Maturity-Onset Diabetes of the Young Type 1 (MODY1)-Associated Mutations R154X and E276Q in Hepatocyte Nuclear Factor 4{alpha} (HNF4{alpha}) Gene Impair Recruitment of p300, a Key Transcriptional Coactivator

Jérôme Eeckhoute, Pierre Formstecher and Bernard Laine

Unité 459 INSERM Laboratoire de Biologie Cellulaire Université H. Warembourg Lille, France F 59045


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a nuclear receptor involved in glucose homeostasis and is required for normal ß-cell function. Mutations in the HNF4{alpha} gene are associated with maturity-onset diabetes of the young type 1. E276Q and R154X mutations were previously shown to impair intrinsic transcriptional activity (without exogenously supplied coactivators) of HNF4{alpha}. Given that transcriptional partners of HNF4{alpha} modulate its intrinsic transcriptional activity and play crucial roles in HNF4{alpha} function, we investigated the effects of these mutations on potentiation of HNF4{alpha} activity by p300, a key coactivator for HNF4{alpha}. We show here that loss of HNF4{alpha} function by both mutations is increased through impaired physical interaction and functional cooperation between HNF4{alpha} and p300. Impairment of p300-mediated potentiation of HNF4{alpha} transcriptional activity is of particular importance for the E276Q mutant since its intrinsic transcriptional activity is moderately affected. Together with previous results obtained with chicken ovalbumin upstream promoter-transcription factor II, our results highlight that impairment of recruitment of transcriptional partners represents an important mechanism leading to abnormal HNF4{alpha} function resulting from the MODY1 E276Q mutation. The impaired potentiations of HNF4{alpha} activity were observed on the promoter of HNF1{alpha}, a transcription factor involved in a transcriptional network and required for ß-cell function. Given its involvement in a regulatory signaling cascade, loss of HNF4{alpha} function may cause reduced ß-cell function secondary to defective HNF1{alpha} expression. Our results also shed light on a better structure-function relationship of HNF4{alpha} and on p300 sequences involved in the interaction with HNF4{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a transcription factor required for normal early embryogenesis (1, 2). HNF4{alpha} plays a central role in the coordination of a complex transcription factor network that defines the hepatocyte phenotype (3, 4, 5) and in the regulation of normal pancreatic endocrine function (6). It controls expression of the transcription factors HNF1{alpha} and HNF6 (5, 6, 7, 8, 9); the former is required for pancreatic endocrine cell function and differentiation (10, 11), whereas the latter plays a central role in pancreatic endocrine differentiation at precursor stage (12).

HNF4{alpha} is a member of the hormone nuclear receptor superfamily (13). It has a modular structure comprising a DNA binding domain (DBD) located in the C domain and two activation function modules AF-1 (residues 1–24, located in the A domain) and AF-2 (residues 360–366, located in the E domain) (14, 15). The HNF4{alpha} AF-2 module does not exhibit an autonomous transactivation activity and requires the sequence 128–366 (located in D–E domains) for its full activity (15). Protein-protein interactions and synergies with other transcription factors such as chicken ovalbumin upstream promoter transcription factors (COUP-TFs) (16) and nuclear transcription factor Y (NPY) (17) or with coactivators of the p160 family (18, 19) and the CREB binding protein (CBP) (19, 20, 21, 22) enhance the transcriptional activity of HNF4{alpha}. CBP has been recently shown to be required for HNF4{alpha} function: through acetylation of HNF4{alpha}, which is required for its nuclear retention, CBP increases HNF4{alpha} DNA binding and transcriptional activities (22). CBP and the closely related p300 protein (together referred to as CBP/p300) (23) serve as coactivators for other hormone nuclear receptors. CBP/p300 contains three LXXLL motifs located in the N-terminal receptor interaction domain (RID), near the C/H1 domain and in the C-terminal sequence, respectively (24) (scheme in Fig. 1CGo). Requirement of the RID in CBP/p300- mediated activation of nuclear receptors depends on nuclear receptors. The RID, which was initially shown to interact with thyroid hormone receptor (TR) and retinoic acid receptor (RAR) homodimers (24), is required to enhance activity of the peroxisome proliferator-activated receptor{gamma} (PPAR{gamma})-retinoid X receptor (RXR) heterodimer (25) but is dispensable for enhanced transactivation by TR-RXR and RAR-RXR heterodimers (26, 27). CBP/p300-mediated transcriptional potentiation of the activities of these two latter heterodimers requires steroid receptor coactivator-1 (SRC-1) (26). On the other hand, CBP-mediated activation of HNF4{alpha}, which acts as a homodimer (28), does not require SRC-1 (21). The N-terminal third of CBP (sequence 1–771) forms a much stronger complex with full-length HNF4{alpha} than its C terminus (21). Regions of HNF4{alpha} interacting with CBP have been mapped to the AF-1 and to the sequence required for full activity of the AF-2 [amino acids (aa) 128–366 (21)]. For convenience, this latter sequence will be named D–E domains hereafter.



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Figure 1. Effect of R154X Mutation on the Functional Cooperation and Physical Interaction between HNF4{alpha} and p300

A, Transactivation activity of R154X, HEK 293T cells were transfected with 1 µg of HNF1{alpha} promoter and 50 ng of HNF4{alpha} expression vectors. Fold induction refers to the activity with no HNF4{alpha} derivative. Results are means ± SD of three independent experiments performed in triplicate. B, Enhancement of HNF4{alpha} transcriptional activity by p300 on the HNF1{alpha} promoter. Cells were transfected as in A along with 500 ng of pCMVß p300 (+) or the empty expression vector (-). For convenience, activities of the promoter in the presence of HNF4{alpha}2 alone or R154X alone were set to 100 to better visualize the difference in their potentiation by p300. Results are means ± SD of three independent experiments performed in triplicate. **, Value statistically different from that observed for wild-type HNF4{alpha} (P < 0.01). C, Schematic representation of the p300 coactivator and the three GST-p300 chimeras, GST-p300(1–595), GST-p300(1–340), and GST-p300(340–528), used in pull-down experiments. RID and C/H stand for receptor interaction domain and cysteine/histidine-rich domain, respectively; circles represent the LXXLL motifs. D, Physical interaction of R154X with p300. [35S]methionine- labeled HNF4{alpha}2 or R154X was incubated with GST or GST-p300 proteins depicted in panel C and immobilized on glutathione-Sepharose 4B beads. HNF4{alpha} proteins retained on beads after extensive washing were analyzed by SDS-PAGE and PhosphorImager. Shown are a PhosphorImage and a graph representing means ± SD of the R154X binding relative to that of wild-type HNF4{alpha} from two independent experiments performed in duplicate. Inputs (10% of the in vitro synthesized proteins used in each incubation) were taken into account for binding quantification. To better visualize faint bands corresponding to R154X, the intensity of the bottom part of the PhosphorImage was enhanced. The GST proteins used in these experiments were visualized on a Coomassie blue-stained SDS-polyacrylamide gel (inset in the frame).

 
HNF4{alpha} is involved in glucose homeostasis, and loss of HNF4{alpha} function results in the decreased expression of genes involved in glucose transport and metabolism (6, 29) and in insulin secretion (6). Mutations in the HNF4{alpha} gene are associated with type 1 maturity-onset diabetes of the young (MODY1), a monogenic form of diabetes characterized by abnormal pancreatic ß-cell function (30). Studies of functional properties of several MODY1-associated mutations of HNF4{alpha} have shown that mutations can have variable effects on the ability of HNF4{alpha} to transactivate a reporter gene (29, 31, 32, 33, 34, 35, 36, 37). We have previously shown that mutations E276Q and R154X impair HNF4{alpha} functions. Mutation E276Q affects the interaction and synergy with COUP-TFII, which enhances HNF4{alpha} activity on the HNF1{alpha} promoter (33). Mutation R154X decreases the transcriptional activity of HNF4{alpha}, the decrease being more pronounced in pancreatic ß-cells compared with non-ß-cells. In addition, in the former cells, R154X exhibits a slight dominant-negative behavior (36). HNF4{alpha} R154X has intact AF-1 and DNA binding domains but lacks the E domain, which is involved in interaction with CBP/p300. The E276Q mutation is located in this E domain. Taking into account the crucial role of CBP/p300, we investigated the effects of these two mutations on the physical interaction and functional cooperation between HNF4{alpha} and p300.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effect of the R154X Mutation on p300 Recruitment by HNF4{alpha}
We have previously shown that the R154X mutation resulted in a strong impairment of HNF4{alpha} transcriptional activity on several promoters in various cell lines. Interestingly, the transcriptional activity of R154X remained equal to one-third that of wild-type HNF4{alpha} on the HNF1{alpha} promoter in HEK 293T cells (Ref. 36 and Fig. 1AGo). We investigated whether this low transcriptional activity could be enhanced by the coactivator p300. Cotransfection of p300 and HNF4{alpha}2 increased HNF1{alpha} promoter activity 3.8-fold over the activation by HNF4{alpha}2 alone (Fig. 1BGo). Coexpression of p300 and R154X stimulated HNF1{alpha} promoter activity only 2.3-fold over the activation by R154X alone (Fig. 1BGo). Transfection of p300 in the absence of transfected HNF4{alpha} did not increase the activity of the HNF1{alpha} promoter in HEK 293T cells (data not shown), indicating that the effect of p300 is mediated through HNF4{alpha}.

These results led us to compare interactions of wild-type and mutated HNF4{alpha} with p300 by pull-down assays using the three glutathione-S-transferase (GST)-p300(1–595), GST-p300(1–340), and GST-p300(340–528) chimeras. GST-p300(1–595) contains two LXXLL motifs located in the receptor interaction domain (RID) and near the C/H1 domain, respectively, whereas GST-p300(1–340) and GST-p300(340–528) contain only one of these LXXLL motifs (Fig. 1CGo) (24). HNF4{alpha}2 and R154X were retained on both p300(>NOREF>1–595) and p30040–528) fragments (Fig. 1DGo, lanes 5, 6, 9, and 10). Nevertheless, quantification of intensities of bands indicated that, compared with wild-type HNF4{alpha}2, R154X bound three times less efficiently to these two p300 fragments (Fig. 1DGo, graph). The specificity of these complexes was ascertained by the inability of GST alone to bind HNF4{alpha}2 and R154X (Fig. 1DGo, lanes 3 and 4). HNF4{alpha}2 and R154X were not bound by the p300(1–340) fragment (Fig. 1DGo, lanes 7 and 8) although GST-p300(1–340) was readily expressed (Fig. 1DGo, inset).

A chimera comprising two copies of the HNF4{alpha} AF-1 module fused to GAL4 DBD has been shown to interact with fragment 1–771 of CBP (14). R154X binding to p300 is likely mediated through the AF-1 module located in the sequence 1–24 since it has been shown that a HNF4{alpha} fragment spanning residues 45–142 does not interact with CBP (21). To ascertain this hypothesis, the mutation F19D, which abolishes the transcriptional activity of the AF-1 module (14), was introduced in R154X. This amino acid residue is located within the sequence (aa 13–24) involved in the interaction between HNF4{alpha} AF-1 and CBP (14). Results presented in Fig. 2AGo show that mutation F19D strongly impaired the transactivation activity of R154X. The drop in R154X-F19D activity is not due to its decreased expression (Fig. 2AGo, inset). In addition, R154X-F19D activity was poorly enhanced by p300: the activity was enhanced 1.17-fold whereas that of R154X was enhanced 2.30-fold (Fig. 2BGo). By pull-down experiments, we observed that the F19D mutation nearly abolished interaction of R154X with p300 (Fig. 2CGo). These results confirm that AF-1 is involved in R154X transcriptional activity and in potentiation of this latter by p300.



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Figure 2. R154X Activities Are Markedly Decreased by Mutation of the AF-1 Module

A, HEK 293T cells were transfected as in Fig. 1AGo with the indicated HNF4{alpha} expression vectors. Fold induction by R154X-F19D is expressed in percentage relative to that by R154X. Results are means ± SD of three independent experiments performed in triplicate. *** Denotes a significant difference (P < 0.001) compared with R154X value. Inset, Control of protein expression. B, Enhancement of R154X activity by p300 is impaired by mutation in the AF-1 module. HEK 293T cells were transfected as in A together with 0.5 µg of p300 or empty expression vector. For convenience, activities of the promoter in the presence of R154X alone or R154X-F19D alone were set to 100 to better visualize the difference in -fold enhancement by p300. Results are means ± SD of three independent experiments performed in triplicate. ** Denotes a significant difference (P < 0.01) compared with R154X value. C, Physical interactions between [35S] methionine-labeled R154X or R154X-F19D and GST-p300(340–528) immobilized on glutathione-Sepharose beads. HNF4{alpha} mutants retained on beads after extensive washing were analyzed by SDS-PAGE and PhosphorImager. Shown are a PhosphorImage and a graph representing means ± SD of the R154X-F19D binding relative to that of R154X from two independent experiments performed in duplicate. Inputs (5% of the in vitro synthesized proteins used in each incubation) were taken into account for binding quantification.

 
Next we introduced the F19D mutation in full-length HNF4{alpha} to investigate the AF-1 contribution toward the potentiation of full-length HNF4{alpha} activity by p300. F19D mutation resulted in a drop of 50% in the transactivation activity of HNF4{alpha}2 (Fig. 3AGo). Conversely, the F19D mutation did not alter the p300-mediated enhancement of HNF4{alpha}2 transcriptional activity (Fig. 3BGo) despite a decrease in interaction with p300(340–528) (Fig. 3CGo). These results show that the AF-1 is required for the full intrinsic activity of HNF4{alpha} (without exogenously supplied coactivators) but exhibits a secondary role in the p300-mediated potentiation of this transcription factor.



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Figure 3. Contribution of HNF4{alpha}2 AF-1 to p300 Recruitment

A, HEK 293T cells were transfected as in Fig. 1AGo with HNF4{alpha}2 (WT) or HNF4{alpha}2-F19D (F19D) expression vectors. Fold induction by HNF4{alpha}2-F19D is expressed in percentage relative to that by HNF4{alpha}2. ** Denotes a significant difference (P < 0.01) compared with wild-type HNF4{alpha} value. Inset, control of protein expression. B, HEK 293T cells were transfected as in panel A together with 0.5 µg of p300 or empty expression vector. For convenience, activities of the promoter in the presence of HNF4{alpha}2 alone or HNF4{alpha}2-F19D alone were set to 100 to better visualize the difference in -fold enhancement by p300. Results are means ± SD of three independent experiments performed in triplicate. C, Physical interactions between [35S]methionine-labeled HNF4{alpha}2 or HNF4{alpha}2-F19D and GST-p300(340–528) immobilized on glutathione-Sepharose beads. HNF4{alpha} proteins retained on beads after extensive washing were analyzed by SDS-PAGE and PhosphorImager. Shown are a PhosphorImage and a graph representing means ± SD of the HNF4{alpha}2-F19D binding relative to that of wild-type HNF4{alpha}2 from three independent experiments performed in duplicate. Inputs (10% of the in vitro synthesized proteins used in each incubation) were taken into account for binding quantification.

 
Effect of the E276Q Mutation on p300 Recruitment by HNF4{alpha}
In HEK 293T cells, HNF4{alpha}2 and HNF4{alpha}2-E276Q, which were expressed at the same levels (Fig. 4AGo, inset), exhibited similar transactivation activities on the HNF1{alpha} promoter (Fig. 4AGo). On the other hand, the p300-mediated enhancement of these activities was markedly different: it was much higher for HNF4{alpha}2 than for its E276Q mutant (Fig. 4BGo), thus demonstrating that the E276Q mutation impairs the cooperation between HNF4{alpha} and p300. We then investigated whether this impairment could be correlated with a decrease in physical interaction between HNF4{alpha} and its coactivator by pull-down experiments. In these experiments we used GST-p300(1–595) and GST-p300(340–528) but not GST-p300(1–340), which could not bind HNF4{alpha} (see Fig. 1DGo). We have previously shown that wild-type and E276Q HNF4{alpha}2 do not bind GST (33). The E276Q mutation significantly impaired the HNF4{alpha}-p300 interaction as evidenced with both GST-p300(1–595) and GST-p300(340–528) proteins (Fig. 4CGo).



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Figure 4. Effect of E276Q Mutation on Functional Cooperation and Physical Interaction between HNF4{alpha}2 and p300

A, Transactivation activity of E276Q, HEK 293T cells were transfected as in Fig. 1AGo. Fold induction refers to the activity with no HNF4{alpha} derivative. Results are means ± SD of three independent experiments performed in triplicate. Inset, Control of protein expression. B, Enhancement of HNF4{alpha} transcriptional activity by p300 on the HNF1{alpha} promoter. Cells were transfected as in panel A along with 500 ng of pCMVß p300 (+) or the empty control vector (-). Results are means ± SD of three independent experiments performed in triplicate. *** Denotes a significant difference (P < 0.001) compared with wild-type HNF4{alpha} value. C, Pull-down assays were carried out as in Fig. 1DGo using [35S]methionine-labeled HNF4{alpha}2 or its E276Q mutant to analyze their interactions with GST-p300(1–595) and GST-p300(340–528). Shown are a PhosphorImage and a graph representing means ± SD of the E276Q binding relative to that of wild-type HNF4{alpha} from two independent experiments performed in duplicate. Inputs (10% of the in vitro synthesized proteins used in each incubation) were taken into account for binding quantification.

 
The E276Q Mutation Affects the Cooperation between p300 and the D–E Domains Required for Full Activity of the HNF4{alpha} AF-2 Activation Function
The HNF4{alpha} AF-2 activation function does not exhibit an autonomous transcriptional activity and requires the sequence spanning residues 128–366 for its full activity (15). The E276Q mutation is located within this sequence, which encompasses the HNF4{alpha} D–E domains. We therefore investigated the effect of the E276Q mutation on the activity of the D–E domains. To this end, wild-type or mutated HNF4{alpha} fragments spanning the sequence 128–369 fused to the GAL4 DBD were expressed. These chimeras correspond to GAL4-HNF4{alpha}(128–369) and GAL4-HNF4{alpha}(128–369)-E276Q and were denoted WT and E276Q, respectively, in Fig. 5Go. In HEK 293T cells, p300 alone elicited a 3-fold activation of the (UAS)2xtk promoter containing two GAL4 response elements (UAS) (Fig. 5AGo, lane b). GAL4-HNF4{alpha}(128–369) and GAL4-HNF4{alpha}(128–369)-E276Q exhibited indistinguishable activities on this promoter when p300 was not cotransfected (Fig. 5AGo, lanes c and e). On the other hand, cotransfection of p300 resulted in a greater enhancement of activity for GAL4-HNF4{alpha}(128–369) than for GAL4-HNF4{alpha}(128–369)-E276Q (Fig. 5AGo, compare lanes d and f). These experiments were also carried out in Hela cells in which the p300-mediated enhancement of HNF4{alpha} activity was previously documented (21). Again in these cells, the transactivation activities of GAL4-HNF4{alpha}(128–369) and GAL4-HNF4{alpha}(128–369)-E276Q were similar when p300 was not cotransfected (Fig. 5BGo, lanes c and e), and cotransfection of p300 resulted in a higher enhancement of activity for GAL4-HNF4{alpha}(128–369) than for GAL4-HNF4{alpha}(128–369)-E276Q (Fig. 5BGo, compare lanes d and f). Therefore, in both cell lines, the E276Q mutation markedly impaired the cooperation between p300 and the HNF4{alpha} D–E domains. This impairment was correlated with a decrease in physical interaction between p300 and HNF4{alpha} D–E domains, as evidenced by pull-down assays (Fig. 5CGo).



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Figure 5. E276Q Mutation Affects the Functional Cooperation and Physical Interaction between HNF4{alpha} D-E Domains and p300

HEK 293T cells (A) and HeLa cells (B) were transfected with 500 ng of (UAS)2x tk Luc, 100 ng of pCMVß p300 (+), or its empty expression vector (-). In columns a and b, cells were cotransfected with 50 ng of empty pGAP vector, whereas in columns c–f, cells were cotransfected with 50 ng of pGAP HNF4{alpha}(128–369) or its E276Q mutant, denoted WT and E276Q, respectively. Numbers above bars indicate activities of the promoter relative to that with no added p300 and HNF4{alpha} derivatives (lane a). Results are means ± SD of three independent experiments performed in triplicate. ** Denotes a significant difference (P < 0.01) compared with value obtained with wild-type HNF4{alpha} derivative in the presence of p300. C, Pull-down assays were carried out as in Fig. 1DGo using [35S]methionine-labeled GAL4-HNF4{alpha}(128–369) or its E276Q mutant, which were incubated with GST-p300(1–595) or GST-p300(340–528). Shown are a PhosphorImage and a graph representing means ± SD of the GAL4-HNF4{alpha}(128–369)-E276Q binding relative to that of GAL4-HNF4{alpha}(128–369) from two independent experiments performed in duplicate. Inputs (10% of the in vitro synthesized proteins used in each incubation) were taken into account for binding quantification.

 
Mapping of the Sequences of p300 Required for Interaction with HNF4{alpha}
In the course of this work, we observed that HNF4{alpha} bound efficiently to GST-p300(1–595) and GST-p300(340–528) but not to GST-p300(1–340) (Fig. 1DGo). To determine more precisely the sequence of p300 required for interaction with HNF4{alpha}, we compared the abilities of GST-p300(340–528) and GST-p300(355–528) to interact with wild-type HNF4{alpha}. In this experiment, we also analyzed binding of R154X and GAL4-HNF4{alpha}(128–369), which contain only one of the activation function modules: AF-1 and AF-2, respectively. Removal of 15 amino acid residues at the N terminus of the p300(340–528) fragment resulted in a dramatic decrease in its interaction with full-length HNF4{alpha} and GAL4-HNF4{alpha}(128–369) (Fig. 6AGo, lanes 3 and 9 compared with lanes 2 and 8, respectively) but did not affect binding of R154X (lanes 5 and 6 in Fig. 6AGo). The faint bands of full-length HNF4{alpha} and GAL4-HNF4{alpha}(128–369) retained on GST-p300(355–528) were not due to a low expression of this latter protein (Fig. 6AGo, inset). These results indicate that the p300 sequence 340–354 is required for efficient binding of full-length HNF4{alpha} and HNF4{alpha}(128–369) but is dispensable for binding of R154X. To confirm the role of this sequence in p300 recruitment by HNF4{alpha}, in a functional context ex vivo, we tested whether the p300(302–443) fragment (Fig. 6BGo), containing this sequence 340–354 but lacking the domain involved in histone acetyltransferase (HAT) activity, has a dominant negative effect on the cooperation between p300 and HNF4{alpha}. In this assay, the fragment p300(355–443) (Fig. 6BGo) was used as a control. When cotransfected with HNF4{alpha} and full-length p300, the fragment p300(302–443) exhibited a dominant negative effect that was not observed with the fragment p300(355–443) (Fig. 6Go C). Taken together, results of in vitro protein-protein interaction and ex vivo transactivation experiments obviously demonstrated the involvement of the sequence 340–354 of p300 in the potentiation of HNF4{alpha} activity.



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Figure 6. The Sequence 340–354 of p300 Is Involved in Recruitment of This Coactivator by HNF4{alpha}

A, Pull-down assays were carried out as in Fig. 1DGo using [35S]methionine- labeled HNF4{alpha}2 (left), R154X (middle), or GAL4-HNF4{alpha}(128–369) (right) and either GST-p300(340–528) or GST-p300(355–528). Due to the weak binding of R154X to GST-p300 fragments (see Fig. 1DGo), a larger amount (8 µl vs. 5 µl) of labeled R154X was used to better visualize the bands. The GST proteins used in these experiments were visualized on a Coomassie blue-stained SDS-polyacrylamide gel (inset). B, Schematic representation of the p300 fragments used in panel C. The LXXLL motif at position 342–346 is depicted by a circle. C, HEK 293T cells were transfected by wild-type HNF4{alpha}2 and the HNF1{alpha} promoter as in Fig. 1AGo along with the indicated p300 expression vectors. Results are means ± SD of three independent experiments performed in triplicate. * Denotes a significant difference (P < 0.05) compared with value obtained with full-length p300 alone.

 
It has been previously documented that interaction between the CBP (1–771) fragment and HNF4{alpha} is AF-2 independent (21). Nevertheless, results in Fig. 6AGo indicated that p300 (340–354) efficiently interacted with HNF4{alpha} constructs containing the AF-2 module [full-length HNF4{alpha} and HNF4{alpha}(128–369)] but did not interact with R154X lacking the AF-2. The presence of a LXXLL motif within the p300 sequence 340–354 (at position 342–346) and involvement of LXXLL motifs in the interaction between coactivators and AF-2 modules of nuclear receptors (27) led us to reevaluate the role of the HNF4{alpha} AF-2 module in binding to p300(340–528). To this aim, we compared binding to GST-p300(340–528) of HNF4{alpha}-{Delta}F and HNF4{alpha}-{Delta}AF-2, which only differ by the lack of AF-2 in the latter (Fig. 7A). Deletion of the AF-2 resulted in a significant decrease in binding to p300(340–528) (Fig. 7BGo), thus demonstrating the crucial role of the HNF4{alpha} AF-2 in binding to this p300 fragment.



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Figure 7. Involvement of HNF4{alpha} AF-2 in Interaction with p300(340–528) Fragment

A, Schematic representation of HNF4{alpha}-{triangleup}F and HNF4{alpha}-{triangleup}AF-2 constructs. B, Pull-down assays were carried out as in Fig. 1DGo using [35S]methionine-labeled HNF4{alpha}-{triangleup}F and HNF4{alpha}-{triangleup}AF-2 depicted in panel A and GST-p300(340–528). Shown are a PhosphorImage and a graph representing means ± SD of the HNF4{alpha}-{triangleup}AF-2 binding relative to that of HNF4{alpha}-{triangleup}F from three independent experiments performed in duplicate. Inputs (10% of the in vitro synthesized proteins used in each incubation) were taken into account for binding quantification.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our results showed that MODY1-associated mutations R154X and E276Q in the HNF4{alpha} gene impair the functional cooperation and physical interaction between HNF4{alpha} and p300. In the case of the R154X mutant, this impairment can be ascribed to the lack of AF-2, which plays a crucial role in the recruitment of p300 as discussed below. In the case of the E276Q mutant, this impairment is ascribed to an alteration of the interaction and cooperation between p300 and the D–E domains, which are required for full activity of the HNF4{alpha} AF-2 activation function. Impairment of the p300-mediated potentiation of HNF4{alpha} transcriptional activity is of particular importance for the E276Q mutant of which the intrinsic transcriptional activity (without exogenously supplied coactivators) is moderately affected (33, 35) by comparison with that of the R154X mutant (35, 36). Another MODY1-associated HNF4{alpha} mutant, R127W, was shown recently to have no effect on the p300-mediated enhancement of HNF4{alpha} activity (37). The localization of this mutation in the HNF4{alpha} DBD (aa 50–142), which does not interact with CBP/p300 (21), may account for the lack of change in CBP/p300 recruitment.

Many authors who study the interaction and cooperation between nuclear receptors and their coactivators assume that they act synergistically when their combined effects on enhancement of transcription of the target promoter are higher than the sum of their individual effects. Nevertheless, we cannot rule out that other models of interaction may be involved in p300 recruitment by HNF4{alpha}. Whatever the mechanism involved in this interaction, our results clearly showed that recruitment of p300, which has been shown to be essential for transcription by HNF4{alpha}, was impaired by the studied HNF4{alpha} mutations.

The ability of CBP/p300 to interact with the HNF4{alpha} AF-1 in the context of the chimera GAL4 (AF-1)2x has been shown previously (14). CBP/p300 interacts with the HNF4{alpha} fragments 1–128 (21) and 1–153 (present work), which contain the AF-1. Our results showing significant decreases in interaction and cooperation between R154X and p300 by the mutation F19D indicate unambiguously that the AF-1 is actually involved in p300 recruitment to this truncated HNF4{alpha}. In addition, our results extend these findings by demonstrating for the first time that the HNF4{alpha} AF-1 activity can be enhanced by p300.

Mutation F19D resulted in a 50% decrease of full-length HNF4{alpha}2 activity despite the presence of the AF-2, a result consistent with that of Green et al. (14) and which confirms the crucial role of the AF-1 in the transcriptional activity of full-length HNF4{alpha}. The AF-1 role in full-length HNF4{alpha} activity does not depend on p300 recruitment since the F19D mutation did not alter the p300-mediated enhancement of full-length HNF4{alpha} transcriptional activity. This result fits with those obtained by Dell and Hadzopoulou-Cladaras (21) using a complete deletion of the AF-1. It appears therefore that, even if p300-mediated potentiation of truncated HNF4{alpha} lacking the AF-2, i.e. R154X, is exerted through the AF-1, p300-mediated potentiation of HNF4{alpha} proteins containing the AF-2 is exerted mainly through this latter activation function. This suggests that, in the context of full-length HNF4{alpha}, the p300-mediated enhancement of AF-2 activity is strong enough to compensate for the decrease in cooperation between p300 and the AF-1 by the F19D mutation. This hypothesis is supported by the drop of 60% in cooperation between p300 and full-length HNF4{alpha} due to the E276Q mutation located in the D–E domains required for full activity of the AF-2. The role of the AF-1 in the CBP/p300 action is much more crucial for other nuclear receptors than for HNF4{alpha}. Indeed, CBP/p300-mediated potentiation of AF-1 plays a major role in the transcriptional activities of androgen receptor, estrogen receptor-{alpha}, and estrogen receptor-ß since mutations in AF-1 cause reductions in ligand-induced functional synergism between their AF-1 and AF-2 mediated by CBP/p300 (38, 39).

Previous mapping of CBP sequences interacting with full-length or truncated HNF4{alpha} used either a CBP fragment spanning the sequence 271–451 (20) and corresponding to sequence 251–436 of p300 (23) or a CBP fragment spanning the sequence 1–771 (14, 21). We have shown that the p300 sequence 340–528 is sufficient for binding full-length HNF4{alpha} as well as HNF4{alpha} fragments encompassing sequences 1–153 and 128–369 containing the AF-1 and AF-2, respectively. Interestingly, we have observed that within the p300 fragment spanning residues 340–528, the sequence 340–354, which contains a LXXLL motif, is required for efficient interaction with the full-length HNF4{alpha} and the HNF4{alpha}(128–369) fragment containing the AF-2. Our results thus allow a more precise mapping of sequences of p300 involved in its interaction with HNF4{alpha}.

We have observed that the HNF4{alpha} AF-2 plays a crucial role in interaction with the p300(340–528) fragment, whereas Dell and Hadzopoulou-Cladaras have shown that interaction of the CBP(1–771) fragment with HNF4{alpha} is AF-2 independent (21) (referred to as AF-2 AD in this reference). This discrepancy is not due to the presence of the RID in the CBP(1–771) fragment since both our results and those of Yoshida et al. (20) showed that the RID is dispensable for interaction of CBP/p300 with HNF4{alpha}. Nevertheless, we cannot exclude a slight difference of behavior between CBP and p300 despite their very close structures (23).

Cooperation between HNF4{alpha} and other transcription factors or coactivators is crucial in HNF4{alpha}-mediated transcriptional activation (16, 17, 18, 40, 41, 42, 43, 44). Some of these transcriptional partners interact directly with HNF4{alpha} (16, 17, 18, 44). In addition to alteration in p300 recruitment by R154X and E276Q mutations, we have previously shown that alteration in synergy with the transcription factor COUP-TFII by the E276Q mutation strongly impairs HNF4{alpha} function (33). Alteration in recruitments probably occurs with other HNF4{alpha} partners since the R154X and E276Q mutations also impair the in vitro interaction with the GRIP-1 (glucocorticoid receptor interacting protein-1) coactivator (J. Eeckhoute, unpublished results). Therefore, our results emphasize that impairment of recruitment of transcriptional partners by MODY1-associated HNF4{alpha} mutations most likely represents a key mechanism leading to HNF4{alpha} loss of function.

The impairment of p300-mediated enhancement of HNF4{alpha} activity by MODY1 mutations has been observed on the HNF1{alpha} promoter. HNF1{alpha} is essential for insulin gene transcription and also regulates expression of genes involved in glucose transport and metabolism and in insulin secretion (11). In addition, loss of HNF1{alpha} function results in defective insulin secretion responses to glucose (10, 11), and mutations in the HNF1{alpha} gene are associated with another form of MODY, MODY3 (45). Interestingly, patients with MODY1 and MODY3 exhibit similar clinical profiles (46). Also, recent results showed that the phenotype and gene expression patterns of INS-1 ß-cells expressing a dominant negative HNF4{alpha} are strikingly similar to those of INS-1 ß-cells expressing a dominant negative HNF1{alpha} (6), which led the authors to suggest that loss of HNF4{alpha} function may cause abnormal ß-cell function secondary to defective HNF1{alpha} function. Therefore, impairment by R154X and E276Q mutations in the p300 recruitment leading to a reduced activity of the HNF1{alpha} promoter is of major interest. Nevertheless, as for other MODY1-associated HNF4{alpha} mutations, further studies are needed to link our mechanistic information to comprehension of ß-cell dysfunction and MODY1 physiopathology. Indeed, whatever the mechanisms underlying HNF4{alpha} loss of function (either deletion of the AF-2 activation function for the Q268X and R154X mutations or impaired recruitment of HNF4{alpha} partners such as COUP-TF and p300 for the E276Q mutation), the scientific community is still unable to explain clearly how these HNF4{alpha} mutations lead to ß-cell abnormal function and to development of diabetes. These mutations, as well as mutations in the HNF1{alpha} gene, are supposed to cause haploinsufficiency or reduced gene dosage (35, 47) since they do not result in proteins exhibiting dominant negative activities on the wild-type protein expressed from the unaffected allele [except for a slight dominant negative behavior of R154X in HIT-T15 cells (36)]. Strikingly, although the Q268X exhibits a complete lack of transcriptional activity, its expression in ß-cells did not result in deficient insulin secretion (6). Furthermore, the huge variability in the clinical characteristics of patients with MODY1 diabetes suggests the existence of other factors (environmental, genetic) influencing the phenotype of MODY1 patients (46). Advances in clinical studies and cell biology of Langerhans islets, together with functional studies of HNF4{alpha} mutants, are clearly required for a better understanding of the physiopathology of MODY1 diabetes. The present work, exploring the mechanisms by which the mutations E276Q and R154X could affect the function of HNF4{alpha}, represents one contribution to achieve this goal. This work also sheds new light on the crucial roles played by the glutamic acid residue at position 276 and by the AF-2 activation function module in recruitment of the p300 coactivator and therefore in the structure-function relationship of this nuclear receptor.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA Constructs
Wild-type human HNF4{alpha}2 and its mutants E276Q, -{Delta}F, and -{Delta}AF-2 cloned in the expression vector pcDNA3 were described previously (33, 36, 48). The wild-type and R154X human HNF4{alpha}2 cloned in the vector pcDNA3.1/HisB were described in Ref. 36 . Plasmids pcDNA3 HNF4{alpha}2-F19D and pcDNA3.1HisB HNF4{alpha}-R154X-F19D were cloned using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) and primers spanning nucleotides 42–78 (taking A of ATG of initiation methionine at position 1) with mutations T->G and T->A at positions 55 and 56, respectively. The plasmids pCMVß-NHA p300 and its pCMVß control vector, pCMVmycNLS p300(302–443), pCMVmycNLS p300(355–443), and pGEX2TK-p300(1–595), -p300(1–340), -p300(340–528), and -p300(355–528) were generous gifts from Dr. S. R. Grossman. The pGAP HNF4{alpha}(128–369) vector expressing wild-type HNF4{alpha}(128–369) fused to the GAL4 DBD (aa 1–147) corresponds to plasmid GAL AF-2 described in Ref. 48 ; its name has been modified to comply with the AF-2 terminology now restricted to the highly conserved module in helix 12 of nuclear receptors. The plasmid pGAP HNF4{alpha}(128–369)-E276Q was prepared by a similar method using pcDNA3 HNF4{alpha}2-E276Q as template. The mouse HNF1{alpha} promoter-luciferase reporter construct was described in Ref. 33 . The (UAS)2xtkLUC containing two copies of the GAL4-binding element upstream of the thymidine kinase (TK) promoter was a gift from V.K.K. Chatterjee. All constructs were verified by DNA sequencing.

Cell Culture and Transient Transfection Assays
HEK 293T and Hela cells (2 x 105 cells and 1.5 x 105 cells per 12-well dishes, respectively) were grown and transfected as in Ref. 33 except that 1 µg of reporter plasmid, 50 ng of HNF4{alpha} expression plasmid, and 500 ng of p300 expression vector were used unless otherwise specified in the figure legends. Luciferase activities were measured using the SteadyGlo buffer (Promega Corp., Madison, WI) and the Lumicount apparatus (Packard Instruments, Meriden, CT).

Western Blot Assays
Western blot assays were carried out as described previously (33).

In Vitro Protein-Protein Interaction
Pull-down assays were performed as described previously (33) using immobilized GST-p300 proteins and [35S] labeled wild-type or mutated HNF4{alpha}2 which were synthesized in vitro using reticulocyte lysates (Promega Corp.). Binding of proteins was quantified by the PhosphorImager apparatus using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

Data Analysis
Statistical analysis was performed by Student’s t test for unpaired data using the Prism software. The significance has been considered at *** P < 0.001; ** P < 0.01; * P < 0.05.


    ACKNOWLEDGMENTS
 
Acknowledgements

The authors are indebted to S. Grossman and M. P. Calos for providing plasmids expressing the p300 coactivator and HEK 293T cells, respectively. They acknowledge P. Lefebvre for critically reading the manuscript and I. Briche for her skillful technical assistance.


    FOOTNOTES
 
Address requests for reprints to: B. Laine, Ph.D., U459 INSERM, Laboratoire de Biologie Cellulaire, Université H. Warembourg, 1 Place de Verdun, F 59045 Lille, France. E-mail: blaine{at}lille.inserm.fr

Received for publication November 13, 2000. Revision received March 26, 2001. Accepted for publication March 29, 2001.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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