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Endocrinology, doi:10.1210/en.2003-1188
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Endocrinology Vol. 145, No. 6 2918-2928
Copyright © 2004 by The Endocrine Society

Pancreas Duodenum Homeobox-1 Transcriptional Activation Requires Interactions with p300

Violeta Stanojevic, Joel F. Habener and Melissa K. Thomas

Laboratory of Molecular Endocrinology (V.S., J.F.H., M.K.T.) and Diabetes Unit (M.K.T.), Massachusetts General Hospital, Howard Hughes Medical Institute (V.S., J.F.H.), and Harvard Medical School (J.F.H., M.K.T.), Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Melissa K. Thomas, M.D., Ph.D., Laboratory of Molecular Endocrinology and Diabetes Unit, Massachusetts General Hospital, Wellman 340, 50 Blossom Street, Boston, Massachusetts 02114. E-mail: mthomas1{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The homeodomain transcription factor, pancreas duodenum homeobox (PDX)-1, is essential for pancreas development, insulin production, and glucose homeostasis. Mutations in pdx-1(ipf-1) are associated both with maturity-onset diabetes of the young and type 2 diabetes. PDX-1 interacts with multiple transcription factors and coregulators, including the coactivator p300, to activate the transcription of the insulin gene and other target genes within pancreatic ß-cells. In characterizing the protein-protein interactions of PDX-1 and p300, we identified mutations in PDX-1 that disrupt its function and are associated with increased or decreased interactions with p300. Several mutant PDX-1 proteins that are associated with heritable forms of diabetes in humans, in particular the mutant P63fsdelC, exhibited increased binding to a carboxy-terminal segment of p300 in the setting of decreased DNA-binding activities, suggesting that sequestration of p300 by mutant PDX-1 proteins may be an additional mechanism by which insulin gene expression is reduced in heterozygous carriers of pdx-1(ipf-1) mutations. The introduction of the point mutations S66A/Y68A in the highly conserved amino-terminal PDX-1 transactivation domain reduced the ability of PDX-1 to interact with p300, substantially diminished the transcriptional activation of PDX-1, and reduced the synergistic activation of glucose-responsive insulin promoter enhancer sequences by PDX-1, E12, and E47. We propose that interactions of PDX-1 with p300 are required for the transcriptional activation of PDX-1 target genes. Impairment of interactions between PDX-1 and p300 in pancreatic ß-cells may limit insulin production and lead to the development of diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IDENTIFICATION OF associations between mutations in transcriptional regulators of the insulin gene and heritable forms of diabetes in humans has stimulated considerable interest in the transcriptional regulation of insulin production in pancreatic ß-cells. The homeodomain transcription factor pancreas duodenum homeobox (PDX)-1 functions as a master regulator of pancreas development and differentiated pancreatic ß-cell function. The pdx-1 gene (1) was cloned by several laboratories and also is known as idx-1 (2), ipf-1 (3), and stf-1 (4). Homozygous disruption of the pdx-1(ipf-1) gene results in pancreatic agenesis in mice and in humans (1, 5, 6).

PDX-1 regulates the transcription of several genes that are essential for glucose-sensing and insulin production in pancreatic ß-cells, including insulin, glucokinase, and glucose transporter-2. The PDX-1 protein includes an amino-terminal transcriptional activation domain and a central highly conserved homeodomain that confers DNA-binding activity. In the rat and human insulin promoters PDX-1 binds to conserved glucose-responsive enhancers known as A boxes, in synergy with the binding of the basic helix-loop-helix transcription factors Beta-2/neuroD1, E12, and E47 to adjacent conserved E boxes, to provide substantial pancreatic ß-cell selectivity and glucose-responsiveness (7, 8, 9, 10, 11). The synergistic activation of the insulin promoter results from multiple protein-protein interactions among PDX-1 and E12/E47, Beta-2/neuroD1, and coactivators including Bridge-1, CBP, and p300 that form multiprotein complexes resulting in chromatin remodeling and the recruitment of basal transcription machinery (12, 13, 14, 15, 16).

PDX-1 function is essential for normal glucose metabolism and insulin production in humans and rodents. The heterozygous inheritance of the inactivating mutation P63fsdelC in the human pdx-1(ipf-1) gene is associated with maturity-onset diabetes of the young (MODY4) (17). Heterozygous missense mutations in the human pdx-1(ipf-1) gene confer a predisposition to the development of late-onset (type 2) or gestational diabetes (18, 19, 20, 21). Similar metabolic phenotypes are observed in mice, in which the heterozygous disruption of the pdx-1 allele results in impaired glucose tolerance (22, 23) and diminished glucose-stimulated insulin secretion (24). The disruption of PDX-1 expression after the pancreas develops in mice results in diabetes (22, 25, 26).

A variety of human pdx-1(ipf-1) mutations are found in individuals with hyperglycemia, including P63fsdelC (17), D76N (18, 19), A140T (27), and InsCCG243 (18). Metabolic dysfunction is thought to arise either from a loss of pdx-1 gene dosage and/or dominant-negative functions of mutant PDX-1 proteins. For example, the P63fsdelC mutation encodes two mutant PDX-1 proteins, the smaller of which encodes a truncated amino-terminal protein lacking a DNA-binding domain and a nuclear localization signal but including an unique carboxy-terminal sequence arising from the frame shift (28). The larger mutant PDX-1 protein is derived from internal translation and lacks the amino-terminal transactivation domain but includes the DNA-binding homeodomain and the carboxy-terminal domain, providing one mechanism for dominant-negative function via DNA-binding of an inactive PDX-1 mutant protein (28).

Here we evaluated the importance of the interaction of PDX-1 with the coactivator p300 in determining the transcriptional activation of PDX-1 and the functional properties of PDX-1 proteins harboring mutations associated with heritable forms of human type 2 diabetes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids and transfections
INS-1 (from C. Wollheim, University Medical Center, Geneva, Switzerland) and HeLa (from R. Stein, Vanderbilt University School of Medicine, Nashville, TN) cells were cultured as previously described (15, 25). Transient transfections were conducted with Lipofectamine (Invitrogen Life Technologies, Carlsbad, CA) according to published methods (29). FarFlat-chloramphenicol transferase (CAT) (from L. G. Moss, Tufts University School of Medicine, Boston, MA) encodes pentamerized glucose-responsive E and A box enhancers Far and Flat and a CAT reporter. FarFlat-luciferase (from M. German, University of California San Francisco, San Francisco, CA) encodes pentamerized Far and Flat enhancers and a luciferase reporter in the previously described vector pFoxluc1.prl (7, 12). The Gal4-CAT (pG5-CAT) or Gal4-luciferase reporter (pFR-Luc) plasmids were purchased from Clontech (Palo Alto, CA) and Stratagene (La Jolla, CA), respectively. The pCMVß-p300 wild-type and dominant-negative mutant ({Delta}CH/3, del 1737–1836) plasmids were purchased from Upstate Biotechnology (Lake Placid, NY). Glutathione-S-transferase (GST)-p300 fusion protein expression plasmids were obtained from H. Lu (Oregon Health and Science University, Portland, OR) and D. Livingston (Dana-Farber Cancer Institute, Boston, MA). The pCMV-E1A wild-type and mutant {Delta}2–36 plasmids were from R. Stein. Expression plasmids for rat E12, E47, PDX-1, and GST-PDX-1 have been described previously (2, 25). To generate the Gal4-PDX-1 constructs, full-length rat PDX-1 cDNA was cloned in frame within the multiple cloning site of the Gal4(1–147) expression vector pM (Clontech). Gal4-PDX-1(1–78) and (1–143) were constructed by engineering premature stop codons in the Gal4-PDX-1 vector by QuikChange site-directed mutagenesis (Stratagene) conducted according to the manufacturer’s instructions. Point mutagenesis to generate premature stop codons or sequence changes was conducted with the QuikChange site-directed mutagenesis kit. All constructs generated by cloning and mutagenesis experiments were confirmed by automated sequence analyses.

The following oligonucleotides and their respective reverse complements were used for site-directed mutagenesis of rat PDX-1 expression vectors: PDX-1(1–38), 5'-CGTGCCTGTACATGTGACGCCAGCCCCC-AC-3'; PDX-1(1–78), 5'-GATGACCCGGCTTGAGCGCACCTCCACC-3'; PDX-1(1–143), 5'-CAGCAGAACCGTAGGAGAATAAGAGGACCCG-3'; PDX-1(1–206), 5'-GGAAGAAAGAGGAAGATTAGAAAC-GTAGTAGCGG-3'; Y14A, 5'-CCACACAGCTGCCAAGGACCCGTGG-CG-3'; S29G/N31A, 5'-CGGTGCCAGAGTTCGGTGCTGCT CCCCCT-GCGTGC-3'; S61G, 5'-CTGGAACAGGGAGGTCCCCCGGACATC-3'; S66A, 5'-CCCCCGGACATCGCCCCATACGAAG-3'; Y68A, 5'-CGGACATCTCCCCAGCCGAAGTGCCCCCGCTC-3'; S66A/Y68A, 5'-CGGACATCGCCCCAGCCGAAGTGCCCCCGCTC-3'; S124A/T125A, 5'-CCTTTCCCGTGGATGAAAGCCGCCAAAGCTCACG-3'. To generate the PDX-1(143–283) construct for in vitro transcription/translation studies, a XhoI site was created in the GST-PDX-1 vector at amino acid position 143 by site-directed mutagenesis using the sense oligonucleotide 5'-GGTGCATACGCAGCAGAACTCGAGGAGAATAAGAG-3' and its reverse complement. A XhoI fragment containing the cDNA encoding the PDX-1 homeodomain and carboxy terminus was cloned into the XhoI site within the multiple cloning site of a pET16B (Novagen, Madison, WI) vector. Expression vectors for in vitro transcription/translation of human PDX-1 mutant proteins were generated by excising the PDX-1 coding sequences by EcoRI digestion of previously constructed pCMV5-PDX-1 vectors (18, 27) and cloning them into an EcoRI site within the multiple cloning region of the pcDNA3 (Invitrogen) vector. Luciferase and CAT assays were conducted using standard methods as described (15, 29). Western blots of extracts derived from transfected cells were conducted according to previously developed methods (25).

GST pull-down assays
Recombinant GST fusion proteins were synthesized in bacteria as described (2). Normalization of GST fusion protein input was determined by assessing recombinant protein yields at serial dilutions on Coomassie blue-stained sodium dodecyl sulfate (SDS) polyacrylamide gels. [35S]methionine radiolabeled in vitro-translated proteins were synthesized from rabbit reticulocyte lysates according to the manufacturer’s instructions (Promega, Madison, WI) and as previously described (15). Normalization of in vitro-translated protein input was determined by autoradiography of SDS-polyacrylamide gels with consideration given for the expected incorporation of radiolabeled methionine. Protein-protein interaction assays were conducted with glutathione Sepharose 4B beads at 4 C in PBS at pH 7.4 with 150 mM NaCl, 0.1% Tween 20, 5 µg/ml antipain, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 ng/ml pepstatin, and 5 µg/ml soybean trypsin inhibitor using standard methods outlined by the manufacturer (Amersham Biosciences, Piscataway, NJ).

DNA-binding assays
DNA-binding assays were conducted with gel-purified double-stranded [32P]-radiolabeled oligonucleotide probes as described previously (28, 30). In all cases free probe and empty expression vector control reactions were conducted. In vitro-translated rat and human PDX-1 proteins and empty expression vector control samples were prepared in rabbit reticulocyte lysates according to standard methods (Promega). Expression levels of synthesized PDX-1 proteins were determined by Western blots to normalize amounts of PDX-1 protein input for DNA-binding assays. Rabbit reticulocyte lysate content also was normalized for all DNA-binding reactions. The following double-stranded oligonucleotide probes encompassing known PDX-1 binding sites (A boxes) were employed in DNA-binding assays: rat insulin I promoter (–197 to –225) Flat probe, 5'-GATCTTGTTAATAATCTAATTACCCTAGGTCT-A-3' and 3'-AACAATTATTAGATTAATGGGATCCAGATCTAG-5'; P1 probe (–59 to –103), 5'-GATCCTACCTACCCCTCCTAGAGCCCTTAATGGGCCAAACGGCAAA-3' and 3'-GATGGATGGGGAGGAT-CTCGGGAATTACCCGGTTTGCCGTTTCTAG-5'; human insulin promoter (–201 to –230) CT2 probe, 5'-GATCCCCCTGGTTAAGAC-TCTAATGACCCGCTGG-3' and 3'-GGGGACCAATTCTGAGATTA-CTGGGCGACCCTAG-5'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The transcriptional activation potential of PDX-1 is enhanced by p300
The activation of highly conserved glucose-responsive enhancers in the insulin gene promoters is dependent on the synergistic interaction of the homeodomain transcription factor PDX-1 and heterodimers of basic helix-loop-helix transcription factors including E12 or E47 (31). We assessed the function of PDX-1 in transcriptional activation in HeLa cells using an established experimental model system of synergistic activation of the glucose-responsive elements Far and Flat derived from the rat insulin I promoter (25, 28) (Fig. 1AGo). The combination of E12, E47, and PDX-1 resulted in the synergistic activation of the FarFlat reporter construct in transient transfections. The adenoviral protein E1A represses the activation of glucose-responsive enhancers derived from either the rat insulin I or II promoters in insulin-producing cells (16, 32). Similarly, in HeLa cells the addition of the adenoviral protein E1A suppressed the activation of the FarFlat enhancers by over 90% (Fig. 1AGo). E1A is known to act in part by binding and sequestering coactivator proteins like p300. Notably an E1A mutant that is unable to bind p300 (E1A{Delta}2–36) (33) did not disrupt the observed synergistic activation in HeLa cells analogous to results observed in HIT-T15 insulinoma cells (16).



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  		FIG. 1. The transcriptional activation of PDX-1 is enhanced by p300. A, E1A inhibition of the synergistic activation of the rat insulin I glucose-responsive enhancers by PDX-1, E12, and E47 is dependent on interactions with endogenous p300. HeLa cells were transiently transfected with 50 ng pcDNA3-E12, 50 ng pcDNA3-E47, 125 ng pCMV5-PDX-1, 300 ng pCMV-E1A, or 300 ng pCMV-E1A{Delta}2–36 and 500 ng FarFlat-CAT reporter as indicated. Empty expression vectors were added to normalize total amounts of DNA in all transfections. Results shown are the mean ± SEM of data normalized to the activity of E12, E47, and PDX-1 in combination (n = 4 transfections conducted in duplicate; ***, P < 0.001). B, E1A inhibition of PDX-1 transcriptional activation is dependent on endogenous p300. HeLa cells were transiently transfected with 500 ng of the Gal4 expression vector pM or Gal4-PDX-1 in the presence or absence of 50 ng pCMV-E1A or pCMV-E1A{Delta}2–36 and 500 ng of a Gal4-CAT reporter as indicated. Results shown are the mean ± SEM of data normalized to the activity of Gal4-PDX-1 (n = 3 transfections conducted in duplicate). C, Exogenous p300 increases the transcriptional activation of PDX-1. HeLa cells were transiently transfected with 500 ng of the Gal4 expression vector pM or Gal4-PDX-1 as indicated in the presence or absence of 1000 ng of pCMV-p300 and 500 ng of a Gal4-CAT reporter. Results shown are the mean ± SEM (n = 4 transfections conducted in duplicate; *, P < 0.05). D, p300-mediated increases in PDX-1 transcriptional activation are dose dependent. HeLa cells were transiently transfected in duplicate with 500 ng of Gal4-PDX-1 in the presence of increasing amounts of 0–2000 ng pCMV-p300 as indicated and 500 ng of a Gal4-CAT reporter. Empty expression vector was added to normalize total amounts of DNA in all transfections. A fluorescence image of a thin layer chromatogram from a representative CAT assay is shown (upper panel) with the fluorescent substrate (S) and the acetylated products (*) indicated. Western blot analysis of Gal4-PDX-1 (PDX-1) expression in the corresponding transfected cell extracts is shown (lower panel).

 
To determine the effect of the adenoviral E1A protein on the transcriptional activation function of PDX-1 alone, we employed a Gal4-PDX-1 fusion protein construct encoding the full-length rat PDX-1 protein and assessed the ability of Gal4-PDX-1 to activate a CAT reporter construct regulated by multimerized Gal4 DNA-binding sites in HeLa cells (Fig. 1Go, B–D). Gal4-PDX-1 activated the Gal4 reporter substantially in comparison with the transcriptional activation by the Gal4 protein alone. The addition of E1A suppressed the transcriptional activation of PDX-1 by 70% under conditions in which the expression levels of the Gal4-PDX-1 fusion proteins were unchanged (as determined by Western blots, data not shown). In contrast, the addition of the E1A mutant E1A{Delta}2–36 resulted in minimal suppression of the transcriptional activation potential of the Gal4-PDX-1 fusion protein (Fig. 1BGo). These findings indicate that a substantial component of the observed E1A-mediated suppression of PDX-1 transcriptional activation likely is mediated by the sequestration of the coactivator p300 by E1A proteins.

The addition of p300 directly to the Gal4-PDX-1 fusion protein increased the transcriptional activation of the Gal4-CAT reporter by 3- to 5-fold (Fig. 1Go, C and D). The extent of activation of PDX-1 that we observed with the addition of exogenous p300 was greater than that observed in insulin-producing cells (16) and may reflect differences in endogenous levels of p300 expressed in different cell types. The Gal4-PDX-1 construct increased the activation of the Gal4-CAT reporter in a dose-dependent manner with the addition of p300 as shown by increasing CAT enzyme activity (Fig. 1DGo, upper panel) under conditions in which the expression levels of the Gal4-PDX-1 fusion protein were not changed (as determined by Western blots, Fig. 1DGo, lower panel).

Interactions of PDX-1 and p300
To map the interaction domains between p300 and PDX-1, we combined in vitro-translated full-length rat PDX-1 protein with GST fusion proteins encompassing cysteine- and histidine-rich protein-protein interaction domains of human p300 (34), C/H1 (amino acids 1–595), C/H2 (amino acids 744-1571), and the carboxy-terminal C/H3 and Q-rich domains (amino acids 1572–2370) in GST pull-down assays (Fig. 2AGo). We observed the strongest binding of PDX-1 to the GST fusion protein encoding amino acids 1572–2370 and encompassing the C/H3 and Q-rich domains of human p300 (Fig. 2BGo). The wild-type in vitro-translated p300 protein interacted efficiently with GST-PDX-1, and a similar interaction was observed with mutant p300 lacking the C/H3 domain (amino acids 1737–1836 deleted, data not shown). These findings are supported by a previous report suggesting that one binding site for PDX-1 is located between amino acids 1947 and 2414 of p300 (16). Surprisingly we observed a second reproducible but lower affinity protein-protein interaction between PDX-1 and the GST fusion protein encoding amino acids 744-1571 of human p300 that was not observed between PDX-1 and the GST protein alone (Fig. 2BGo). These findings suggest that PDX-1 can interact with at least two distinct binding sites in p300. The stronger interaction site apparently lies between amino acids 1836 and 2370, and the weaker interaction site is located between amino acids 744 and 1571.



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  		FIG. 2. The amino terminus of PDX-1 interacts with carboxy-terminal domains of p300. A, Schematic models of PDX-1 and p300 with depiction of functional domains and numbered amino acids indicated. C/H denotes cysteine- and histidine-rich domains. The p300 model is adapted from Ref.34 . B, PDX-1 interacts strongly with the C/H3 and Q-rich domains and weakly with the C/H2 domain of p300. Radiolabeled in vitro-translated PDX-1 (+) or control reticulocyte lysates (–) were incubated with glutathione Sepharose fusion proteins encoding the C/H1 (amino acids 1–595), C/H2 (amino acids 744-1571), and the C/H3 and Q-rich (amino acids 1572–2370) domains of p300 (GST-p300) or GST alone (GST) in GST pull-down assays followed by separation on SDS-polyacrylamide gels and autoradiography. An autoradiogram from a representative experiment is shown with the migration position of [35S]PDX-1 (arrow) and 5% of input proteins indicated. C, The carboxy-terminal segment of p300 interacts with the amino-terminal domain but not the homeodomain and carboxy-terminal domain of PDX-1. GST-p300 (1572–2370) fusion proteins (+) or GST alone (pGEX5X-1) (–) control proteins were incubated with radiolabeled in vitro-translated PDX-1 proteins including the amino-terminal domain (amino acids 1–143), the homeodomain and carboxy-terminal domain (amino acids 143–283), and the amino-terminal domain and homeodomain (amino acids 1–206) as indicated. GST pull-down assays were separated by SDS-PAGE. Autoradiograms of SDS-polyacrylamide gels from a representative experiment are shown with GST pull-down assays (left panels) and 2% of input proteins (right panel). D, The PDX-1 amino-terminal transcriptional activation domain interacts with the carboxy-terminal segment of p300. GST-p300 (1572–2370) fusion proteins were incubated with radiolabeled in vitro-translated PDX-1 proteins encoding amino-terminal domains of PDX-1 [amino acids 1–78 (1 ), 1–143 (2 ), or full-length PDX-1 (WT, 3 )]. Autoradiograms from a representative experiment are shown with GST pull-down assays using GST-p300 (1572–2370) (left, GST-p300) or GST alone (right, GST).

 
We further explored the p300 binding sites within PDX-1 by conducting GST pull-down assays with GST fusion proteins encoding amino acids 1572–2370 of p300 and in vitro-translated PDX-1 mutant proteins (Fig. 2CGo). The PDX-1 mutant lacking the carboxy-terminal domain (amino acids 1–206) retained a strong interaction with the GST-p300 fusion protein spanning amino acids 1572–2370, whereas a PDX-1 mutant encompassing the homeodomain and carboxy-terminal domain, but lacking the amino-terminal 142 amino acids, did not. In multiple experiments the PDX-1(1–206) mutant demonstrated a stronger interaction with the p300 (1572–2370) region than did the PDX-1(1–143) mutant or the wild-type PDX-1 (data not shown). These data suggest that the PDX-1 carboxy-terminal domain may contribute to conformational states of PDX-1 that regulate interactions with p300. A PDX-1(1–143) mutant protein in which both the carboxy-terminal and homeodomain regions were deleted retained the ability to interact with the GST-p300 (1572–2370) fragment, albeit less well than did the PDX-1(1–206) mutant protein. The in vitro-translated PDX-1(1–78) mutant protein interacted with the GST-p300 (1572–2370) fragment although stronger interactions were observed with the PDX-1(1–143) mutant protein (Fig. 2DGo). Similar weak interactions with wild-type in vitro-translated p300 were observed with GST-PDX-1 mutant proteins encoding the amino-terminal transactivation domains (amino acids 1–38 or 1–78) in contrast to the strong interaction observed with full-length PDX-1 (data not shown). These studies demonstrated that a binding site for p300 exists within the amino-terminal domain of PDX-1 between amino acids 1 and 143 consistent with proposed binding sites for both p300 and its homolog CBP (13, 16).

Human PDX-1 mutant proteins associated with heritable diabetes interact strongly with p300
Several mutations in the human gene for pdx-1(ipf-1) confer a heritable predisposition to the development of diabetes (17, 18, 19, 20). We sought to determine whether PDX-1 mutant proteins known to be associated with type 2 diabetes might have altered binding affinities for p300. We used site-directed mutagenesis to generate a series of mutations in human PDX-1 cDNA associated with heritable forms of early-onset or type 2 diabetes. Radiolabeled mutant human PDX-1 proteins D76N (18, 19), A140T (27), InsCCG243 (18), and P63fsdelC (17) were synthesized in vitro and tested for their binding to the GST-p300 (1572–2370) fragment (Fig. 3Go, A and B). All of the human PDX-1 mutant proteins bound to GST-p300 (1572–2370) at least as well as the wild-type PDX-1. Strikingly the P63fsdelC PDX-1 mutant protein demonstrated a greater than 4.5-fold binding for GST-p300 (1572–2370), compared with wild-type PDX-1 on autoradiograms from GST pull-down assays (Fig. 3BGo), despite a substantial disadvantage in incorporated marker radioactivity in the input P63fsdelC PDX-1 protein (Fig. 3AGo).



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  		FIG. 3. The human PDX-1 mutant protein P63fsdelC exhibits a stronger interaction with the p300 (1572–2370) fragment than does the wild-type PDX-1 protein. A, Radiolabeled in vitro-translated human PDX-1 proteins were synthesized with the wild-type (WT) coding sequence or mutations known to be associated with diabetes in humans as indicated. PDX-1 proteins were incubated with GST-p300 (1572–2370), and GST pull-down assays were separated by SDS-PAGE. Autoradiograms from a representative experiment are shown with GST pull-down assays in the left panel (1572–2370) and the 2% input of radiolabeled proteins shown in the right panel. B, A series of three GST pull-down experiments including that shown in A were conducted with radiolabeled in vitro-translated human PDX-1 mutant proteins and GST-p300 (1572–2370). Autoradiograms were scanned, and quantitative results were normalized to the interaction seen with wild-type (WT) PDX-1 protein. Results shown are the mean ± SEM (n = 3; *, P < 0.05). C, Human PDX-1 mutant proteins have impaired binding to a human insulin promoter CT2 enhancer element oligonucleotide probe. An autoradiogram from a representative DNA-binding assay is shown. The specificity of the protein-DNA complexes is indicated by the attenuation of DNA-binding activity following incubation with anti-PDX-1 antiserum (± {alpha}-PDX-1). D, Dose-dependent (upper panel) and competition (lower panel) DNA-binding assays highlight the impaired binding of human PDX-1 mutant proteins to the human insulin promoter CT2 enhancer element. In vitro-translated human PDX-1 proteins were synthesized by coupled in vitro transcription/translation reactions, and comparable expression levels were determined by Western blots. Increasing amounts of human PDX-1 proteins (a, 1-fold; b, 2-fold; c, 3-fold) were incubated with a radiolabeled CT2 oligonucleotide probe in DNA-binding assays (upper panel). Normalized in vitro-translated human PDX-1 proteins were incubated for 10 min with a radiolabeled CT2 oligonucleotide probe in the absence (d) or presence of an increasing excess (e, 25-fold; f, 50-fold; g, 100-fold) of unlabeled CT2 oligonucleotide probe as indicated. Corresponding autoradiograms are shown.

 
The DNA-binding activities of the in vitro-translated human PDX-1 mutant proteins were decreased relative to wild-type PDX-1 (Fig. 3CGo). Quantitative analysis by densitometric scanning of autoradiograms derived from four independent DNA-binding experiments revealed the following significant (P < 0.05) reduced levels of binding to a human insulin promoter CT2 element for each of the following PDX-1 mutants relative to the binding of the wild-type protein (100%): D76N, 67.8 ± 14.6%; A140T, 60 ± 16.8%; InsCCG243, 67.9 ± 10%. In addition, these results were confirmed by dose-dependence and competition DNA-binding assays (Fig. 3DGo).

Point mutations in PDX-1 that impair interaction with p300 also impair transcriptional activation
Because PDX-1 can be phosphorylated by a wide variety of kinases in vitro (data not shown) and PDX-1 function is known to be regulated by a range of extracellular signals (reviewed in Ref.35), we reasoned that potential PDX-1 phosphorylation sites may be important candidates to regulate PDX-1 conformation and protein-protein interactions. By inspection of the amino-terminal sequence of PDX-1, we identified several conserved potential phosphorylation sites. In an attempt to more precisely identify regions of PDX-1 essential for interaction with p300 and transcriptional activation, we selected a series of conserved serine, threonine, or tyrosine residues within the first 143 amino acids of PDX-1 for site-directed mutagenesis. We generated the following point mutations in rat GST-PDX-1: Y14A, S29G/N31A, S61G, S66A, S66A/Y68A, and S124A/T125A. Mutant GST-PDX-1 fusion proteins were generated, and the input of the mutant proteins was normalized on Coomassie-blue stained SDS-polyacrylamide gels before GST pull-down assays with radiolabeled wild-type in vitro-translated p300. With equivalent protein input, several of the mutant GST-PDX-1 proteins demonstrated a significantly diminished interaction with full-length p300 (Fig. 4AGo), as illustrated by the mutant GST-PDX-1 S66A/Y68A (Fig. 4BGo). These reductions in interaction with p300 represented the sum of decreased interactions at one or more of multiple contact sites for PDX-1 within p300. We then studied the PDX-1 mutants in the context of their selective interaction with the p300 (1572–2370) region by evaluating the capacity of GST-p300 (1572–2370) to bind radiolabeled in vitro-translated mutant PDX-1 proteins (Fig. 4CGo). Although many of the PDX-1 mutants tested showed a trend toward diminished interactions with the GST-p300 (1572–2370) fusion protein, we found a significant 40% reduction in the interaction of the GST-p300 (1572–2370) fusion protein only with the S66A/Y68A PDX-1 mutant (Fig. 4Go, C and D), suggesting potential regional selectivity of interactions between the p300 (1572–2370) segment and the PDX-1 S66 and Y68 amino acids.



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  		FIG. 4. Point mutations in PDX-1 reduce the interaction with p300. A, Multiple mutant GST-PDX-1 proteins exhibit decreased binding to p300. GST-PDX-1 proteins with a series of point mutations as indicated were synthesized and assessed for interaction with radiolabeled in vitro-translated p300 in GST pull-down assays. Autoradiograms were scanned and quantitative results were normalized to the interaction seen with wild-type (WT) GST-PDX-1 protein. Results shown are the mean ± SEM (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001). B, GST-PDX-1 S66A/Y68A mutant proteins have decreased binding to p300. A representative autoradiogram is shown from an experiment in which wild-type GST-PDX-1 (WT), the mutant GST-PDX-1 S66A/Y68A, or GST alone were evaluated in GST pull-down assays for the ability to interact with radiolabeled in vitro-translated p300. The migration position of [35S]p300 is indicated (arrow). C, Mutant PDX-1 proteins exhibit decreased binding to GST-p300 (1572–2370). Radiolabeled in vitro-translated PDX-1 wild-type or mutant proteins were incubated with GST-p300 (1572–2370) in GST pull-down assays and subjected to SDS-PAGE. Autoradiograms were scanned and quantitative results were normalized to the interaction seen with wild-type (WT) PDX-1 protein. Results shown are the mean ± SEM (n = 3; **, P < 0.01). D, Radiolabeled PDX-1 S66A/Y68A mutant proteins have reduced binding to GST-p300 (1572–2370). Representative autoradiograms are shown of an SDS-polyacrylamide gel from an experiment in which radiolabeled in vitro-translated wild-type PDX-1 (WT) or PDX-1 S66A/Y68A proteins were incubated with GST-p300 (1572–2370) (+) or GST alone (–) in GST pull-down assays (left panels) and from the corresponding protein 2% input gel (right panels).

 
Next we evaluated the impact of the PDX-1 mutations on transcriptional activation and DNA-binding activity. The rat PDX-1 mutant proteins had comparable decrements in DNA-binding activity relative to the wild-type PDX-1 proteins on radiolabeled oligonucleotide probes encoding either Flat or P1 enhancer elements derived from the rat insulin I promoter, as illustrated by a representative DNA-binding assay with a radiolabeled Flat oligonucleotide probe (Fig. 5AGo). Quantitative analysis by densitometric scanning of autoradiograms derived from four independent DNA-binding experiments, two conducted with a Flat oligonucleotide probe and two conducted with a P1 oligonucleotide probe, showed the following significantly (P < 0.005) reduced DNA-binding activities for each of the following PDX-1 mutants relative to the binding of the wild-type protein (100%): S29G/N31A, 66.4 ± 9.2; S61G, 64.7 ± 5.7; S66A/Y68A, 49.5 ± 12.5; S124A/T125A, 42.6 ± 12.5.



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  		FIG. 5. The PDX-1 S66A/Y68A mutation impairs the transcriptional activation of PDX-1 in insulin-producing cells. A, PDX-1 amino-terminal mutations decrease DNA-binding to rat insulin I promoter enhancer sequences. In vitro-translated PDX-1 mutant (as indicated) or wild-type (WT) proteins and in vitro transcription/translation reactions conducted with empty expression vector (vector) were incubated with a radiolabeled rat insulin I promoter enhancer element Flat oligonucleotide probe followed by native gel electrophoresis and autoradiography. A representative autoradiogram is shown. B, The mutation S66A/Y68A reduces the transcriptional activation of PDX-1. INS-1 cells were transiently transfected in 11.1 mM glucose with 750 ng of the Gal4 expression plasmid pM, Gal4-PDX-1(1–143), Gal4-PDX-1(1–143) S61G, Gal4-PDX-1(1–143) S66A, Gal4-PDX-1(1–143) S61G/S66A, Gal4-PDX-1(1–143) S66A/Y68A, or Gal4-PDX-1(1–143) S124A/T125A and 500 ng of a Gal4-luciferase reporter. Transfected cells were cultured in 2 mM glucose for 24 h and then incubated with 2 mM glucose or 16.7 mM glucose (as indicated) for 6 h before harvest. Results shown are the mean ± SEM of data normalized to the activity of Gal4-PDX-1(1–143) in cells cultured in 2 mM glucose (n = 3–5 transfections conducted in duplicate; *, P < 0.05; ***, P < 0.001). C, The S66A/Y68A mutation reduces the transcriptional activation of PDX-1 to a similar extent as that seen by the addition of E1A. INS-1 cells were cultured in 11.1 mM glucose and transiently transfected with 500 ng of the Gal4 expression plasmid pM, Gal4-PDX-1(1–78), Gal4-PDX-1(1–78) S66A/Y68A, or 25 ng pCMV-E1A as indicated and 500 ng of a Gal4-luciferase reporter. Results shown are the mean ± SEM (n = 2–3 transfections conducted in duplicate). D, The S66A/Y68A mutation reduces the synergistic activation of rat insulin I promoter glucose-responsive FarFlat elements by PDX-1, E12 and E47 (E2A) proteins. HeLa cells were transiently transfected with 50 ng pcDNA3-E12 and 50 ng pcDNA3-E47 (E2A), 125 ng pcDNA3-PDX-1 encoding wild-type (PDX-1) or mutant rat PDX-1 cDNA, or pcDNA3 alone (vector) as indicated (+) in the presence of 500 ng FarFlat-luciferase reporter. Empty expression vectors were added to normalize total amounts of DNA in all transfections. Results shown are the mean ± SEM of data normalized to the activity of E12, E47, and wild-type PDX-1 in combination (n = 6 transfections conducted in duplicate; ns, no significant difference with P = 0.09; **, P < 0.005).

 
To study the transcriptional activation of PDX-1, Gal4-PDX-1 fusion proteins were evaluated for their abilities to activate a Gal4 reporter construct. Mutant Gal4-PDX-1(1–143) fusion constructs encoding the PDX-1 transcriptional activation domain (amino acids 1–143) were generated by site-directed mutagenesis. We generated the following point mutations in rat Gal4-PDX-1(1–143): Y14A, S29G/N31A, S61G, S66A, S66A/Y68A, and S124A/T125A. Clonal insulin-producing INS-1 cells were transiently transfected with Gal4-PDX-1(1–143) constructs and a reporter with multimerized Gal4 DNA-binding sites. In either the presence of 2 mM or 16.7 mM glucose, the Gal4-PDX-1(1–143) S66A/Y68A mutation significantly decreased the transcriptional activation of the Gal4 reporter construct by approximately 50% (Fig. 5BGo). Thus the S66A/Y68A point mutation in PDX-1 that consistently reduced the interaction of PDX-1 with the GST-p300 (1572–2370) fusion protein also diminished the transcriptional activation of PDX-1 in insulin-producing cells. In the context of a Gal4-PDX-1(1–78) fusion protein that contains a shorter segment of the PDX-1 transcriptional activation domain, the S66A/Y68A point mutations reduced the activation of the Gal4 reporter construct by 65% (Fig. 5CGo). The reduction in the transcriptional activation of PDX-1 as a result of this mutation was similar in magnitude to that observed for the wild-type PDX-1 with the addition of E1A protein expression to promote the sequestration of p300.

To evaluate the functional impact of the S66A/Y68A mutation in the context of a known transcriptional target of PDX-1, we used the experimental model system of synergistic activation of the glucose-responsive elements Far and Flat derived from the rat insulin I promoter. We compared the abilities of wild-type or mutant PDX-1 to activate a FarFlat-luciferase reporter construct and to synergize with E12 and E47 (Fig. 5DGo). In the absence of E12 and E47, the S66A/Y68A PDX-1 mutant showed a trend toward reduced activation of the reporter, compared with wild-type PDX-1, that was not statistically significant (wild-type PDX-1 16.4 ± 4.1%, compared with S66A/Y68A 9.6 ± 3.2%; n = 6, P = 0.09). The combination of the S66A/Y68A PDX-1 mutant with E12 and E47 did result in the synergistic activation of the FarFlat reporter. However, the maximal synergistic activation of the insulin promoter enhancer sequences was reduced by over 40% with the introduction of the S66A/Y68A mutation even though mutations in PDX-1 would not be expected to disrupt known interactions between p300 and E47 (14).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mutations in the pancreatic homeodomain transcription factor pdx-1(ipf-1) gene are associated with defects in pancreas development, insulin production, and glucose homeostasis in humans. Regulated interactions of PDX-1 with the coactivator p300 are likely to be important for the transcriptional activation of PDX-1 target genes (13, 16). Here we identified the point mutations S66A/Y68A located within the transcriptional activation domain of PDX-1 that impair both the transcriptional activation of PDX-1 and its ability to interact with the coactivator p300. In addition, we discovered that the previously identified mutation P63fsdelC in the human pdx-1(ipf-1) gene, associated with pancreatic agenesis in a homozygous carrier (6) and MODY4 in heterozygous carriers (17), yields a mutant PDX-1 protein with increased binding affinity for p300.

We propose that mutations in the PDX-1 protein that alter its conformation resulting in either increased or decreased interactions with the coactivator p300 may result in metabolic dysfunction (Fig. 6Go). For PDX-1 mutations in which binding to p300 is preserved or enhanced but DNA-binding affinity for target genes is reduced, the mutant PDX-1 may sequester p300 and impair the necessary chromatin remodeling and assembly of basal transcription machinery required for target gene activation (Fig. 6BGo). In individuals who are heterozygous for pdx-1(ipf-1) mutations, such a mechanism could result in competition between the wild-type and mutant PDX-1 proteins for interaction with p300. We find that the smaller amino-terminal protein derived from the pdx-1(ipf-1) P63fsdelC mutation has at least a 4.5-fold greater capacity to bind p300, compared with that of wild-type PDX-1. This finding suggests the possibility that an additional dominant-negative mechanism may be operative in which p300 is sequestered in the cytoplasm by the PDX-1 mutant protein, thereby depleting the nuclear p300 available for target gene activation. The intracellular distribution of rate-limiting levels of coactivators like p300 is important in the control of signal transduction (34, 36). It is noteworthy that among the pdx-1(ipf-1) mutations we studied, the P63fsdelC mutation is associated with the most severe clinical phenotype as manifest by the earliest age of onset of diabetes (MODY4).



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  		FIG. 6. Models of impairment of PDX-1 and p300 interactions in diabetes. A, Model of active PDX-1 target gene (INSULIN) transcription in pancreatic ß-cells in which PDX-1 in conjunction with other transcription factors (E12 and E47) recruits p300 to enhance the assembly of basal transcription machinery (POL II) to activate gene transcription. B, Model of impaired PDX-1 target gene transcription in which mutant PDX-1 proteins fail to bind to target gene enhancer sequences but bind p300 with high affinity, sequestering p300 and limiting the activation of gene transcription even in the presence of wild-type PDX-1 (WT). C, Model of impaired PDX-1 target gene transcription in which mutant PDX-1 proteins bind p300 poorly and limit the activation of gene transcription. D, Model of impaired PDX-1 target gene transcription in which p300 expression is rate limiting with limited activation of gene transcription.

 
A second possible mechanism by which mutations in PDX-1 may diminish the transcriptional activation of target genes such as insulin and result in metabolic dysfunction is illustrated by the mutation S66A/Y68A that results in decreased protein-protein interactions of p300 and PDX-1 (Fig. 6CGo). Although to date we have not encountered a human mutation in the pdx-1(ipf-1) gene that fits this pattern, our mutagenesis studies indicate that this type of mutation in PDX-1 would be expected to disrupt target gene activation and diminish insulin production. A third mechanism by which metabolic dysfunction associated with abnormal interactions between PDX-1 and p300 may occur would not require a mutation in PDX-1 (Fig. 6DGo). In pathophysiologic settings in which expression levels of p300 are diminished in pancreatic ß-cells, it is conceivable that insufficient p300 protein would be available for appropriate PDX-1 target gene activation. In a transgenic mouse model of Huntington’s disease that progressively develops insulin deficiency and diabetes, we observed age-dependent reductions in the expression levels of both PDX-1 and p300 in pancreatic islets that correlated with progressive reductions in insulin gene expression and glucose tolerance (37).

The interactions between PDX-1 and p300 are likely to be more complex than previously appreciated. Our studies indicate that multiple potential interaction domains for PDX-1 lie within the full-length p300 molecule. We found a high-affinity binding site in the carboxy-terminal domain of p300 supported by an earlier report (16) and identified a new lower affinity binding site between amino acids 744 and 1571, encompassing the C/H2 domain implicated in a wide range of protein-protein interactions. The disruption of the transcriptional activation of PDX-1 by the S66A/Y68A mutation suggests that either the S66 and Y68 amino acids within PDX-1 represent direct contact points for p300 or alternatively that these amino acids are important regulators of PDX-1 conformation to promote interaction with p300 at other sites. Despite the apparent function of amino acids 66 and 68 in PDX-1 to facilitate interactions with p300 and promote the transcriptional activation of PDX-1 target genes, multiple regions within the PDX-1 amino-terminal transactivation domain may represent sites for interactions with p300.

The importance of the coactivator p300 in the regulation of pancreatic ß-cell function extends beyond the regulation of PDX-1 functions. Mutations in other transcriptional regulators important in the regulation of glucose metabolism have been reported to impair interactions with p300. For example, the MODY6 mutation 206+C in the Beta-2/neuroD1 gene encodes a mutant protein that fails to bind a p300 fragment known to interact with native Beta-2/neuroD1 (38). Similarly, the MODY1 mutations R154X and E276Q interfere with p300 recruitment and transcriptional activation by HNF4{alpha} (39).

Previously we proposed that reduced PDX-1 function, even in the absence of inherited mutations, can limit insulin production and lead to the development of hyperglycemia, particularly in aging populations (25). Our findings suggest that appropriately regulated interactions between PDX-1 and p300 will be an important component in the pursuit of therapeutic approaches to maintain and restore PDX-1 transcriptional activation functions in patients with diabetes. The mapping of protein-protein interaction surfaces between PDX-1 and p300 should facilitate future efforts to develop small molecule approaches to enhance the transcriptional activation of PDX-1 and restore insulin production.


   Acknowledgments
 
The authors thank M. German, D. Livingston, H. Lu, L. G. Moss, R. Stein, and C. Wollheim for their generous gifts of cells and reagents. We thank the members of the Laboratory of Molecular Endocrinology for input and suggestions during the course of these studies. We appreciate the expert administrative assistance of Kimberly MacDonald in the preparation of this manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK58783 and DK02476 (to M.K.T.) and DK55365 (to J.F.H.). J.F.H. is an investigator with the Howard Hughes Medical Institute.

Abbreviations: CAT, Chloramphenicol transferase; GST, glutathione- S-transferase; MODY, maturity-onset diabetes of the young; pCMV, polyclonal cytomegalovirus; PDX-1, pancreas duodenum homeobox-1; SDS, sodium dodecyl sulfate.

Received September 9, 2003.

Accepted for publication February 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BLM, Wright CVE 1996 PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122:983–995[Abstract]
  2. Miller CP, McGehee RE, Habener JF 1994 IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene. EMBO J 13:1145–1156[Medline]
  3. Ohlsson H, Karlsson K, Edlund T 1993 IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J 12:4251–4259[Medline]
  4. Leonard J, Peers B, Johnson T, Ferreri K, Lee S, Montminy MR 1993 Characterization of somatostatin transactivating factor-1, a novel homeobox factor that stimulates somatostatin expression in pancreatic islet cells. Mol Endocrinol 7:1275–1283[Abstract/Free Full Text]
  5. Jonsson J, Carlsson L, Edlund T, Edlund H 1994 Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371:606–609[CrossRef][Medline]
  6. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF 1997 Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 coding region. Nat Genet 15:106–110[CrossRef][Medline]
  7. German MS, Wang J, Chadwick RB, Rutter WJ 1992 Synergistic activation of the insulin gene by a LIM-homeodomain protein and a basic helix-loop-helix protein: building a functional insulin minienhancer complex. Genes Dev 6:2165–2176[Abstract/Free Full Text]
  8. Petersen HV, Serup P, Leonard J, Michelsen BK, Madsen OD 1994 Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through CT boxes. Proc Natl Acad Sci USA 91:10465–10469[Abstract/Free Full Text]
  9. Melloul D, Ben-Neriah Y, Cerasi E 1993 Glucose modulates the binding of an islet-specific factor to a conserved sequence within the rat I and the human insulin promoters. Proc Natl Acad Sci USA 90:3865–3869[Abstract/Free Full Text]
  10. Marshak S, Totary H, Cerasi E, Melloul D 1996 Purification of the ß-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells. Proc Natl Acad Sci USA 93:15057–15062[Abstract/Free Full Text]
  11. Johnson JD, Zhang W, Rudnick A, Rutter WJ, German MS 1997 Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity. Mol Cell Biol 17:3488–3496[Abstract]
  12. Ohneda K, Mirmira RG, Wang J, Johnson JD, German MS 2000 The homeodomain of PDX-1 mediates multiple protein-protein interactions in the formation of a transcriptional activation complex on the insulin promoter. Mol Cell Biol 20:900–911[Abstract/Free Full Text]
  13. Asahara H, Dutta S, Kao HY, Evans RM, Montminy M 1999 Pbx-Hox heterodimers recruit coactivator-corepressor complexes in a isoform-specific manner. Mol Cell Biol 19:8219–8225[Abstract/Free Full Text]
  14. Qiu Y, Sharma A, Stein R 1998 p300 Mediates transcriptional stimulation by the basic helix-loop-helix activators of the insulin gene. Mol Cell Biol 18:2957–2964[Abstract/Free Full Text]
  15. Thomas MK, Yao KM, Tenser MS, Wong GG, Habener JF 1999 Bridge-1, a novel PDZ-domain coactivator of E2A-mediated regulation of insulin gene transcription. Mol Cell Biol 19:8492–8504[Abstract/Free Full Text]
  16. Qiu Y, Guo M, Huang S, Stein R 2002 Insulin gene transcription is mediated by interactions between the p300 coactivator and PDX-1, BETA2, and E47. Mol Cell Biol 22:412–420[Abstract/Free Full Text]
  17. Stoffers DA, Ferrer J, Clarke WL, Habener JF 1997 Early-onset type-II diabetes mellitus (MODY4) linked to IPF-1. Nat Genet 17:138–139[CrossRef][Medline]
  18. Hani EH, Stoffers DA, Chevre JC, Durand E, Stanojevic V, Dina C, Habener JF, Froguel P 1999 Defective mutations in the insulin promoter factor-1 (IPF-1) gene in late-onset type 2 diabetes mellitus. J Clin Invest 104:R41–R48
  19. Macfarlane WM, Frayling TM, Ellard S, Evans JC, Allen LI, Bulman MP, Ayres S, Shepherd M, Clark P, Millward A, Demaine A, Wilkin T, Docherty K, Hattersley AT 1999 Missense mutations in the insulin promoter factor-1 gene predispose to type 2 diabetes. J Clin Invest 104:R33–R39
  20. Weng J, Macfarlane WM, Lehto M, Gu HF, Shepherd LM, Ivarsson SA, Wibell L, Smith T, Groop LC 2001 Functional consequences of mutations in the MODY4 gene (IPF1) and coexistence with MODY3 mutations. Diabetologia 44:249–258[CrossRef][Medline]
  21. Weng J, Ekelund M, Lehto M, Li H, Ekberg G, Frid A, Aberg A, Groop LC, Berntorp K 2002 Screening for MODY mutations, GAD antibodies, and type 1 diabetes-associated HLA genotypes in women with gestational diabetes mellitus. Diabetes Care 25:68–71[Abstract/Free Full Text]
  22. Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H 1998 ß-Cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the ß-cell phenotype and maturity onset diabetes. Genes Dev 12:1763–1768[Abstract/Free Full Text]
  23. Dutta S, Bonner-Weir S, Montminy M, Wright C 1998 Regulatory factor linked to late-onset diabetes? Nature 392:560[Medline]
  24. Brissova M, Shiota M, Nicholson WE, Gannon M, Knobel SM, Piston DW, Wright CV, Powers AC 2002 Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion. J Biol Chem 277:11225–11232[Abstract/Free Full Text]
  25. Thomas MK, Devon ON, Lee JH, Peter A, Schlosser DA, Tenser MS, Habener JF 2001 Development of diabetes mellitus in aging transgenic mice following suppression of pancreatic homeoprotein IDX-1. J Clin Invest 108:319–329[CrossRef][Medline]
  26. Holland AM, Hale MA, Kagami H, Hammer RE, MacDonald RJ 2002 Experimental control of pancreatic development and maintenance. Proc Natl Acad Sci USA 99:12236–12241[Abstract/Free Full Text]
  27. Hansen L, Urioste S, Petersen HV, Jensen JN, Eiberg H, Barbetti F, Serup P, Hansen T, Pedersen O 2000 Missense mutations in the human insulin promoter factor-1 gene and their relation to maturity-onset diabetes of the young and late-onset type 2 diabetes mellitus in Caucasians. J Clin Endocrinol Metab 85:1323–1326[Abstract/Free Full Text]
  28. Stoffers DA, Stanojevic V, Habener JF 1998 Insulin promoter factor-1 gene mutation linked to early-onset type 2 diabetes mellitus directs expression of a dominant negative isoprotein. J Clin Invest 102:232–241[Medline]
  29. Thomas MK, Rastalsky N, Lee JH, Habener JF 2000 Hedgehog signaling regulates insulin production by pancreatic ß-cells. Diabetes 49:2039–2047[Abstract/Free Full Text]
  30. Thomas MK, Lee JH, Rastalsky N, Habener JF 2001 Hedgehog signaling regulation of homeodomain protein IDX-1 expression in pancreatic ß cells. Endocrinol 142:1033–1040[Abstract/Free Full Text]
  31. Peers B, Leonard J, Sharma S, Teitelman G, Montminy MR 1994 Insulin expression in pancreatic islet cells relies on cooperative interactions between the helix loop helix factor E47 and the homeobox factor STF-1. Mol Endocrinol 8:1798–1806[Abstract/Free Full Text]
  32. Stein RW, Whelan J 1989 Insulin gene enhancer activity is inhibited by adenovirus 5 E1a gene products. Mol Cell Biol 9:4531–4534[Abstract/Free Full Text]
  33. Stein RW, Corrigan M, Yaciuk P, Whelan J, Moran E 1990 Analysis of E1A-mediated growth regulation functions: binding of the 300-kildalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity. J Virol 64:4421–4427[Abstract/Free Full Text]
  34. Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG, Juguilon H, Montminy M, Evans RM 1996 Role of CBP/p300 in nuclear receptor signaling. Nature 383:99–103[CrossRef][Medline]
  35. Thomas MK, Habener JF 2004 IDX1 and pancreatic agenesis and type 2 diabetes. In: Epstein CJ, Erickson RP, Wynshaw-Boris A, eds. Inborn errors of development: the molecular basis of clinical disorders of morphogenesis. Oxford, UK: Oxford University Press; 552–556
  36. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[CrossRef][Medline]
  37. Andreassen OA, Dedeoglu A, Stanojevic V, Hughes DB, Browne SE, Leech CA, Ferrante RJ, Habener JF, Beal MF, Thomas MK 2002 Huntington’s disease of the endocrine pancreas: insulin deficiency and diabetes mellitus due to impaired insulin gene expression. Neurobiol Dis 11:410–424[CrossRef][Medline]
  38. Malecki MT, Jhala US, Antonellis A, Fields L, Doria A, Orban T, Saad M, Warram JH, Montminy M, Krolewski AS 1999 Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus. Nat Genet 23:323–328[CrossRef][Medline]
  39. Eeckhoute J, Formstecher P, Laine B 2001 Maturity-onset diabetes of the young type 1 (MODY1)-associated mutations R154X and E276Q in hepatocyte nuclear factor 4alpha (HNF4{alpha}) gene impair recruitment of p300, a key transcriptional coactivator. Mol Endocrinol 15:1200–1210[Abstract/Free Full Text]



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