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Molecular Endocrinology 18 (1): 142-149
Copyright © 2004 by The Endocrine Society

Neurogenin3 Activates the Islet Differentiation Program while Repressing Its Own Expression

Stuart B. Smith, Hirotaka Watada and Michael S. German

Diabetes Center (S.B.S., H.W., M.S.G.), Hormone Research Institute and Department of Medicine (M.S.G.), University of California San Francisco, San Francisco, California 94143-0534

Address all correspondence and requests for reprints to: Michael S. German, Hormone Research Institute, University of California San Francisco, 513 Parnassus Avenue, San Francisco, California 94143-0534. E-mail: mgerman{at}biochem.ucsf.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of the proendocrine factor Neurogenin3 determines which progenitor cells in the developing pancreas will differentiate into the endocrine cells of the islets of Langerhans. To better understand how Neurogenin3 directs endocrine differentiation, we examined the mechanisms by which Neurogenin3 regulates the promoters of three transcription factor genes expressed in endocrine precursor cells: the nkx2.2 gene, the PAX4 gene, and the NEUROG3 gene, the human gene encoding Neurogenin3 itself. The function of all three promoters depends on at least one critical E box, a common DNA sequence that forms a binding site for basic helix-loop-helix proteins like Neurogenin3. Neurogenin3 bound to and effectively activated transcription through the nkx2.2 and PAX4 E boxes. In contrast, Neurogenin3 strongly repressed the NEUROG3 promoter, although a proximal E box was required for activity in the absence of Neurogenin3, suggesting that a ubiquitous transcriptional activator may bind to this site, and that Neurogenin3 could act as a competitive inhibitor of this activator. This hypothesis was supported by the lack of evidence for significant intrinsic transcriptional repression capacity in the Neurogenin3 protein, and by the ability of isolated DNA-binding basic helix-loop-helix domains to repress the NEUROG3 promoter. Neurogenin3 produced additional repression, however, when the protein included an intact transcriptional activation domain, suggesting that it may also induce the expression of a downstream transcriptional repressor. In summary, while Neurogenin3 orchestrates islet cell differentiation by activating islet cell transcription factor genes, it simultaneously represses its own promoter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AS THE PANCREAS develops from a cluster of multipotent epithelial progenitor cells into the distinct populations of mature cells that form the adult pancreas, a diverse group of transcription factors orchestrate the determination of cell-type fates and the progression of these cells through specific lineages (for review see Ref. 1). Key among these factors is the basic helix-loop-helix (bHLH) factor Neurogenin3. The expression of Neurogenin3 is both necessary and sufficient (2, 3, 4) to drive undifferentiated progenitor cells to an endocrine fate, but only initiates the islet differentiation program because it is extinguished before final differentiation of the cells (3, 5). Additional factors, such as the bHLH factor neuroD1 (6) and the homeodomain factors Pax4 (7), Pax6 (8, 9), nkx2.2 (10), nkx6.1 (11), isl1 (12), and PDX1 (13, 14, 15) are also necessary for this process of differentiation and for maintenance of the mature, differentiated cells.

Subsequent to the identification of Neurogenin3 as a pancreatic proendocrine gene, establishing the factors that lie directly upstream and downstream has stimulated keen interest. Studies of the human NEUROG3 promoter have demonstrated that fragments of the promoter down to the proximal few hundred base pairs are highly active in transformed cell lines irrespective of their tissue of origin, demonstrating the presence of universal activators working through the proximal promoter. In contrast, longer promoter constructs can direct expression of a transgene specifically to progenitor cells in the pancreas, gut, and neural tissues (16). Pancreatic transcriptional activators of the HNF6 (17), HNF1 and HNF3/FoxA (16) families all bind to this longer NEUROG3 promoter. In pancreatic cells not fated to become islet cells, signals through the notch receptor activate the expression of the transcriptional repressor HES-1, which binds to and prevents the activation of the NEUROG3 promoter (4, 16, 18). The mechanisms by which the expression of Neurogenin3 is extinguished in progenitor cells before final differentiation are unknown. In addition, the downstream genes through which Neurogenin3 activates the islet cell differentiation program are largely unknown. Proposed targets for Neurogenin3 include the genes encoding the islet transcription factors NeuroD1 (19), Pax4 (20, 21), and Nkx2.2 (22). Neurogenin3 has been shown to cooperate with HNF1 homeodomain factors in activating both the PAX4 promoter and the chromosomal pax4 gene (21), and with FoxA winged-helix factors in activating the nkx2.2 1A promoter (22), but otherwise little is known about how Neurogenin3 may activate these genes.

In the current study, we set out to understand more clearly how Neurogenin3 expression is controlled, and to understand the function of Neurogenin3 on a molecular level. We found that Neurogenin3 can function as a potent transcriptional activator when bound to E elements from the PAX4 and nkx2.2 gene promoters; and one hybrid analysis mapped this activation capacity to the carboxyl terminus of the Neurogenin3 protein but did not detect significant intrinsic transcriptional repression capacity. The proximal NEUROG3 promoter itself contains a critical E box; but surprisingly, exogenously expressed bHLH activating factors including Neurogenin3 itself repress the activity of the NEUROG3 promoter. We propose that Neurogenin3 can act as a transcriptional repressor of its own promoter by competing with a ubiquitous transcriptional activator, possibly in combination with the induction of a transcriptional repressor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A previous study demonstrated by transient transfection assay that NEUROG3 promoter constructs as short as 520 bp are active in a variety of cell lines including nonpancreatic lines (16). To identify important promoter elements within this proximal promoter, we constructed additional 5' promoter deletions driving expression of a luciferase reporter gene. A promoter containing only 207 bp upstream of the transcription start site still maintained a significant level of activity. This promoter contains a potentially important E box (a sequence element with the consensus CANNTG that binds to transcription factors in the bHLH family, including Neurogenin3 itself) located at -149 bp that is also conserved in the mouse promoter. To test the importance of the -149-bp E box, we introduced a 2-bp mutation in essential bases of the E box consensus and found that the mutation abolished the activity of the proximal promoter in all cell lines tested (Fig. 1Go).



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  		Fig. 1. NEUROG3 Promoter Activity Is Dependent upon an E Box Located at -149 bp

The three cell lines shown were transfected with reporter plasmids containing the firefly luciferase gene under the control of the either the wild-type -207-bp NEUROG3 promoter, or the -207-bp NEUROG3 promoter containing a 2-bp mutation in the E box (-207-bp ME). Luciferase activities of all samples were determined 48 h after transfection and are expressed relative to the activity of the promoterless backbone vector (pFOXluc1). Results are expressed as the mean ± SEM of data from experiments performed in triplicate on at least three separate occasions.

 
Given the importance of this conserved E box within the proximal promoter, we tested whether Neurogenin3, alone or in conjunction with its heterodimeric partner E47, could influence the activity of its own promoter (Fig. 2Go). Surprisingly, Neurogenin3 and E47 repressed the NEUROG3 promoter constructs, but not the control RSV promoter or promoterless constructs. The promoter construct with the mutant E box was not repressed by Neurogenin3 (data not shown), but this promoter was already inactive in the absence of Neurogenin3. In addition, two copies of a short minienhancer containing the proximal NEUROG3 E box also was active in both ßTC3 and NIH 3T3 cells, dependent on the E box sequence, and repressed by Neurogenin3 (Fig. 3Go and data not shown).



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  		Fig. 2. Neurogenin3 Represses Its Own Promoter

NIH3T3 cells were transfected with reporter plasmids containing the firefly luciferase gene either with no promoter or under the control of the various length NEUROG3 promoters indicated or the Rous sarcoma virus LTR (RSV). Cells were cotransfected with expression plasmids containing the cytomegalovirus (CMV) early gene promoter driving the expression of either no cDNA or the cDNAs for Neurogenin3 and its heterodimeric partner E47. Luciferase activities of all samples were determined 48 h after transfection and are expressed relative to the activity of the promoterless backbone vector (pFOXluc1) cotransfected with the expression plasmid containing no cDNA. Results are expressed as the mean ± SEM of data from experiments performed in triplicate on at least three separate occasions.

 


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  		Fig. 3. E Box Activity Is Conferred upon a Heterologous Promoter

ßTC3 cells were transfected with reporter plasmids containing the firefly luciferase gene under the control of the herpes simplex virus thymidine kinase minimal promoter either by itself (TK) or linked to two copies of the NSE minienhancer which contains the sequences from -105 to -158 bp from the NEUROG3 promoter including the proximal E box or, in panel A, two copies of the N3mE minienhancer with a 2-bp mutation of the E box. In panel B, cells were cotransfected with either a control plasmid expressing no cDNA or two plasmids expressing the E47 and Neurogenin3 cDNAs under the control of the cytomegalovirus (CMV) early gene promoter. Luciferase activities of all samples were determined 48 h after transfection and are expressed relative to the activity in cells transfected with the vector with the isolated TK promoter (TK). Results are expressed as the mean ± SEM of data from experiments performed in triplicate on at least three separate occasions.

 
Next, we tested the affinity of the pancreatic bHLH proteins Neurogenin3, neuroD1, and E47 for the E box of the NEUROG3 promoter. EMSAs were performed using isolated E boxes from the NEUROG3, nkx2.2, PAX4, and rat insulin I gene promoters as probes in conjunction with in vitro produced proteins Neurogenin3, NeuroD1, and their dimeric partner E47 (Fig. 4Go). E47 bound to each of the elements with comparable affinity; and, unexpectedly for class B bHLH factors, homodimers of neuroD1 and Neurogenin3 bound to the E box of the nkx2.2 promoter. Heterodimers of E47 with Neurogenin3 or neuroD1 bound to all four probes tested, with differing relative binding affinities in the order from the NEUROG3 E box (weakest), to the nkx2.2 E3 element, the rat Insulin I E2 element, and the PAX4 E box (strongest).



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  		Fig. 4. bHLH Factors Bind Pancreatic Promoter E Boxes

EMSA were used to test the ability of E47, Neurogenin3, and neuroD1 to bind to labeled, double-stranded oligonucleotides containing the E box sequences from the rat insulin I and mouse nkx2.2 promoters (A) or the human PAX4 and NEUROG3 promoters (B). One microliter of each in vitro-translated protein was incubated with the indicated probes, either individually or in the combination shown. Results are typical of experiments done on three occasions.

 
To test the function of the different E boxes from the four genes in isolation, six copies of each E box were ligated upstream of the minimal herpes simplex virus (HSV)-thymidine kinase (TK) promoter driving the luciferase reporter gene. The activity of the resulting heterologous promoter was determined in the absence and presence of cotransfected factors (Fig. 5Go). Unlike the other three constructs, the NEUROG3 E box construct was not activated by any of the transcription factor combinations. It should be noted that in contrast to this minienhancer construct made from six copies of a 16-bp E box sequence, the larger, 54-bp NEUROG3 E box minienhancer used in Fig. 3Go was active, and was repressed by Neurogenin3. The higher activity of the larger minienhancer suggests that the ubiquitous activator that requires the NEUROG3 E box either requires a larger binding site, or interacts with factor(s) binding adjacent to the E box.



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  		Fig. 5. The NEUROG3 E Box Is Not Activated by the Proendocrine bHLH Factors

NIH3T3 cells were transfected with reporter plasmids containing the firefly luciferase gene under the control of the herpes simplex virus thymidine kinase minimal promoter either by itself (TK) or linked to six copies of the indicated E boxes. Cells were cotransfected with expression plasmids containing the cytomegalovirus (CMV) early gene promoter driving the expression of either no cDNA, the E47 cDNA or the cDNA combinations shown. Luciferase activities of all samples were determined 48 h after transfection and are expressed relative to the activity of the vector with the isolated TK promoter (TK) cotransfected with the expression plasmid containing no cDNA. Results are expressed as the mean ± SEM of data from experiments performed in triplicate on at least three separate occasions.

 
To determine whether transcriptional repression is an inherent property of Neurogenin3 and whether Neurogenin3 thereby could repress its own promoter by a direct mechanism, one hybrid analysis was used to study the transactivating properties of the Neurogenin3 protein (Fig. 6Go). Various regions of the protein were fused to the DNA binding domain of GAL4, and the ability of the resulting protein to affect transcription from a promoter containing five GAL4 binding sites was determined. Several regions of the Neurogenin3 protein were able to stimulate transcription, the most potent activation coming from regions encompassing the carboxyl terminus of the protein (constructs 128–214 and 190–214), which was a more potent effect than that exhibited by the pax6 activation domain that served as a positive control. Only one region of the protein, the isolated bHLH domain, was able to weakly repress transcription. A similar construct containing the equivalent region of the neuroD1 protein acted in a comparable manner, suggesting that this weak repression is likely to be a common characteristic of an isolated bHLH domain and not an idiosyncrasy of Neurogenin3.



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  		Fig. 6. One Hybrid Analysis Maps Activation Domains within Neurogenin3

In panel A, a low background reporter construct comprised of five copies of the Gal4 consensus binding site (UAS) ligated upstream of the E1b viral promoter driving luciferase expression was transfected into NIH3T3 cells. The reporter construct was cotransfected with a plasmid expressing a fusion protein comprised of the Gal4 DNA binding domain and the indicated portion of Neurogenin3 protein; a similar construct containing the previously characterized pax6 activation domain was included as a positive control. In panel B, a high background reporter construct comprised of five copies of the Gal4 UAS ligated upstream of the HSV-TK promoter driving luciferase was transfected into NIH3T3 cells. The reporter construct was cotransfected with a plasmid expressing the GAL4 DNA binding domain fused to the bHLH domain of Neurogenin3 or NeuroD1/ß2 or to the pax6 activation domain. Luciferase activities of all samples were determined 48 h after transfection and are expressed relative to the activity of the luciferase vector cotransfected with the expression plasmid containing the Gal4 DNA binding domain alone. Results are expressed as the mean ± SEM of data from experiments performed in triplicate on at least three separate occasions. In panel C, a diagram outlines the functional domains of the Neurogenin3 protein.

 
Due to the apparent inability of Neurogenin3 to act directly as a transcriptional repressor, we hypothesized that Neurogenin3 may repress its own promoter by competing for binding with another activator or by inducing the expression of a transcriptional repressor that in turn represses the promoter. If the mechanism was purely the first model, then any factor able to bind the E box should be able to compete and thereby repress in a similar manner. The second model would necessitate that the protein contain a transcriptional activation domain to induce expression of a downstream repressor. To test these two possibilities, we cotransfected the -325-bp NEUROG3 promoter reporter construct with plasmids expressing various E box-binding bHLH proteins. We used the -325-bp NEUROG3 promoter because longer promoters contain additional E boxes.

All combinations repressed the NEUROG3 promoter, with the single exception of E47 in combination with the muscle bHLH protein myoD (Fig. 7Go). Consistent with the competition model for transcriptional repression of the NEUROG3 promoter, a truncated version of E47 [E47({Delta}1–598)] containing only the DNA-binding bHLH domain and lacking any activation domain repressed the promoter as efficiently as the wild-type protein, thus suggesting that E47 repression is not due to the activation of an additional gene, but may simply result from competition with an activator for binding to the E box. In contrast, the isolated Neurogenin3 bHLH domain, which should not bind by itself to the NEUROG3 E box (Fig. 4Go), did not repress the NEUROG3 promoter. Consistent with the downstream repressor model, however, inclusion of the activation domain allowed Neurogenin3 to repress the NEUROG3 promoter. Because Neurogenin3 probably normally exits as a heterodimer with E47 or other ubiquitous class A bHLH proteins, the heterodimer may both activate a downstream repressor and compete with an activator. Consistent with this possibility, the greatest repression of the NEUROG3 promoter was produced by the combination of E47 and Neurogenin3.



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  		Fig. 7. Pancreatic bHLH Factors Repress the NEUROG3 Promoter

NIH3T3 cells were transfected with a reporter plasmids containing the firefly luciferase gene under the control of the -325-bp NEUROG3 promoter. Cells were cotransfected with expression plasmids containing the cytomegalovirus (CMV) early gene promoter driving the expression of either no cDNA or the wild-type or truncated bHLH cDNAs indicated. Luciferase activities of all samples were determined 48 h after transfection and are expressed relative to the activity of the luciferase vector cotransfected with the expression plasmid containing no cDNA. Results are expressed as the mean ± SEM of data from experiments performed in triplicate on at least three separate occasions.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Neurogenin3 activates a cascade of genes involved in islet cell differentiation but is itself inactivated before differentiation, so that it initiates but does not complete the differentiation program. The closely related bHLH gene neuroD1 is activated by Neurogenin3 (19) and persists in the mature islet cells where it plays a role in completing and maintaining the differentiated state (6) by driving the expression of such genes as insulin (23) and glucagon (24). To prevent persistent expression of Neurogenin3 once its task of initiating differentiation is achieved, some mechanism must limit the expression of Neurogenin3 in the differentiating cells. Our data suggest that autorepression by Neurogenin3 of its own expression contributes at least part of this mechanism.

Other genes also utilize autorepression to limit or modulate their own expression. The pancreatic transcription factors pax4 (20) and HES1 (25) bind to and directly inhibit their own promoters. Both pax4 and HES1 are themselves potent transcriptional repressors, and binding to their own promoters presumably results in direct transcriptional repression through the recruitment of corepressors and inhibition of the transcription complex. Because Neurogenin3 lacks a significant transcriptional repression domain, it must work by a different mechanism. Interestingly, the NEUROG3 promoter contains a critical E box that neither Neurogenin3 nor neuroD1 can activate. Instead, it appears that the pancreatic bHLH proteins may compete with some ubiquitous activator that binds to the E box, so that expression of Neurogenin3 and neuroD1 in the differentiating progenitor could displace that activator and inactivate the NEUROG3 gene.

The NEUROG3 E box is a perfect consensus binding site for the basic leucine zipper protein adaptor protein complex-4 (26) or a close match to the site of bHLH protein cMyc (27), both of which function as transcriptional activators. A role for cMyc as the ubiquitous activator is particularly appealing, as its expression is associated with cell division, as is seen in the Neurogenin3-expressing precursors, but not in the differentiated, neuroD1-expressing islet cells (3, 5).

In addition, however, Neurogenin3 may do more than simply displace an activator from the NEUROG3 promoter. In combination with E47, Neurogenin3 is a more potent repressor of the NEUROG3 promoter than the other bHLH proteins tested, and this additional repression depends on the transcriptional activation domain of the Neurogenin3 protein. These data suggest that Neurogenin3 may activate the transcription of some other gene that in turn inhibits the NEUROG3 promoter. The identity of this hypothetical downstream repressor is unknown, but members of the HES family of transcriptional repressors are possible candidates given the remarkable ability of HES1 to extinguish the NEUROG3 promoter (16). In studies in vitro, we have found that Neurogenin3 does not increase HES1 expression, but it does induce the expression of other members of the HES family (Gasa, R., C. Mrejen, and M. S. German, unpublished data). Furthermore, it must be considered that the intact NEUROG3 gene, which certainly contains important regulatory domains outside the limited promoter constructs used in these studies, may have additional mechanisms for silencing.

Finally, it must be noted that the NEUROG3 gene remains off after Neurogenin3 disappears from mature endocrine cells. Our data do not directly address how the NEUROG3 gene stays off because the mechanisms that silence an active gene may be quite different from those that maintain gene silencing. Neurogenin3 alters the expression of many genes, and these changes persist after Neurogenin3 disappears. For example, the nkx2.2 and neuroD1 genes, both direct targets of Neurogenin3, remain active after Neurogenin3 is gone. NeuroD1, although not as potent a repressor of the NEUROG3 promoter as Neurogenin3 itself, may be sufficient to keep the promoter off. Similarly, a downstream repressor could also persist after Neurogenin3 is gone. In addition, the gene expression changes initiated by Neurogenin3 could include the permanent loss of activators of the NEUROG3 promoter such as HNF6 (17).

In summary, the proendocrine factor Neurogenin3 functions as a transcriptional activator, initiating the cascade of gene expression events that leads to the differentiation of pluripotent progenitor cells into mature islet cells in the pancreas. Once this chain of events is initiated, however, Neurogenin3 represses its own expression, possibly by both competing with an activator and activating a repressor, allowing differentiation to proceed autonomously.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
EMSAs
Single-stranded oligonucleotides were 5'-end labeled using ({gamma}-32P)ATP and T4 polynucleotide kinase. An excess of complementary strand was then annealed to form a duplex strand that was column purified. EMSA buffers and electrophoresis conditions were as described previously (28) using 500 ng of poly(deoxyinosine-deoxycytidine):poly(deoxyinosine-deoxycytidine) per 10 µl binding mix. For in vitro-produced protein, 1 µl of the 50-µl total reaction volume was used per binding mix.

Oligonucleotides used were as follows (coding strand shown from each double-stranded pair): NEUROG3 promoter E element, 5'-ctttgtccggaatccagctgtgccctgcgggggag-3'; rat insulin I promoter E2 element, 5'-ctgcttcatcaggccatctggccccttgttaataa-3'; PAX4 promoter E element, 5'-tgtataattgtgagcagatggcgggggctggcggc-3'; nkx2.2 promoter E3 element 5'-ttattaccgctgaacatatggccaatattttgact-3'. EMSA results are representative of those seen on at least three occasions.

In Vitro Protein Production
The cloning and construction of in vitro expression vectors containing the cDNAs encoding E47, Neurogenin3, and neuroD1 ligated downstream of the T7 phage promoter have been previously described (20). Proteins were produced using the TNT-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer’s instructions to provide a total reaction volume of 50 µl from 1 µg of DNA template.

Luciferase Reporter Constructs
The longer NEUROG3 promoter luciferase constructs have been described previously (16). The shorter promoter fragments were generated by PCR and ligated upstream of the luciferase gene in the plasmid pFOXLuc1. A 2-bp mutation was introduced into the proximal NEUROG3 promoter E box in the intact -207-bp promoter by a PCR-based technique whereby two complementary primers corresponding to the region, and containing the mutation, were used as PCR primers to amplify the entire plasmid (29) using Pfu Turbo polymerase (Stratagene, La Jolla, CA). The positive strand primer sequence (mutation in bold type) was 5'-gccctttgtccggaatctggctgtgccctgcggggga-3'.

The minienhancers containing two copies of the proximal E box used in Fig. 3Go were ligated upstream of the TK minimal promoter in the plasmid pFOXLuc1TK (30). Oligonucleotides used were as follows (coding strand shown from each double-stranded pair): N3E (-105 to -158 of NEUROG3 promoter) 5'-gatcttccggaatccagctgtgccctgcgggggaggagcgggctcgcgtggcgcggcccg-3', N3mE 5'-gatcttccggaatctggctgtgccctgcgggggaggagcgggctcgcgtggcgcggcccg-3'. The minienhancers used in Fig. 5Go contained six copies of a 16-bp repeat containing each E box, and were constructed by ligating two copies of the following oligonucleotides containing three copies of the respective E boxes (16 bp) upstream of the TK minimal promoter in pFOXLuc1TK: Pax4 5'-gatctgtgagcagatggcggggtgagcagatggcggggtgagcagatggcggg-3'; Nkx2.2 5'-gatctctgaacatatggccaactgaacatatggccaactgaacatatggccaag- 3'; Ngn3 5'-gatctgaatccagctgtgcccgaatccagctgtgcc cgaatccagctgtgcccg-3'.

One-Hybrid Analysis
One-hybrid expression vectors were constructed by amplifying the appropriate coding fragments of Neurogenin3 by PCR and then ligating into the EcoRI and BamHI sites of the Gal4DBD vector (CLONTECH, Palo Alto, CA). Two reporter vectors were constructed carrying DNA binding sites for the GAL4 protein. The low background vector to test for activation was constructed from pFOXluc1 (31) with five copies of the GAL4 upstream activating sequence (UAS) ligated upstream of the adenovirus E1b promoter driving the expression of firefly luciferase. The high background vector used to test for repression was constructed from pFOXluc2 (30) with five copies of the GAL4 UAS ligated upstream of the HSV-TK promoter driving expression of firefly luciferase. Two micrograms of reporter construct and 200 ng of the GAL4DBD vector were transfected into one million cultured cells using Transfast lipid reagent (Promega) according to the manufacturer’s instructions and luciferase activity was determined 48 h after transfection using the Promega assay system according to the manufacturer’s instructions.

Cell Culture and Transfection
The mouse ß-cell line, ßTC3, and the mouse {alpha}-cell line, {alpha}TC1.6, were grown in DMEM supplemented with 2.5% fetal bovine serum and 15% horse serum. NIH3T3 mouse fibroblast cells were grown in DMEM supplemented with 10% fetal bovine serum. In preparation for transfection, cells were split into six-well plates 24 h before transfection, one million cells per well were used for {alpha}TC1.6 and ßTC3 transfection and 50 thousand per well for NIH3T3 cells. Two micrograms of reporter construct were used per well, and 50 ng of any cotransfected transcription factor cDNA was used per well. Transfast (Promega) cationic lipid agent was used for all transfections according to the manufacturer’s instructions. Cells were harvested 48 h after transfection and luciferase assays performed with 5 µg of total protein as previously described (20). Transfections were performed on at least three occasions, all data are expressed as mean ± SEM.


   ACKNOWLEDGMENTS
 
We thank members of German, Hebrok, and Vaisse laboratories for helpful comments.


   FOOTNOTES
 
This work was supported by a grant from the Nora Eccles Treadwell Foundation and NIH Grants DK553401 and DK21344. S.S. and H.W. are both recipients of Juvenile Diabetes Research Foundation International Postdoctoral Fellowships and Juvenile Diabetes Research Foundation International Advanced Postdoctoral Fellowships.

Present address for H.W.: Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.

Abbreviations: bHLH, Basic helix-loop-helix; HSV, herpes simplex virus; TK, thymidine kinase; UAS, upstream activating sequence.

Received for publication February 4, 2003. Accepted for publication September 29, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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