull-down assays were performed using in vitro-translated DRIP205. 35S-Labeled DRIP205.wt interacted only modestly with the HNF4
LBD construct (2.2%) just as it did with the VDR LBD in the absence of ligand (1.6%; Fig. 3A
, lanes 3 and 4). In contrast, addition of 1,25-(OH)2D3 significantly enhanced binding to the VDR LBD (21.5%; lane 5) and served as a positive control.

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Figure 3. The Polyamine Spermine Enhances Binding of DRIP205.wt to HNF4 LBD
A, Pull-down assay as described in Materials and Methods with in vitro-synthesized 35S-labeled DRIP205.wt and immobilized GST fusion proteins of the HNF4 and VDR LBD (see Fig. 1 ) in the absence and presence of 4 µM 1,25-(OH)2D3 (+D3) dissolved in ethanol (EtOH) as indicated. Shown is an autoradiograph of SDS-PAGE quantified by phosphor imaging. 10% input, 10% of amount of lysate used in the reaction; % bound, percentage of total input bound normalized to binding to the GST control (GST). B, Pull-down assay as in A with 35S-labeled DRIP205.wt and immobilized GST.HNF4.LBD in the presence of 0, 1, 0.1, and 0.01 mM spermine as indicated. Arrow, Full-length DRIP205.wt. Brackets, Partial translation products of DRIP205.wt. 10% input, 10% of amount of lysate used in the reaction; % bound, percentage of input of full-length DRIP205 bound, normalized to GST control (GST).
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To resolve the apparent discrepancy between the ability of the HNF4
LBD to bring down the DRIP complex from cell extracts and the relative inability of the LBD to interact with in vitro-translated DRIP205.wt, we incubated the 35S-labeled DRIP205.wt with cell extracts and repeated the pull-down assay. We found that under these conditions the DRIP205.wt could indeed interact with the HNF4
LBD but that the addition of the cell extracts was not required for the effect, only the addition of the extract buffer (data not shown). Because the most unusual components of the nuclear extraction buffer were the positively charged polyamines spermine and spermidine, we tested them in the pull-down assay. We found that physiological concentrations of spermine (1 mM) markedly enhanced the interaction between 35S-labeled DRIP205.wt and the HNF4
LBD, from 1.5% to 22%, but had only a negligible affect on background binding to the GST control (Fig. 3B
, compare arrow in lane 5 with lane 3, and lane 4 with lane 2). Less spermine (0.1 mM) resulted in less binding (3.5%), indicating a dose effect (lane 7). Interestingly, truncated 35S-labeled DRIP205 products present in the lysate bound relatively well to the HNF4
LBD, irrespective of the addition of spermine (lanes 3, 5, 7, 9, bracket). Similar effects were observed with spermidine although to a lesser degree (data not shown).
In investigating the nature of the effect of spermine on the HNF4
-DRIP205 interaction, we found several properties that suggest that the effect is a specific one. For example, spermine did not enhance binding of DRIP205.wt to a GST HNF4
fusion construct lacking the AF-2 (C360), indicating that the AF-2 is required (Fig. 4A
, top panel, compare lane 6 with lane 3). Spermine also only marginally affected the ability of the HNF4
LBD to interact with another protein, namely 35S-labeled HNF4
1 (Fig. 4A
, bottom panel, compare lane 7 with lane 4; 1.4-fold effect vs. an 8.3-fold effect on binding DRIP205), and this effect did not require the presence of the AF-2 (compare lane 6 with lane 3). The effect of spermine also required the presence of the NR boxes of DRIP205 as pull-downs with the full-length DRIP205 containing mutations in the two NR boxes (DRIP205.mut) were not affected by the addition of spermine (Fig. 4B
, middle panel, compare lane 7 with lane 4). Likewise, interaction with a small region of DRIP205, which encompasses just the NR boxes (DRIP205.box), was not greatly affected by the addition of spermine (compare lane 7 with lane 4, bottom panel). These results indicate that whereas the effect of spermine was dependent on the presence of the AF-2 and the NR boxes, it was not due to an increased interaction between just these two regions. This is not surprising in that spermine is a charged molecule and the AF-2-NR box interaction is primarily a hydrophobic one.

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Figure 4. Spermine Enhances Binding of DRIP205.wt to NR LBDs in an AF-2- and NR Box-Dependent Manner
A, Pull-down assay as in Fig. 3 with 35S-labeled DRIP205.wt or HNF4 1 (full length) and immobilized GST fusion proteins (C360, GST.HNF4.360; LBD, GST.HNF4.LBD; see Fig. 1 ) in the absence and presence of 1 mM spermine as indicated. 10% input, 10% of amount of lysate used in the reaction; % bound, percentage of total input bound normalized to binding to the GST control (GST). B, Pull-down assay as in A with 35S-labeled DRIP205 constructs (DRIP205.wt, DRIP205.mut, DRIP205.box; see Fig. 1 ) and immobilized GST fusion proteins (DBD, GST.HNF4.DBD; LBD, GST.HNF4.LBD; see Fig. 1 ) in the absence and presence of 1 mM spermine as indicated. 10% input, 10% of amount of lysate used in the reaction; % bound, percentage of total input bound normalized to binding to the GST control (GST). C, Pull-down assay as in B with 35S-labeled DRIP205 constructs (DRIP205.wt, DRIP205.box; see Fig. 1 ) and immobilized GST.VDR.LBD fusion protein (VDR.LBD) in the absence and presence of 1 mM spermine and 1,25-(OH)2D3 (+D3) or ethanol control (EtOH) as indicated. 10% input, 10% of amount of lysate used in the reaction; % bound, percentage of total input bound normalized to binding to the GST control (GST).
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Additional studies support the notion that the effect of the spermine may be due to more than simply an enhancement of the AF-2-NR box interaction and may in fact involve the entire DRIP205 molecule. Because other coactivators have been found to interact with the DNA binding domain (DBD) of nuclear receptors (23, 24), we sought to determine whether DRIP205 might also interact with the HNF4
DBD and whether that interaction was affected by the addition of spermine. Whereas the HNF4
DBD bound remarkably well to both the wt DRIP205 (67%) and the DRIP205 containing the mutated NR boxes (54.5%; Fig. 4B
, top and middle panels, lane 3), spermine greatly decreased both of those interactions (lane 6). In contrast, the isolated DRIP205 NR box did not bind well to the HNF4
DBD either in the absence or presence of spermine (bottom panel, lanes 3 and 6). Whereas it is possible that the decrease in binding of the full-length DRIP205 to the HNF4
DBD is not related to the increased in binding to the LBD, it is equally possible that the two effects are related. For example, spermine could induce a conformational change in DRIP205.wt that causes a decrease in the interaction with the HNF4
DBD and an increase in the interaction with the LBD, perhaps as a result of increased access to the NR boxes. Such a conformational change could also explain why the HNF4
LBD binds the truncated DRIP205 products better than the full-length DRIP205 and why that binding is not affected by sperminein the truncated products, the NR boxes may already be fully accessible (Fig. 3B
).
If spermine is primarily affecting DRIP205 and not HNF4
, then one might expect to see a similar affect on the interaction with other nuclear receptors. This is exactly what we found with the VDR LBD: the presence of spermine enhanced the interaction with DRIP205.wt from 15.4% binding to a remarkable 71.4% binding in the presence of 1,25-(OH)2D3 (Fig. 4C
, top panel, compare lane 7 with lane 4). It also enhanced binding to the unliganded VDR, although that binding was still considerably less than in the presence of ligand (Fig, 4C
, top panel, lane 6 vs. lane 3). Once again, the affect of spermine was evident only on the full-length DRIP205, as there was no appreciable effect on the isolated NR boxes (Fig. 4C
, bottom panel, lane 7 vs. lane 4).
GRIP1: Decreased Binding in the Presence of Polyamines and Synergy with DRIP205
To determine whether polyamines could increase the interaction between nuclear receptors and any NR box containing protein, we examined the effect of spermine on binding to p160 family member GRIP1. Much to our surprise, addition of spermine to the pull-down reaction reproducibly decreased the interaction of 35S-labeled GRIP1 with both the HNF4
and the VDR LBD (Fig. 5A
, top and bottom panels, compare lane 7 with lane 4).
Because spermine decreased the interaction with GRIP1 but increased the interaction with DRIP205, this suggested that the two coactivator complexes could act together in a single pathway with spermine acting as a switch between the two complexes. If this is the case, then one might expect to see a synergistic effect between the two coactivators. In contrast, if GRIP1 and DRIP205 act independently in two parallel pathways, then one would expect to see only an additive effect. Transient cotransfection analysis showed that DRIP205 activated a Gal4 fusion construct containing the HNF4
LBD approximately 2.2-fold (Fig. 5B
, compare lane 6 with lane 5) and that GRIP1 activated it 2.6-fold (compare lane 7 with lane 5). In contrast, when both GRIP1 and DRIP205 were present, the activation was more than additive (8.8-fold over the Gal4.LBD alone; compare lane 8 with lane 5), indicating a synergistic effect of these coactivators and supporting the notion that they act on the same pathway.
In Vivo Evidence of a Role for Polyamines in Activated Transcription
To determine whether the effects of spermine seen in vitro also played a role in vivo, the transient transfection assay was performed in the presence of a specific inhibitor of polyamine biosynthesis,
-difluoromethylornithine (DFMO), which is known to decrease the levels of intracellular polyamines such as spermidine and spermine by blocking the activity of ornithine decarboxylase (Ref. 25 and Fig. 6A
). A 24-h treatment with DFMO reduced the intracellular levels of both spermidine and spermine in the 293T cells (to
37% and
77%, respectively; Fig. 6B
), and decreased the HNF4
1-mediated transcription by nearly 50% (Fig. 6C
, top, compare lane 3 with lane 1 and lane 4 with lane 2). The effect on the HNF4
1-mediated transcription appeared to be specific in that DFMO slightly increased the level of background transcription from the reporter construct but did not decrease the HNF4
1 protein levels as evidenced by immunoblot analysis (Fig. 6C
, bottom, compare lane 3 with lane 2). Because the addition of polyamines to a gel shift reaction decreased the ability of HNF4
1 to bind DNA in vitro (Seidel, S., and F. M. Sladek, unpublished data), it is unlikely that the decrease in HNF4
-mediated transcription in the DFMO-treated cells is due to a decrease in DNA binding. Rather, the results suggest that blocking synthesis of polyamines such as spermine specifically decreased the ability of HNF4
1 to activate transcription.
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DISCUSSION
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In the current study, we show for the first time that the orphan receptor HNF4
interacts with a complex containing DRIP proteins in vitro and responds to DRIP205 in vivo in the absence of exogenously added ligand (Fig. 2
). We also show that full-length in vitro-translated DRIP205.wt does not interact well with the HNF4
LBD unless the polyamine spermine is added to the reaction (Fig. 3
). The enhancement by spermine appears to be specific in that it was dependent on the presence of the AF-2 of HNF4
and the NR boxes of DRIP205 (Fig. 4
). Spermine also enhanced the interaction of DRIP205 with the VDR LBD but decreased the interaction with the HNF4
DBD (Fig. 4
). An in vivo role for spermine in activated transcription was observed when the specific inhibitor of polyamine biosynthesis, DFMO, decreased HNF4
1-mediated transcription but not basal transcription (Fig. 6
). Finally, spermine decreased the interaction of both HNF4
and VDR with coactivator GRIP1, and GRIP1 synergized with DRIP205 in vivo to activate HNF4
-mediated transcription (Fig. 5
). We and others (5, 6, 7, 26, 27) have proposed previously that the p160 and p300 coactivators and the DRIP complex act in a sequential fashion. Our current work supports that model and allows us to propose a mechanism by which this might occurnamely, that polyamines may play a role in switching from one coactivator complex to the other (Fig. 7
).

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Figure 7. Proposed Model for a Role of Polyamines in Transcriptional Activation
Polyamines such as spermine may decrease the binding of activators such as HNF4 and VDR to coactivators such as GRIP1 (a p160 family member) but increase the binding to DRIP205 (shaded molecules). This would allow for a switch between the p160/p300 complex and the DRIP complex, both of which interact with nuclear receptors via a common docking surface, the AF-2 (black dot). As described in the text, one potential source of the polyamines could be the chromatin.
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Polyamines are small, ubiquitous, organic polycations found at relatively high concentrations in the cell, approximately 12 mM. They have been implicated in a wide variety of physiological functions and have been found to bind proteins as well as nucleic acids (reviewed in Refs. 28, 29, 30). Perhaps one of the most intriguing, if elusive, roles of polyamines is in the regulation of gene expression. Polyamines are implicated in the formation of higher order chromosomal fibers, facilitating condensation of chromatin fragments and interacting specifically with nucleosome core particles and DNA (31). Whereas this would suggest that an increase in polyamine levels would globally repress transcription, in fact it results in an increase in expression of many genes (32), suggesting that polyamines regulate gene expression by additional mechanisms. One such mechanism could be via affecting the interaction of transcriptional activators, such as HNF4
, with coactivators, such as the DRIP complex.
The data presented in this study indicate that p160 family member GRIP1 and DRIP205 can synergistically activate transcription (Fig. 5B
), suggesting that they act in a single pathway but raising the question of how the switch is made from one coactivator to the other. Our data also show that the polyamine spermine can decrease the binding of at least two different nuclear receptors to GRIP1 (Fig. 5A
) and can increase the binding to DRIP205 (Figs. 3
and 4
), suggesting that polyamines may play a role in that switch. Support for that role in vivo is observed when blocking the biosynthesis of polyamines resulted in a decrease in HNF4
-mediated transcription (Fig. 6
). If polyamines were acting at the level of chromatin only, and not at the level of coactivators, then one would expect a decrease in polyamine levels to decondense the chromatin and result in an increase, not a decrease, in transcription. Therefore, the in vivo as well as the in vitro results suggest that polyamines may play a role in transcription activation other than altering the state of the chromatin. However, it must be noted that other interpretations of the in vivo data are possible and that additional experiments are required to definitively prove that polyamines modulate the synergy between GRIP1 and DRIP205 in vivo.
The model of polyamines playing a role in the switch between different coactivator complexes is intriguing but provocative, and raises several important questions. One is how do polyamines affect binding of coactivators to activators? Whereas the mechanism is not known, our results suggest that it is not due to a simple enhancement of the interaction between the AF-2 of the nuclear receptors and the NR boxes of DRIP205 (Fig. 4
). Rather, because truncated DRIP205 products bound the HNF4
LBD equally well in the presence and absence of spermine (Fig. 3
), we favor a model in which polyamines induce a conformational change in DRIP205 that exposes otherwise buried NR boxes. There is at least one example already of a conformational change in a coactivator that is important for its function (33). Another important question is where do the polyamines come from? Most of the polyamines in the cell are not free but are bound to negatively charged molecules such as chromatin (28, 29, 30). Because others have shown that the hyperacetylation of histones inhibits the ability of polyamines to condense the chromatin (31), it is possible that acetylation of histones by coactivators such as p300 or p300/CBP-associated factor may also cause a release of polyamines from the chromatin. If this were the case, then upon activator binding DNA and recruiting a HAT-containing coactivator complex, there would be an increase in the local polyamine concentration that could then mediate the switch from the HAT-containing complex to the DRIP complex. Some evidence already exists that polyamine levels can affect histone acetylation by influencing the action of histone acetyltransferases and deacetylases (32). Finally, an attractive feature of the model is that it could apply not only to nuclear receptors but to any transcriptional activator that recruits both HAT-containing coactivators and the DRIP/TRAP/ARC/mediator complex, of which there are several examples (4, 7).
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MATERIALS AND METHODS
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Materials
1,25-(OH)2D3 was a generous gift from A. Norman (University of California, Riverside, CA). Spermine was purchased from Sigma (St. Louis, MO) and DFMO was provided by Ilex Oncology (San Antonio, TX).
Plasmids and Transient Transfection Assays
The following constructs have been previously described: pMT7 expression vectors containing full-length rat HNF4
1 (pMT7.HNF4
1) and amino acids (aa) 1374 (HNF4.N1C374); a pcDNA3.1 vector containing aa 1360 of HNF4
1 (HNF4.N1C360) (12); HNF4
GST fusion constructs containing aa 127374 (GST.HNF4.LBD), aa 127360 (GST.HNF4.360), and aa 45125 (GST.HNF4.DBD); the reporter construct ApoB.-8547.E4.Luc; a Gal4 DBD fusion construct containing the HNF4
LBD (Gal4.HNF4.LBD) (34); the GST fusion construct containing aa 110427 of the human VDR (GST.VDR.LBD) (17); pcDNA3.1 expression vectors containing the full-length wt human DRIP205 cDNA (DRIP205.wt), a small region spanning the two NR boxes of DRIP205 (aa 527714) (DRIP205.box), and a full-length DRIP205 with two mutated NR boxes (LXXLL to LXXAA; DRIP205.mut) (8); pSG5.GRIP1 containing full-length mouse GRIP1 kindly provided by M. Stallcup (University of Southern California) (Ref. 35 and see Fig. 1
). Human embryonic kidney 293T and Saos-2 cells were maintained and transfected with Lipofectin (Life Technologies, Inc., Gaithersburg, MD) as previously described (34). DNA mixtures typically contained 2 µg luciferase reporter; 0.1 µg pMT7.HNF4
1; 5.0 µg of the DRIP expression vectors, pSG5.GRIP1, or appropriate empty vector; and 2 µg RSV.ßgal as indicated.
GST Pull-Down Assay
In vitro protein-protein interaction assays were performed using GST fusion proteins as previously described (34). In general, 20 µl of beads containing 1020 µg protein were incubated at 4 C with 25 µl in vitro-translated [35S] methionine-labeled protein in rabbit reticulocyte lysate (TNT, Promega Corp., Madison, WI) for 1 h with gentle agitation before extensive washing and elution with SDS buffer and analysis by 10% SDS-PAGE followed by autoradiography and phosphor imaging. GST.HNF4.DBD beads were prewashed in buffer containing 1 M NaCl to remove contaminating DNA. Pull-downs with Namalwa B cell nuclear extracts were performed as previously described (17). For pull-downs with ligand or polyamines, 1,25-(OH)2D3 or spermine was added to 14 µM or 1 mM, respectively, to the GST beads at the same time as the lysate or extract. The concentration of Tris-HCl was increased from 20 mM to 50 mM for experiments with polyamines to maintain the pH at 8.0.
Measurement of Intracellular Polyamine Levels
Intracellular polyamine levels were measured by ion-exchange chromatography followed by reverse-phase HPLC as previously described (36). Briefly, 3 x 106 293T cells plated in a 100-mm plate 1 d in advance were treated with 1 mM DFMO in DMEM when the cells were approximately70% confluent. Twenty-four hours later, the cells were washed extensively with PBS and lysed in 400 µl 0.2 N perchloric acid followed by sonication. The extracted material was then subjected to chromatography.
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ACKNOWLEDGMENTS
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We thank E. Martinez for critical reading of the manuscript and S. D. Seidel for discussions.
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FOOTNOTES
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This work was supported by NIH Grants CA-79909 (to C.V.B.), DK-45460 (to L.P.F.), and DK-53892 (to F.M.S.) and fellowships to Y.M. from the Japan Society for the Promotion of Science and University of California Toxic Substances Research and Teaching Program.
1 Current address: Institut National de la Santé et de la Recherche Médicale Unité 459, Faculte de Medicine H. Warembourg, 1 Place de Verdun, 59045 Lille cedex, France. 
Abbreviations: aa, Amino acids; AF-2, activation function 2; ARC, activator recruited cofactor; CBP, cAMP response element binding protein-binding protein; DBD, DNA binding domain; DFMO,
-difluoromethylornithine; 1,25-(OH)2D3, 1,25-dihydroxy vitamin D3; DRIP, VDR-interacting protein; GRIP1, glucocorticoid receptor interacting protein 1; GST, glutathione S-transferase; HAT, histone acetyl transferase; HNF4
, hepatocyte nuclear factor 4
; LBD, ligand binding domain; NR, nuclear receptor; PCAF, p300/CBP associated factor; SRC1, steroid receptor coactivator; TRAP, thyroid hormone receptor associated protein; VDR, vitamin D receptor; wt, wild-type.
Received for publication October 5, 2001.
Accepted for publication March 14, 2002.
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