help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eeckhoute, J.
Right arrow Articles by Laine, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eeckhoute, J.
Right arrow Articles by Laine, B.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
Endocrinology Vol. 144, No. 5 1686-1694
Copyright © 2003 by The Endocrine Society

Hepatocyte Nuclear Factor 4{alpha} Isoforms Originated from the P1 Promoter Are Expressed in Human Pancreatic ß-Cells and Exhibit Stronger Transcriptional Potentials than P2 Promoter-Driven Isoforms

J. Eeckhoute1, E. Moerman1, T. Bouckenooghe, B. Lukoviak, F. Pattou, P. Formstecher, J. Kerr-Conte, B. Vandewalle and B. Laine

Institut National de la Santé et de la Recherche Médicale Unit 459 (J.E., E.M., P.F., B.L.); and Diabetes Cell Therapy Unit, ERIM 106 Institut National de la Santé et de la Recherche Médicale (T.B., B.L., F.P., J.K.-C., B.V.), Faculté H. Warembourg, Lille F 59045, France

Address all correspondence and requests for reprints to: B. Laine, Institut National de la Santé et de la Recherche Médicale Unit 459, Laboratoire de Biologie Cellulaire, Université H. Warembourg, 1 Place de Verdun, F 59045 Lille, France. E-mail: blaine{at}lille.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nuclear receptor hepatocyte nuclear factor (HNF) 4{alpha} is involved in a transcriptional network and plays an important role in pancreatic ß-cells. Mutations in the HNF4{alpha} gene are correlated with maturity-onset diabetes of the young 1. HNF4{alpha} isoforms result from both alternative splicing and alternate usage of promoters P1 and P2. It has recently been reported that HNF4{alpha} transcription is driven almost exclusively by the P2 promoter in pancreatic islets. We observed that transcripts from both P1 and P2 promoters were expressed in human pancreatic ß-cells and in the pancreatic ß-cell lines RIN m5F and HIT-T15. Expression of HNF4{alpha} proteins originating from the P1 promoter was confirmed by immunodetection. Due to the presence of the activation function module AF-1, HNF4{alpha} isoforms originating from the P1 promoter exhibit stronger transcriptional activities and recruit coactivators more efficiently than isoforms driven by the P2 promoter. Conversely, activities of isoforms produced by both promoters were similarly repressed by the corepressor small heterodimer partner. These behaviors were observed on the promoter of HNF1{alpha} that is required for ß-cell function. Our results highlight that expression of P1 promoter-driven isoforms is important in the control of pancreatic ß-cell function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HEPATOCYTE NUCLEAR FACTOR (HNF) 4{alpha} is an orphan nuclear receptor (nuclear receptor 2A1, NR2A1) that activates the expression of genes involved in the transport and metabolism of many nutrients including lipids and glucose (1, 2). HNF4{alpha} is required for normal hepatic function (3). It also plays an important role in pancreatic ß-cells: HNF4{alpha} directly activates the insulin gene promoter (4) and is required for glucose-induced insulin secretion (5). Mutations in HNF4{alpha} gene are correlated with maturity-onset diabetes of the young 1, characterized by an autosomal dominant mode of inheritance, early onset (usually before the age of 25 yr), and impaired glucose-induced insulin secretion due to dysfunction of pancreatic ß-cells (6). HNF4{alpha} is involved, together with other transcription factors including HNF1{alpha}, in a complex transcription factor network that is thought to be crucial for the function of pancreatic ß-cells (7, 8, 9).

In mammals, HNF4 is encoded by two different genes: HNF4{alpha} and HNF4{gamma} (10). The HNF4{alpha} gene potentially encodes nine distinct isoforms. Their structure and nomenclature are presented in Ref. 1 . HNF4{alpha} isoforms result from both alternate promoter usage and alternative splicing. As shown in Fig. 1AGo, promoter P1 initiates transcripts that contain exon 1A (isoforms {alpha}1–{alpha}6), whereas promoter P2 initiates transcripts that contain exon 1D (isoforms {alpha}7–{alpha}9; Refs. 11 and 12). Isoforms {alpha}2 and {alpha}8 are the alternatively spliced variants of isoforms {alpha}1 and {alpha}7, respectively. They contain a 30-bp insert in their 3' sequence (Fig. 1Go, A and B). Compared with these four isoforms, isoforms {alpha}3 and {alpha}9, which have a completely different 3' sequence, are much less expressed, whereas isoforms {alpha}4–{alpha}6 are very poorly expressed or have not yet been identified in vivo (1, 13). The expression of HNF4{alpha} isoforms varies with development, differentiation, and tissue origin (1, 14, 15, 16). The expression of HNF4{alpha} mainly initiates at the P1 promoter in adult liver and kidney (15). Recently, two different groups documented the expression of transcripts of the P2 promoter in insulinoma INS-1 cells, as well as in mouse and human pancreatic islets, but did not detect transcripts of the P1 promoter, leading to the conclusion that HNF4{alpha} transcription is driven almost exclusively by the P2 promoter in the endocrine pancreas (11, 12, 17). Strongly contrasting with these data, we observed that HNF4{alpha} transcripts initiated at the P1 promoter are substantially expressed, alongside P2 promoter transcripts, in human pancreatic ß-cells and in the rodent pancreatic ß-cell lines RIN m5F and HIT-T15. Only HNF4{alpha} isoforms initiated at the P1 promoter contain the activation function module AF-1, which is encoded by exon 1A. The AF-1 plays a key role in HNF4{alpha} transcriptional potential and in the recruitment of coactivators that are essential for HNF4{alpha} function (18, 19, 20). We show that lack of the AF-1 in HNF4{alpha} isoforms initiated at the P2 promoter markedly decreases both the transcriptional potential and interaction with several coactivators. Conversely, lack of the AF-1 does not affect the repression of HNF4{alpha} activity by small heterodimer partner (SHP). This finding highlights the functional consequences of expression of the HNF4{alpha} P1 promoter transcripts in pancreatic ß-cells.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. Expression of HNF4 transcripts in human pancreatic ß-cells. A, Schematic representation of the HNF4{alpha} gene showing promoters P1 and P2 (black boxes) with their respective first exon named 1A and 1D (hatched boxes). Lines linking exons indicate splicing events; splicing of exon 1A to exon 2 yields isoforms {alpha}1–{alpha}3, whereas splicing resulting in insertion of exon 1B (dotted lines) yields isoforms {alpha}4–{alpha}6. Use of exon 1C, which has not been demonstrated, is not presented. Splicing of exon 1D to exon 2 yields isoforms {alpha}7–{alpha}9. Use of a second donor site at the 3' end of exon 9 yields a 30-bp insert (squares in exon 9) in isoforms {alpha}2 and {alpha}8. B, Schematic representation of mature transcripts of the HNF4{alpha} isoforms {alpha}1, {alpha}2, {alpha}7, and {alpha}8 showing the 30-bp insert in isoforms {alpha}2 and {alpha}8. For clarity’s sake, isoforms {alpha}3–{alpha}6 and {alpha}9, which are expressed at low levels, are not shown. Positions of primers used in RT-PCR are indicated. C, RT-PCR analysis of HNF4{alpha} transcripts containing exon 1D, using primers d and b. D, RT-PCR analysis of HNF4{alpha} transcripts containing exon 1A, using primers a and b. E, Determination of the relative amounts of isoforms {alpha}1 and/or {alpha}7 vs. isoforms {alpha}2 and/or {alpha}8 transcripts, using primers e and f. F, RT-PCR analysis of HNF4{gamma} transcript. G, RT-PCR of the control ß-actin. C–G, Lane 1, molecular mass markers (in base pairs); lane 2, human pancreatic islets; lane 3, isolated human pancreatic ß-cells; lane 4, human exocrine pancreas; lane 5, rat insulinoma RIN m5F cells; lane 6, hamster insulinoma HIT-T15 cells. For all RT-PCR analyses, a negative control is shown (lane 7). H, Lack of cross-amplification of P2 promoter transcripts by primers a+b (1 and 2, PCR was performed with cloned HNF4{alpha}2 and -{alpha}8, respectively). I, Control of the specificity of amplification of reverse-transcribed RNAs from human pancreatic islets by primers a+b. 1, RT-PCR was performed as in D; 2, RT-PCR was performed as in D except that the step of reverse transcription was carried out in the absence of Moloney murine leukemia virus reverse transcriptase. RNA from three donors yielded identical results. J, Expression of P1 promoter transcripts using primers h and g designed to specifically amplify exons 1A and 2. 1, Reverse-transcribed RNA; 2, non-reverse-transcribed RNA from human pancreatic islets.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Insulinoma cell lines
Cells were obtained from the American Type Culture Collection (Manassas, VA). RIN m5F cells of rat origin (ATCC CRL-2058) were grown as described in Ref. 21 . HIT-T15 cells of hamster origin (ATCC CRL-1777) were grown as described in Ref. 22 .

Human pancreas processing
Human pancreases (n = 5) were harvested from adult brain-dead donors in accord with French Regulations and with the local Institutional Ethical Committee. Pancreatic islets were isolated after ductal distension of the pancreas and digestion of the tissue with liberase (Roche Molecular Biochemicals, Mannheim, Germany) as described in Ref. 23 . Exocrine fraction was obtained as described in Ref. 24 .

Islet enrichment and single ß-cell preparation
Pancreatic islet-enriched fractions (purity 90 ± 5%) were obtained from semipurified preparations by "handpicking" the dithizone-stained islets under a binocular microscope. Single ß-cell suspensions were obtained by gentle pipetting of islets for 4–6 min in an enzymatic dissociation buffer containing 3.6 g/liter papain (Splittix, Bio Media, Boussens, France). The reaction was stopped by 0.2 vol of the enzyme-inhibiting solution consisting of Splitstop (Bio Media) plus fetal calf serum (3/1 vol/vol, respectively), when about 80% of islet cells appeared as single cells. Cell sorting was then achieved as described in Ref. 25 .

Semiquantitative RT-PCR
Total RNA was prepared using the Macherey Nagel (Düren, Germany) RNA extraction kit according to manufacturer’s protocol. Reverse transcription and PCR were performed as described in Ref. 21 in conditions that were adjusted to maintain the amplification within the exponential range. Primers used for HNF4 transcripts are shown in Table 1Go, and primers for the coactivator peroxisome proliferator activated receptor {gamma} coactivator (PGC)-1 transcripts have the following sequences: sense, 5'-gtggatgaagacggattgcc-3'; antisense, 5'-ttctagttgtctagagtcttgg-3'. PCR products were electrophoresed in 2% agarose gel, and band intensity was analyzed on a Kaiser camera equipped with the Gel Analyst 3.01 software (both provided by Vasse SARL, Templemars, France).


View this table:
[in this window]
[in a new window]
  		Table 1. Primers used in RT-PCR

 
Plasmid constructs
Plasmid pcDNA3 human HNF4{alpha}2 was described in Ref. 26 . Plasmid pcDNA3 human HNF4{alpha}2 Y6D/F19D was obtained by site-directed mutagenesis from pcDNA3 human HNF4{alpha}2 F19D (20). The human HNF4{alpha}8 cDNA was obtained by PCR using human HNF4{alpha}2 cDNA as PCR matrix. The sense primer encompassed the sequence +1/+49 of exon 1D (11) and a sequence hybridizing to the first 23 nucleotides of exon 2, which is common to both {alpha}2 and {alpha}8 HNF4{alpha} isoforms. The antisense primer hybridized to sequence 1412–1433 of HNF4{alpha}2 cDNA, taking adenosine of the ATG codon of initiation methionine as position 1 (27). This cDNA was introduced in the EcoRI and XbaI sites of pcDNA3 to generate the expression plasmid pcDNA3 human HNF4{alpha}8. Constructs were verified by DNA sequencing. The human HNF1{alpha} promoter cloned in pGL3 was a gift from Dr. G. Bell (Howard Hughes Medical Institute, Chicago, IL). Plasmids expressing the fusion proteins glutathione-S-transferase (GST)-PGC-1 (36–797), GST-p300 (340–528), GST-glucocorticoid receptor interacting protein (GRIP)-1(1122–1462), and GST-full-length SHP were gifts from Drs. Spiegelman (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA), Grossman (Dana-Farber Cancer Institute), Stallcup (University of Southern California, Los Angeles, CA), and Gustafsson (Karolinska Institute, Huddinge, Sweden), respectively. The expression plasmids pCMVß-NHA p300, its pCMVß control vector, and pSG5 SHP were gifts from Drs. Grossman and Gustafsson, respectively.

EMSAs
When used for control of specificity, HNF4{alpha}2 and -{alpha}8 were synthesized in vitro as described in Ref. 26 . EMSAs were performed as described in Ref. 28 using the 32P-labeled HNF4{alpha} response element of the HNF1{alpha} promoter and either in vitro-synthesized proteins or nuclear extracts of HIT-T15 and HepG2 cells prepared as described in Ref. 28 . Supershifts were performed by incubating proteins with either the {alpha}455 antiserum (1) or the H4/55 monoclonal antibody (15) before addition of the labeled probe.

Transient transfections
RIN m5F cells were transiently transfected with the LipofectAmine reagent (3 µl/µg of DNA) according to Invitrogen (SARL, Cergy Pontoise, France) recommendations. HIT-T15 and HeLa cells were transiently transfected with the polyethylenimine reagent (4 µl/µg of DNA) according to Euromedex (Souffelweyersheim, France) recommendations. Normalization for differences in transfection efficiencies was performed as previously described using either the Renilla luciferase reporter plasmid in RIN m5F and HeLa cells (29) or the ß-galactosidase reporter plasmid in HIT-T15 cells (30). Luciferase assays were performed with the Dual Luciferase kit (Promega Corp., Madison, WI) for RIN m5F and HeLa cells and as described in Ref. 20 for HIT-T15 cells. Statistical analysis was performed using Student’s t test for unpaired data using the Prism software (Intuitive Software for Science, San Diego, CA).

Western blot analysis
Western assays were performed as described in Ref. 26 and revealed with the {alpha}455 antiserum (1).

In vitro protein-protein interaction assays
GST pull-down assays were performed as described previously (26) using 35S-methionine-labeled in vitro-synthesized HNF4{alpha} and bacterially expressed GST fusion proteins. Interactions were quantified using the ImageQuant software on a PhosphorImager apparatus (both from Molecular Dynamics, Inc., Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcripts initiated at the P1 HNF4{alpha} promoter are readily expressed in human pancreatic ß-cells
Figure 1AGo depicts a schematic representation of HNF4{alpha} gene with P1 and P2 promoters. Expression of P1 and P2 promoter transcripts was analyzed by RT-PCR. Promoter P2 transcripts were selectively amplified with a sense primer hybridizing to exon 1D and an antisense primer hybridizing to exon 5 (primers d and b in Fig. 1Go, A and B, and Table 1Go). Transcripts from promoter P1 were selectively amplified with the sense primer a hybridizing with exon 1A and primer b (Fig. 1Go, A and B, and Table 1Go). As expected from data obtained with pancreatic islets and INS-1 cells (11, 12, 17), we detected transcripts from promoter P2 in human islets, isolated human pancreatic ß-cells, and RIN m5F and HIT-T15 insulinoma cells (Fig. 1CGo). In exocrine pancreas, the band corresponding to promoter P2 transcripts was barely detectable (Fig. 1CGo, lane 4), although the band of ß-actin had an intensity similar to that obtained from endocrine cells (Fig. 1GGo). Interestingly, promoter P1 transcripts from human islets and isolated human pancreatic ß-cells yielded highly visible bands, indicating that HNF4{alpha} promoter P1 transcripts are substantially expressed in human endocrine pancreas (Fig. 1DGo, lanes 2 and 3, respectively). Reproducible data were obtained from five different donors. Similar results were obtained with RIN m5F and HIT-T15 RNA (Fig. 1DGo, lanes 5 and 6). Again, in human exocrine pancreas, promoter P1 transcripts were expressed at very low levels (Fig. 1DGo, lane 4). Two other groups did not detect P1 promoter transcripts in pancreatic islets and INS-1 cells (11, 12, 17), but the following controls confirmed the accuracy of our results showing expression of the P1 promoter transcripts. First, we checked the specificity of amplification of P1 promoter transcripts with the a+b primer set: cloned HNF4{alpha}2 yielded a highly visible band, but no band could be detected with cloned HNF4{alpha}8 (Fig. 1HGo, lanes 1 and 2, respectively). Second, using cloned HNF4{alpha}2 and HNF4{alpha}8, we verified that primer sets a+b and d+b exhibited similar amplification efficiency (data not shown). Third, using human pancreatic islets, we made sure that the band obtained for P1 transcripts did not result from a genomic or plasmidic contamination during RNA isolation or reverse transcription: no amplicon could be detected when the step of reverse transcription of RNAs was performed in the absence of Moloney murine leukemia virus reverse transcriptase (Fig. 1IGo). Conversely, use of another set of primers (h and g), designed to specifically amplify exon 1A and 2, yielded with RNAs from human islets a band that was not obtained when reverse transcription was omitted (Fig. 1JGo), thus confirming that transcripts of the HNF4{alpha} P1 promoter are readily expressed in endocrine pancreas.

Human pancreatic ß-cells express HNF4 isoforms {alpha}1, {alpha}2, {alpha}7, {alpha}8, and HNF4{gamma}
HNF4{alpha} transcripts from promoter P1 mainly represent isoforms {alpha}1 and {alpha}2, and transcripts from promoter P2 mainly represent isoforms {alpha}7 and {alpha}8, because we determined that very low levels of isoforms {alpha}3–{alpha}6 and {alpha}9 were expressed in the cells tested. Indeed, when using primers that specifically amplify isoforms {alpha}3 + {alpha}6 + {alpha}9 or isoforms {alpha}4 + {alpha}5 + {alpha}6 (couples of primers P6 + P7 and P9 + P10, respectively, described in Ref. 14), only very faint bands could be detected (data not shown). In addition, when analyzing transcripts from the P1 promoter, we failed to detect a band of 612 bp, the expected size of amplicons of transcripts containing exon 1B (Fig. 1DGo). This result confirms the very low, if any, expression of isoforms {alpha}4–{alpha}6.

Because the insertion of 10 amino acid residues in the carboxy-terminal sequence of HNF4{alpha}2 was shown to facilitate recruitment of coactivators (31), we analyzed the expression of isoforms containing the 30-bp insert in ß-cells. Primers e and f, hybridizing to exons 9 and 10, respectively, were designed to amplify two fragments, the larger one containing the 30-bp insert of isoforms {alpha}2 and {alpha}8 (Fig. 1Go, A and B). The levels of expression of isoforms {alpha}2 and/or {alpha}8 were equivalent to those of isoforms {alpha}1 and/or {alpha}7 in human pancreatic islets and isolated human pancreatic ß-cells (Fig. 1EGo, lanes 2 and 3). Similar results were obtained with RIN m5F and HIT-T15 RNA (Fig. 1EGo, lanes 5 and 6).

Next we analyzed HNF4{gamma} expression using primers that specifically amplify the carboxy-terminal domain of this isoform (Table 1Go). Results presented in Fig. 1FGo show that HNF4{gamma} is well expressed in human pancreatic islets, isolated human pancreatic ß-cells, and RIN m5F and HIT-T15 cells but is poorly expressed in human exocrine pancreas.

Immunodetection of HNF4{alpha} proteins originated from the P1 promoter in pancreatic ß-cells
To ascertain the expression of HNF4{alpha} proteins encoded by the P1 promoter transcripts containing exon 1A, we used the monoclonal antibody H4/55, which specifically recognizes the amino-terminal sequence of isoforms {alpha}1 and {alpha}2 but not isoforms {alpha}7 and {alpha}8 (15). Because this antibody is not suitable in Western blot assays (Ryffel, G. U., personal communication), HNF4{alpha} expression was studied in EMSA performed with the HNF4{alpha} response element of the HNF1{alpha} promoter (HNF1{alpha} probe). The specificity of the H4/55 monoclonal antibody was confirmed using in vitro-synthesized HNF4{alpha}2 and HNF4{alpha}8: it supershifted the complex formed with HNF4{alpha}2 but not that formed with HNF4{alpha}8 (Fig. 2AGo, lanes 3 and 6). Conversely, complexes formed with both HNF4{alpha} isoforms could be supershifted by the {alpha}455 antiserum raised against their identical carboxy-terminal sequence (Fig. 2AGo, lanes 2 and 5). Nuclear extracts of HepG2 cells, which predominantly express HNF4{alpha}1 and {alpha}2, yielded a complex that could be supershifted by both the {alpha}455 antiserum and the H4/55 monoclonal antibody (Fig. 2BGo, lanes 5–7). Pancreatic ß-cells express much lower amounts of HNF4{alpha} than HepG2 cells (32), which compelled us to use 4-fold higher amounts of HIT-T15 nuclear proteins than HepG2 nuclear proteins. This accounts for the unspecific background observed when analyzing the former material (Fig. 2BGo, lanes 1–4). Nevertheless, with HIT-T15 nuclear extracts, we could easily detect a retarded band (Fig. 2BGo, lane 1) corresponding to a complex mainly formed with HNF4{alpha}: this complex exhibited the same electrophoretic mobility as that yielded by HNF4{alpha} proteins from HepG2 cells; it could be supershifted by the {alpha}455 antiserum (Fig. 2BGo, lane 2) but could not be obtained using a mutated HNF1{alpha} probe that is unable to bind HNF4{alpha} (data not shown). Addition of the H4/55 monoclonal antibody resulted in a concomitant decrease in the retarded band and formation of a strong supershifted band that could not be obtained by an antibody raised against an irrelevant peptide sequence, the Xpress tag (Fig. 2BGo, lanes 3 and 4). This result unambiguously shows endogenous expression of HNF4{alpha}1 and/or -{alpha}2 proteins and confirms that HNF4{alpha} P1 promoter transcripts are readily expressed in the pancreatic ß-cell line HIT-T15. This cell line also expresses HNF4{gamma}, which, when complexed with DNA, comigrates with complexes formed between DNA and HNF4{alpha}1 and -{alpha}2 (33). HNF4{gamma} is not recognized by the {alpha}455 and H4/55 antibodies; this could account for the partial supershift observed in lanes 2 and 3, as previously observed with nuclear extracts of intestinal villi and crypts (33).



View larger version (47K):
[in this window]
[in a new window]
  		Figure 2. Immunodetection of HNF4{alpha} isoforms derived from the P1 promoter in the pancreatic ß-cell line HIT-T15. EMSAs were performed with the HNF4{alpha} response element of the HNF1{alpha} promoter and either in vitro-synthesized HNF4{alpha}2 and HNF4{alpha}8 (A) or nuclear extracts of HIT-T15 (20 µg) and HepG2 (5 µg) cells (B). DNA-protein complexes were incubated with either the {alpha}455 antiserum, which recognizes the common C-terminal end of isoforms {alpha}1, {alpha}2, {alpha}7, and {alpha}8, or the monoclonal H4/55 antibody, which recognizes the N-terminal sequence of isoforms {alpha}1 and {alpha}2, or the unrelated Xpress antibody. Arrow and arrowhead denote DNA-HNF4{alpha} shifted complexes and supershifted complexes, respectively.

 
Weak transcriptional potentials of HNF4{alpha}8 produced from the P2 promoter in pancreatic ß-cells
HNF4{alpha}1 and -{alpha}2, whose transcripts include exon 1A, contain the activation function AF-1 module at their amino-terminal end (Fig. 3AGo). This module includes structural motifs characteristic of acidic activators and plays a key role in the transcriptional potential of HNF4 isoforms {alpha}1 and {alpha}2 (18;19). The amino-terminal sequence of HNF4{alpha}7 and -{alpha}8, encoded by exon 1D, does not contain these motifs and seems to lack an activation function AF-1 module. We addressed the consequences of this sequence difference on the HNF4{alpha} transcriptional potential in pancreatic ß-cell lines, by comparing the transcriptional activities of HNF4 isoforms {alpha}2 and {alpha}8 in RIN m5F and HIT-T15 insulinoma cells. To better define the effect of lack of the AF-1 on the HNF4{alpha} transcriptional potential in these cells, we also analyzed the activity of HNF4{alpha}2 Y6D/F19D, which has a mutated AF-1 module (Fig. 3AGo). The mutated residues are crucial for the AF-1 function in hepatoma HepG2 cells (18) and in embryonal kidney HEK 293T cells (Eeckhoute, J., unpublished results). In RIN m5F cells, the HNF1{alpha} promoter was activated 6.3-fold by HNF4{alpha}2 but only 4.8-fold by HNF4{alpha}8 and 4.2-fold by HNF4{alpha}2 Y6D/F19D (Fig. 3BGo). In HIT-T15 cells, only HNF4{alpha}2 activated the HNF1{alpha} promoter (Fig. 3CGo). The differences in transactivation were not due to low expression of HNF4{alpha}8 or HNF4{alpha}2 Y6D/F19D (Fig. 3DGo). These results support the hypothesis that the lower transcriptional activity of HNF4{alpha}8 is due to lack of a AF-1 module.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Lack or mutation of the AF-1 activation function decreased HNF4{alpha} transcriptional potential in pancreatic ß-cells. A, Schematic representation of HNF4{alpha}2 with its AF-1 module (black box), HNF4{alpha}2 Y6D/F19D with the mutated AF-1 (black box with white diamonds), and HNF4{alpha}8 lacking the AF-1. The sequence 1–16 of isoform {alpha}8 (hatched box) does not share any homology with sequence 1–29 of isoform {alpha}2 (11 ). RIN m5F and HIT-T15 cells (B and C, respectively) were transiently transfected with 300 ng of human HNF1{alpha} promoter-Firefly luciferase-reporter plasmid, 25 ng of the indicated HNF4{alpha} expression plasmids, and 10 ng of either pGL3-Renilla luciferase (in RIN m5F) or pCMV-ß-galactosidase (in HIT-T15). Fold induction refers to activities without any HNF4{alpha} derivative (empty expression vector). Error bars indicate SD values of Renilla luciferase or ß-galactosidase-normalized Firefly luciferase activities from three experiments performed in quadruplicate. Significance of the difference with values obtained for HNF4{alpha}2 is indicated: ***, P < 0.001; and **, P < 0.01. D, Western blotting of overexpressed HNF4{alpha} proteins revealed with the {alpha}455 antiserum.

 
HNF4{alpha}8 driven from the P2 promoter exhibits reduced interaction and cooperation with several coactivators
As for other nuclear hormone receptors, the HNF4{alpha} transcriptional potential is modulated by coactivators or corepressors. The HNF4{alpha} AF-1 interacts with the coactivator p300 (18, 19, 20), as do the AF-1 of other nuclear receptors with p300 and GRIP-1 (34). We therefore investigated the effects of lack of the AF-1 in HNF4{alpha}8 and mutation of this module in HNF4{alpha}2 Y6D/F19D on the interaction with coregulators. In this study, we included p300, GRIP-1, and PGC-1, which are HNF4{alpha} coactivators (31, 35, 36, 37), and SHP, a HNF4{alpha} corepressor (38). GRIP-1, p300, and SHP were reported to be expressed in the endocrine pancreas (7, 39, 40), and PGC-1 expression in human pancreatic ß-cells and in insulinoma cell lines was evidenced by RT-PCR (Fig. 4AGo). HNF4{alpha}8 interacted much less efficiently than HNF4{alpha}2 with p300 and GRIP-1 (Fig. 4BGo). Conversely, no difference between both isoforms was observed when studying physical interaction with PGC-1 and SHP (Fig. 4BGo). Results obtained with HNF4{alpha}2 Y6D/F19D support that the lower interaction of isoform {alpha}8 with p300 and GRIP-1 is due to the lack of AF-1 in this isoform.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 4. Effects of lack of the AF-1 activation function or of AF-1 mutation on physical interaction between HNF4{alpha} and coregulators. A, PGC-1 expression analyzed by RT-PCR. Lane 1, Human pancreatic islets; lane 2, human exocrine pancreas; lane 3, isolated human pancreatic ß-cells; lane 4, rat insulinoma RIN m5F cells; lane 5, hamster insulinoma HIT-T15 cells. B, Physical interactions with coregulators. In vitro-synthesized 35S-methionine-labeled HNF4{alpha}2, HNF4{alpha}8, or HNF4{alpha}2 Y6D/F19D were incubated with immobilized GST-PGC-1 (36–797), GST-p300 (340–528), GST-GRIP-1 (1122–1462), or GST-full-length SHP. After extensive washing, proteins were eluted with sodium dodecyl sulfate loading buffer and analyzed by SDS-PAGE and PhosphorImager (Molecular Dynamics, Inc.). Values under photographs indicate binding of HNF4{alpha}2 Y6D/F19D and HNF4{alpha}8, relative to that of HNF4{alpha}2 from three independent experiments. Inputs, corresponding to 10% of amounts of labeled proteins used in the assays, were taken into account for binding quantifications.

 
Next we studied the effect of lack of the AF-1 on the cooperation between HNF4{alpha} and p300 that is required for activation of transcription (35). To circumvent technical difficulties due to cotransfection of multiple expression plasmids in pancreatic cells and to avoid interference with endogenous HNF4{alpha} and HNF4{gamma}, this study was carried out in HeLa cells. When p300 was not overexpressed, the HNF1{alpha} promoter was activated 5.3-fold by HNF4{alpha}2 but only 2.6-fold by HNF4{alpha}8 and HNF4{alpha}2 Y6D/F19D (Fig. 5AGo). The marked difference in transactivation potentials of HNF4 isoforms {alpha}2 and {alpha}8 was not due to a difference in expression of the proteins (Fig. 5CGo). The coactivator p300 had no effect on the activity of the HNF1{alpha} promoter in the absence of HNF4{alpha} but enhanced the transcriptional potential of HNF4{alpha}2 2-fold vs. only 1.4-fold and 1.2-fold for HNF4{alpha}8 and HNF4{alpha}2 Y6D/F19D, respectively (Fig. 5AGo). Therefore, when the AF-1 module was either lacking or mutated, both the physical interaction and the functional cooperation between p300 and HNF4{alpha} were decreased. As expected from results obtained in pull-down assays, the transcriptional activities of isoforms {alpha}2 and {alpha}8 were repressed to similar extents by SHP (Fig. 5BGo).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. The transcriptional potentials of HNF4{alpha}2 and -{alpha}8 are differently enhanced by the coactivator p300 but similarly repressed by the corepressor SHP. HeLa cells were transiently transfected with 250 ng of human HNF1{alpha} promoter-Firefly luciferase-reporter plasmid, 10 ng of Renilla luciferase expression plasmid, and either 12.5 ng of the indicated HNF4{alpha} expression plasmids together with 250 ng of p300 expression plasmid (A) or 25 ng of the indicated HNF4{alpha} expression plasmids together with 250 ng of SHP expression plasmid (B). The total amounts of transfected DNA was equalized with the corresponding empty vectors (-). Fold induction refers to the activity with no HNF4{alpha} and mediator. Error bars indicate SD values of Renilla luciferase normalized Firefly luciferase activities from three experiments performed in triplicate. The extent of activation or repression is presented (values above each bar) relative to activity of each isoform or mutant (set to 100) without cotransfected mediator. C, Western blotting of overexpressed HNF4{alpha} proteins revealed with the {alpha}455 antiserum.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent studies, having failed to detect transcripts from the P1 promoter in pancreatic islets and in insulinoma INS-1 cells, the authors concluded that HNF4{alpha} transcription is driven almost exclusively by the P2 promoter in pancreatic islets (11, 12, 17). But until now, HNF4{alpha} expression was not documented in human pancreatic ß-cells. In our study on human islets and isolated pancreatic ß-cells, we observed that transcripts of the P1 promoter were significantly expressed, as were those of the P2 promoter. This indicates that HNF4{alpha} expression does not exclusively initiate at the P2 promoter in human pancreatic ß-cells. Similar results were observed in the rat RIN m5F and hamster HIT-T15 pancreatic ß-cell lines. Several controls confirmed the accuracy of our results. Importantly, we confirmed expression of P1 transcripts in the pancreatic ß-cell line HIT-T15 by immunodetecting HNF4{alpha} proteins containing the amino-terminal sequence encoded by exon 1A. Lack of detection of promoter P1 transcripts in islets by Ferrer’s group (12, 17) may be explained by a low amplification efficiency of the P1 promoter transcripts. This may account for the weak intensity they obtained for the exon 1A+2 band in liver, relative to that of exon 1D+2 band in islets (Fig. 2bGo in Ref. 17), although isoforms {alpha}1 and {alpha}2 are by far more abundant in liver than isoforms {alpha}7 and {alpha}8 in islets. In concordance with the considerable difference in expression (32), primers amplifying exons 8–10 contained in the four isoforms {alpha}1, {alpha}2, {alpha}7 and {alpha}8 yielded bands of much stronger intensity for isoforms {alpha}1 + {alpha}2 in liver than for isoforms {alpha}7 + {alpha}8 in islets (Fig. 1cGo in Ref. 17).

A mutation in the HNF4{alpha} P1 promoter significantly decreasing the promoter’s activity is associated with diabetes (41). This suggests that promoter P1 transcription may be important for normal pancreatic ß-cell function. In addition, a HNF4{alpha}2 clone was isolated from a human pancreatic islet cDNA library, also arguing for the expression of HNF4{alpha} P1 promoter transcripts in endocrine pancreas (Furuta, H., and G. Bell, personal communication).

Controversial results were obtained concerning HNF4{alpha} expression in exocrine pancreas (12, 32). We confirmed results described in Ref. 32 stating that HNF4{alpha} was poorly expressed in the exocrine pancreas.

Compared with isoform {alpha}7, isoform {alpha}1 is a stronger transcriptional activator in nonpancreatic ß-cells except on promoters of genes that are expressed early in liver (15, 16, 42). Our functional studies showed that the transcriptional potential of HNF4{alpha}2 was significantly stronger than that of isoform {alpha}8 in models of pancreatic ß-cells. Interestingly, these results were obtained on the promoter of HNF1{alpha}, which plays a crucial role in these cells (7, 9, 43, 44). Because HNF1{alpha} expression is regulated by HNF4{alpha} in pancreatic ß-cells (5, 45), we suggest that expression of the HNF4{alpha} P1 promoter—producing isoforms {alpha}1 and {alpha}2—is most probably of major importance in pancreatic ß-cells. From our data obtained with HNF4{alpha}2 containing a mutated AF-1, we can infer that the difference in transcriptional potential between isoforms {alpha}2 and {alpha}8 was due to the lack of the AF-1 module in the latter isoform.

Furthermore, lack of AF-1 in HNF4{alpha} resulted in a decreased interaction with p300 and GRIP-1 coactivators. Interaction with p300 was studied using a fragment (amino acids 340–528) that interacts with both the HNF4{alpha} AF-1 and AF-2 (20). Despite interaction through the AF-2, we observed a marked decrease in binding of HNF4{alpha} lacking the AF-1, thus highlighting the substantial contribution of AF-1 to the interaction with p300. Consistent with this result, we observed that p300 enhanced the transcriptional potential of isoform {alpha}2 more efficiently than that of isoform {alpha}8 and HNF4{alpha}2 with a mutated AF-1. Interestingly, the AF-1 module is also required for the synergistic action of p300 and GRIP-1 to enhance the HNF4{alpha} transcriptional activity (42).

In contrast to these coactivators, the corepressor SHP interacted similarly with HNF4{alpha}2 and {alpha}8 and repressed their transcriptional activity to similar extents. This strongly suggests that SHP-mediated recruitment of transcriptional inhibitor(s) is not AF-1 dependent. Silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), another HNF4{alpha} corepressor (46), was shown to interact similarly with HNF4{alpha}1 and HNF4{alpha}7 (42). It appears therefore that the HNF4{alpha} AF-1 interacts only with coactivators whereas the AF-2 interacts with both coactivators and corepressors in a mutually exclusive fashion. Because the balance between coactivators and corepressors is crucial for HNF4{alpha} transcriptional activity (38, 46), lack of the AF-1 in isoforms {alpha}7 and {alpha}8 most probably results in a shift toward a lower ability to recruit coactivators and a subsequent lower coactivator-mediated enhancement of HNF4{alpha} transcriptional potential. SHP is highly expressed in human pancreatic islets (Moerman, E., unpublished results) and may serve as an important checkpoint to balance the activity of the transcriptional network of pancreatic ß-cells (7, 8). In these cells in which the HNF4{alpha} AF-2 has a high probability to be occupied by SHP, the transcriptional activity of isoforms containing the AF-1 may be more efficiently enhanced by coactivators. Thus, even if the AF-1-dependent cofactor-mediated modulations of HNF4{alpha} transcriptional activity observed in this study and in Ref. 42 remain to be shown in pancreatic ß-cells, our results strongly argue for an essential role of the promoter P1-driven HNF4{alpha} isoforms in these cells.

A cross-regulatory loop between HNF1{alpha} and HNF4{alpha} has been evidenced in pancreatic ß-cells, as reviewed in Refs. 8 and 9 . In the model proposed by Ferrer (9), activation of the HNF1{alpha} promoter by HNF4{alpha} is crucial to maintain HNF1{alpha} expression above the threshold level required to hold the HNF1{alpha}/HNF4{alpha} circuit in the switch-ON state and, consequently, to avoid ß-cell dysfunction and development of maturity-onset diabetes of the young (9). Expression of the HNF4{alpha} P1 promoter transcripts in pancreatic ß-cells is probably crucial to maintain this circuit in the switch-ON state. Indeed, HNF4{alpha} is expressed at a limiting amount in ß-cells, and, among isoforms {alpha}1, {alpha}2, {alpha}7, {alpha}8, and {gamma}, only the first two contain the AF-1 module, which confers a stronger transcriptional potential and leads to a more efficient recruitment of coactivators. Thus, expression of isoforms {alpha}1 and {alpha}2 is likely of crucial importance in the transcriptional network controlling pancreatic islet function.


   Acknowledgments
 
Professors Ryffel, Spiegelman, Gustafsson, Stallcup, Bell, and Grossman are acknowledged for providing the H4/55 monoclonal antibody, pGEX-PGC1, pGEX-SHP, pGEX-GRIP-1, pGL3-HNF1{alpha}, and pCMCß and pGEX-p300 plasmids, respectively. We are indebted to Dr. C. Brand for helpful discussion and to L. Touzet for proofreading.


   Footnotes
 
This work was supported by a grant from the European Foundation for the Study of Diabetes.

1 J.E. and E.M. contributed equally to this work. Back

Abbreviations: AF, Activation function; GRIP, glucocorticoid receptor interacting protein; GST, glutathione-S-transferase; HNF, hepatocyte nuclear factor; PGC, peroxisome proliferator activated receptor {gamma} coactivator; SHP, small heterodimer partner.

Received November 11, 2002.

Accepted for publication December 24, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sladek FM, Seidel SD 2001 Hepatocyte nuclear factor 4{alpha}. In: Burris TP, McCabe ERB, eds. Nuclear receptors and genetic disease. San Francisco: Academic Press; chap 9, pp. 309–361
  2. Stoffel M, Duncan SA 1997 The maturity-onset diabetes of the young (MODY1) transcription factor HNF-4{alpha} regulates expression of the genes required for glucose transport and metabolism. Proc Natl Acad Sci USA 94:13209–13214[Abstract/Free Full Text]
  3. Hayhurst GP, Lee YH, Lambert G, Ward JM, Gonzalez FJ 2001 Hepatocyte nuclear factor 4{alpha} (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis. Mol Cell Biol 21:1393–1403[Abstract/Free Full Text]
  4. Bartoov-Shifman R, Hertz R, Wang H, Wollheim CB, Bar-Tana J, Walker MD 2002 Activation of the insulin gene promoter through a direct effect of hepatocyte nuclear factor 4{alpha}. J Biol Chem 277:25914–25919[Abstract/Free Full Text]
  5. Wang H, Maechler P, Antinozzi PA, Hagenfeldt KA, Wollheim CB 2000 HNF4{alpha} regulates the expression of pancreatic ß-cell genes implicated in glucose metabolism and nutrient-induced insulin secretion. J Biol Chem 275:35953–35959[Abstract/Free Full Text]
  6. Fajans SS, Bell GI, Polonsky KS 2001 Molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. N Engl J Med 345:971–980[Free Full Text]
  7. Shih DQ, Screenan S, Munoz KN, Philipson L, Pontoglio M, Yaniv M, Polonsky KS, Stoffel M 2001 Loss of HNF-1{alpha} function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism. Diabetes 50:2472–2480[Abstract/Free Full Text]
  8. Shih DQ, Stoffel M 2001 Dissecting the transcriptional network of pancreatic islets during development and differentiation. Proc Natl Acad Sci USA 98:14189–14191[Free Full Text]
  9. Ferrer J 2002 A genetic switch in pancreatic ß-cells: implications for differentiation and haploinsufficiency. Diabetes 51:2355–2362[Abstract/Free Full Text]
  10. Drewes T, Senkel S, Holewa B, Ryffel GU 1996 Human hepatocyte nuclear factor 4 isoforms are encoded by distinct and differentially expressed genes. Mol Cell Biol 16:925–931[Abstract]
  11. Thomas H, Jaschkowitz K, Bulman M, Frayling TM, Mitchell SM, Roosen S, Lingott-Frieg A, Tack CJ, Ellard S, Ryffel GU, Hattersley AT 2001 A distant upstream promoter of the HNF-4{alpha} gene connects the transcription factors involved in maturity-onset diabetes of the young. Hum Mol Genet 10:2089–2097[Abstract/Free Full Text]
  12. Boj SF, Parrizas M, Maestro MA, Ferrer J 2001 A transcription factor regulatory circuit in differentiated pancreatic cells. Proc Natl Acad Sci USA 98:14481–14486[Abstract/Free Full Text]
  13. Furuta H, Iwasaki N, Oda N, Hinokio Y, Horikawa Y, Yamagata K, Yano N, Sugahiro J, Ogata M, Ohgawara H, Omori Y, Iwamoto Y, Bell GI 1997 Organization and partial sequence of the hepatocyte nuclear factor-4{alpha} MODY1 gene and identification of a missense mutation, R127W, in a Japanese family with MODY. Diabetes 46:1652–1657[Abstract]
  14. Suaud L, Joseph B, Formstecher P, Laine B 1997 mRNA expression of HNF-4 isoforms and of HNF-1{alpha}/HNF-1ß variants and differentiation of human cell lines that mimic highly specialized phenotypes of intestinal epithelium. Biochem Biophys Res Commun 235:820–825[CrossRef][Medline]
  15. Nakhei H, Lingott A, Lemm I, Ryffel GU 1998 An alternative splice variant of the tissue specific transcription factor HNF4{alpha} predominates in undifferentiated murine cell types. Nucleic Acids Res 26:497–504[Abstract/Free Full Text]
  16. Torres-Padilla ME, Fougere-Deschatrette C, Weiss MC 2001 Expression of HNF4{alpha} isoforms in mouse liver development is regulated by sequential promoter usage and constitutive 3' end splicing. Mech Dev 109:183–193[CrossRef][Medline]
  17. Hansen SK, Parrizas M, Jensen ML, Pruhova S, Ek J, Boj SF, Johansen A, Maestro MA, Rivera F, Eiberg H, Andel M, Lebl J, Pedersen O, Ferrer J, Hansen T 2002 Genetic evidence that HNF-1{alpha}-dependent transcriptional control of HNF-4{alpha} is essential for human pancreatic ß cell function. J Clin Invest 110:827–833[CrossRef][Medline]
  18. Green VJ, Kokkotou E, Ladias JAA 1998 Critical structural elements and multitarget protein interactions of the transcriptional activator AF-1 of hepatocyte nuclear factor 4. J Biol Chem 273:29950–29957[Abstract/Free Full Text]
  19. Kistanova E, Dell H, Tsantili P, Falvey E, Cladaras C, Hadzopoulou-Cladaras M 2001 The activation function-1 of hepatocyte nuclear factor-4 is an acidic activator that mediates interactions through bulky hydrophobic residues. Biochem J 356:635–642[CrossRef][Medline]
  20. Eeckhoute J, Formstecher P, Laine B 2001 MODY1 diabetes-associated mutations R154X and E276Q in HNF4{alpha} gene impair recruitment of p300, a key transcriptional coactivator. Mol Endocrinol 15:1200–1210[Abstract/Free Full Text]
  21. Riachy R, Vandewalle B, Kerr-Conte J, Moerman E, Sacchetti P, Lukowiak B, Gmyr V, Bouckenooghe T, Dubois M, Pattou F 2002 1,25-Dihydroxyvitamin D3 protects RINm5F and human islet cells against cytokine-induced apoptosis. Implication of the antiapoptotic protein A20. Endocrinology 143:4809–4819[Abstract/Free Full Text]
  22. Laine B, Eeckhoute J, Suaud L, Briche I, Furuta H, Bell GI, Formstecher P 2000 Functional properties of the R154X HNF-4{alpha} protein generated by a mutation associated with maturity-onset diabetes of the young, type 1. FEBS Lett 479:41–45[CrossRef][Medline]
  23. Linetsky E, Bottino R, Lehmann R, Alejandro R, Inverardi L, Ricordi C 1997 Improved human islet isolation using a new enzyme blend, liberase. Diabetes 46:1120–1123[Abstract]
  24. Gmyr V, Kerr-Conte J, Vandewalle B, Proye C, Lefebvre J, Pattou F 2001 Human pancreatic ductal cells: large-scale isolation and expansion. Cell Transplant 10:109–121[Medline]
  25. Lukowiak B, Vandewalle B, Riachy R, Kerr-Conte J, Gmyr V, Belaich SLJ, Pattou F 2001 Identification and purification of functional human ß-cells by a new specific zinc-fluorescent probe. J Histochem Cytochem 49:519–527[Abstract/Free Full Text]
  26. Suaud L, Hemimou Y, Formstecher P, Laine B 1999 Functional study of the E276Q mutant hepatocyte nuclear factor-4{alpha} found in type 1 maturity-onset diabetes of the young, impaired synergy with chicken ovalbumin upstream promoter transcription factor II on the hepatocyte nuclear factor-1 promoter. Diabetes 48:1162–1167[Abstract]
  27. Chartier FC, Bossu JP, Laudet V, Fruchart JC, Laine B 1994 Cloning and sequencing of cDNA encoding the human hepatocyte nuclear factor 4 indicate the presence of two isoforms in human liver. Gene 147:269–272[CrossRef][Medline]
  28. Bossu J-P, Chartier FL, Fruchart J-C, Auwerx J, Staels B, Laine B 1996 Two regulatory elements of similar structure and placed in tandem account for the repressive activity of the first intron of the human apolipoprotein A-II gene. Biochem J 318:547–553
  29. Oxombre B, Moerman E, Eeckhoute J, Formstecher P, Laine B 2002 Mutations in hepatocyte nuclear factor 4{alpha} gene associated with diabetes result in greater loss of HNF4{alpha} function in pancreatic ß-cells than in nonpancreatic ß-cells and in reduced activation of the apolipoprotein CIII promoter in hepatic cells. J Mol Med 80:423–430[CrossRef][Medline]
  30. Suaud L, Formstecher P, Laine B 1999 The activity of the activation function 2 of the human hepatocyte nuclear factor 4 (HNF-4) is differently modulated by F domains from various origins. Biochem J 340:161–169
  31. Sladek FM, Ruse MD, Nepomuceno L, Huang SM, Stallcup MR 1999 Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4{alpha}1. Mol Cell Biol 19:6509–6522[Abstract/Free Full Text]
  32. Miquerol L, Lopez S, Cartier N, Tulliez M, Raymondjean M, Kahn A 1994 Expression of the L-type pyruvate kinase gene and the hepatocyte nuclear factor 4 transcription factor in exocrine and endocrine pancreas. J Biol Chem 269:8944–8951[Abstract/Free Full Text]
  33. Sauvaget D, Chauffeton V, Citadelle D, Chatelet FP, Cywiner-Golenzer C, Chambaz J, Pincon-Raymond M, Cardot P, Le Beyec J, Ribeiro A 2002 Restriction of apolipoprotein A-IV gene expression to the intestine villus depends on a hormone-responsive element and parallels differential expression of the hepatic nuclear factor 4{alpha} and {gamma} isoforms. J Biol Chem 277:34540–34548[Abstract/Free Full Text]
  34. Kumar R, Thompson EB 2003 Transactivation functions of the N-terminal domains of nuclear hormone receptors: protein folding and coactivator interactions. Mol Endocrinol 17:1–10[Abstract/Free Full Text]
  35. Soutoglou E, Katrakili N, Talianidis I 2000 Acetylation regulates transcription factor activity at multiple levels. Mol Cell 5:745–751[CrossRef][Medline]
  36. Wang JC, Stafford JM, Granner DK 1998 SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4. J Biol Chem 273:30847–30850[Abstract/Free Full Text]
  37. Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G, Stafford J, Kahn CR, Granner DK, Newgard CB, Spiegelman BM 2001 Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413:131–138[CrossRef][Medline]
  38. Lee YK, Dell H, Dowhan DH, Hadzopoulou-Cladaras M, Moore DD 1999 The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: two mechanisms for repression. Mol Cell Biol 2000 20:187–195
  39. Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160-kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J 15:3667–3675[Medline]
  40. Ban N, Yamada Y, Someya Y, Miyawaki K, Ihara Y, Hosokawa M, Toyokuni S, Tsuda K, Seino Y 2002 Hepatocyte nuclear factor-1{alpha} recruits the transcriptional co-activator p300 on the GLUT2 gene promoter. Diabetes 51:1409–1418[Abstract/Free Full Text]
  41. Price JA, Fossey SC, Sale MM, Brewer CS, Freedman BI, Wuerth JP, Bowden DW 2000 Analysis of the HNF4{alpha} gene in Caucasian type II diabetic nephropathic patients. Diabetologia 43:364–372[CrossRef][Medline]
  42. Torres-Padilla ME, Sladek FM, Weiss MC 2002 Developmentally regulated N-terminal variants of the nuclear receptor HNF4{alpha} mediate multiple interactions through coactivator and corepressor/HDAC complexes. J Biol Chem 277:44677–44687[Abstract/Free Full Text]
  43. Pontoglio M, Sreenan S, Roe M, Pugh W, Ostrega D, Doyen A, Pick AJ, Baldwin A, Velho G, Froguel P, Levisetti M, Bonner-Weir S, Bell GI, Yaniv M, Polonsky KS 1998 Defective insulin secretion in hepatocyte nuclear factor 1{alpha}-deficient mice. J Clin Invest 101:2215–2222[Medline]
  44. Hagenfeldt-Johansson KA, Herrera PL, Wang H, Gjinovci A, Ishihara H, Wollheim CB 2001 ß-Cell-targeted expression of a dominant-negative hepatocyte nuclear factor-1{alpha} induces a maturity-onset diabetes of the young (MODY)3-like phenotype in transgenic mice. Endocrinology 142:5311–5320[Abstract/Free Full Text]
  45. Gragnoli C, Lindner T, Cockburn BN, Kaisaki PJ, Gragnoli F, Marozzi G, Bell GI 1997 Maturity-onset diabetes of the young due to a mutation in the hepatocyte nuclear factor-4{alpha} binding site in the promoter of the hepatocyte nuclear factor-1{alpha} gene. Diabetes 46:1648–1651[Abstract]
  46. Ruse Jr MD, Privalsky ML, Sladek FM 2002 Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4{alpha}1: modulation by the F domain. Mol Cell Biol 22:1626–1638[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 DiabetesHome page
L. W. Harries, J. M. Locke, B. Shields, N. A. Hanley, K. P. Hanley, A. Steele, P. R. Njolstad, S. Ellard, and A. T. Hattersley
The Diabetic Phenotype in HNF4A Mutation Carriers Is Moderated By the Expression of HNF4A Isoforms From the P1 Promoter During Fetal Development
Diabetes, June 1, 2008; 57(6): 1745 - 1752.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
C. Gates Goodyer, Z. Rhani, and H. Zheng
Expression of the Hepatic Specific V1 Messenger Ribonucleic Acid of the Human Growth Hormone Receptor Gene Is Regulated by Hepatic Nuclear Factor (HNF)-4{alpha}2 and HNF-4{alpha}8
Mol. Endocrinol., February 1, 2008; 22(2): 485 - 500.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. Leng, S. Lu, Y. Yao, Z. Kan, G. S. Morris, B. R. Stair, M. A. Cherny, and D. D. Black
Hepatocyte nuclear factor-4 mediates apolipoprotein A-IV transcriptional regulation by fatty acid in newborn swine enterocytes
Am J Physiol Gastrointest Liver Physiol, August 1, 2007; 293(2): G475 - G483.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
G. Benoit, A. Cooney, V. Giguere, H. Ingraham, M. Lazar, G. Muscat, T. Perlmann, J.-P. Renaud, J. Schwabe, F. Sladek, et al.
International Union of Pharmacology. LXVI. Orphan Nuclear Receptors
Pharmacol. Rev., December 1, 2006; 58(4): 798 - 836.
[Abstract] [Full Text] [PDF]