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Am J Physiol Gastrointest Liver Physiol 285: G62-G72, 2003. First published March 19, 2003; doi:10.1152/ajpgi.00074.2003
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MUCOSAL BIOLOGY

HNF-1{alpha} and endodermal transcription factors cooperatively activate Fabpl: MODY3 mutations abrogate cooperativity

Joyce K. Divine,1,2 Sean P. McCaul,2 and Theodore C. Simon1,2

1Division of Biology and Biomedical Sciences and 2Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110

Submitted 14 February 2003 ; accepted in final form 11 March 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocyte nuclear factor (HNF)-1{alpha} plays a central role in intestinal and hepatic gene regulation and is required for hepatic expression of the liver fatty acid binding protein gene (Fabpl). An Fabpl transgene was directly activated through cognate sites by HNF-1{alpha} and HNF-1{beta}, as well as five other endodermal factors: CDX-1, C/EBP{beta}, GATA-4, FoxA2, and HNF-4{alpha}. HNF-1{alpha} activated the Fabpl transgene by as much as 60-fold greater in the presence of the other five endodermal factors than in their absence, accounting for up to one-half the total transgene activation by the group of six factors. This degree of synergistic interaction suggests that multifactor cooperativity is a critical determinant of endodermal gene activation by HNF-1{alpha}. Mutations in HNF-1{alpha} that result in maturity onset diabetes of the young (MODY3) provide evidence for the in vivo significance of these synergistic interactions. An R131Q HNF-1{alpha} MODY3 mutant exhibits complete loss of synergistic activation in concert with the other endodermal transcription factors despite wild-type transactivation ability in their absence. Furthermore, whereas wild-type HNF-1{alpha} exhibited pairwise cooperative synergy with each of the other five factors, the R131Q mutant could synergize only with GATA-4 and C/EBP{beta}. Selective loss of synergy with other endodermal transcription factors accompanied by retention of native transactivation ability in an HNF-1{alpha} MODY mutant suggests in vivo significance for cooperative synergy.

HEPATOCYTE NUCLEAR FACTOR-1 (HNF-1) family members have been proposed as key regulators of gene expression in endodermal and genitourinary tissues (60). The two HNF-1 family members are HNF-1{alpha} and HNF-1{beta}, which share highly homologous homeodomain DNA-binding motifs that recognize the same DNA sequence (34, 45). The HNF-1 factors bind to DNA as heterodimers or homodimers and are found in the liver, kidney, small intestine, pancreas, and genitourinary tissues (13). Many genes in these tissues are activated by HNF-1{alpha} (50). Genes activated by HNF-1{alpha} may also be activated by HNF-1{beta}, or HNF-1{beta} may inhibit HNF-1{alpha} activation (6). Mice with targeted disruption of both HNF-1{alpha} alleles exhibit Fanconi syndrome, enlarged fatty liver, defects in bile acid metabolism, and diabetes (31, 43, 44). Altered expression of numerous genes occurs in the livers of these animals (50). Loss of one HNF-1{alpha} allele in humans results in renal dysfunction (26, 35) and pancreatic {beta}-cell defects that give rise to diabetes, termed maturity onset diabetes of the young (MODY) (66).

MODY is characterized by onset between 10 and 60 years of age, with a defect in insulin secretion (7, 17). Mutations in HNF-1{beta}, HNF-4{alpha}, and PDX-1 also result in MODY with phenotype similar to that of HNF-1{alpha} mutations (40). The exact targets of these transcription factors that result in MODY are unknown. These transcription factors along with factors of the FTF, FOXA (formerly HNF-3), GATA, and HNF-6 families comprise a genetic network critical in endodermal development (16, 42, 70). MODY due to haploinsufficiency of HNF-1{alpha} is termed MODY3, and complete loss of HNF-1{alpha} activity at one allele causes the disease (64). However, two-thirds of the ~80 defined HNF-1{alpha} gene MODY3 mutations are missense mutations that result in a full-length protein containing a single amino acid change (17, 46). Other MODY3 HNF-1{alpha} mutations result in early protein truncation, a loss of transactivation potential, or dominant-negative activity against HNF-1{alpha} target genes in cellular transfection assays (46). However, some MODY3 missense mutants retain significant ability to transactivate HNF-1 target genes (63). The defects in gene regulation resulting from HNF-1{alpha} MODY mutations are largely unknown. In addition to MODY3 HNF-1{alpha} mutations that are autosomal dominant for a severe phenotype, a non-MODY HNF-1{alpha} mutation has been described that is a risk factor for type 2 diabetes but does not result a dominant phenotype (59).

The rat liver fatty acid binding protein gene (Fabpl) has been utilized as an experimental model to study gene regulation in endoderm-derived tissues (10, 52, 56). Rat Fabpl is highly expressed in hepatocytes and enterocytes, and expression is primarily regulated at the transcriptional level (3). A transgene constructed of Fabpl nucleotides –596 to +21 relative to the start site of transcription is active in murine hepatocytes, all small intestinal epithelial cells, renal proximal tubular epithelial cells, and the urinary tract (47, 52, 56). Two HNF-1 binding sites were noted in the Fabpl promoter, and mice with targeted disruption of HNF-1{alpha} exhibit complete loss of Fabpl expression in the liver (1). We found functional binding sites for five additional endodermal transcription factor families in the proximal Fabpl promoter and examined the interaction between HNF-1{alpha} and these other endodermal transcription factors to determine why HNF-1{alpha} is essential for Fabpl expression. Experiments demonstrated that multifactor cooperativity is a critical determinant of Fabpl activation by HNF-1{alpha} and that HNF-1{alpha} MODY3 mutations result in loss of multifactor cooperativity but not individual Fabpl activating ability.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sequence analysis. Transcription factor binding sites in the Fabpl promoter (GenBank accession no. M13501 [GenBank] ) were identified with the transcription element search system (48), and by direct examination using binding site matrices from transcription factor database (TRANSFAC) (21).

Plasmids. An Fabpl transgene was constructed from Fabpl nucleotides –596 to +21, relative to the start site of transcription, linked to the entire human growth hormone (hGH) gene lacking regulatory sequences. The Fapbl promoter was released from pEPLFABP (56) by cleaving with EcoRI and BamHI and inserted into pBluescript II SK+ cut with the same enzymes to produce pTS9. The entire hGH gene was released from pBShGH (52) by BamHI digestion and ligated into the BamHI site of pTS9 to create pTS10. pTS10 was digested with XbaI and religated, deleting an 18-nucleotide fragment containing the BamHI site distal to the hGH gene and creating pTS154. A glucocorticoid receptor site in the first intron of the hGH gene (39) was destroyed by site-directed mutagenesis to create pTS245. Site-directed mutagenesis was performed with a commercial kit (QuikChange; Stratagene, La Jolla, CA). The mutation changed hGH nucleotides 5268–5269 (GenBank accession no. J03071 [GenBank] ) from TG to GT, using complimentary oligonucleotides with sense strand sequence 5'-CTAAAATCCCTTTGGGCACAATGgtTCCTGAGGGGAGAGGCAGCG-3'. The presence of this mutation and the absence of other mutations were confirmed between the AvrII and BamHI sites of the mutated pTS154. This fragment was released by endonuclease digestion and ligated into pTS154 digested with the same enzymes, yielding PTS245.

Potential transcription factor binding sites in Fabpl were destroyed by site-directed mutagenesis of pTS10 as described above. These sites are indicated in Fig. 1, and the sense sequence from one of each complementary oligonucleotide pair with changed bases after mutagenesis in lower case is: HNF-4 –55 5'-ATCGACAATCACTGAaaTATGGaaTATATTTGAGGAGGAA-3'; Cdx –78/–82 and C/EBP –78 overlapping sites, 5'-GGAGTTAATGTTTGATCCTGGCCATggAGggATCGACAATCACTGACCTATGGCC-3'; FoxA –94, 5'-GACCATTGCTCTCAGGAGTTAATGaTcGAcCCTGGCCATA-3'; HNF-1 –95, 5'-GACCATTGCTCTCAGGAGggccTGTTTGATCCTGGCCATA-3'; GATA –128/–130, 5'-CTTCTGCCTTGCCCATTCTacTTTTTAgtGTTGACCATTGC-3', FOXA –155/–169; 5'-CCTTGATTGGACTCACTAAgGcTTtCTGAATTAGAACAggCTTCTGCC-3'; GATA –229, 5'-ACTCTTATTTCATGAGCGGTacTAAGACACCAAAAATGC-3'; and GATA –557, 5'ACAGCTTTAGGGACTacTAAAATATATGTAAAATTATGT-3'.



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  		Fig. 1. Nucleotides –596 to +21 of the rat fatty acid binding protein gene (Fabpl) promoter contain binding sites for 6 transcription factor families important in endoderm. Shown is the rat Fabpl promoter sequence numbered relative to the start site of transcription (designated +1). Transcription factor binding sites are boxed and designated by the name of the factor family and most proximal nucleotide of the consensus binding site.

 

Targeted mutations were confirmed by sequencing the entire Fabpl sequence and a functional hGH reporter verified by protein production in cultured cells (see below). Promoters with multiple mutations were created by sequential rounds of mutagenesis. Plasmids created by site-directed mutagenesis were termed: pTS146, HNF-1 –95 site mutated; pTS147, HNF-4 –55 site mutated; pTS179, Cdx –78/–82 and C/EBP –78 overlapping sites mutated; pTS187, GATA –128/–130/–229/–557 sites mutated; and pTS211, FoxA –94/–155/–169 sites mutated. The hGH reporter with mutagenized glucocorticoid receptor binding site was released from pTS245 by digestion with BamHI and NotI and was ligated into pTS146, pTS147, pTS179, pTS187, or pTS211 digested with the same enzymes to form pTS255, pTS247, pTS256, pTS246, and pTS248, respectively.

Transcription factor expression plasmids were produced by inserting the transcription factor coding sequences into pSG5 (Stratagene), a mammalian expression vector containing the early SV40 promoter. The murine C/EBP{beta} open reading frame was released from MSV/EBP{beta} (provided by Steve McKnight) by digestion with EcoRI and BamHI and inserted into the EcoRI/BamHI sites in pSG5 to form pTS142. The murine HNF-1{beta} coding sequence was released from pBJ5-HNF-1{beta} [provided by Peter Traber, Baylor University Medical School, Houston, TX (67)] with EcoRI/NotI and ligated into the BamHI site in pSG5 to form pTS156 after blunting the ends of both fragments with DNA bacteriophage T4 DNA polymerase. The murine HNF-1{alpha} coding sequence was released from pBJ-HNF-1{alpha} (provided by Peter Traber) with EcoRI/EcoRV and ligated into pSG5 digested with BglII (blunted) then EcoRI to form pTS258. The murine GATA-4 was released from pMT2615A [provided by David Wilson, Washington University, St. Louis, MO (4)] by EcoRI digestion and inserted into the EcoRI site of pSG5 to form pTS186. The rat FoxA2 open reading frame was released from pHNF3{beta} [provided by Robert Costa, University of Illinois, Chicago, IL (41)] with EcoRI digestion, and this sequence was inserted into the pSG5 EcoRI site to form pTS190. The human CDX-1 protein coding sequence was released from pCDX1 [provided by Beatrice Levy-Wilson, Palo Alto Research Foundation, Palo Alto, CA (30)] with EcoRI and cloned into the pSG5 EcoRI site to form pTS197. Sequences containing the coding sequence for human HNF-4{alpha}2 from pHNF-4{alpha}2 [provided by Gerhart Ryffel, Institut für Zellbiologie, Essen, Germany (15)] was isolated with HindIII/NotI digestion and cloned into the pSG5 EcoRI site to create pTS276 after blunting both fragments.

Expression constructs for HNF-1{alpha} MODY3 mutations were derived by site-directed mutagenesis of pTS158 using primers as previously described (63). The entire open reading frame of each HNF-1{alpha} mutant was sequenced to ensure that no additional mutations were introduced.

Cell culture and transfections. Caco-2 and HepG2 cells were from American Type Culture Collection (Manassas, VA) and were maintained as recommended, and HeLa cells were a kind gift from Alan Schwartz. Transient transfections were performed with calcium phosphate precipitation as follows. All plasmids utilized in transfection assays were purified with a commercial kit that yields reduced endotoxin contamination (Qiagen, Valencia, CA). Each assay contained an Fabpl reporter plasmid, transcription factor expression plasmids, and plasmids to control for expression efficiency. Fabpl reporter gene plasmids pTS10 or pTS245 and their mutagenized derivatives were used interchangeably with equivalent results. Transfection efficiency was monitored by including identical amounts in each assay of pSV40{beta}-galactosidase (Promega, Madison, WI) or pGL3 (Promega), which constitutively express bacterial {beta}-galactosidase or Photinus pyralis luciferase, respectively. The amount of DNA (5–9 µg per well) was kept constant in each experiment by the addition of pSG5 plasmid. Enough DNA for three wells was diluted with water to a volume of 157.5 µl. An equal volume of 0.5 M CaCl2 was added, then 315 µl BES-buffered saline (50 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4) was added. A precipitate was allowed to form for 20 min at room temperature before adding the DNA solution to the cells. Cells were in six-well plates at 30–50% confluence at the time of transfection, and one-tenth volume (200 µl) of each transfection solution was added to three separate wells. Cells were washed twice with 2 ml phosphate-buffered saline after overnight incubation and then covered with the appropriate culture medium. Culture media were renewed on the following day, and media and cells were harvested 24 h later. hGH was detected in the media using a specific radioimmunoassay (Nichols Institute). Dilutions with media were utilized when necessary to remain in the linear assay range. Transfection efficiency was assayed by using either a {beta}-galactosidase assay kit (Promega) or luciferase assay kit (Promega). Values were calculated as the average of the three wells for each DNA solution, and error was calculated as SD or propagated error for calculated values. Values are reported as fold activation over the activity of the native Fabpl reporter with no added transcription factor expression plasmids. All experiments were repeated at least twice with similar results.

Nuclear extract preparation and electrophoretic mobility shift assays. Nuclear extracts were prepared from Caco-2 cells after transfection with transcription factor expression plasmids. Caco-2 cells at 40% confluence in a single 75-cm2 flask were transfected by calcium phosphate precipitation exactly as for the expression studies, except that precipitates of 48 µg of either pTS158 (expressing HNF-1{alpha}), pTS156 (expressing HNF-1{beta}), or pSG5 in a larger volume were utilized. Transfection efficiency was monitored by inclusion of 16 µg pXGH5 (Nichols Institute). pXGH5 expresses hGH from the metallothionine promoter. Nuclear extracts were prepared by using a commercial kit (NE-PER kit; Pierce, Rockford, IL). Approximately 50 µl of packed cells were obtained from each flask. Nuclear extract protein concentration was determined with a commercial protein assay kit (Bio-Rad, Hercules, CA).

EMSAs were performed as previously described (51), except 1.5 µg nuclear extract, 1 µg herring sperm DNA, and no dIdC were included in each 20-µl reaction. Reactions were incubated 18 min at room temperature before electrophoresis, except that supershift assays contained 2 µg antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and were incubated for 32 min. The radiolabeled probe was a double-stranded oligonucleotide derived from the putative Fabpl HNF-1 recognition sequence shown in Fig. 3A. Competitors were either a double-stranded oligonucleotide with the mutagenized Fabpl sequence noted in Fig. 3 or an authentic {beta}-fibrinogen HNF-1 site (11).



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  		Fig. 3. EMSA demonstrates that the Fabpl element binds HNF-1{alpha} and HNF-1{beta}. A: Sequence of the rat Fabpl HNF-1 binding site utilized as a probe with base changes to abrogate factor binding indicated as targeted mutation. B: EMSA using the probe indicated in A and nuclear extracts from Caco-2 cells transfected with an expression construct for HNF-1{alpha} (lanes 6–10), an expression construct for HNF-1{beta} (lanes 10–13), or an empty expression construct (lanes 2–5). Lane 1 is the probe incubated without nuclear extract. Three complexes designated 1–3 in lane 2 did not form in the presence of a 128-fold molar excess of an oligonucleotide with the sequence of an authentic HNF-1 site in the {beta}-fibrinogen gene promoter (lane 3). These complexes did not form in the presence of a 128-fold molar excess of unlabeled probe (lane 4) but are not affected by a 128-fold molar excess of mutagenized probe. The Fabpl HNF-1 probe formed a prominent complex with nuclear extracts from cells transfected with an HNF-1{alpha} expression construct (lane 6), and this complex migrated at the same mobility as complex 1 in extracts from cells transfected with a control vector. The addition of competitor oligonucleotides resulted in the same competition pattern as for complexes 1–3. Identical results were obtained with nuclear extract from cells transfected with an HNF-1{beta} expression construct, except that a prominent complex formed with the same mobility as complex 3. C: EMSAs were performed with nuclear extracts from cells transfected with an HNF-1 expression construct (lanes 1–3) or an HNF-1{beta} expression construct (lanes 4–6). Included in the incubation were specific antibodies to HNF-1{alpha} (lanes 2 and 5) and HNF-1{beta} (lanes 3 and 6). The mobility of the prominent specific complex in each reaction was shifted by the corresponding antibody (supershift).

 


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Fabpl proximal promoter contains binding sites for numerous endodermal transcription factor families. A transgene constructed from rat Fabpl nucleotides –596 to +21 is expressed in mice in all small intestinal epithelial cells, in all proximal colonic epithelial cells, in hepatocytes, and in proximal tubular epithelial cells (56, 58). Endogenous Fabpl is active only in small intestinal enterocytes and hepatocytes (56). Deletions and modifications of this promoter result in striking changes in cellular expression patterns (51, 52). A search for the transcription factors that mediate these effects was undertaken. The HNF-1 binding site (1) and a peroxisome proliferator-activated receptor element coincident with the HNF-4 binding site (52) have been previously identified. The transcription element search system (48) as well as direct sequence evaluation were used to identify potential transcription factor binding sites in the Fabpl promoter (Fig. 1). Potential binding sites for factors of the CDX, C/EBP, FOXA, GATA, HNF-1, and HNF-4 families were identified and are designated by the most proximal base of the consensus binding site relative to the start site of transcription.

Endodermal transcription factors directly activate Fabpl through interactions with cognate sites. Transient transfection assays were utilized to determine the potential function of the transcription factor binding sites identified through Fabpl promoter sequence analysis. A transgene was constructed from rat Fabpl nucleotides –596 to +21 linked to a reporter consisting of the entire hGH gene minus its regulatory regions. The Fabpl transgene was active when transfected into Caco-2 cells or HepG2 cells (Fig. 2, "native + control" in all panels). These cell lines were chosen to resemble enterocytes and hepatocytes, respectively. The potential of transcription factor families with cognate binding sites in the Fabpl promoter to transactivate the Fabpl transgene was assessed by cotransfection with an expression plasmid for one transcription factor from each family. CDX-1, C/EBP{beta}, FoxA2, GATA-4, HNF-1{alpha}, and HNF-4{alpha} all activate the native Fabpl transgene (Fig. 2). To demonstrate that activation by these factors was direct and through their cognate sites, transgenes were created with all sites for each factor family mutagenized to destroy binding. For example, to test for indirect activation by FoxA2, a transgene was created with all three FoxA sites mutagenized (Fig. 1). This mutagenized transgene was active in both cell lines but was not stimulated by FoxA2 (compare mutant control and mutant + factor in Fig. 2). This result indicates the Fabpl transgene activation by FoxA2 is mediated by interaction with these three sites. Similar transgenes were created to test for indirect activation by each other factor family. Mutagenesis of potential binding sites essentially eliminated activation by every factor except C/EBP{beta}, which displayed reduced activity. A second potential HNF-1 binding site in the Fabpl promoter has been described at –355 (1), but this site did not mediate Fabpl transgene transactivation (data not shown). These results indicate that the six factor families with binding site(s) in the Fabpl promoter activate Fabpl through interaction with their cognate site(s).



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  		Fig. 2. Transient transfection assays demonstrate direct Fabpl activation by CDX, C/EBP, FOXA, GATA, hepatocyte nuclear factor (HNF)-1, and HNF-4 factor families through cognate sites in the proximal promoter. A transgene constructed from Fabpl nucleotides –596 to +21 linked to hGH was active in Caco-2 cells and HepG2 cells (native + control in all panels). This transgene was stimulated by CDX-1, C/EBP{beta}, FoxA2, GATA-4, HNF-1{alpha}, and HNF-4{alpha} (native + factor). Transgene activity was normalized to the control activity of the native transgene in each cell line. Mutant transgenes were created with all the potential binding sites for a particular factor family mutagenized. All the mutant transgenes were active in both cell lines (mutant + control). Mutagenesis of all potential binding sites for each factor family eliminated activation by each respective factor except C/EBP{beta}, which displayed reduced activity (compare mutant + factor with native + factor). Data from one experiment with each factor is displayed as normalized activity, with error indicating SD from the mean of values obtained with 3 separate wells of cells. Experiments were repeated at least 3 times with similar results.

 

HNF-1{alpha} and HNF-1{beta} interact with the cognate Fabpl binding sequence in vitro. HNF-1{alpha} has been proposed as a critical regulator of intestinal epithelial gene regulation (57) and to directly regulate Fabpl in hepatocytes (1). However, because the proposed Fabpl HNF-1 binding site differs from the consensus at one nucleotide (Fabpl –97 is G not A; Fig. 1), interaction of HNF-1{alpha} and HNF-1{beta} with the Fabpl site was tested in vitro. Nuclear extracts were prepared from Caco-2 cells transfected with either an expression construct for HNF-1{alpha}, an expression construct for HNF-1{beta}, or a control construct. EMSAs were performed with these nuclear extracts and a radiolabeled double-stranded oligonucleotide probe derived from the putative Fabpl HNF-1 recognition site (Fig. 3A). Three specific complexes formed with extracts from Caco-2 cells transfected with the control vector (Fig. 3B). Formation of these complexes was prevented by competition with a 128-fold molar excess of unlabeled probe or a 128-fold molar excess of an oligonucleotide with the sequence of an authentic HNF-1 binding site from the {beta}-fibrinogen promoter (11). Complex formation was not affected by inclusion of a 128-fold molar excess of the mutagenized Fabpl HNF-1 binding site that lacked activity in the transient transfection assay. The three specific complexes that form between Caco-2 extracts and the Fabpl binding site have mobilities similar to those identified for HNF-1{alpha}/HNF-1{beta} homo- and heterodimers (45). The slowest-moving complex is the HNF-1{alpha} homodimer, the fastest-moving complex is the HNF-1{beta} homodimer, and the middle complex is the heterodimer. These EMSA with nuclear extracts from cells transfected with HNF-1{alpha} or HNF-1{beta} expression constructs demonstrated that abundant binding protein is produced with each expression construct in Caco-2 cells and is consistent with the complex identification for extracts from cells transfected with the control vector. Supershift EMSA confirmed the identity of the complexes (Fig. 3C). These experiments demonstrate that the Fabpl HNF-1 site readily forms complexes with HNF-1{alpha} and HNF-1{beta} despite differing from the consensus sequence.

HNF-1{alpha} and HNF-1{beta} transactivate Fabpl individually and together without interference or cooperation. Because HNF-1{alpha} and HNF-1{beta} both bind to the cognate Fabpl site in vitro, the transactivation potential of both factors was determined in transient transfection assays (Fig. 4, A and B). Both HNF-1{alpha} and HNF-1{beta} transactivated the Fabpl transgene in both cell lines, and this activation was eliminated by specific mutagenesis of the HNF-1 binding site. Transfection with 2 µg of expression vector for both factors resulted in greater activation by HNF-1{alpha} than HNF-1{beta} in both cell lines. Adding 4 µg expression plasmid resulted in significant activation by HNF-1{beta} in both cell lines (Fig. 4, C and D). Because HNF-1{beta} has been reported to interfere with HNF-1{alpha} transactivation of other genes (6, 22, 54), the interaction of HNF-1{alpha} and HNF-1{beta} in Fabpl transgene activation was determined. Transient transfections were performed with various ratios of expression plasmids for each factor (Fig. 4, C and D). Fabpl activation with any combination of HNF-1{alpha} and HNF-1{beta} in both cell lines exhibited no interference or cooperativity.



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  		Fig. 4. HNF-1{alpha} and HNF-1{beta} transactivate Fabpl individually and together without interference or cooperation. Transient transfections were performed as described in Fig. 2 in Caco-2 and HepG2 cells. The native Fabpl transgene or an Fabpl transgene with a mutagenized HNF-1 binding site were stimulated with expression constructs for HNF-1{alpha} and HNF-1{beta}. A and B: HNF-1{alpha} and HNF-1{beta} activated the native but not mutagenized Fabpl transgene in both cell lines, and HNF-1{alpha} induced greater transactivation than HNF-1{beta}. C and D: transient transfection in both cell lines was performed with the native Fabpl transgene and varying mixtures of expression constructs for HNF-1{alpha} and HNF-1{beta}. Total added expression plasmid in each well was 4 µg and consisted of different ratios of HNF-1{alpha} (µg {alpha}) to HNF-1{beta} (µg {beta}) as indicated. At this level of added expression vector, both HNF-1{alpha} and HNF-1{beta} significantly stimulated the Fabpl transgene, and mixtures of the two factors show no evidence of cooperativity or interference in activation. Data is expressed as fold activation over unstimulated activity of the native transgene in each cell line, and error is mean ± SD of triplicate wells.

 

Endodermal transcription factors exhibit strong cooperative synergy. HNF-1{alpha} or HNF-1{beta} Fabpl transgene activation was assayed in concert with a mixture of five transcription factors, consisting of one member from each of the other five endodermal transcription factor families with functional binding sites in the Fabpl promoter (Fig. 5). HNF-1{alpha} by itself stimulated the Fabpl transgene eightfold in Caco-2 cells. A mixture of HNF-1{alpha} plus the other five endodermal transcription factors stimulated the transgene 157-fold, whereas the five-factor mix alone stimulated the transgene 68-fold. Thus addition of HNF-1{alpha} to the five factors resulted in an 89-fold increase in transgene activity relative to the unstimulated activity. These results reveal that HNF-1{alpha} activated the transgene in Caco-2 cells 11 times better in the presence of the other factors than by itself (89- vs. 8-fold). The result of cooperation between HNF-1{alpha} with the five-factor group resulted in transgene activation 2.1-fold compared with activation by the five factors together plus activation of HNF-1{alpha} by itself. Similar results were obtained in HepG2 cells in which HNF-1{alpha} activated the Fabpl transgene 187-fold in the presence of the five factors vs. threefold by itself. Cooperative contribution of HNF-1{alpha} to the factor mix in HepG2 cells is 1.3-fold. Activation with the mixture of five factors plus HNF-1{alpha} activated the transgene 157-fold in Caco-2 cells, compared with a calculated additive value of 23-fold. Calculated additive values were derived from the sum of the values obtained for transgene activation by the individual factors in the mix. In HepG2 cells, five factors plus HNF-1{alpha} activated the transgene 740-fold vs. a calculated additive value of 67-fold relative to the unstimulated transgene. These results demonstrate that cooperative synergy among all the factors is quantitatively more important for Fabpl gene expression in these cells than activation by any single factor. This degree of synergistic interaction suggests that multifactor cooperativity is a critical determinant of endodermal gene activation by HNF-1{alpha}. In contrast to HNF-1{alpha}, HNF-1{beta} obstructed Fabpl activation by the other five endodermal factors in HepG2 cells and to a lesser extent in Caco-2 cells (Fig. 5).



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  		Fig. 5. HNF-1{alpha} but not HNF-1{beta} exhibits cooperative Fabpl activation with transcription factors from 5 other families. The Fabpl transgene was activated by HNF-1{alpha} or HNF-1{beta} in the presence or absence of a mixture of Cdx-1, C/EBP{beta}, FoxA2, GATA-4, and HNF-4{alpha} (5 factors) in Caco-2 and HepG2 cells. The actual transgene activity in the presence of each factor or factor mix is indicated by black bars. Gray bars indicate the calculated additive value for factor mixtures derived from the sum of the values obtained for transgene activation by the individual factors in the mix. Fabpl transgene activation by the 5-factor mix was significantly greater than the calculated additive activation, and HNF-1{alpha} but not HNF-1{beta} exhibited considerably greater activation in the presence of the 5-factor mix. Cells were transfected with expression plasmids for various transcription factors and the native Fabpl transgene as noted in Fig. 2. One microgram of each transcription factor expression plasmid was included in each well. Data is expressed as fold activation over the activity of the native transgene, and error is SD from the mean of triplicate wells or propagated error for calculated values.

 

Pairwise cooperative interaction between the HNF-1 factors and each of the other five endodermal transcription factors were evaluated (Fig. 6). The actual activation of the Fabpl transgene by each factor pair together was compared with the calculated additive value for transgene stimulation by each factor separately. Cooperative interactions of twofold or greater were observed between HNF-1{alpha} and CDX-1, C/EBP{beta}, GATA-4, FoxA2, and HNF-4{alpha} in Caco-2 cells relative to the unstimulated transgene, consistent with the extensive cooperative interaction between HNF-1{alpha} and these factors as a mixture (Fig. 5). In HepG2 cells, HNF-1{alpha} exhibited significant cooperative activation with all factors except FoxA2. In contrast, HNF-1{beta} did not exhibit significant synergy with any factor in Caco-2 cells (Fig. 6). In HepG2 cells, HNF-1{beta} had greater than twofold cooperative activation with C/EBP{beta} and GATA-4 but significant anergy with HNF-4{alpha}. Lack of pairwise interactions between HNF-1{beta} and other factors is consistent with the lack of cooperative interaction between HNF-1{beta} and the factors as a group (Fig. 5).



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  		Fig. 6. Pairwise interactions between HNF-1{alpha} or HNF-1{beta} and 5 transcription factors. Cell transfections were performed as described in Fig. 2. Black bars indicate observed Fabpl transgene activity. Gray bars indicate calculated additive activity for HNF-1 and the second factor, which are derived from the sum of the values obtained for transgene activation individually by each factor (data not shown). Top: results in Caco-2 cells in which HNF-1{alpha} but not HNF-1{beta} exhibited cooperative activation of twofold or greater with all 5 other factors. Bottom: transfections performed in HepG2 cells in which HNF-1{alpha} exhibited significant cooperative activation with all factors except FoxA2, whereas HNF-1{beta} had cooperative activation with C/EBP{beta} and GATA-4 but anergy with HNF-4{alpha}. Data is expressed as fold activation over the activity of the native transgene, and error is SD from the mean of triplicate wells or propagated error for calculated values.

 

HNF-1{alpha} MODY3 mutants exhibit a selective defect in cooperative activation with other endodermal transcription factors. HNF-1{alpha} MODY3 point mutations have been reported that result in a full-length protein with significant transactivation ability and no dominant-negative activity (63). Because cooperative multifactor interactions are more important in target gene activation than the action of any one factor (Fig. 5), HNF-1{alpha} MODY mutants with significant individual activation ability were examined for defects in cooperative interactions. Five of 10 HNF-1{alpha} MODY3 mutations examined in the original report retained significant transactivation ability for a synthetic target gene consisting of HNF-1 binding sites upstream of a minimal promoter (63). Each of these five mutants (Y122C, R131Q, R159Q, K205Q, R272H) was able to transactivate the Fabpl transgene to varying degrees in Caco-2 and HepG2 cells (data not shown). In contrast, these five HNF-1{alpha} MODY3 mutants exhibited loss of cooperative synergy with the group of five endodermal transcription factors in both cell lines (data not shown). Two MODY3 mutants, R131Q and Y122C, were particularly informative. Both of these mutants are in the HNF-1{alpha} DNA binding domain, and both proteins localize to the nucleus and form complexes with a canonical HNF-1 binding site in EMSA despite a reduced halflife (63). The R131Q MODY3 mutant retained wild-type transactivation ability for the Fabpl transgene in HeLa cells, and the Y122C mutant retained 63% of the wild-type activity (Fig. 7A). In HepG2 cells, both mutants showed significantly less ability to transactivate the Fabpl transgene (Fig. 7B). In the presence of the five-factor mix, wild-type HNF-1{alpha} shows a dramatic cooperative synergy (Figs. 5 and 7C). Neither MODY3 mutant exhibited cooperative synergy with the five other factors, but both actually inhibited activation by the five factors (Fig. 7C). The G319S HNF-1{alpha} mutant does not result in MODY or a dominant phenotype (59) and has wild-type ability to transactivate the Fabpl transgene in HeLa or HepG2 cells (Fig. 7, A and B). Furthermore, this mutant did not display a defect in cooperative synergistic activation with the other endodermal factors (Fig. 7C). HepG2 cells are known to endogenously express many endodermal transcription factors, whereas HeLa cells do not (14). The decrease in Fabpl activation in HepG2 cells compared with HeLa cells only in those factors deficient in cooperative synergy may reflect a contribution of synergy by the endogenous factors. These HNF-1{alpha} MODY3 mutants exhibited defects in specific pairwise interactions with the other endodermal transcription factors (Fig. 7D), whereas the non-MODY G319S mutant did not. The wild-type HNF-1{alpha} exhibited synergy with CDX-1, C/EBP{beta}, GATA-4, and HNF-4{alpha} in HepG2 cells, whereas the R131Q MODY3 HNF-1{alpha} mutant lost cooperative activation with CDX-1 and HNF-4{alpha} and exhibited anergy with FoxA2. The Y122C MODY3 HNF-1{alpha} lost cooperative activation with CDX-1, C/EBP{beta}, and HNF-4{alpha} and exhibited anergy with FoxA2. The G319S displayed the same pairwise cooperative interactions as the wild-type HNF-1{alpha}. Similar results were obtained for the R131Q and Y122C mutants with Caco-2 cells and HeLa cells with the five-factor mix (data not shown).



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  		Fig. 7. HNF-1{alpha} maturity onset diabetes of the young (MODY)3 mutants exhibit a selective defect in cooperative activation with other endodermal transcription factors compared with wild-type HNF-1{alpha} and a non-MODY HNF-1{alpha} mutant. Transient transfections were carried out in HeLa or HepG2 cells as described in Fig. 2, except that 0.67 µg transcription factor expression plasmid was used for experiments shown in A–C. All data is expressed as fold activation over the unstimulated activity of the Fabpl transgene in each cell line, and error is SD from the mean of triplicate wells or propagated error for calculated values. A: comparison of Fabpl transactivation in HeLa cells by wild-type HNF-1{alpha}, MODY3 HNF-1{alpha} mutants R131Q and Y122C, and non-MODY G319S HNF-1{alpha} mutant. B: identical experiment to that in A performed in HepG2 cells. R131Q transactivates Fabpl at least as well as wild-type in HeLa cells, which do not endogenously express significant levels of endodermal transcription factors. R313Q exhibits a significant defect in Fabpl transactivation in HepG2 cells, which endogenously express numerous endodermal transcription factors. In contrast, G319S shows 90% of wild-type activity in both cell lines. C: the effect of the MODY3 mutations on cooperative synergy between HNF-1{alpha} and the other 5 endodermal transcription factors was determined with transfection assays. The 5-factor mix activated the transgene 907-fold in HepG2 cells. In the presence of the 5-factor mix, wild-type HNF-1{alpha} increased activation an additional 277-fold. However, addition of the R131Q or Y122C HNF-1{alpha} mutants actually decreased Fabpl activation by the 5-factor mixture, but the non-MODY G319S mutant showed no defect in cooperative synergy compared with wild-type HNF-1{alpha}. D: pairwise interactions in HepG2 cells between the 5 endodermal transcription factors and wild-type or mutant HNF-1{alpha} factors. Black bars indicate observed Fabpl transgene activity. Gray bars indicate calculated additive activity for HNF-1 and the second factor, which are derived from the sum of the values obtained for transgene activation individually by each factor (data not shown). Wild-type HNF-1{alpha} exhibited synergy with CDX-1, C/EBP{beta}, GATA-4, and HNF-4{alpha}. The R131Q MODY3 HNF-1{alpha} mutant lost cooperative activation with CDX-1 and HNF-4{alpha}. The Y122C MODY3 HNF-1{alpha} lost cooperative activation with CDX-1, C/EBP{beta}, and HNF-4{alpha} and exhibited anergy with FoxA2. However, the G319S HNF-1{alpha} non-MODY mutant with no defect in cooperative synergy with the 5-factor mix (C) exhibited identical pairwise cooperative synergy to wild-type HNF-1{alpha}.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Fabpl transgene activation in cultured cells by multifactor synergistic cooperation is quantitatively more important than activation by any factor by itself. Transgene activation by the group of six factors was as much as 11 times greater than activation by the sum of the individual factors alone. HNF-1{alpha} exhibits extensive cooperative synergy with other endodermal transcription factors. The target gene is activated up to 60 times greater by HNF-1{alpha} in the presence of the other factors than in their absence, with HNF-1{alpha} contributing up to one-half the total activation. Hepatocytes contain numerous transcription factors capable of transactivating Fabpl, including C/EBP, FOXA, GATA, and HNF-4 family factors (12), but mice with HNF-1{alpha} gene null mutations exhibit complete loss of Fabpl expression in the liver (1). The lack of Fabpl expression in the liver of HNF-1{alpha}-null mice despite the presence of the other activating factors is consistent with a central role for HNF-1{alpha} in multifactor synergy.

Pairwise cooperative interactions occur between HNF-1{alpha} and each of the other endodermal transcription factors that activate the Fabpl transgene. These multiple cooperative interactions between pairs of factors may combine to yield the observed multifactor cooperative synergy. HNF-1{alpha} has been reported to interact pairwise to cooperatively activate genes besides Fabpl with these same factors: FoxA2 (9, 61), C/EBP{alpha} (8, 68, 69), and HNF-4{alpha} (23). Cooperative activation is dependent, at least in part, on the target gene, because no synergy in activation between HNF-1{alpha} and HNF-4{alpha} is observed with some targets (33) and actual anergy occurs with other targets (28, 29). HNF-1{alpha} cooperatively interacts with GATA-4 and Cdx-2 to activate the sucrase-isomaltase gene (5). HNF-1{alpha} has also been reported to exhibit pairwise synergy to activate genes besides Fabpl with other factor family members, including FoxA3 (61), GATA-4 (62), GATA-5 (27), and Cdx2 (38), plus additional factors important in endoderm: DBP (2), HNF-6 (20), Oct-1 (24), COUP-TF (32), and HOXC11 (37). The mechanism for most of these synergies is unknown, but protein-protein interaction has been described between HNF-1{alpha} and Cdx-2 (21) or GATA-5 (27). Synergistic gene activation through recruitment of multiple factors has been hypothesized to result from increased efficiency of assembly of a competent RNA polymerase II initiation complex through multiple mechanisms (36). We observed results similar to those shown for HepG2 cells in Fig. 7C in HeLa cells, indicating that the mechanism of cooperative synergy is not unique to endodermal cells.

Direct relevance for the significance of cooperative synergy in vivo is obtained from experiments with the MODY3 HNF-1{alpha} mutations. MODY3 is an autosomal dominant disease in which loss of one copy of the HNF-1{alpha} gene is sufficient to disturb pancreatic gene expression (17). It is perhaps surprising that haploin-sufficiency of one transcription factor results in disease when genes are typically activated by numerous factors. Haploinsufficiency of transcription factors frequently results in disease, and it has been suggested this may be due to loss of transcriptional synergy (65). The R131Q MODY3 mutation exhibits wild-type target gene activation alone but results in a severe MODY phenotype with average age of onset at 14 years (7, 18, 25). This phenotype compares with an average age of onset of 24 yr for all MODY3 mutations (49), indicating that selective loss of synergy results in a disease at least as severe as that caused by other mutations. The R131Q mutant activates the Fabpl target gene at least as well as wild type in HeLa cells but only 47% in HepG2 cells. This difference can be explained by cooperative synergy between HNF-1{alpha} and endogenous endodermal transcription factors present in the HepG2 but not HeLa cells (14).

The R131Q mutant and to a greater extent the Y122C mutant inhibited target gene activation by the other five transcription factors (Fig. 7C). R131Q has wild-type ability to transactivate the Fabpl transgene, and Y122C retains 63% of the wild-type target gene transactivation (Fig. 7A). The decrease in Y122C target gene transactivation may be a result of the reported decreased stability of these mutants compared with the wild-type protein (63). However, the significant inhibition of Fabpl activation by the five factors in the presence of Y122C is difficult to attribute solely to a decrease in stability. It is interesting that a loss of cooperation with some but not all of the five factors tested with the HNF-1{alpha} mutants results in a complete loss of synergy with the entire group (Fig. 7, C and D). These results could explain the in vivo MODY phenotype in which the numerous interactions that might occur on the target gene promoters could be disrupted by loss of a few critical interactions. The G319S does not have any loss of cooperative interactions compared with wild-type HNF-1{alpha} and does not lead to the autosomal dominant phenotype observed with the MODY mutants but rather to a more subtle phenotype that manifests as a risk factor for type 2 diabetes (59).

We describe a MODY mutation that results in loss of interaction with multiple endodermal transcription factors. Another MODY mutation has been described that results in loss of pairwise synergy between HNF-4{alpha} E276Q MODY1 and COUP-TFII to activate the HNF-1{alpha} promoter (55). This finding suggests that other MODY mutations may also function through loss of synergy and that this effect may be amplified through the genetic network of endodermal transcription factors. All the MODY transcription factors are constituents of a genetic network for transcriptional activation in endoderm that also encompasses FTF, FOXA, GATA, and HNF-6 (16, 19, 42, 70). Each transcription factor may activate the gene for another factor and/or autoactivate its own gene. Thus loss of one transcription factor may lead to endodermal defects through failure to activate other factors. Synergistic activation of the transcription factor genes would amplify the loss of any one factor and contribute to the loss of direct activation of target genes in differentiated tissue.


   ACKNOWLEDGMENTS
 
We thank Katherine Lee, Lilia Rissman, and Joshua Rissman for technical assistance and David Wilson and Jonathan Gitlin for review of the manuscript. We are grateful to Robert Costa, James Darnell, Beatrice Levy-Wilson, Steven McKnight, Gerhart Ryffel, Peter Traber, and David Wilson for sharing transcription factor expression plasmids.

This work was supported by grants from the March of Dimes Foundation (to T. C. Simon) and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56361 (to T. C. Simon) and P30-DK-52574 (to the Washington University Digestive Disease Research Core Center).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. C. Simon, Washington University School of Medicine, Dept. of Pediatrics, Campus Box 8208, St. Louis, MO 63110 (E-mail: simon_t{at}kids.wustl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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