The PDX-1 Activation Domain Provides Specific Functions Necessary for Transcriptional Stimulation in Pancreatic ß-Cells

Mina Peshavaria1, Michelle A. Cissell, Eva Henderson, Helle V. Petersen and Roland Stein

Department of Molecular Physiology and Biophysics (M.P., M.A.C., E.H., R.S.) Vanderbilt University Medical School Nashville, Tennessee 37232
Hagedorn Research Institute (H.V.P.) DK-2820 Gentofte, Denmark


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PDX-1 is a homeodomain transcription factor whose targeted disruption results in a failure of the pancreas to develop. Mutations in the human pdx-1 gene are linked to an early onset form of non-insulin-dependent diabetes mellitus. PDX-1 binds to and transactivates the promoters of several physiologically relevant genes within the ß-cell, including insulin, glucose transporter 2, glucokinase, and islet amyloid polypeptide. This study focuses on the mechanisms by which PDX-1 activates insulin gene transcription. To evaluate the role of PDX-1 in transcription of the insulin gene, a chloramphenicol acetyltransferase reporter construct was designed with a single yeast GAL4-DNA binding site in place of the A3/PDX-1 binding element in the rat insulin II enhancer. In the presence of GAL4:PDX chimeras containing N-terminal transactivation domain sequences, this GAL4-substituted insulin construct was active in PDX-1-expressing ß-cells and not non-ß-cells. PDX-1 activation was mediated through three highly conserved segments of the transactivation domain. In addition, when cotransfected together with the GAL4-substituted insulin enhancer reporter gene in glucose-responsive MIN-6 ß-cells, glucose-induced activation is observed with GAL4:PDX-1 but not with fusions of the heterologous activation domains from herpes virus VP16 or adenovirus-5 E1A proteins. Using A3 element-substituted GAL4 insulin enhancer reporter constructs containing mutations in two additional key control elements, E1 and C1, we also show that full activation requires cooperative interactions between other enhancer-bound factors, particularly the E1 element activators. In contrast, the activity of the VP16 activation factor was not dependent on the activators of either the E1 or C1 sites. These results suggest that the PDX-1 transactivation domain is specifically required for appropriate regulation of insulin enhancer function in ß-cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin gene expression is tightly restricted to the pancreatic ß-cells within the islets of Langerhans. Tissue specificity is conferred by a highly conserved enhancer region located within approximately 350 bp of the insulin gene transcription start site; multiple positively and negatively acting cis-elements within the enhancer coordinately modulate insulin gene transcription (1, 2, 3, 4). Detailed mutagenesis studies conducted on this region have identified several key control elements, including the C2 (-317/-311 bp) (2), A3 (-201 to -196 bp) (5, 6), C1/RIPE3b (-118 and -107 bp) (7, 8) and E1 (-100 to -91 bp) (9, 10, 11) elements. The core sequence motifs for each of these elements are also found within the transcription unit of all characterized mammalian insulin genes (12), suggesting that similar transcriptional regulatory mechanisms are used in control.

The A3 element contributes to both ß-cell-selective (5, 6, 10, 13) and glucose-regulated transcription (13, 14, 15) of the insulin gene. The ß-cell-enriched homeodomain factor, PDX-1 (pancreatic and duodenal homeobox protein 1; also known as STF-1, IDX-1, IPF-1, IUF-1), controls A3 element-directed transcription (6, 13, 16, 17, 18). Another PDX-1 binding site is present within the insulin transcription unit at -81 to -77 bp; however, this site appears to be much less important than A3 in control (6). PDX-1 also activates expression of other islet genes, including GLUT2 (19), islet amyloid polypeptide (IAPP) (20, 21), somatostatin (22, 23), glucokinase (24), and HB-EGF (25).

The expression pattern of PDX-1 implicates this transcription factor in the development of ß-cell identity. During embryogenesis, PDX-1 expression is observed in the ventral and dorsal walls of the primitive foregut where the pancreatic buds will form (16). As development proceeds, PDX-1 is detectable in islet cell precursors and, transiently, in exocrine and ductal cells (26). In the adult pancreas, PDX-1 expression becomes restricted to the islets (6, 16, 22, 23, 26), where it is found in almost all (>90%) ß-cells as well as a subset of {alpha} (3%) and {delta} (15%) cells (6). PDX-1 expression is also observed in the submucosal epithelium of the duodenum (22, 23, 26) and antral stomach (27).

Germline deletion of the mouse pdx-1 gene also supports a critical role for this protein in pancreatic development. Homozygous knockout of the mouse pdx-1 locus results in pancreatic agenesis (28, 29) and malformations of the rostral duodenum (29). Furthermore, selective removal of the pdx-1 gene in islet ß-cells of mice has also clearly demonstrated that PDX-1 plays a key role in vivo in generating functional ß-cells, at least in part, through its actions on insulin, IAPP, and GLUT2 expression (30). In this experiment, a critical region of the endogenous pdx-1 gene, flanked by loxP sites, was conditionally removed in ß-cells by expression of insulin enhancer/promoter-driven Cre recombinase. Mice carrying this ß-cell-specific pdx-1 deletion developed diabetes (30). In humans, an inactivating mutation in the ipf1/pdx-1 locus is linked to a form of early-onset diabetes when inherited in a heterozygous state (31). Moreover, a patient that is homozygous for this pdx-1 mutation failed to develop a pancreas (32).

PDX-1 is analogous to most other transcription factors in possessing a modular structure composed of separable functional domains. The highly conserved homeodomain within the middle of the protein confers both DNA-binding and nuclear translocation functions (33, 34), whereas the N-terminal region provides most of the transactivation potential (17, 33, 35), although a cryptic activation domain in the C terminus may also be involved in stimulating somatostatin gene expression in {delta}-cells (33). Within the N terminus, at least three evolutionarily conserved subdomains are critical for transactivation (35). PDX-1 mutants lacking these subdomains act as dominant negative inhibitors due to their ability to translocate to the nucleus and bind DNA but not stimulate transcription (35, 36).

Our current understanding of the amino acid sequences within the N-terminal region of PDX-1 that are required for transcriptional activation has been derived primarily from analysis of PDX-1 in non-ß-cells or from GAL4:PDX-1 fusion proteins tested with simple yeast GAL4 element-driven constructs in ß-cells (17, 33, 35, 37, 38). These artificial constructs consisted of multimerized GAL4 binding sites driven by the E1b promoter and allowed the mapping of the activation domain in both ß- and non-ß-cell types. Although these approaches have provided useful information about the general sequences involved in activation, it is important to note that these experiments do not measure transcriptional regulation within the context of the native insulin enhancer in ß-cells. To define the factors regulating PDX-1-activated transcription in this environment, the A3 element of the rat insulin II enhancer was replaced with a single binding site for the yeast protein, GAL4. This assay system allows us to study the mechanisms involved in PDX-1 (i.e. GAL4:PDX-1) activation of the rat insulin II enhancer in the ß-cell without interference from the endogenous factor. The results from this study demonstrate that the PDX-1 activation domain is specifically required for ß-cell-selective and glucose-stimulated activation of the insulin gene by functionally interacting with other insulin enhancer-bound factors. We discuss the possible significance of these findings to other PDX-1-activated genes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GAL4:PDX-1 Only Stimulates -217 GAL Activity in ß-Cells
An insulin reporter gene in which the PDX-1 control region between -205 to -189 bp was replaced with a single yeast GAL4 DNA binding site was used to study how PDX-1 activates insulin gene transcription (Fig. 1AGo; -217 GAL). Replacement of the A3 element with the GAL4 binding site reduced insulin enhancer-mediated activity to the same extent as a site-directed A3 mutant that prevented PDX-1 binding (Ref. 6 and data not shown), demonstrating that this mutation does not influence the activity of other immediate 5'- or 3'-control elements. To test whether GAL4:PDX-1 fusion proteins could stimulate insulin enhancer activity, a fusion construct representing the full-length [GAL4:PDX(FL)] and N-terminal activation domain region [GAL4:PDX(1–149)] of rat PDX-1 was cotransfected with -217 GAL into either a ß- (HIT T-15, ßTC3, INS-1) or non-ß- (HeLa, BHK) cell line. GAL4:PDX(FL) and GAL4:PDX(1–149) potentiated -217 GAL activity, but only in ß-cell lines (Fig. 1Go). The GAL4:PDX constructs were equally active, stimulating approximately 3- to 8-fold, depending upon the ß-cell line (Fig. 1Go). We have previously shown that GAL4:PDX(FL) and GAL4:PDX(1–149) are effectively expressed, and at comparable levels, in both HIT-T15 and HeLa cells (35), demonstrating that their inactivity in non-ß-cells is not due simply to the lack of PDX-1.



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Figure 1. GAL4:PDX-1 Only Activates -217 GAL Activity in ß-Cells

A, Schematic of the -217 to +2 bp region of the rat insulin II gene. The enhancer domain (-217 to -91 bp) contains a binding site for the PDX-1 (A3, -201 to -196 bp), RIPE3b1 (C1, -115 to -107 bp), and BETA2/NeuroD1:E47 (E1, -100 to -91 bp) activators. The A3 element was replaced by a single GAL4 binding site (-217 GAL); the insulin sequences are linked to the CAT reporter gene. The sequence of the A3 element (bold) is shown in comparison to the GAL4-substituted element (bold and underlined). A total of 16 nucleotides were changed by the GAL4 substitution. B, The GAL4-substituted insulin enhancer construct, -217 GAL, was cotransfected with vector alone (-) or with vectors encoding fusions of the GAL4 DNA-binding domain with full-length [GAL4:PDX(FL)] or amino acids 1 through 149 [GAL4:PDX(1–149)] of PDX-1. Transfections were performed in ß (HIT T-15, ßTC3, and INS-1) and non-ß lines (HeLa and BHK). The CAT activity in each sample was normalized to the LUC activity from the cotransfected pGL2RSV plasmid. Results are presented as relative to -217 GAL cotransfected with the GAL4 DBD vector alone ± SD; the -217 GAL alone point was arbitrarily set to a value of 1.0. Each transfection represents an average of at least four separate experiments.

 
To further explore the ß-cell specificity of PDX-1, we directly compared in ß (HIT T-15) and non-ß (HeLa) cells the ability of GAL4:PDX-1 fusion proteins to activate GAL4 target genes in three distinct promoter contexts: 1) a single GAL4 binding site within the insulin enhancer (-217 GAL LUC); 2) a single GAL4-binding site linked to the minimal E1b promoter [(GAL4)1E1b LUC]; and, 3) five GAL4 binding sites linked to the E1b promoter [(GAL4)5E1b LUC]. GAL4:PDX-1 is a very poor transactivator of (GAL4)1E1b LUC in either HIT-T15 or HeLa cells, whereas (GAL4)5E1b LUC was transcriptionally active in both ß- and non-ß-cells (though to higher level in ß-cells) (Fig. 2Go). In contrast, GAL4:PDX-1 activated -217 GAL LUC effectively only in ß-cells (Fig. 2Go). These results clearly demonstrate that a single PDX-1 binding unit cannot support cell type-specific transcription in isolation, whereas a single PDX-1 site is sufficient in an insulin enhancer context.



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Figure 2. Functional Interactions between PDX-1 and a ß-Cell- Enriched Activator(s) of -217 GAL Are Crucial for Transcriptional Stimulation

The -217 GAL LUC, (GAL4)1E1b LUC, or (GAL4)5E1b LUC vectors were cotransfected into HIT-T15 and HeLa cells either alone or with GAL4DBD, GAL4:PDX(FL), or GAL4:PDX(1–149). The LUC activity of each sample was normalized to the CAT activity of cotransfected pRSVCAT. The relative activity ± SD is calculated as the activity of reporter vector plus the GAL4 expression construct divided by the reporter vector alone. Note that the overall stimulatory pattern of GAL4:PDX with -217 GAL LUC and (GAL4)5E1b LUC is identical to that observed with the analogous CAT reporter constructs [-217 GAL, Fig. 1Go; (GAL4)5E1b CAT (17 35 )], although the magnitude of the response differs.

 
Collectively, the data suggest that the activity of PDX-1 transactivation domain per se is not ß-cell specific, but requires other ß-cell-restricted enhancer-bound factors to promote selective expression of the insulin gene. The following experiments were designed to identify the sequences within PDX-1 that mediate insulin enhancer activation, and to determine the role of this region of PDX-1 in glucose-inducible insulin gene transcription. In addition, the ß-cell-enriched activators that cooperate with PDX-1 to stimulate insulin enhancer activity were identified.

The PDX-1 Transactivation Domain Mediates GAL4:PDX-1 Activity
To define the sequences of PDX-1 required for insulin enhancer activation in ß-cells, N- and C-terminal GAL4 fusion mutants of rat PDX-1 and the Xenopus homolog, XlHbox8, were assayed for their ability to stimulate -217 GAL activity in HIT T-15 ß-cells (Fig. 3Go). The rat and Xenopus proteins share 100% and approximately 50% identity in their homeodomains [amino acids (aa) 146–206] and N-terminal regions (aa 1–145), respectively, whereas there is little conservation within the C-terminal region (35). The activation domain of these proteins was localized to aa 1–79 using a multimerized GAL4 binding site-reporter-driven construct [(GAL4)5E1b; (17, 35)]. Transactivation is mediated by three highly conserved sequences that span aa 13–22 (subdomain A), 32–38 (subdomain B), and 60–73 (subdomain C) (35). Deletion of all three subdomains [GAL4:PDX(1–149{Delta}ABC)] results in 98% reduction of activity of the (GAL4)5E1b reporter vector in HeLa cells (35). Each of the GAL4:PDX fusion proteins analyzed here is expressed at equivalent levels in transfected HIT T-15 (Ref. 35 and data not shown).



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Figure 3. The N-Terminal Regions of PDX-1 Are Crucial for Transcriptional Activation

Diagrammatic representation of rat PDX-1 and Xenopus XlHbox8 (not drawn to scale). XlHbox8 sequences and homeodomain sequences (HD, 146–206) are indicated by hatched and gray boxes, respectively. The activation domain (AD) is located between amino acids 1–79. The deletion end points of PDX-1 and XlHbox8 in the GAL4 chimeras are indicated. The transcriptional activities of the full-length (FL) and mutant PDX-1 and XlHbox8 GAL4 fusion proteins with -217 GAL were examined in HIT T-15 cells. The normalized CAT activity from -217 GAL plus GAL4:PDX-1 is reported relative to -217 GAL cotransfected with pSG424 alone. Each value represents an average of at least three transfections ± the SE of the data.

 
GAL4:PDX-1 and GAL4:XlHbox8 proteins lacking N-terminal sequences [GAL4:PDX(76–284) and GAL4:XlHbox8(91–271)] were unable to potentiate -217 GAL (Fig. 3Go). In contrast, the GAL4 constructs containing conserved N-terminal sequences of PDX–1 or XlHbox8 [GAL4:PDX/XlHbox8(1–79/1–75), GAL4:PDX/XlHbox8(1–149/1–150), GAL4:PDX/XlHbox8(1–210/1–210), and GAL4:PDX/XlHbox8(FL/FL)] stimulated reporter gene activity effectively. GAL4:PDX(FL) and GAL4:XlHbox8(FL) stimulate (GAL4)5E1b chloramphenicol acetyltransferase (CAT) activity poorly relative to GAL4:PDX(1–149) (17, 35, 38), yet the homeodomain and C-terminal regions of PDX-1 or XlHbox8 did not have any significant effect on -217 GAL activation (Fig. 3Go). To test this proposal more thoroughly, the nonconserved C-terminal sequences of Xenopus XlHbox8 were substituted for those of rat PDX-1 in GAL:PDX(FL). As expected, activation by this GAL4 chimera was similar to deletion mutants lacking the C terminus, such as GAL4:PDX(1–210) (data not shown).

Mutants within the conserved N-terminal subdomains of PDX-1 were next tested for their ability to activate -217 GAL. This analysis included aa 13–22, 32–38, 60–73, and 81–85 (i.e. subdomain D) deletion mutants, as well as site-directed mutants within aa 117–126 (i.e. subdomain E) that eliminate the potential interaction of PDX-1 with PBX-1 or phosphorylation by protein kinase C. Deletion of the A, B, or C subdomains individually reduced GAL4:PDX(1–149) activity by 25–35% (Fig. 4Go). In contrast, little or no effect on -217 GAL activation was found upon deletion of subdomain D or by preventing PBX-1 interaction or serine/threonine phosphorylation within the aa 117–126 region. The latter result is consistent with a previous observation demonstrating that the interaction of PDX-1 with PBX-1 is not required for insulin transcription in ß-cells, although it does affect somatostatin expression in islet {delta}-cells (39). An N-terminal mutant spanning aa 76–149 was unable to transactivate -217 GAL (Fig. 4Go), further emphasizing the requirement for PDX-1 activation domain sequences in this process. Thus, it was not surprising that stimulation by GAL4:PDX(1–149{Delta}ABC) was severely impaired (i.e. by 70%; Fig. 4Go). However, GAL4:PDX(1–149{Delta}ABC) activation of -217 GAL was not entirely lost, as it was with (GAL4)5E1b CAT (35), suggesting that as-yet-unidentified interactions, within either less conserved sequences and/or subdomains D and E, are uniquely required for transactivation of the insulin enhancer.



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Figure 4. Contribution of the Transactivation Subdomains to the Activity of GAL4:PDX(1–149)

The conserved amino acid segments (A, 13–22; B, 32–38; C, 60–73; D, 87–91; Pbx, 119–123; PKC, 124–125) and homeodomain sequences (HD, 146–149) are indicated by black and gray boxes, respectively. The deleted subdomains are indicated with an X. HIT-T15 cells were transfected with -217 GAL vector alone and either pSG424 (GAL4DBD), GAL4:PDX(1–149) or a mutant form of GAL4:PDX(1–149). The results are calculated as a percentage of the normalized CAT activity of the GAL4:PDX(1–149) expression vector ± SEM.

 
Glucose Stimulates PDX-1 Transactivation Domain Function
Glucose-stimulated transcription of the insulin gene is mediated through multiple enhancer elements, including A3 (13, 14, 15, 40), C1 (8, 41), and E1 (8, 41, 42). The A3 element of the insulin enhancer contributes to glucose responsiveness in part through increased PDX-1 binding activity (13, 15, 40). To examine the contribution of the PDX-1 transactivation domain to glucose induction, GAL4:PDX(1–149) stimulation of -217 GAL was compared with GAL4 fusions of potent, but unrelated, viral activation domains [GAL4:VP16 and GAL4:E1A(121–223)] in MIN-6 ß-cells. The wild-type insulin enhancer (-238 WT CAT) was induced 4-fold in cells grown in 20 mM (high) vs. 3 mM (low) glucose (Fig. 5Go). As expected, replacement of the A3 element with the GAL4 binding site reduced the level of glucose induction (Fig. 5Go). GAL4:PDX(1–149) fully restored glucose induction of -217 GAL to wild-type levels (Fig. 5Go). In contrast, the transactivation domains of the herpes virus VP16 and adenovirus E1A proteins were incapable of inducing -217 GAL activity in glucose-stimulated ß-cells. These results indicate that the PDX-1 transactivation domain specifically potentiates insulin enhancer activity in response to ß-cell activator factors and the glucose signaling pathway.



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Figure 5. Transactivation by PDX-1 Is Potentiated Selectively in Glucose-Stimulated ß-Cells

MIN6 cells were transfected with -238 WT, the wild-type insulin-driven CAT reporter, or -217 GAL alone or plus GAL4:PDX(1–149), GAL4:VP16, or GAL4:E1A(121–223) (GAL4:E1A). The results are calculated as the ratio of the normalized CAT activity at 20 mM glucose divided by the activity at 3 mM ± SEM.

 
Transactivation by PDX-1 Is Mediated by the Insulin Gene E1 Activators
The ß-cell specificity of GAL4:PDX-1 activation suggested that another insulin enhancer factor(s) was required for stimulation by PDX-1. To explore this hypothesis further, site-directed mutations within the C1 and E1 elements were introduced into -217 GAL. The binding of the BETA2/NeuroD1:E2A (43) and RIPE3b1 (7, 8) activators was prevented in the mutants of E1 and C1, respectively. GAL4:PDX(FL) and GAL4:PDX(1–149) activation was reduced by 10-fold in the E1 mutant, -217GAL(E1mt), relative to -217GAL in HIT T-15 cells, whereas GAL4:VP16 stimulation was only inhibited by 2-fold (Fig. 6Go). In contrast, GAL4:PDX stimulation was potentiated by 2-fold in the C1 element mutant, -217GAL(C1mt) (Fig. 6Go). GAL4:VP16 activation was unaffected by the C1 mutation. Activation by the GAL4:PDX-1 fusion proteins was completely abolished in the C1/E1 double mutant, -217GAL(C1/E1mt), although stimulation by GAL4:VP16 was only reduced by 2-fold (Fig. 6Go). Analogous results were obtained when these constructs were assayed in ßTC3 cells (data not shown). The data indicate that functional interactions between PDX-1 and the E1-bound activators, BETA2/NeuroD1:E2A, are particularly critical to insulin enhancer-mediated activation.



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Figure 6. Effect of Mutating the C1 and E1 Elements in -217 GAL on GAL4:PDX-1 Activation

The wild-type, E1 mutant (-217 GAL(E1mt)), C1 mutant (-217 GAL(C1mt)), or C1/E1 double mutant (-217 GAL(C1/E1mt)) -217 GAL reporter plasmid was co-transfected into HIT T-15 cells in the absence (-) or presence of GAL4:PDX expression vectors. The normalized CAT activity from wild-type or mutant -217 GAL plus GAL4:PDX-1 is reported relative to the insulin:GAL plasmid cotransfected with pSG424 alone. The normalized CAT activity of -217 GAL, -217 GAL(E1mt), -217 GAL(C1mt), and -217 GAL(C1/E1mt)) transfected with pSG424 was 159,031 ± 14,667, 5,347 ± 330, 11,379 ± 1,741, and 6,474 ± 1,033 cpm, respectively.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PDX-1 activates transcription of genes associated with ß-cell identity, including insulin, GLUT2, glucokinase, and IAPP. The transactivation potential of PDX-1 has primarily been analyzed by fusing the yeast GAL4 DNA-binding domain to various regions of the PDX-1 protein and assaying the ability of these chimeric proteins to transactivate an artificial promoter composed of multiple GAL4 binding sites (17, 35, 37). In this study, the key PDX-1 binding site in the insulin enhancer was replaced with a single GAL4 binding site and the functional properties of chimeric GAL4:PDX-1 proteins were assessed in this GAL4-substituted insulin enhancer-dependent transcription system. Our results indicate that specific features of the PDX-1 activation domain are required for appropriate regulation of ß-cell target genes.

When the insulin enhancer A3 element is replaced with a single GAL4 binding site, the transcriptional activity of the enhancer is reduced to a level that is consistent with that observed in an enhancer carrying an A3 mutation (6). The chimeric GAL4:PDX-1 protein restores activity of the GAL4-substituted enhancer in ß-cell lines, but not non-ß-cells (Fig. 1Go). This observation contrasts with the results from the multimerized GAL4 element reporter gene that is activated by GAL4:PDX-1 in both HIT T-15 and HeLa cells (17, 35). Thus, a single PDX-1 transactivation domain is not sufficient to activate transcription from the insulin enhancer but requires other ß-cell-enriched factors. This conclusion is further emphasized by the inability of GAL4:PDX-1 to activate expression from reporters driven by either a single GAL4 binding site linked to a heterologous promoter or the GAL4-substituted insulin enhancer lacking E1 and C1 activator function. These results also suggest that the PDX-1 transactivation domain per se is not ß-cell specific, but requires other ß-cell-enriched factors to drive cell type-specific insulin gene transcription.

The observation that a single enhancer-bound transactivator is unable to activate transcription or mediate a response to a particular stimulus is not uncommon. Many genes require "enhanceosomes" or "response units" composed of multiple, distinct DNA-bound factors to generate a complete transcriptional response (44, 45); it is proposed that the various enhancer factors arrayed on a particular promoter cooperatively create a unique surface that, in turn, recruits the transcriptional machinery. For example, the interferon-ß (IFNß) enhanceosome, which mediates transcriptional induction in response to viral infection, requires three transcription factors—NF{kappa}B, ATF2/c-jun, IRF-1—as well as the chromosomal protein HMG-I/Y bound to adjacent elements for synergistic activation of the enhancer (46, 47). In vitro, the enhanceosome stabilizes formation of a preinitiation complex composed of general transcription factors (TFIID, TFIIA, and TFIIB) and the cofactor USA (47) through the specific recruitment of TFIIB (48). In addition, the RNA pol II holoenzyme is also targeted to the IFNß promoter by an interaction between the enhanceosome and the transcriptional coactivator CREB-binding protein (CBP)/p300 (48, 49). Importantly, although the enhanceosome transcription factors can each individually associate with CBP/p300 in vitro, in the context of the IFNß enhancer, transactivation domains from all of the bound factors are necessary for CBP/p300 recruitment (49). The need to assemble a complex, multiprotein enhanceosome for efficient activation of IFNß transcription ensures that the gene will only be expressed when the multiple signals mediating the response to viral infection converge on the promoter.

Similarly, the assembly of a PDX-1-dependent enhanceosome might require other ß-cell-enriched factors for activity. Under such circumstances, the aberrant expression of insulin in a PDX-1 expressing nonislet cell types (i.e. epithelial cells of the duodenum) would be precluded, despite the availability of PDX-1, by the lack of other ß-cell-enriched factors. Since the E1 mutation in -217 GAL drastically impaired PDX-1 activation (Fig. 6Go), the insulin enhanceosome appears to minimally depend on the PDX-1 and E1 activators. The E1 activator is a dimeric complex composed of basic helix-loop-helix factors that are islet enriched [BETA2/NeuroD1 (43)] and generally distributed [HEB (50) or the E2A encoded proteins, E12, E47, or E2/5 (7, 51, 52, 53)], which is also incapable of supporting significant activation of insulin gene transcription when isolated from other insulin enhancer binding factors (7, 43, 54). Notably, stably transfected PDX-1 can induce ectopic insulin expression in cultured {alpha}-cells that normally express the E1 element activators but not in fibroblast cells that have no detectable E1 binding activity (55). E1 activator function is known to be mediated through interactions of CBP/p300 with both BETA2/NeuroD1 (56, 57, 58) and the E47/HEB proteins (58). Similarly, PDX-1 physically associates through N-terminal conserved sequences with CBP/p300 in GST pull-down in vitro and in vivo immunoprecipitation experiments (Ref. 59 and data not shown). In addition, recent in vitro experiments suggest that the PDX-1 homeodomain region may also interact directly with the E1 activators (60). Together, the data indicate that much like the IFNß gene, the assembly of an insulin enhanceosome requires both activator:activator and activator:CBP/p300 co-activator interactions. The results presented here stress the importance of the N-terminal PDX-1 activation domain in communicating signals through CBP/p300 to BETA2/NeuroD1 and the RNA polymerase II transcriptional machinery.

The PDX-1 transactivation domain was dissected for the purpose of identifying the motif(s) necessary for PDX-1 function in the context of the insulin enhancer. Several studies have shown that the PDX-1 transactivation domain resides in the N-terminal region of the PDX-1 protein (17, 33, 35). As previously reported for the multimerized GAL4 binding site-driven construct (35), N-terminal sequences spanning amino acids 13–22, 32–38, and 60–73 are required for full transactivation of the insulin enhancer. Whereas the C terminus and homeodomain interfere with the transactivation potential of the N terminus when assayed on the multimerized GAL4 construct (17, 35, 38), these regions are largely dispensable for transactivation of the insulin enhancer by the chimeric GAL4:PDX-1 proteins (Fig. 3Go). This result further confirms that a C-terminal cryptic activation domain that functions in activation of the somatostatin PDX-1 binding site (33) is not required for insulin enhancer expression.

The A3 element of the insulin enhancer contributes to glucose responsiveness in part through increased PDX-1 binding activity (14, 37, 61). Recently, it has also been shown that the PDX-1 transactivation domain fused to the GAL4 DNA-binding domain can confer glucose induction to a heterologous promoter composed of five GAL4 binding sites (37, 38). This suggests that, in addition to effects on DNA binding, glucose also stimulates the activation potential of PDX-1. In the current study, substitution of the PDX-1 cis-element with a GAL4 binding site reduces glucose induction of the insulin enhancer by 50%; the residual glucose effect is conferred by the glucose-responsive C1 and E1 activator factors (8, 42). The glucose induction is fully restored to wild-type levels by the addition of a GAL4:PDX-1 transactivation domain-expressing construct, but not by GAL4:activation domain fusions with the potent, but distinct, E1A or VP16 transactivating proteins. These results confirm and extend the previous conclusion by demonstrating that the PDX-1 transactivation domain provides an essential and unique function to the insulin enhancer in response to glucose that is independent of its ability to bind the A3 element.

The mechanism of PDX-1 activity in native promoters appears to depend on the context of its cognate DNA-binding element as well as on interactions with other cell-specific factors. For example, PDX-1 activation of the TSEII element in the somatostatin enhancer is potentiated by cooperative binding of a second homeodomain cofactor, PBX (39). The activity of another PDX-1 binding site in the somatostatin promoter, TSEI, is strongly enhanced by binding of PBX1a/Prep1 heterodimers to an adjacent cis-element (62). PDX-1 activation of the elastase-1 gene is similarly dependent on cell context. In acinar cells, PDX-1 function in the elastase enhancer requires formation of a trimeric complex between PDX-1, PBX1b, and MRG1; furthermore, a multimerized PDX-1/PBX1b/MRG-1 binding site from the elastase gene is inactive in acinar cells, indicating a dependence on additional enhancer elements for PDX-1 function (63). In contrast, PDX-1 cannot activate the elastase enhancer in ß-cells due to the lack of PBX1b and MRG-1, although transcription from the multimerized PDX-1 element of the elastase gene can be stimulated by uncomplexed PDX-1 in ß-cells (63). Importantly, PBX does not influence the activity of PDX-1 on the A element of the insulin enhancer (39), suggesting that PDX-1 activation of the insulin gene may require a distinct set of functional interactions.

The data in this report support the hypothesis that PDX-1 activation in the context of the insulin gene functions mainly through a conserved transactivation domain in the extreme N-terminal region and requires interaction with factors bound to adjacent elements, in particular the E1 activators. The activity of the PDX-1 transactivation domain, which has thus far been shown to be active in expression from the insulin A3 element (this report and Ref. 17), the somatostatin TAAT1 element (33), and a multimerized heterologous DNA binding site (GAL4) (17, 35, 38), is likely to also be generally applicable to the mechanism of PDX-1 activation of additional target genes such as GLUT2, glucokinase, IAPP, and others. Our results provide further evidence indicating that the interactions that the PDX-1 transactivation domain make with additional DNA bound factors are flexible and depend heavily on the context of the PDX-1 binding site in a target promoter. For example, PDX-1 activation of the IAPP promoter may not be dependent on an interaction with the E1 activators, just as PDX-1-mediated activation of the insulin promoter does not require PBX interactions. We speculate that in other PDX-1 regulated promoters, transcriptional synergy will depend upon recruitment of common bridging factors, like CBP/p300, to the promoter by PDX-1 and other DNA-bound factors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transfections
Monolayer cultures of HIT T-15 (64), ßTC-3 (54), MIN-6 (37), HeLa (64), and BHK (65) cells were grown under conditions described previously. INS-1 cells were cultured and transfected in RPMI 1640 (-glucose) medium supplemented with 10% (vol/vol) FCS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 µM ß-mercaptoethanol, and glucose to a final concentration of 4 mM. All media were supplemented with 50 µg/ml each of streptomycin and penicillin. The insulin:CAT constructs were transiently transfected by the calcium phosphate coprecipitation [HIT T-15, BHK, and HeLa, (64), MIN-6 (37)] or lipofectamine (ßTC3 and INS-1; 1 x 106 cells) procedures (LipofectAMINE Reagent, Life Technologies, Inc., Gaithersburg, MD). The precipitates (12.5 µg total) used for the HIT T-15, MIN-6, HeLa, and BHK cell transfections contained 1.25 µg CAT reporter vector, 2.5 µg GAL4 fusion protein expression vector or CMV4 plasmid (66), and 1.25 µg pGL2RSV. The Rous sarcoma virus (RSV) enhancer-driven luciferase (LUC) expression plasmid, pGL2RSV (Promega Corp., Madison WI), was used as a recovery marker. HIT T-15 and BHK cells were treated with 20% glycerol (2 min) 4 h after the addition of the calcium phosphate DNA precipitate. After transfection, the MIN6 cells were grown in RPMI 1640 (10% FCS) containing either 3 or 20 mM glucose for 16 h. ßTC3 and INS-1 cells were transfected with a ratio of 6 µl of LipofectAMINE reagent to 1 µg DNA using the conditions described by the manufacturer (i.e. 1 µg CAT reporter vector, 2 µg GAL4 fusion protein expression vector or CMV4 plasmid, and 1 µg pGL2RSV). Extracts were prepared either 16 (MIN6) or 48 h after transfection and chloramphenicol acetyltransferase (CAT) (67) and luciferase (68) enzymatic assays performed. The CAT activity from the reporter construct was normalized to the LUC activity of the cotransfected internal control plasmid. Each experiment was carried out at least three times with two or more independently isolated plasmid preparations.

Plasmid DNAs
The -238 WT CAT and -217 WT CAT vectors contain rat insulin II gene sequences from -238 bp to +8 bp or -217 bp to +2 bp, respectively (69). PCR was used in the construction of -217 GAL (70); the insulin A3 element (-205 to -189 bp) in -217 WT was replaced with a single palindromic GAL4 DNA binding site (5'-CGGAGGACTGTCCTCC-3'). This mutant exchanges 16 nucleotides of insulin sequence with a GAL4 binding element of the same length and causes a mutation in the core A3 element binding site (i.e. TAATT) as well as 4 nucleotides 5' and 7 nucleotides 3' to the core. The -217 CAT (E1mt) (69) and -238 CAT (C1mt) (8) plasmids served as the PCR templates in generating -217 GAL(C1mt), -217 GAL(E1mt), and -217 GAL(C1/E1mt) plasmids. The -217 GAL LUC vector was made by subcloning the insulin GAL4 sequences from -217 GAL just upstream of the LUC gene in pSV0ARPL2L (71). The (GAL4)1E1b LUC vector was a gift of M. Daniels and D. K. Granner (Vanderbilt University, Nashville TN). All of the GAL4:PDX-1 or GAL4:XlHbox8 fusion protein expression vectors and their derivatives were previously reported (35) except the GAL4:PDX(–149{Delta}Pbx) and GAL4:PDX(1–149{Delta}PKC) vectors that were prepared in GAL4:PDX(1–149) by PCR using an oligonucleotide spanning the site of mutagenesis at either the Pbx interaction motif (aa 119–123 FPWMK mutated to AAGGQ) (5'-GCGCAAGCTTACGCGTGAGCTTTGGTGGACTGGCCGCCGGCGGCAGGGAGATGAACGCGGCT-3') or the potential PKC phosphorylation site (aa 124–125 ST mutated to AA) (5'-GCTTTTCCACGCGTGAGCTTTGGCGGCTTTCATCCACGGGAAAGGGAG-3'). The GAL4:VP16 (72), GAL4:E1A(121–223) (73), and the GAL4 DNA binding domain [termed GAL4 DBD; pSG424 (73)] expression vectors were previously described. Cloning and isolation of plasmids were performed by standard protocols. The sequence of all plasmid constructs was confirmed by DNA sequence analysis.


    ACKNOWLEDGMENTS
 
The authors thank Mr. K. Gerrish and Drs. M. Gannon, Y. Qiu, S. Samaras, and C. Wright for assistance and scientific discussions.


    FOOTNOTES
 
Address requests for reprints to: Dr. Roland Stein, Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Light Hall, Room 708, Nashville, Tennessee 37232. E-mail: roland.stein{at}mcmail.vanderbilt.edu

This work was supported by a grant from the NIH (NIH RO1 DK-50203 to R.S.) as well as partial support from the Vanderbilt University Diabetes Research and Training Center Molecular Biology Core Laboratory (Public Health Service Grant P60 DK-20593 from the NIH).

1 Present address: Ontogeny Inc., 45 Moulton Street, Cambridge, Massachusetts 02138-1118. Back

Received for publication December 9, 1999. Revision received August 16, 2000. Accepted for publication August 22, 2000.


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 DISCUSSION
 MATERIALS AND METHODS
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