Hepatocyte Nuclear Factor 4 enhances the Hepatocyte Nuclear Factor 1-m
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《核酸研究医学期刊》
INSERM Unit 459, Laboratoire de Biologie Cellulaire, Faculté de Médecine H. Warembourg, 1 Place de Verdun, F 59045 Lille, France
*To whom correspondence should be addressed. Tel: +33 3 20 62 69 51; Fax: +33 3 20 62 68 84; Email: blaine@lille.inserm.fr
ABSTRACT
Hepatocyte Nuclear Factor 1 (HNF1) and Hepatocyte Nuclear Factor 4 (HNF4) are two liver-enriched transcription factors coexpressed in specific tissues where they play a crucial role through their involvement in a complex cross-regulatory network. HNF1 down regulates HNF4-mediated activation of transcription via a direct protein–protein interaction. Here we show that HNF4 enhances the transcriptional activity of HNF1 in a DNA binding independent manner, thus indicating that it behaves as a HNF1 coactivator. Using mutations in the ligand binding domain (LBD) of HNF4, we confirmed the involvement of the Activation Function 2 module and demonstrated the requirement of the integrity of the LBD for the interaction with HNF1. Moreover, we show that HNF4 cooperates with p300 to achieve the highest HNF1-mediated transcription rates. Our findings highlight a new way by which HNF4 can regulate gene expression and extend our knowledge of the complexity of the transcriptional network involving HNF4 and HNF1.
INTRODUCTION
Hepatocyte Nuclear Factor 1 (HNF1) and Hepatocyte Nuclear Factor 4 (HNF4) are two liver-enriched transcription factors that are also expressed in kidney, intestine and endocrine pancreas (1). They are involved in complex cross-regulatory networks that determine the phenotype of hepatocytes and pancreatic ?-cells (1–3). HNF1 is a homeodomain-containing transcription factor (1) whereas HNF4 belongs to the hormone nuclear receptor superfamily (4). Both transcription factors are highly conserved during evolution (4,5). HNF1 and HNF4 directly modulate the expression of a large number of genes (4,6,7). They can also modulate transcription indirectly through the above mentioned transcription factor network, including a HNF1-mediated negative regulation of genes activated by HNF4 (8,9). The roles of these transcription factors in vivo have been confirmed by defects linked to the invalidation of their genes in mice (10–14). Further underscoring the importance of these transcription factors, mutations in HNF1 and HNF4 genes have been identified in patients with Maturity Onset Diabetes of the Young, MODY3 and MODY1, respectively (15,16). MODY1 and MODY3 mutations result in loss of function of these transcription factors (17). Moreover, it has been reported that expression of a HNF1 dominant negative mutant linked to MODY3 led to an impaired function of pancreatic ?-cells (18).
HNF1 contains two domains involved in DNA binding (an atypical homeodomain and a POU-like domain), an N-terminal dimerization domain and a C-terminal transactivation domain (1). Several proteins interact with various HNF1 domains and play crucial roles in HNF1 function. DcoH (Dimerization Cofactor of HNF1), is a small protein, which binds to the HNF1 dimerization domain and is involved in dimer stabilization (19). The ability of various HNF1 domains to interact with multiple coactivators allows formation of a platform for recruitment of a transcriptional complex, leading to a strong enhancement of transcription. CBP/p300 interacts with both the DNA binding domain and the activation domain of HNF1 while P/CAF, SRC-1 and RAC3 interact with the HNF1 activation domain (20,21). Each of these coactivators can independently increase activation of transcription by HNF1. In addition, they cooperate with each other to further enhance the HNF1-mediated activation of transcription (21). HNF1 can also interact with GATA 4, GATA 5 and Cdx-2 transcription factors (22). The interactions between HNF1, GATA5 and Cdx-2 lead to a cooperative enhancement of HNF1-mediated activation of transcription (22). A synergy between HNF1 and neurogenin 3 was also recently reported (23). HNF1 down regulates HNF4-mediated activation of transcription via a direct interaction of these transcription factors (8,9).
In this work, we show that HNF4 enhances the transcriptional activity of HNF1, and that the cooperation between both factors can be further enhanced by p300 recruitment.
MATERIALS AND METHODS
DNA constructs
Plasmids pcDNA3 HNF42 and pcDNA3 HNF42-E276Q were previously described (24). Plasmid pcDNA3 HNF42-D126Y was described by Oxombre et al. (25). Plasmids pcDNA3 HNF42-E262A and pGEX2TK HNF42 were described by Eeckhoute et al. (26). Plasmids pcDNA3 HNF1 and pcDNA3 HNF1-P291fsinsC, here named HNF1-AD, were generous gifts from Drs M. Yaniv and A. Abderrahmani, respectively. Plasmids pcDNA3 HNF42-A223F and pcDNA3 HNF42-AF2mut were obtained by site-directed mutagenesis using the QuickChangeTM kit (Stratagene) to introduce the A223F or the three mutations E363K, L365Q and L366Q, respectively. Plasmid pM3-VP16 was from Clontech and pcDNA3-RXR? encoding the full-length human RXR? was a generous gift from Dr R. Polakowska. Plasmid pGEX2TK HNF1 was prepared by a strategy identical to that used for cloning pGEX2TK COUP-TFII (24) by inserting a PCR fragment encompassing the human HNF1 cDNA. Plasmid pCMV?-NHA p300, PGEX2TK p300(1–595), PGEX2TK p300(340–528) and PGEX2TK p300(1572–2370) were kindly provided by Dr S. R. Grossman. Expression plasmids for VP16, Np300-VP16 and Cp300-VP16 were gifts from Dr D. Hum (27). Plasmid pGL3 (–96/+11) LPK was a generous gift from Dr M. Vasseur-Cognet (28). Plasmid pGL3 (–341/+183) human HNF1 was a gift from Dr G. Bell. Plasmid (GAL4)x5 TATA Luc was described by Chang and Gralla (29). Plasmid pGL3 HNF1-TATA-Luc was cloned by inserting a double-stranded oligonucleotide encompassing the HNF1 binding site (–56/ –35) of the SRC promoter into the SacI/NheI sites of the pGL3 basic vector in which the TATA box of the adenovirus major late promoter had been previously cloned as described by Suaud et al. (24). All constructs were verified by DNA sequencing.
GST pull-down assays
GST pull-down assays were performed as described previously (24) using methionine-labelled in vitro synthesized HNF1 or HNF4 and bacterially expressed GST-fusion proteins indicated in legends to figures.
Cell culture and transient transfection assays
HeLa cells (5.5 x 104 cells per 24-well dish) were grown and transfected as described by Eeckhoute et al. (30) with plasmid amounts indicated in figure legends. Luciferase activities were measured using the Bright-Glo Luciferase assay system (Promega).
Western blotting
Western blotting performed from whole-cell extracts and using the 455 HNF4 antiserum was carried out as indicated by Eeckhoute et al. (26).
Data analysis
Statistical analyses were based on Student’s t-test for unpaired data using Prism software. Statistical significance was set at ***P < 0.001, **P < 0.01 and *P < 0.05.
RESULTS
Enhancement of HNF1-mediated activation of transcription by HNF4
HNF1 and HNF4 can positively regulate one another’s expression in cell types that endogenously express these two factors (3,6). To avoid any interference with the endogenous proteins, the effect of HNF4 on the transactivation activity of HNF1 was analysed in HeLa cells. The experiments were performed on a synthetic promoter consisting of one HNF1 binding site located upstream of the TATA box (HNF1-TATA promoter) and on the natural liver pyruvate kinase promoter (–96/+11) LPK containing one HNF1 binding site (site L1, position –96/–72) but lacking the HNF4 response element (28). As expected, both promoters were activated by HNF1 but not by HNF4 (Fig. 1A and B). Cotransfection of HNF1 and HNF4 resulted in a marked increase in the HNF1-mediated activation of these promoters (2.8- and 3.4-fold activation of the synthetic and LPK promoters, respectively) (Fig. 1A and B). The enhancement of the HNF1 activity was not impaired when introducing the D126Y mutation in HNF4, which significantly decreases its DNA binding and transactivation activities (25) (data not shown), thus confirming that the synergy between HNF1 and HNF4 does not require the DNA binding-dependent activities of HNF4 (Fig. 1A and B). This induction was not an artefact linked to expression of a protein since cotransfection of an unrelated factor failed to enhance the HNF1-mediated activation of transcription (compare the second and last columns in Fig. 1A and B).
Figure 1. HNF4 enhanced the HNF1-dependent activation of transcription. HeLa cells were transiently transfected with 250 ng of the HNF1-TATA promoter (A) or the (–96/+11) LPK promoter (B), 15 ng of HNF1 and 20 ng of HNF4 or RXR? expression plasmids, as indicated. (C) HNF4 was unable to cooperate with the VP16 activation domain. HeLa cells were transiently transfected with 250 ng of the (GAL4)x5 TATA promoter, 5 ng of GAL4-VP16 and 20 ng of HNF4 expression vectors, as indicated. In (A), (B) and (C), the total amounts of transfected DNA were equalized with the empty expression plasmids (–). Fold induction refers to the basal activities of promoters (left columns). Results are means ± S.E. of three experiments performed in triplicate. The extent of HNF4-mediated enhancement of HNF1-dependent activation of transcription is indicated (values above bars). (D) In vitro interaction between HNF1 and HNF4 analysed by pull-down assays. A schematic representation of the HNF1 and HNF1-AD proteins is presented, AD denotes activation domain. methionine-labelled HNF1 or HNF1-AD was incubated with immobilized GST or GST-HNF4. Bound proteins were analysed by SDS–PAGE and PhosphorImager (Molecular Dynamics). Inputs correspond to 10% of amounts of labelled proteins used in the assays. (E) Requirement of the activation domains of HNF1 for the cooperation between HNF4 and HNF1. HeLa cells were transfected as in (A) with the indicated HNF1 constructs. Fold induction refers to the basal activity of the promoter (left column). Results are means ± S.E. of three experiments performed in triplicate.
To control the possibility that HNF4 does not act as a transcriptional partner of another transcription factor, we analysed the effect of HNF4 on the VP16 activation domain fused to the GAL4 DBD (construct GAL4-VP16). HNF4 was unable to enhance the VP16-mediated activation of transcription (Fig. 1C), thus indicating the selectivity of the cooperation between HNF4 and HNF1.
As predicted from the results of Ktistaki and Talianidis (9), we observed that full-length HNF1 and HNF4 physically interact (Fig. 1D). Interestingly, pull-down experiments also showed that HNF1 lacking its activation domain (HNF1-AD) efficiently interacted with HNF4 (Fig. 1D). This result indicates that the sequence 1–291 of HNF1, containing the dimerization and DNA binding domains, is able to interact with HNF4. This prompted us to investigate whether coexpression of HNF4 and HNF1-AD could activate the HNF1-TATA promoter. Despite their efficient interaction, HNF1-AD and HNF4 were unable to activate transcription (Fig. 1E), thus indicating that cooperation between HNF1 and HNF4 requires the HNF1 activation domain.
AF2 is not the only sequence within the HNF4 LBD required for interaction and cooperation with HNF1
The repressive action of HNF1 on HNF4 was unambiguously shown to require the HNF4 LBD (9). However, using truncated fragments of HNF4, Ktistaki and Talianidis mapped the sequence involved in the interaction with HNF1 between residues 337 and 368, leading to the conclusion that the HNF4 AF2 activation function is necessary and sufficient for interaction with HNF1 (9). In fact, this sequence does not contain solely the AF2 (sequence 358–366 corresponding to helix 12 of the LBD), but also a fragment forming part of the large helix termed H10–H11, according to conventional nomenclature (31). Indeed, H10 and H11 are contiguous in HNF4 and form a helix that plays a key role in HNF4 function (31). Furthermore, the integrity of this helix is crucial for protein conformation (32). These recent findings begged a re-evaluation of the HNF4 sequence involved in interaction with HNF1. Using full-length HNF4, we analysed the effects of point mutations located in the HNF4 LBD (Fig. 2A) on the interaction and cooperation with HNF1. The E262A mutation was shown to affect HNF4 dimerization (26). The E276Q mutation does not alter the intrinsic HNF4 transcriptional activity but impairs recruitment of HNF4 transcriptional partners (24,33). Owing to the bulky side chain of phenylalanine, the A223F mutation was hypothesized to affect occupancy of the ligand binding pocket by fatty acids that act as structural cofactors rather than as conventional ligands (31). Accord ingly, A223F mutation mildly impaired HNF4 transcriptional activity and interaction with coactivators (data not shown). In the AF2 module, the acidic group of the amino acid residue side chain at position 363 and the hydrophobic nature of the amino acid residue side chain at position 365 are strictly conserved and play a key role in nuclear receptor function (34). Residue L366 is also important since the L366Q mutation alters HNF4 transcriptional activity (35). Pull-down assays showed that introducing three mutations E363K, L365Q and L366Q in the AF2 (HNF4-AF2mut construct) altered interaction between HNF4 and HNF1 (Fig. 2B). Note however that the decrease in interaction was mild. This result confirms the involvement of the AF2 in the interaction between HNF1 and HNF4 but indicates that this module is not the unique sequence required for interaction between these proteins. Interaction with HNF1 was also significantly decreased by the A223F and E276Q mutations while mutation E262A had no effect (Fig. 2B). Consistent with data from pull-down assays, the HNF4-mediated enhancement of HNF1-dependent transcriptional activation of the (–96/+11) LPK promoter was not impaired by the E262A mutation but was significantly decreased with the mutants A223F, E276Q and HNF4-AF2mut (Fig. 2C). We controlled so that mutant and wild-type HNF4 were expressed at a similar level in transfected cells as shown in Figure 2D for mutants A223F and HNF4-AF2mut and in (24,26) for the mutant E276Q and E262A, respectively. It appears therefore that the interaction and cooperation between HNF4 and HNF1 depend on both the AF2 module (helix 12) and the integrity of the HNF4 LBD.
Figure 2. The AF2 is not the only sequence within the HNF4 LBD required for interaction and cooperation with HNF1. (A) Positions of mutations used in this study. A scheme of HNF4 structure with the various domains is given: DBD, DNA binding domain; LBD, ligand binding domain; AF2, activation function 2 module. (B) Effects of HNF4 mutations on the physical interaction with HNF1. methionine-labelled wild-type or mutated HNF4 were incubated with immobilized GST-HNF1. Bound proteins were analysed by SDS–PAGE and PhosphorImager. Values under photographs indicate binding of HNF4 mutants relative to that of wild-type HNF4 from three independent experiments. Inputs, corresponding to 5% of amounts of labelled proteins used in the assays, were taken into account for binding quantifications. (C) Effects of HNF4 mutations on the cooperation between HNF4 and HNF1 on the (–96/+11) LPK promoter. HeLa cells were transfected as in Figure 1B. Fold induction refers to the basal activity of the promoter (left column). Results are means ± S.E. of three experiments performed in triplicate. Statistical significance of differences with values obtained with wild-type HNF42 is indicated by stars (P < 0.01). (D) Western blotting of wild-type (WT), A223F and AF2mut HNF4 expressed in HeLa cells using the 455 HNF4 antiserum (43). Position of HNF4 is indicated. The star denotes a non-specific band obtained from HeLa cells transfected with the empty vector (–). The number on the left indicates the molecular mass of a marker size in kDa.
HNF4 cooperates with the coactivator p300 to enhance HNF1 transcriptional activity
Because CBP/p300 are key coactivators of HNF1, we hypothesized that cooperation between HNF4 and HNF1 could involve p300. In the absence of HNF1, the coactivator p300 alone or coexpressed with HNF4 was unable to activate the HNF1-TATA promoter (Fig. 3A). On this promoter, p300 slightly enhanced HNF1 transcriptional activity (Fig. 3A). Note that this moderate increase is due at least in part to the low ratio of p300/HNF1 expression vectors used in our experiments (3:1) compared to that used in other studies performed on other promoters . We have verified that by using a higher ratio, we could obtain a stronger enhancement of HNF1 activity by p300 alone (data not shown). Interestingly, in the presence of HNF4, p300 synergistically enhanced HNF1-mediated activation of transcription: the activity of the promoter was increased 18.4-fold, which is 4.7-fold higher than the additive value for the induction of HNF1 activity by p300 and HNF4 alone (1.8 and 2.1, respectively). The marked p300-mediated enhancement of cooperation between HNF1 and HNF4 could also be observed on the (–96/+11) LPK promoter (Fig. 3B). On both promoters, the enhanced cooperation between HNF1 and HNF4 was significantly impaired by mutations in the HNF4 AF2 module (Fig. 3A and B). These results strongly argue for the involvement of HNF4 in the p300-mediated activation of these promoters despite the absence of HNF4 response element in their sequences. The result obtained with the HNF4-AF2mut led us to investigate whether mutations in AF2 affect p300 binding. Pull-down experiments clearly showed a much weaker interaction of p300 with HNF4-AF2mut than with wild-type HNF4 (Fig. 3C). This result is in line with our previous observation that deletion of the AF2 resulted in a decrease in interaction (about 70%) between HNF4 and p300 (33). Therefore, we cannot exclude that the impaired cooperativity of HNF4-AF2mut was also due to reduced p300 recruitment. Because it has been documented that HNF4 and CBP/p300 form a transcriptionally active complex (36), we verified whether in our experimental conditions this complex was sufficient to achieve the strong cooperation. This question was addressed by the use of human HNF1 promoter containing a HNF4 response element but lacking a HNF1 binding site. Using the same p300/HNF4 expression vector amounts used in Figure 3A and B, cotransfection of p300 failed to strongly enhance the HNF4-mediated activation of transcription (Fig. 3D). It appears therefore that the strong activation of transcription observed in Figure 3A and B requires a ternary complex including HNF1, HNF4 and p300.
Figure 3. Involvement of p300 in the cooperation between HNF1 and HNF4. The cooperation was analysed on the HNF1-TATA and (–96/+11) LPK promoters, (A) and (B), respectively. HeLa cells were transiently transfected with 250 ng of promoter plasmids, 10 ng of HNF1, 15 ng of HNF4 or HNF4-AF2mut and 30 ng of p300 expression plasmids, as indicated. Fold induction refers to the basal activity of the promoter (left column). Values above bars indicate the enhancement of HNF1- mediated activation of transcription by HNF4 or p300 alone or in cooperation. Results are means ± S.E. of three experiments performed in triplicate. (C) Effect of the mutations in the AF2 of HNF4 on its interaction with p300 analysed by pull-down assay. Methionine-labelled HNF4 or HNF4-AF2mut were incubated with immobilized GST-p300(340–528). Bound proteins were analysed by SDS–PAGE and PhosphorImager. (D) HeLa cells were transiently transfected with expression vector amounts indicated in (A) and 250 ng of the human HNF1 promoter. Fold induction, calculated from three experiments performed in triplicate refers to the basal activity of the promoter.
With the aim to investigate the mechanism underlying the p300-mediated enhancement of the cooperation between HNF1 and HNF4, we surveyed the p300 domains involved in interactions with these transcription factors. Using a double hybrid approach, it was previously shown that the sequences 180–662 and 1818–2079 of p300 interact with the HNF1 transactivation domain (20). Using GST pull-down assays, we mapped more precisely the amino-terminal sequence of p300 involved in interaction with full-length HNF1 between residues 340 and 528 (Fig. 4B). We also confirmed that full-length HNF1 interacts with the carboxy-terminal region of p300 (Fig. 4C). We have previously shown the ability of HNF4 to interact with the amino-terminal domain of p300 (33). HNF4 also interacted with the p300 carboxy-terminal region as shown in Figure 4C. The presence of multiple interaction surfaces between these proteins led us to analyse the effect of HNF4 on the recruitment of the amino- and carboxy-terminal regions of p300 to HNF1. The strategy consisted of determining whether HNF4 affects the cooperation between HNF1 and the amino- or carboxy-terminal fragments of p300 fused to the VP16 activation domain named Np300-VP16 and Cp300-VP16, respectively (Fig. 4A). Expression of the VP16 activation domain affected neither the basal activity of the promoter nor its activity in the presence of expression vectors for HNF1 alone or together with HNF4 (Fig. 4D, left part). Expression of Np300-VP16 failed to affect the basal activity of the promoter and the HNF1-mediated activation of this promoter (Fig. 4D, middle). Similar results were obtained with expression of Cp300-VP16 (Fig. 4D, right). Conversely, in the presence of both HNF1 and HNF4, Np300-VP16 and Cp300-VP16 markedly enhanced the promoter activity (Fig. 4D). These results indicate that, in a cellular context, p300 recruitment by the HNF1–HNF4 complex can be mediated through both its amino- and carboxy-terminal regions and reinforce data obtained in Figure 3 suggesting that HNF4 improves p300 recruitment to a promoter containing a HNF1 binding site (Fig. 4E).
Figure 4. HNF4 enhanced the cooperation between HNF1 and p300 through interactions with both the amino- and carboxy-terminal sequences of the coactivator. (A) Schematic representation of p300 fragments fused to either GST or the VP16 activation domain used in interaction studies with HNF1 and HNF4. (B) Interaction of HNF1 with the amino-terminal domain of p300 analysed by pull-down assays. Methionine-labelled HNF1 was incubated with immobilized GST, GST-p300(1–595) or GST-p300(340–528). (C) Interaction of HNF1 and HNF4 with the carboxy-terminal domain of p300 analysed by pull-down assays. Methionine-labelled HNF1 or HNF4 were incubated with immobilized GST or GST-p300(1572–2370). In (B) and (C), bound proteins were analysed by SDS–PAGE and PhosphorImager. (D) HNF4 potentiates enhancement of HNF1 activity by Np300- and Cp300-VP16. HeLa cells were transiently transfected with 250 ng of HNF1-TATA promoter, 10 ng of HNF1, 15 ng of HNF4 and 60 ng of VP16 or its empty vector (left), VP16 or Np300-VP16 (middle) and VP16 or Cp300-VP16 (right) expression plasmids. In each case, the total amount of transfected DNA was kept constant using empty pcDNA3. Fold induction refers to the basal activity of the promoter (left columns). Results are means ± S.E. of at least three experiments performed in triplicate. (E) Mechanisms by which HNF4 could enhance HNF1 transcriptional activity. The amino- and/or carboxy-terminal domains of p300 can be involved in p300-mediated activation of transcription. HNF4 could enhance p300 recruitment through direct binding to these p300 fragments and/or through an indirect effect on the HNF1–p300 interactions. HNF4 could also help recruitment of other coactivators to the transcriptional complex.
DISCUSSION
It has been previously shown that HNF1 acts as a repressor of HNF4-mediated activation of transcription (8,9). In this study, we show that HNF4 can serve as a coactivator for HNF1 since HNF4 is able to enhance HNF1 activity in a DNA-binding independent manner. A synergy between HNF1 and HNF4 can be inferred from data obtained in undifferentiated Caco-2 cells on a promoter containing binding sites for these two HNFs. Since mutation of the HNF4 binding site did not affect this synergy, the authors suggested that this effect probably did not involve HNF4 DNA binding (37). In light of our results, these data can now most probably be explained by the ability of HNF4 to directly act as a HNF1 coactivator. Analysis of the physiological role of this HNF4 coactivator function in cell types that endogenously express HNF1 and HNF4 cannot be realized by conventional targeting of HNF4 expression since HNF4 also positively regulates HNF1 expression. Therefore, this concern will first require development of an experimental model that permits to distinguish between HNF4 effects linked to regulation of HNF1 expression on one hand and to regulation of HNF1 transcriptional activity on the other hand (Fig. 5).
Figure 5. Cross-talk between HNF1 and HNF4. The cross-talk involves both expression activation, through binding to promoters (solid lines), and modulation of the transcriptional potential, through protein–protein interactions (dotted lines), of the other HNF transcription factor.
Interplays between two transcription factors in both activation and repression of transcription have already been documented. Indeed, cross-talk between HNF6 and HNF3?, two other liver-enriched transcription factors, also results in opposite effects on the activities of these proteins (38).
Despite their efficient interaction, HNF4 and HNF1 lacking its activation function (HNF1-AD) were unable to cooperate. Such a behaviour is reminiscent of that of CBP and P/CAF with HNF1 (1–280) (21). Only the simultaneous overexpression of CBP and P/CAF together with that of the truncated HNF1 could activate transcription (21). This points to a critical role for the HNF1 activation domain, which could be the recruitment of other required coactivators and/or stabilization of the transcriptional complex recruited by HNF1.
Our results indicate that the HNF4 AF2 is involved in interaction with HNF1, but also that other regions of the HNF4 LBD are required for full interaction between these proteins. Ktistaki and Talianidis previously stated that the HNF4 AF2 is sufficient for interaction with HNF1 (9). The discrepancy between their conclusion and our results is likely explained by the fact that these authors used deletion of a fragment (residues 337–368), which includes not only the AF2 but also part of the large helix H10–H11, the integrity of which has meanwhile been shown to be crucial for the protein conformation (32). We observed that two-point mutations in the HNF4 LBD, A223F and E276Q, significantly decreased interaction and cooperation with HNF1 thus demonstrating that the integrity of the HNF4 LBD is required for the synergy with HNF1.
The similarity of phenotypes exhibited by diabetic patients carrying MODY1 and MODY3 mutations has been ascribed to the mutual control of transcription of HNF1 and HNF4. The ability of HNF4 to serve as an HNF1 coactivator could provide an additional explanation to the closely related phenotypes of these MODY forms of diabetes. Impairment of interaction and of enhancement of HNF1 transcriptional activity by the HNF4 E276Q mutation may be physiologically relevant since this mutation is correlated with MODY1. Unfortunately, the low transfection efficiency and the presence of large amounts of endogenous HNF4 in pancreatic ?-cells prevented us from analysing the effects of this mutation on the synergy between HNF4 and HNF1 in these cells.
Our results support a model that involves the combined action of HNF4 and the coactivator p300 to achieve the highest rate of transcription mediated by HNF1. Our results also strongly suggest that HNF4 improves p300 recruitment. The synergy between HNF4 and p300 may be mediated in two ways. The first involves simultaneous interactions of HNF4 and p300 with HNF1 (Fig. 4E). In this case, HNF4 could indirectly improve HNF1–p300 interaction through induction of a HNF1 conformational change. Such a mechanism is reminiscent of that of the CBP-mediated improvement of the interaction between HNF1 and P/CAF (21). Similarly, upon docking to PPAR, PGC-1 undergoes a conformational change that permits binding of SRC-1 and p300 (39). The second involves docking of p300 by HNF4, which is bound to HNF1 (Fig. 4E). The recruitment of p300 to the HNF1–HNF4 complex could be mediated by its amino- and/or carboxy-terminal regions, which contain the C/H1 and C/H3 domains, respectively. These domains are known to be involved in protein–protein interactions (40). The presence of multiple interaction surfaces in HNF1, HNF4 and p300 most probably facilitates formation of a ternary complex formed with these proteins (Fig. 4E). The remaining synergy between HNF1 and HNF4 after mutations in the HNF4 AF2 (Fig. 3) may be accounted for by the ability of p300 to interact and activate the HNF4 AF1 (33,41) and by the fact that the two above mentioned mechanisms may not be mutually exclusive.
In conclusion, our results highlight a new way by which HNF4 can regulate gene expression. HNF4 not only directly binds to promoters but also, through interaction with other transcription factors already bound to DNA, can facilitate coactivator recruitment to further enhance transcription. Recently, it has been shown that HNF4 also serves as coactivator for Sterol Regulatory Element-Binding protein-2 (42). Furthermore, our results yield insights into a higher complexity of the transcriptional network and on the primordial relationship between HNF1 and HNF4 (Fig. 5). In the regulatory loop between HNF1 and HNF4, HNF4 can activate both HNF1 expression and transcriptional activity. In contrast to these additive processes, the negative effect of HNF1 on HNF4 transcriptional activity can attenuate the HNF1-mediated activation of expression. These regulatory feedback mechanisms will have to be taken into account considering that HNF1 and HNF4 are involved in a large diversity of pathways controlling function of multiple organs, notably liver and endocrine pancreas.
ACKNOWLEDGEMENTS
Drs M. Yaniv, A. Abderrahmani, M. Vasseur-Cognet, R. Polakowska, S. Grossman, D. W. Hum and G. Bell are acknowledged for generous gifts of plasmids. We acknowledge Dr P. Sacchetti for helpful discussions and I. Briche for skillful technical assistance.
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*To whom correspondence should be addressed. Tel: +33 3 20 62 69 51; Fax: +33 3 20 62 68 84; Email: blaine@lille.inserm.fr
ABSTRACT
Hepatocyte Nuclear Factor 1 (HNF1) and Hepatocyte Nuclear Factor 4 (HNF4) are two liver-enriched transcription factors coexpressed in specific tissues where they play a crucial role through their involvement in a complex cross-regulatory network. HNF1 down regulates HNF4-mediated activation of transcription via a direct protein–protein interaction. Here we show that HNF4 enhances the transcriptional activity of HNF1 in a DNA binding independent manner, thus indicating that it behaves as a HNF1 coactivator. Using mutations in the ligand binding domain (LBD) of HNF4, we confirmed the involvement of the Activation Function 2 module and demonstrated the requirement of the integrity of the LBD for the interaction with HNF1. Moreover, we show that HNF4 cooperates with p300 to achieve the highest HNF1-mediated transcription rates. Our findings highlight a new way by which HNF4 can regulate gene expression and extend our knowledge of the complexity of the transcriptional network involving HNF4 and HNF1.
INTRODUCTION
Hepatocyte Nuclear Factor 1 (HNF1) and Hepatocyte Nuclear Factor 4 (HNF4) are two liver-enriched transcription factors that are also expressed in kidney, intestine and endocrine pancreas (1). They are involved in complex cross-regulatory networks that determine the phenotype of hepatocytes and pancreatic ?-cells (1–3). HNF1 is a homeodomain-containing transcription factor (1) whereas HNF4 belongs to the hormone nuclear receptor superfamily (4). Both transcription factors are highly conserved during evolution (4,5). HNF1 and HNF4 directly modulate the expression of a large number of genes (4,6,7). They can also modulate transcription indirectly through the above mentioned transcription factor network, including a HNF1-mediated negative regulation of genes activated by HNF4 (8,9). The roles of these transcription factors in vivo have been confirmed by defects linked to the invalidation of their genes in mice (10–14). Further underscoring the importance of these transcription factors, mutations in HNF1 and HNF4 genes have been identified in patients with Maturity Onset Diabetes of the Young, MODY3 and MODY1, respectively (15,16). MODY1 and MODY3 mutations result in loss of function of these transcription factors (17). Moreover, it has been reported that expression of a HNF1 dominant negative mutant linked to MODY3 led to an impaired function of pancreatic ?-cells (18).
HNF1 contains two domains involved in DNA binding (an atypical homeodomain and a POU-like domain), an N-terminal dimerization domain and a C-terminal transactivation domain (1). Several proteins interact with various HNF1 domains and play crucial roles in HNF1 function. DcoH (Dimerization Cofactor of HNF1), is a small protein, which binds to the HNF1 dimerization domain and is involved in dimer stabilization (19). The ability of various HNF1 domains to interact with multiple coactivators allows formation of a platform for recruitment of a transcriptional complex, leading to a strong enhancement of transcription. CBP/p300 interacts with both the DNA binding domain and the activation domain of HNF1 while P/CAF, SRC-1 and RAC3 interact with the HNF1 activation domain (20,21). Each of these coactivators can independently increase activation of transcription by HNF1. In addition, they cooperate with each other to further enhance the HNF1-mediated activation of transcription (21). HNF1 can also interact with GATA 4, GATA 5 and Cdx-2 transcription factors (22). The interactions between HNF1, GATA5 and Cdx-2 lead to a cooperative enhancement of HNF1-mediated activation of transcription (22). A synergy between HNF1 and neurogenin 3 was also recently reported (23). HNF1 down regulates HNF4-mediated activation of transcription via a direct interaction of these transcription factors (8,9).
In this work, we show that HNF4 enhances the transcriptional activity of HNF1, and that the cooperation between both factors can be further enhanced by p300 recruitment.
MATERIALS AND METHODS
DNA constructs
Plasmids pcDNA3 HNF42 and pcDNA3 HNF42-E276Q were previously described (24). Plasmid pcDNA3 HNF42-D126Y was described by Oxombre et al. (25). Plasmids pcDNA3 HNF42-E262A and pGEX2TK HNF42 were described by Eeckhoute et al. (26). Plasmids pcDNA3 HNF1 and pcDNA3 HNF1-P291fsinsC, here named HNF1-AD, were generous gifts from Drs M. Yaniv and A. Abderrahmani, respectively. Plasmids pcDNA3 HNF42-A223F and pcDNA3 HNF42-AF2mut were obtained by site-directed mutagenesis using the QuickChangeTM kit (Stratagene) to introduce the A223F or the three mutations E363K, L365Q and L366Q, respectively. Plasmid pM3-VP16 was from Clontech and pcDNA3-RXR? encoding the full-length human RXR? was a generous gift from Dr R. Polakowska. Plasmid pGEX2TK HNF1 was prepared by a strategy identical to that used for cloning pGEX2TK COUP-TFII (24) by inserting a PCR fragment encompassing the human HNF1 cDNA. Plasmid pCMV?-NHA p300, PGEX2TK p300(1–595), PGEX2TK p300(340–528) and PGEX2TK p300(1572–2370) were kindly provided by Dr S. R. Grossman. Expression plasmids for VP16, Np300-VP16 and Cp300-VP16 were gifts from Dr D. Hum (27). Plasmid pGL3 (–96/+11) LPK was a generous gift from Dr M. Vasseur-Cognet (28). Plasmid pGL3 (–341/+183) human HNF1 was a gift from Dr G. Bell. Plasmid (GAL4)x5 TATA Luc was described by Chang and Gralla (29). Plasmid pGL3 HNF1-TATA-Luc was cloned by inserting a double-stranded oligonucleotide encompassing the HNF1 binding site (–56/ –35) of the SRC promoter into the SacI/NheI sites of the pGL3 basic vector in which the TATA box of the adenovirus major late promoter had been previously cloned as described by Suaud et al. (24). All constructs were verified by DNA sequencing.
GST pull-down assays
GST pull-down assays were performed as described previously (24) using methionine-labelled in vitro synthesized HNF1 or HNF4 and bacterially expressed GST-fusion proteins indicated in legends to figures.
Cell culture and transient transfection assays
HeLa cells (5.5 x 104 cells per 24-well dish) were grown and transfected as described by Eeckhoute et al. (30) with plasmid amounts indicated in figure legends. Luciferase activities were measured using the Bright-Glo Luciferase assay system (Promega).
Western blotting
Western blotting performed from whole-cell extracts and using the 455 HNF4 antiserum was carried out as indicated by Eeckhoute et al. (26).
Data analysis
Statistical analyses were based on Student’s t-test for unpaired data using Prism software. Statistical significance was set at ***P < 0.001, **P < 0.01 and *P < 0.05.
RESULTS
Enhancement of HNF1-mediated activation of transcription by HNF4
HNF1 and HNF4 can positively regulate one another’s expression in cell types that endogenously express these two factors (3,6). To avoid any interference with the endogenous proteins, the effect of HNF4 on the transactivation activity of HNF1 was analysed in HeLa cells. The experiments were performed on a synthetic promoter consisting of one HNF1 binding site located upstream of the TATA box (HNF1-TATA promoter) and on the natural liver pyruvate kinase promoter (–96/+11) LPK containing one HNF1 binding site (site L1, position –96/–72) but lacking the HNF4 response element (28). As expected, both promoters were activated by HNF1 but not by HNF4 (Fig. 1A and B). Cotransfection of HNF1 and HNF4 resulted in a marked increase in the HNF1-mediated activation of these promoters (2.8- and 3.4-fold activation of the synthetic and LPK promoters, respectively) (Fig. 1A and B). The enhancement of the HNF1 activity was not impaired when introducing the D126Y mutation in HNF4, which significantly decreases its DNA binding and transactivation activities (25) (data not shown), thus confirming that the synergy between HNF1 and HNF4 does not require the DNA binding-dependent activities of HNF4 (Fig. 1A and B). This induction was not an artefact linked to expression of a protein since cotransfection of an unrelated factor failed to enhance the HNF1-mediated activation of transcription (compare the second and last columns in Fig. 1A and B).
Figure 1. HNF4 enhanced the HNF1-dependent activation of transcription. HeLa cells were transiently transfected with 250 ng of the HNF1-TATA promoter (A) or the (–96/+11) LPK promoter (B), 15 ng of HNF1 and 20 ng of HNF4 or RXR? expression plasmids, as indicated. (C) HNF4 was unable to cooperate with the VP16 activation domain. HeLa cells were transiently transfected with 250 ng of the (GAL4)x5 TATA promoter, 5 ng of GAL4-VP16 and 20 ng of HNF4 expression vectors, as indicated. In (A), (B) and (C), the total amounts of transfected DNA were equalized with the empty expression plasmids (–). Fold induction refers to the basal activities of promoters (left columns). Results are means ± S.E. of three experiments performed in triplicate. The extent of HNF4-mediated enhancement of HNF1-dependent activation of transcription is indicated (values above bars). (D) In vitro interaction between HNF1 and HNF4 analysed by pull-down assays. A schematic representation of the HNF1 and HNF1-AD proteins is presented, AD denotes activation domain. methionine-labelled HNF1 or HNF1-AD was incubated with immobilized GST or GST-HNF4. Bound proteins were analysed by SDS–PAGE and PhosphorImager (Molecular Dynamics). Inputs correspond to 10% of amounts of labelled proteins used in the assays. (E) Requirement of the activation domains of HNF1 for the cooperation between HNF4 and HNF1. HeLa cells were transfected as in (A) with the indicated HNF1 constructs. Fold induction refers to the basal activity of the promoter (left column). Results are means ± S.E. of three experiments performed in triplicate.
To control the possibility that HNF4 does not act as a transcriptional partner of another transcription factor, we analysed the effect of HNF4 on the VP16 activation domain fused to the GAL4 DBD (construct GAL4-VP16). HNF4 was unable to enhance the VP16-mediated activation of transcription (Fig. 1C), thus indicating the selectivity of the cooperation between HNF4 and HNF1.
As predicted from the results of Ktistaki and Talianidis (9), we observed that full-length HNF1 and HNF4 physically interact (Fig. 1D). Interestingly, pull-down experiments also showed that HNF1 lacking its activation domain (HNF1-AD) efficiently interacted with HNF4 (Fig. 1D). This result indicates that the sequence 1–291 of HNF1, containing the dimerization and DNA binding domains, is able to interact with HNF4. This prompted us to investigate whether coexpression of HNF4 and HNF1-AD could activate the HNF1-TATA promoter. Despite their efficient interaction, HNF1-AD and HNF4 were unable to activate transcription (Fig. 1E), thus indicating that cooperation between HNF1 and HNF4 requires the HNF1 activation domain.
AF2 is not the only sequence within the HNF4 LBD required for interaction and cooperation with HNF1
The repressive action of HNF1 on HNF4 was unambiguously shown to require the HNF4 LBD (9). However, using truncated fragments of HNF4, Ktistaki and Talianidis mapped the sequence involved in the interaction with HNF1 between residues 337 and 368, leading to the conclusion that the HNF4 AF2 activation function is necessary and sufficient for interaction with HNF1 (9). In fact, this sequence does not contain solely the AF2 (sequence 358–366 corresponding to helix 12 of the LBD), but also a fragment forming part of the large helix termed H10–H11, according to conventional nomenclature (31). Indeed, H10 and H11 are contiguous in HNF4 and form a helix that plays a key role in HNF4 function (31). Furthermore, the integrity of this helix is crucial for protein conformation (32). These recent findings begged a re-evaluation of the HNF4 sequence involved in interaction with HNF1. Using full-length HNF4, we analysed the effects of point mutations located in the HNF4 LBD (Fig. 2A) on the interaction and cooperation with HNF1. The E262A mutation was shown to affect HNF4 dimerization (26). The E276Q mutation does not alter the intrinsic HNF4 transcriptional activity but impairs recruitment of HNF4 transcriptional partners (24,33). Owing to the bulky side chain of phenylalanine, the A223F mutation was hypothesized to affect occupancy of the ligand binding pocket by fatty acids that act as structural cofactors rather than as conventional ligands (31). Accord ingly, A223F mutation mildly impaired HNF4 transcriptional activity and interaction with coactivators (data not shown). In the AF2 module, the acidic group of the amino acid residue side chain at position 363 and the hydrophobic nature of the amino acid residue side chain at position 365 are strictly conserved and play a key role in nuclear receptor function (34). Residue L366 is also important since the L366Q mutation alters HNF4 transcriptional activity (35). Pull-down assays showed that introducing three mutations E363K, L365Q and L366Q in the AF2 (HNF4-AF2mut construct) altered interaction between HNF4 and HNF1 (Fig. 2B). Note however that the decrease in interaction was mild. This result confirms the involvement of the AF2 in the interaction between HNF1 and HNF4 but indicates that this module is not the unique sequence required for interaction between these proteins. Interaction with HNF1 was also significantly decreased by the A223F and E276Q mutations while mutation E262A had no effect (Fig. 2B). Consistent with data from pull-down assays, the HNF4-mediated enhancement of HNF1-dependent transcriptional activation of the (–96/+11) LPK promoter was not impaired by the E262A mutation but was significantly decreased with the mutants A223F, E276Q and HNF4-AF2mut (Fig. 2C). We controlled so that mutant and wild-type HNF4 were expressed at a similar level in transfected cells as shown in Figure 2D for mutants A223F and HNF4-AF2mut and in (24,26) for the mutant E276Q and E262A, respectively. It appears therefore that the interaction and cooperation between HNF4 and HNF1 depend on both the AF2 module (helix 12) and the integrity of the HNF4 LBD.
Figure 2. The AF2 is not the only sequence within the HNF4 LBD required for interaction and cooperation with HNF1. (A) Positions of mutations used in this study. A scheme of HNF4 structure with the various domains is given: DBD, DNA binding domain; LBD, ligand binding domain; AF2, activation function 2 module. (B) Effects of HNF4 mutations on the physical interaction with HNF1. methionine-labelled wild-type or mutated HNF4 were incubated with immobilized GST-HNF1. Bound proteins were analysed by SDS–PAGE and PhosphorImager. Values under photographs indicate binding of HNF4 mutants relative to that of wild-type HNF4 from three independent experiments. Inputs, corresponding to 5% of amounts of labelled proteins used in the assays, were taken into account for binding quantifications. (C) Effects of HNF4 mutations on the cooperation between HNF4 and HNF1 on the (–96/+11) LPK promoter. HeLa cells were transfected as in Figure 1B. Fold induction refers to the basal activity of the promoter (left column). Results are means ± S.E. of three experiments performed in triplicate. Statistical significance of differences with values obtained with wild-type HNF42 is indicated by stars (P < 0.01). (D) Western blotting of wild-type (WT), A223F and AF2mut HNF4 expressed in HeLa cells using the 455 HNF4 antiserum (43). Position of HNF4 is indicated. The star denotes a non-specific band obtained from HeLa cells transfected with the empty vector (–). The number on the left indicates the molecular mass of a marker size in kDa.
HNF4 cooperates with the coactivator p300 to enhance HNF1 transcriptional activity
Because CBP/p300 are key coactivators of HNF1, we hypothesized that cooperation between HNF4 and HNF1 could involve p300. In the absence of HNF1, the coactivator p300 alone or coexpressed with HNF4 was unable to activate the HNF1-TATA promoter (Fig. 3A). On this promoter, p300 slightly enhanced HNF1 transcriptional activity (Fig. 3A). Note that this moderate increase is due at least in part to the low ratio of p300/HNF1 expression vectors used in our experiments (3:1) compared to that used in other studies performed on other promoters . We have verified that by using a higher ratio, we could obtain a stronger enhancement of HNF1 activity by p300 alone (data not shown). Interestingly, in the presence of HNF4, p300 synergistically enhanced HNF1-mediated activation of transcription: the activity of the promoter was increased 18.4-fold, which is 4.7-fold higher than the additive value for the induction of HNF1 activity by p300 and HNF4 alone (1.8 and 2.1, respectively). The marked p300-mediated enhancement of cooperation between HNF1 and HNF4 could also be observed on the (–96/+11) LPK promoter (Fig. 3B). On both promoters, the enhanced cooperation between HNF1 and HNF4 was significantly impaired by mutations in the HNF4 AF2 module (Fig. 3A and B). These results strongly argue for the involvement of HNF4 in the p300-mediated activation of these promoters despite the absence of HNF4 response element in their sequences. The result obtained with the HNF4-AF2mut led us to investigate whether mutations in AF2 affect p300 binding. Pull-down experiments clearly showed a much weaker interaction of p300 with HNF4-AF2mut than with wild-type HNF4 (Fig. 3C). This result is in line with our previous observation that deletion of the AF2 resulted in a decrease in interaction (about 70%) between HNF4 and p300 (33). Therefore, we cannot exclude that the impaired cooperativity of HNF4-AF2mut was also due to reduced p300 recruitment. Because it has been documented that HNF4 and CBP/p300 form a transcriptionally active complex (36), we verified whether in our experimental conditions this complex was sufficient to achieve the strong cooperation. This question was addressed by the use of human HNF1 promoter containing a HNF4 response element but lacking a HNF1 binding site. Using the same p300/HNF4 expression vector amounts used in Figure 3A and B, cotransfection of p300 failed to strongly enhance the HNF4-mediated activation of transcription (Fig. 3D). It appears therefore that the strong activation of transcription observed in Figure 3A and B requires a ternary complex including HNF1, HNF4 and p300.
Figure 3. Involvement of p300 in the cooperation between HNF1 and HNF4. The cooperation was analysed on the HNF1-TATA and (–96/+11) LPK promoters, (A) and (B), respectively. HeLa cells were transiently transfected with 250 ng of promoter plasmids, 10 ng of HNF1, 15 ng of HNF4 or HNF4-AF2mut and 30 ng of p300 expression plasmids, as indicated. Fold induction refers to the basal activity of the promoter (left column). Values above bars indicate the enhancement of HNF1- mediated activation of transcription by HNF4 or p300 alone or in cooperation. Results are means ± S.E. of three experiments performed in triplicate. (C) Effect of the mutations in the AF2 of HNF4 on its interaction with p300 analysed by pull-down assay. Methionine-labelled HNF4 or HNF4-AF2mut were incubated with immobilized GST-p300(340–528). Bound proteins were analysed by SDS–PAGE and PhosphorImager. (D) HeLa cells were transiently transfected with expression vector amounts indicated in (A) and 250 ng of the human HNF1 promoter. Fold induction, calculated from three experiments performed in triplicate refers to the basal activity of the promoter.
With the aim to investigate the mechanism underlying the p300-mediated enhancement of the cooperation between HNF1 and HNF4, we surveyed the p300 domains involved in interactions with these transcription factors. Using a double hybrid approach, it was previously shown that the sequences 180–662 and 1818–2079 of p300 interact with the HNF1 transactivation domain (20). Using GST pull-down assays, we mapped more precisely the amino-terminal sequence of p300 involved in interaction with full-length HNF1 between residues 340 and 528 (Fig. 4B). We also confirmed that full-length HNF1 interacts with the carboxy-terminal region of p300 (Fig. 4C). We have previously shown the ability of HNF4 to interact with the amino-terminal domain of p300 (33). HNF4 also interacted with the p300 carboxy-terminal region as shown in Figure 4C. The presence of multiple interaction surfaces between these proteins led us to analyse the effect of HNF4 on the recruitment of the amino- and carboxy-terminal regions of p300 to HNF1. The strategy consisted of determining whether HNF4 affects the cooperation between HNF1 and the amino- or carboxy-terminal fragments of p300 fused to the VP16 activation domain named Np300-VP16 and Cp300-VP16, respectively (Fig. 4A). Expression of the VP16 activation domain affected neither the basal activity of the promoter nor its activity in the presence of expression vectors for HNF1 alone or together with HNF4 (Fig. 4D, left part). Expression of Np300-VP16 failed to affect the basal activity of the promoter and the HNF1-mediated activation of this promoter (Fig. 4D, middle). Similar results were obtained with expression of Cp300-VP16 (Fig. 4D, right). Conversely, in the presence of both HNF1 and HNF4, Np300-VP16 and Cp300-VP16 markedly enhanced the promoter activity (Fig. 4D). These results indicate that, in a cellular context, p300 recruitment by the HNF1–HNF4 complex can be mediated through both its amino- and carboxy-terminal regions and reinforce data obtained in Figure 3 suggesting that HNF4 improves p300 recruitment to a promoter containing a HNF1 binding site (Fig. 4E).
Figure 4. HNF4 enhanced the cooperation between HNF1 and p300 through interactions with both the amino- and carboxy-terminal sequences of the coactivator. (A) Schematic representation of p300 fragments fused to either GST or the VP16 activation domain used in interaction studies with HNF1 and HNF4. (B) Interaction of HNF1 with the amino-terminal domain of p300 analysed by pull-down assays. Methionine-labelled HNF1 was incubated with immobilized GST, GST-p300(1–595) or GST-p300(340–528). (C) Interaction of HNF1 and HNF4 with the carboxy-terminal domain of p300 analysed by pull-down assays. Methionine-labelled HNF1 or HNF4 were incubated with immobilized GST or GST-p300(1572–2370). In (B) and (C), bound proteins were analysed by SDS–PAGE and PhosphorImager. (D) HNF4 potentiates enhancement of HNF1 activity by Np300- and Cp300-VP16. HeLa cells were transiently transfected with 250 ng of HNF1-TATA promoter, 10 ng of HNF1, 15 ng of HNF4 and 60 ng of VP16 or its empty vector (left), VP16 or Np300-VP16 (middle) and VP16 or Cp300-VP16 (right) expression plasmids. In each case, the total amount of transfected DNA was kept constant using empty pcDNA3. Fold induction refers to the basal activity of the promoter (left columns). Results are means ± S.E. of at least three experiments performed in triplicate. (E) Mechanisms by which HNF4 could enhance HNF1 transcriptional activity. The amino- and/or carboxy-terminal domains of p300 can be involved in p300-mediated activation of transcription. HNF4 could enhance p300 recruitment through direct binding to these p300 fragments and/or through an indirect effect on the HNF1–p300 interactions. HNF4 could also help recruitment of other coactivators to the transcriptional complex.
DISCUSSION
It has been previously shown that HNF1 acts as a repressor of HNF4-mediated activation of transcription (8,9). In this study, we show that HNF4 can serve as a coactivator for HNF1 since HNF4 is able to enhance HNF1 activity in a DNA-binding independent manner. A synergy between HNF1 and HNF4 can be inferred from data obtained in undifferentiated Caco-2 cells on a promoter containing binding sites for these two HNFs. Since mutation of the HNF4 binding site did not affect this synergy, the authors suggested that this effect probably did not involve HNF4 DNA binding (37). In light of our results, these data can now most probably be explained by the ability of HNF4 to directly act as a HNF1 coactivator. Analysis of the physiological role of this HNF4 coactivator function in cell types that endogenously express HNF1 and HNF4 cannot be realized by conventional targeting of HNF4 expression since HNF4 also positively regulates HNF1 expression. Therefore, this concern will first require development of an experimental model that permits to distinguish between HNF4 effects linked to regulation of HNF1 expression on one hand and to regulation of HNF1 transcriptional activity on the other hand (Fig. 5).
Figure 5. Cross-talk between HNF1 and HNF4. The cross-talk involves both expression activation, through binding to promoters (solid lines), and modulation of the transcriptional potential, through protein–protein interactions (dotted lines), of the other HNF transcription factor.
Interplays between two transcription factors in both activation and repression of transcription have already been documented. Indeed, cross-talk between HNF6 and HNF3?, two other liver-enriched transcription factors, also results in opposite effects on the activities of these proteins (38).
Despite their efficient interaction, HNF4 and HNF1 lacking its activation function (HNF1-AD) were unable to cooperate. Such a behaviour is reminiscent of that of CBP and P/CAF with HNF1 (1–280) (21). Only the simultaneous overexpression of CBP and P/CAF together with that of the truncated HNF1 could activate transcription (21). This points to a critical role for the HNF1 activation domain, which could be the recruitment of other required coactivators and/or stabilization of the transcriptional complex recruited by HNF1.
Our results indicate that the HNF4 AF2 is involved in interaction with HNF1, but also that other regions of the HNF4 LBD are required for full interaction between these proteins. Ktistaki and Talianidis previously stated that the HNF4 AF2 is sufficient for interaction with HNF1 (9). The discrepancy between their conclusion and our results is likely explained by the fact that these authors used deletion of a fragment (residues 337–368), which includes not only the AF2 but also part of the large helix H10–H11, the integrity of which has meanwhile been shown to be crucial for the protein conformation (32). We observed that two-point mutations in the HNF4 LBD, A223F and E276Q, significantly decreased interaction and cooperation with HNF1 thus demonstrating that the integrity of the HNF4 LBD is required for the synergy with HNF1.
The similarity of phenotypes exhibited by diabetic patients carrying MODY1 and MODY3 mutations has been ascribed to the mutual control of transcription of HNF1 and HNF4. The ability of HNF4 to serve as an HNF1 coactivator could provide an additional explanation to the closely related phenotypes of these MODY forms of diabetes. Impairment of interaction and of enhancement of HNF1 transcriptional activity by the HNF4 E276Q mutation may be physiologically relevant since this mutation is correlated with MODY1. Unfortunately, the low transfection efficiency and the presence of large amounts of endogenous HNF4 in pancreatic ?-cells prevented us from analysing the effects of this mutation on the synergy between HNF4 and HNF1 in these cells.
Our results support a model that involves the combined action of HNF4 and the coactivator p300 to achieve the highest rate of transcription mediated by HNF1. Our results also strongly suggest that HNF4 improves p300 recruitment. The synergy between HNF4 and p300 may be mediated in two ways. The first involves simultaneous interactions of HNF4 and p300 with HNF1 (Fig. 4E). In this case, HNF4 could indirectly improve HNF1–p300 interaction through induction of a HNF1 conformational change. Such a mechanism is reminiscent of that of the CBP-mediated improvement of the interaction between HNF1 and P/CAF (21). Similarly, upon docking to PPAR, PGC-1 undergoes a conformational change that permits binding of SRC-1 and p300 (39). The second involves docking of p300 by HNF4, which is bound to HNF1 (Fig. 4E). The recruitment of p300 to the HNF1–HNF4 complex could be mediated by its amino- and/or carboxy-terminal regions, which contain the C/H1 and C/H3 domains, respectively. These domains are known to be involved in protein–protein interactions (40). The presence of multiple interaction surfaces in HNF1, HNF4 and p300 most probably facilitates formation of a ternary complex formed with these proteins (Fig. 4E). The remaining synergy between HNF1 and HNF4 after mutations in the HNF4 AF2 (Fig. 3) may be accounted for by the ability of p300 to interact and activate the HNF4 AF1 (33,41) and by the fact that the two above mentioned mechanisms may not be mutually exclusive.
In conclusion, our results highlight a new way by which HNF4 can regulate gene expression. HNF4 not only directly binds to promoters but also, through interaction with other transcription factors already bound to DNA, can facilitate coactivator recruitment to further enhance transcription. Recently, it has been shown that HNF4 also serves as coactivator for Sterol Regulatory Element-Binding protein-2 (42). Furthermore, our results yield insights into a higher complexity of the transcriptional network and on the primordial relationship between HNF1 and HNF4 (Fig. 5). In the regulatory loop between HNF1 and HNF4, HNF4 can activate both HNF1 expression and transcriptional activity. In contrast to these additive processes, the negative effect of HNF1 on HNF4 transcriptional activity can attenuate the HNF1-mediated activation of expression. These regulatory feedback mechanisms will have to be taken into account considering that HNF1 and HNF4 are involved in a large diversity of pathways controlling function of multiple organs, notably liver and endocrine pancreas.
ACKNOWLEDGEMENTS
Drs M. Yaniv, A. Abderrahmani, M. Vasseur-Cognet, R. Polakowska, S. Grossman, D. W. Hum and G. Bell are acknowledged for generous gifts of plasmids. We acknowledge Dr P. Sacchetti for helpful discussions and I. Briche for skillful technical assistance.
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