Transcription Factors Recognizing Overlapping C1-A2 Binding Sites Positively Regulate Insulin Gene Expression*

Robert H. HarringtonDagger and Arun SharmaDagger §

From the Dagger  Section of Islet Transplantation & Cell Biology, Joslin Diabetes Center and the § Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215

Received for publication, September 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transcription factors binding the insulin enhancer region, RIPE3b, mediate beta -cell type-specific and glucose-responsive expression of the insulin gene. Earlier studies demonstrate that activator present in the beta -cell-specific RIPE3b1-binding complex is critical for these actions. The DNA binding activity of the RIPE3b1 activator is induced in response to glucose stimulation and is inhibited under glucotoxic conditions. The C1 element within the RIPE3b region has been implicated as the binding site for RIPE3b1 activator. The RIPE3b region also contains an additional element, A2, which shares homology with the A elements in the insulin enhancer. Transcription factors (PDX-1 and HNF-1alpha ) binding to A elements are critical regulators of insulin gene expression and/or pancreatic development. Hence, to understand the roles of C1 and A2 elements in regulating insulin gene expression, we have systematically mutated the RIPE3b region and analyzed the effect of these mutations on gene expression. Our results demonstrate that both C1 and A2 elements together constitute the binding site for the RIPE3b1 activator. In addition to C1-A2 (RIPE3b) binding complexes, three binding complexes that specifically recognize A2 elements are found in nuclear extracts from insulinoma cell lines; the A2.2 complex is detected only in insulin-producing cell lines. Furthermore, two base pairs in the A2 element were critical for binding of both RIPE3b1 and A2.2 activators. Transient transfection results indicate that both C1-A2 and A2-specific binding activators cooperatively activate insulin gene expression. In addition, RIPE3b1- and A2-specific activators respond differently to glucose, suggesting that their overlapping binding specificity and functional cooperation may play an important role in regulating insulin gene expression.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In adult mammals, the insulin gene is expressed only in the pancreatic beta -cells in the islets of Langerhans. In studies using transgenic animals and transient transfection analysis, the proximal 5'-flanking region of the insulin promoter was shown sufficient for directing beta -cell-specific expression of the insulin gene (1-7). Further, mutational analysis of the promoter proximal region identified several cis-acting enhancer elements that are important for insulin expression. The insulin enhancer elements, with the exception of E-box elements, have been classified based on the nucleotide sequence of the element (8). Most of these enhancer elements are well conserved in various species, suggesting the presence of a common regulatory mechanism(s) controlling insulin expression.

Three conserved insulin enhancer elements, A3 (-201 to -196 bp)1 (9-13), RIPE3b/C1-A2 (-126 to -101 bp) (14), and E1 (-100 to -91 bp) (4, 15, 16), play an important role in regulating cell-specific expression of the insulin gene. Factors binding these sites have a very limited cellular distribution. Transcription factors that bind and activate expression from two of the three conserved insulin enhancer elements (A3 and E1) have been cloned. PDX-1, a member of the homeodomain family of transcription factor expressed in cells of the pancreas and duodenum, binds the A3 element (17-22). Heterodimers of ubiquitously distributed basic helix-loop-helix family members (E2A and HEB (23, 24)) and the cell type enriched basic helix-loop-helix member (BETA2 (25, 26)) bind the E1 element. BETA2 is expressed in all pancreatic endocrine cell types, in some intestinal endocrine cells, and in the brain (25-27). Limited but not identical cellular distribution of transcription factors PDX-1 and BETA2 suggests that cell-specific insulin gene expression may result because of the presence of a unique combination of enhancer element binding factors in the pancreatic beta -cells. Interestingly, one of the RIPE3b element-binding complexes is expressed only in insulin-producing cells (14, 28) and may play a critical role in regulating the cell type specificity of insulin gene expression.

The RIPE3b binding activity has been extensively characterized in nuclear extracts from both insulin-producing and non-insulin-producing cell lines (14, 27-31). Two specific RIPE3b-binding complexes have been identified: 1) a cell-specific complex, RIPE3b1, which is detected only in pancreatic beta -cell lines, and 2) the RIPE3b2-binding complex that is detected in nuclear extracts from all cell lines examined to date. The importance of the factor(s) binding to the RIPE3b element in mediating cell type-specific insulin gene expression is further emphasized from transgenic mice studies (5). It was demonstrated that an enhancer construct containing both RIPE3b and E1 elements, RIPE3, could correctly regulate temporal and spatial expression of the transgene in vivo (5), whereas a construct containing the E1 element alone was unable to induce expression in transgenic animals (32). In all RIPE3 transgenic lines, the transgene (growth hormone) expression was detected in beta -cells, whereas in other lines, expression of growth hormone was also noted in alpha -cells (5).

In addition to regulating cell type-specific expression, factors binding to the enhancer elements, A3, E1, and RIPE3b, also regulate glucose-mediated alterations in insulin gene expression (30, 31, 33-36). Our earlier studies demonstrated that the binding activity of the cell-specific RIPE3b1 activator play a key role in regulating this glucose responsiveness (30, 31). Although the binding activity of the RIPE3b1 activator is induced in response to acute change in glucose concentration, under glucotoxic conditions, as observed in HIT T-15 cells cultured chronically in the presence of high concentrations of glucose, the RIPE3b1 and PDX-1 binding activities were inhibited (37-39). PDX-1 protein and mRNA levels were also inhibited in rat islets, following development of diabetes after partial pancreatectomy (40). These observations suggest that in vitro and in vivo glucotoxic conditions regulate the levels and activity of insulin gene transcription factors. Interestingly, the binding activity of the RIPE3b1 factor, but not of PDX-1, is inhibited under glucotoxic conditions in the insulinoma cell line beta TC-6 (41). This suggests that the inhibition of the RIPE3b1 activator binding may be the primary or initial defect under glucotoxic conditions.

The cell type-specific and glucose-responsive transcription factors of the insulin gene also play an important role in pancreatic development and differentiation of beta -cells. Mice homozygous for the null mutation in BETA2/NeuroD have a striking reduction in the number of beta -cells and fail to develop mature islets, develop severe diabetes, and die perinatally (26). The pdx-1 knockout mice have a more profound phenotype, being apancreatic; these animals also develop extreme hyperglycemia and die perinatally (20, 21, 42). During embryonic development, PDX-1 is also expressed in exocrine and ductal epithelial cells, whereas in the adult pancreas expression is predominantly restricted to beta -cells (20-22, 43). However, expression of PDX-1 is induced in the ductal epithelium during pancreatic regeneration in adult animals (44).

Similar to the PDX-1 and BETA2 factors, the RIPE3b1 activator, is an important regulator of cell type-specific and glucose-responsive expression of the insulin gene and may play an important role in pancreatic development and/or beta -cell differentiation. The C1 element (Fig. 1) in the RIPE3b region has been implicated as the binding site for the RIPE3b1 activator (14, 28, 31). Factors binding the C1 element were shown to functionally co-operate with E1 binding factors (BETA2-E12/E47) in regulating insulin gene expression (14, 25, 31, 45). In addition to C1, the RIPE3b region also contains an A element, A2 (Fig. 1 and Refs. 8, 46, and 47). The homeodomain family of transcription factors (PDX-1, HNF1 alpha , and Isl-1) that are important for pancreatic development bind insulin A elements (48-51). Furthermore, factors binding to A elements are implicated in synergistically interacting with other insulin gene transcription factors and regulating gene expression (52-54).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   Top, organization of the rat insulin II enhancer/promoter region with new and old names of different enhancer elements based on the simplified nomenclature. Bottom, suggested DNA binding sequence for the A2/GG1 and the C1 element in the RIPE3b region from Ref. 8.

Hence, in this study, we have further characterized the RIPE3b region to better understand the role of C1 and A2 elements in regulating insulin gene expression. We demonstrate that the presence of both C1 and A2 elements are essential for binding of the RIPE3b1 activator. We also identified factors that require the A2 element but not the C1 element for binding. Interestingly, one of the A2 element-binding complexes, A2.2, is selectively expressed in insulin-producing cell lines. We further demonstrate that two base pairs in the A2 element are critical for the formation of both beta -cell-specific DNA-binding complexes, RIPE3b1 and A2.2. Results from transient transfection analysis indicate that C1-A2 (RIPE3b1) and A2-specific factors positively regulate insulin gene expression. A mutation that prevents binding of both activators shows greater inhibition of insulin gene expression than a mutation that prevents binding of either of these factors alone. Based on these results we suggest that the presence of two beta -cell-specific factors with overlapping DNA-binding sites may provide an important means of regulating insulin gene expression in response to various metabolic/environmental signals.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissue Culture-- The HIT T-15 cell line was obtained from American Type Culture Collection (Manassas, VA), and all experiments were conducted with cells between passage numbers 68 and 80. beta TC-3 and beta TC·TET cell lines have been described earlier (55, 56) and were provided by Dr. Shimon Efrat. Non-insulin-producing cell lines used in this study are: HeLa cells (human cervical carcinoma), baby hamster kidney cells, and IM Duct (immorto -mouse ductal cell line, provided by Dr. Susan Bonner-Weir, Boston, MA). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with Ham's F-12 (22 mM glucose), 10% fetal bovine serum, penicillin, and streptomycin. For the preparation of medium with different glucose concentration, glucose-free Dulbecco's modified Eagle's medium and dialyzed fetal bovine serum (Life Technolgies Inc.) were used. Glucose levels in the medium were adjusted to the desired concentration by addition of filter sterilized glucose.

Extract Preparation-- HIT T-15 nuclear extracts, prepared using a large scale nuclear extract preparation protocol (57), were generously provided by Dr. Larry Moss (Tufts University, Boston, MA). Other insulin-producing and non-insulin-producing cell lines were cultured for 4-6 days, and nuclear extracts were prepared as described (58). To prepare extracts from HIT T-15 cells grown in the presence of different concentrations of glucose, cells were cultured for 4 days in Dulbecco's modified Eagle's medium/Ham's F-12 medium. Medium was then removed, cells were washed twice with phosphate-buffered saline, and medium containing the desired concentration of glucose was added. Cells were harvested after 48 h, and nuclear extracts were prepared (58).

Electrophoretic Mobility Shift Assays-- Oligonucleotides used as probes and competitors (Table I) were synthesized either by Sigma-Genosys (The Woodlands, TX) or by the DNA core facility at the Joslin Diabetes Center. In addition to the oligonucleotides listed in Table I, a series of two base pairs substitution (A left-right-arrow C and G left-right-arrow T) mutations in the RIPE3b (-126 to -101 bp) oligonucleotide were synthesized and named according to the two mutated nucleotides (e.g. -126·125m oligonucleotide is mutated at positions -126 and -125 bp in the RIPE3b oligonucleotide). Double-stranded oligonucleotides were radiolabeled with [alpha -32P]dCTP and the Klenow fragment of DNA polymerase I and used as probes. Binding reaction conditions for the RIPE3b, -139 to -113 and thyroid transcription factor-1 probes were identical (10 mM Tris, pH 7.8, 150 mM NaCl, 2 mM EDTA, 8 mM dithiothreitol, 0.4 mg of poly(dI-dC), 5% glycerol, 50-100 fmol of probe, and 3-5 µg of nuclear extracts). Binding reaction conditions for A3 (FLAT-E) and major late transcription factor probes have been described (31, 37). Competition experiments were performed by simultaneous addition of radiolabeled probe and excess unlabeled competitors to the binding reaction.


                              
View this table:
[in this window]
[in a new window]
 
Table I
This list shows the nucleotide sequences corresponding to the top strand of wild type and mutant oligonucleotides used in EMSA. Underlined nucleotides represents sites of mutated base pairs in the wild type oligonucleotides. A series of two base pair substitution (A iff  C and G iff  T) mutations in the RIPE3b oligonucleotide that were named according to two mutated nucleotides is not included in the table. The position of A3 and MLTF (31,37) and TTF-1 (60) oligonucleotides in the corresponding genes has been described. Oligonucleotides -150 to -125 (A2) and -148 to -122 (A2) represent rat insulin I and human insulin sequence homologous to rat insulin II -139 to -113 sequence.

The anti-IDX-1 (PDX-1) antibody was generously provided by Dr. Joel Habener (Massachusetts General Hospital, Boston, MA), whereas monoclonal anti-Nkx2.2 antibody was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA). For antibody supershift experiments, antibodies were preincubated with the nuclear extract for 20 min at room temperature, followed by another 20-min incubation in the presence of the radiolabeled probe. Binding reactions were then loaded onto a 6% nondenaturing polyacrylamide gel and were run in Tris-glycine-EDTA buffer (31). After completion of the run, gels were dried and scanned on a Molecular Dynamics PhosphorImager (Sunnyvale, CA), and bands were quantitated using Imagequant software.

DNA Constructs-- The insulin reporter construct-238 WT LUC has been described earlier (31). The QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to construct additional plasmids with specific mutations in the insulin enhancer. Double-stranded oligonucleotides containing desired mutations (underlined nucleotides) were synthesized to construct -125·124m LUC (-138 CA GGC AAG TGT TTT TAA ACT GCA GCT TCA GCC CCT CTG G, -100 bp), -122·121m LUC (-135 GC AAG TGT TTG GAC CCT GCA GCT TCA GCC CCT CTG G, -100 bp), and -110·109m LUC (-135 GC AAG TGT TTG GAA ACT GCA GCT TCC TCC CCT CTG GCC, -98 bp). The -238 WT LUC plasmid was used as the template with these oligonucleotides to construct reporter plasmids with mutations at the desired positions in the insulin enhancer. Plasmids were sequenced at the DNA core facility at Joslin Diabetes Center to confirm the construction of each mutant plasmid. To avoid the effect of nonspecific mutations outside the insulin enhancer region on the reporter gene expression, the plasmids were digested with restriction enzymes to release the mutated insulin enhancer fragments. These fragments were then used to replace the wild type insulin enhancer from the -238 WT LUC construct, resulting in the generation of reporter plasmids that differed only at the specific mutations in the insulin enhancer.

Transient Transfections and Luciferase Assays-- Approximately 18 h before transfection, 5 × 105 HIT T-15 or beta TC-3 cells were plated onto 60-mm2 plates. Reporter plasmids (2.5 or 2.0 µg for HIT T-15 or beta TC-3 cell line, respectively) were co-transfected with 0.1 µg of a pRL-Null (Promega, Madison, WI) plasmid, as an internal control. Although the pRL-Null plasmid lacks any of the eukaryotic enhancer elements, significant levels of constitutive Renilla luciferase expression have been observed from this construct (59). We also observed a linear relationship between the reporter gene expression and the amount of the pRL-Null plasmid used (data not shown). Plasmid DNA were transfected using LipofectAMINE (HIT T-15 cells, DNA:LipofectAMINE ratio of 1:4) or LipofectAMINE PLUS (beta TC-3 cells, DNA:LipofectAMINE PLUS ratio of 1:5) as descried earlier (31). To determine reporter gene expression a commercial Dual Luciferase Kit (Promega) was used. Whole cell extracts were prepared 48 h after transfection, using passive lysis buffer. Extracts were used to determine firefly (reporter plasmids) and Renilla (internal control) luciferase expression using the Moonlight 3010 Luminometer (Analytical Luminescence Laboratory, Sparks, MD). The ratio of the firefly:Renilla luciferase activity represents the normalized reporter gene expression. Transfection experiments were repeated several times, with at least two or three different plasmid preparations.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

C1 and A2 Elements Together Constitute the Binding Site for the RIPE3b1 and RIPE3b2 Factors-- Two sequencespecific RIPE3b (-126 to -101 bp) binding complexes have been identified. The RIPE3b1 complex is detected in insulin-producing cells, whereas the RIPE3b2 complex is detected in nuclear extracts from all cell lines examined to date (14, 27-31). Mutating two nucleotides (-112 and -111, a TC to CG substitution) in the RIPE3b probe prevented the formation of both the RIPE3b1 and the RIPE3b2 complexes (14, 28, 31). Methylation interference analysis indicated that the RIPE3b1 complex exerted a strong interference at Gs -107, -108, and -111 and weak interference at G -114 on the bottom strand, suggesting that the binding site may span from -114 to -107 bp (14). Based on these observations and other results, it was suggested that the RIPE3b region may contain two elements, C1 (-116 to -107) (for the binding of RIPE3b1 and RIPE3b2 factors) and A2 element (-126 to -113) (8, 46, 47). The C1 and A2 elements share a 4-bp segment (-116 to -113 bp) that contains the G -114 (bottom strand) implicated in the binding of the RIPE3b1 factor. Hence, to define the binding site for the RIPE3b1 activator as well as to characterize the role of the A2 element in this binding, we made a series of 2-bp substitution mutations in the RIPE3b oligonucleotide and studied the effect of these mutations on the RIPE3b1 binding.

Electrophoretic mobility shift assays (EMSA) were performed using radiolabeled RIPE3b (-126 to -101) probe and HIT T-15 nuclear extracts, in the presence or absence of 50-fold excess of unlabeled competitors (Fig. 2A). As expected, a 50-fold excess of the previously described unlabeled mutant RIPE3b oligonucleotide (having TC to CG substitution at -112 and -111 bp) did not compete for binding to either RIPE3b1 or RIPE3b2 factors, whereas the unlabeled wild type RIPE3b oligonucleotide successfully competed for binding of these factors. Competition between the RIPE3b probe and a series of RIPE3b oligonucleotides with mutations in two consecutive nucleotides showed interesting binding profiles for the RIPE3b1 and RIPE3b2 factors (Fig. 2A). Mutations in the nucleotides from -112 to -107 bp (C1 region) inhibited the ability of excess unlabeled competitors to compete as effectively as the wild type oligonucleotide, confirming the importance of this region for binding of these factors. Interestingly, the oligonucleotides with mutations in the A2 element (-124 to -117 bp) were also ineffective competitors implying a role of A2 element in the binding of the RIPE3b1 and RIPE3b2 factors. Substitution mutations in the four nucleotides -116 to -113 bp (residing between these two regions) had no effect on their ability to compete as effectively as the wild type oligonucleotide. These results indicate that binding of the RIPE3b1 and the RIPE3b2 complexes require sequences present in both the C1 and A2 elements. Our results also suggest that nucleotides at -110 to -109 bp and -122 to -121 bp are most critical for the binding of these factors (Fig. 2B).



View larger version (57K):
[in this window]
[in a new window]
 
Fig. 2.   Identification of the nucleotide sequence critical for DNA binding of the RIPE3b1 and the RIPE3b2 factors. A, HIT T-15 nuclear extract was incubated with radiolabeled rat insulin II RIPE3b (-126 to -101 bp) probe, in the absence (-) or presence of 50-fold excess of wild type RIPE3b (lane W), a previously described mutant (-112.111m) oligonucleotide (lane M) (14, 31), or a series of different mutant oligonucleotides as unlabeled competitors. Binding reactions were analyzed by EMSA, and the positions of the RIPE3b1 and RIPE3b2-binding complexes are indicated. The mutant competitors -126·125m to -102·101m represents the RIPE3b oligonucleotides with mutations in specified consecutive two nucleotides. B, quantitation of EMSA result described for A by PhosphorImager. Results for the RIPE3b1 and RIPE3b2-binding complexes are presented as percentages of remaining activity in the presence of competitor compared with that in the absence of the competitor. C, effect of altering distance between two binding regions (-124 to -117 bp and -112 to -107 bp) on the RIPE3b1 and the RIPE3b2 binding activity. HIT T-15 nuclear extract was incubated with the radiolabeled RIPE3b probe in the absence (-) or presence of 50-fold excess of cold competitor. The cold competitors W and M represent wild type and mutant oligonucleotides as described in A. The Delta 2 represents RIPE3b oligonucleotide with deletion of 2 bp (CT) at positions-114.113 bp, whereas In2 and In5 represent insertion of 2 or 5 bp (CT or CTTAG respectively), between -115 and -114 bp in the RIPE3b oligonucleotide.

Because two distinct elements, C1 and A2, in the RIPE3b region are required for binding of the RIPE3b1 and the RIPE3b2 factors, we next asked whether the exact spacing between these elements was essential for the binding activity. Competitor oligonucleotides were synthesized with an insertion of two nucleotides or a deletion of two or five nucleotides between the C1 and A2 elements. As shown in Fig. 2C, altering the distance between the two elements prevented these oligonucleotides from competing for binding to these factors. Results presented in Fig. 2 suggest that the RIPE3b1 and RIPE3b2 factors bind a large (-107 to -124 bp) DNA-binding element of the insulin enhancer region. The RIPE3b binding activity not only requires the nucleotide sequence present in both the C1 and A2 elements but also requires the exact distance between these two elements. Hence, we suggest that RIPE3b1 and RIPE3b2 factors bind a composite C1-A2 element.

A beta -Cell-specific Factor Selectively Binds the A2 Element-- Because the RIPE3b1 factor binds the C1-A2 element, we were interested in identifying factors that selectively bind either the C1 or A2 elements. Factors that selectively bind one of these elements could have a potential regulatory role in modulating the DNA binding and transcriptional activation mediated by the RIPE3b1 activator. We consistently observed only two specific RIPE3b (-126 to -101 bp) binding complexes when nuclear extracts from insulin-producing cell lines were used. When the C1 element-specific probe (-116 to -101 bp) was used in EMSA with HIT T-15 nuclear extracts, only nonspecific DNA-binding complexes were detected (data not shown). These observations indicate that insulin-producing cells may not contain a C1 element-specific binding factor.

To identify factors that selectively bind the A2 element, we designed a new probe, -139 to -113 bp, that included the A2 element and additional sequences upstream of the RIPE3b region (Fig. 3A). In EMSA using radiolabeled -139 to -113 bp probe and HIT T-15 nuclear extracts, we detected three binding complexes with gel mobilities distinct from both the RIPE3b1 and RIPE3b2 complexes (Fig. 3B). Competition analysis indicated that these complexes bind specifically to the -139 to -113 bp region of the insulin enhancer. Formation of A2 complexes was not inhibited in the presence of excess unlabeled RIPE3b, C1, A2 (-126 to -113) or sequences upstream of the RIPE3b region (-139 to -127) (Fig. 3). These observations suggest that the formation of A2 complexes requires the rat insulin II enhancer region from -139 to -127 bp, in addition to sequence spanning A2 element (-126 to -113). Furthermore, these results demonstrate that the A2-specific complexes selectively bind to the -139 to -113 region of the insulin enhancer, independent of the presence of the C1 element. Results presented in Fig. 3 also demonstrate that formation of the RIPE3b1 and RIPE3b2 complexes is not inhibited in the presence of excess unlabeled C1, A2, or -139 to -113 regions of the insulin enhancer. Taken together, these observations demonstrate the presence of factors with overlapping C1-A2 and A2-specific DNA binding specificity in nuclear extracts from insulin-producing cells.



View larger version (70K):
[in this window]
[in a new window]
 
Fig. 3.   Rat insulin II enhancer region from -126 to -113 bp is essential for the formation of two distinct groups of binding complexes. A, the nucleotide sequence of rat insulin II enhancer region from -139 to -101 bp, and locations of the C1 and the A2 elements are shown. In addition, relative positions of various probes and competitors used in this study are indicated. B, radiolabeled rat insulin II probes, RIPE3b (-126 to -101 bp) and -139 to -113 bp, were incubated with HIT T-15 nuclear extract in the absence (-) or presence of 50-fold excess of cold RIPE3b, -126 to -113 bp (A2), -116 to -101 bp (C1), -139 to -113 bp, and -139 to -127 bp oligonucleotides. The positions of RIPE3b1 and RIPE3b2 complex and three A2-specific complexes (A2.1, A2.2, and A2.3) are indicated. In addition to -126 to -113 bp, formation of RIPE3b and A2-specific complexes requires -113 to -101 bp and -139 to -127 bp regions, respectively.

The region corresponding to the -139 to -113 bp of the rat insulin II gene is highly conserved in the rat insulin I and the human insulin genes (Fig. 4A). Competition analysis was performed to confirm that the A2 factors that bind the rat insulin II -139 to -113 probe would successfully bind the corresponding region from rat insulin I and the human insulin gene (Fig. 4B). EMSA were performed with the rat insulin II -139 to -113 probe and HIT T-15 nuclear extracts in the absence or presence of increasing concentrations of unlabeled excess -139 to -113 bp oligonucleotide or the corresponding region from the rat insulin I and human insulin gene and the RIPE3b oligonucleotide. Formation of A2-specific complexes was inhibited by excess unlabeled -139 to -113 oligonucleotide and its homologous regions from rat I and human insulin gene but not by the RIPE3b oligonucleotide. These observations suggest that the DNA-binding site recognized by A2-specific factors is highly conserved.



View larger version (78K):
[in this window]
[in a new window]
 
Fig. 4.   The A2 element is conserved within human (hIns), rat I (rIns I), and rat II (rIns II) insulin genes. A, DNA sequence from -139 to -113 bp region of rat insulin II enhancer and corresponding sequence from rat insulin I and human insulin genes are shown. The asterisk denotes nucleotides that are identical within these sequences, and + or - symbol represent conserved or distinct nucleotide substitutions, respectively. B, the ability of the A2 elements from rat insulin I and human insulin gene to compete with rat insulin II A2 element binding activity in HIT T-15 nuclear extract was analyzed. HIT T-15 extract was incubated with rat insulin II -139 to -113 bp probe in the absence or presence of excess cold -139 to -113 bp oligonucleotide from rat II or corresponding region from rat I or human insulin genes or rat II RIPE3b oligonucleotide as competitors, and reactions were analyzed by EMSA. Positions of A2-specific complexes A2.1, A2.2, and A2.3 are indicated.

Because tissue- or cell type-specific transcription factors are major regulators of critical genes in a given cell or tissue, we next determined whether A2-specific factors were selectively expressed in insulin-producing cells. Nuclear extracts from insulin-producing and non-insulin-producing cell lines were analyzed for their ability to form A2-specific complexes. Nuclear extracts from HIT T-15, beta TC-3, and beta TC·TET (insulin-producing cell lines) and baby hamster kidney, HeLa, and a pancreatic ductal cell line IM Duct (non-insulin-producing cells) were incubated with radiolabeled -139 to -113 probe from rat insulin II gene and analyzed by EMSA. As shown in Fig. 5, the A2.1 binding activity was detected in all cell lines, whereas the A2.2-binding complex was formed only with the nuclear extracts from insulin-producing cell lines. These results demonstrate that the A2.2 factor is selectively expressed in the insulin-producing cells. In addition, the A2.2 factor binds a conserved region in the insulin enhancer that overlaps with the binding site for RIPE3b1. The presence of two distinct beta -cell-specific DNA-binding factors with overlapping DNA-binding sites suggest an important role for these factors in regulating insulin expression.



View larger version (80K):
[in this window]
[in a new window]
 
Fig. 5.   The A2.2-binding complex is detected only in insulin-producing cell lines. Nuclear extracts from insulin-producing (HIT T-15, beta TC-3, and beta TC·TET) and non-insulin-producing (baby hamster kidney, HeLa, and IM Duct) cell lines were incubated with radiolabeled rat insulin II -139 to -113 probe. Binding reactions were analyzed by EMSA and positions of A2-specific complexes are indicated. A2.2 complex (asterisk) was formed only in the presence of nuclear extract from insulin-producing cell lines has been indicated.

A2-specific Factors Are Distinct from PDX-1 and Nkx2.2-- The A2 element has been classified based on its homology to other A elements. The homeodomain family of transcription factors, such as PDX-1, bind these A elements (48-51). In addition to homology with the A elements, the -139 to -113 bp region of rat insulin II also contains a consensus-binding element (CAAGTG) for the Nkx2 family of transcription factors (60-62). Nkx2.2, a member of Nkx2 family, is important for differentiation of pancreatic beta -cells (63). Hence, we performed competition analysis and antibody supershift assay to determine whether the PDX-1 and Nkx2.2 are present in the A2-specific complexes.

Nuclear extracts from HIT T-15 cells were incubated with radiolabeled A3 or -139 to -113 probes, in the absence or presence of excess cold A3 and -139 to -113 bp oligonucleotide. In addition, anti- PDX-1 antibody was added to binding reactions containing the A3 and -139 to -113 probes, and the reactions were analyzed by EMSA. As shown in Fig. 6A, formation of the A2-binding complex is not inhibited by the presence of excess unlabeled A3 oligonucleotide. In addition, the anti-PDX-1 antibody neither prevented the formation of the A2-specific complexes nor altered their mobility, suggesting that the A2-specific complex does not contain PDX-1. These observations are consistent with the converse experiment, where presence of the excess unlabeled -139 to -113 oligonucleotide did not prevent formation of PDX-1 or other A3-specific complexes (Fig. 6A). We used a similar strategy to identify the presence of Nkx2.2 in A2-specific complexes. Because the Nkx2.2 binding site is not known, we used the binding element from the rat thyroglobulin gene for the thyroid transcription factor-1, a Nk2 family member (60). As shown in Fig. 6B, Nkx2.2 is not part of the A2-specific complexes. Additionally, oligonucleotides with mutation in the Nkx consensus sequence at positions -133 to -132 (-133·132m) competed as effectively as the wild type -139 to -113 oligonucleotide, suggesting this sequence was not required for binding of A2-specific factors. These results support the conclusion that the A2 binding factors are distinct from other homeodomain proteins and the Nkx2 family of transcription factors. These important observations suggest that the beta -cell-specific A2.2 activator may represent a novel factor.



View larger version (51K):
[in this window]
[in a new window]
 
Fig. 6.   The A2 element-binding complexes are distinct from the transcription factors PDX-1 and Nkx2.2. HIT T-15 nuclear extract was incubated with A3 or -139 to -113 probes (A), or -139 to -113 or thyroid transcription factor-1 probes (B) in the absence or presence of alpha PDX-1 (A) or alpha NKX2.2 (B) antibodies or 50-fold excess of indicated cold competitors. Mutant competitor -133·132m contains an AA to CC mutation in Nkx2 binding consensus (CAAGTG) at positions -133 and -132 bp in -139 to -113 oligonucleotide. Positions of A2.2, PDX-1, and Nkx2.2 complex are indicated, and the asterisk denotes migration of supershifted complex in the presence of specific antibody.

The Binding Activity of A2-specific Factors Is Not Regulated by Glucose-- DNA binding activity of the RIPE3b1 activator is induced in response to an increase in glucose concentrations (28, 30, 31). This increased binding of the RIPE3b1 activator to the insulin enhancer may be responsible for the observed induction of insulin gene expression. To test whether glucose can stimulate binding activity of A2-specific factors, nuclear extracts were prepared from HIT T-15 cells grown in the presence of different concentrations of glucose. Equal concentrations of nuclear extracts from HIT T-15 cells grown in 0.2, 1.0, 5.0, and 22.0 mM glucose were incubated with RIPE3b and -139 to -113 probes, and binding reactions were analyzed by EMSA (Fig. 7). As a control, equal concentration of HIT T15 nuclear extracts were also analyzed for binding to Adenoviral major late transcription factor (data not shown). As we showed earlier (31), increasing concentrations of glucose significantly induced the binding of the RIPE3b1 activator (Fig. 7) but had no effect on the binding activity of ubiquitously expressed major late transcription factor (data not shown). However, when the same experiment was performed with -139 to -113 probe under identical conditions, there was no difference in the binding activity of any A2-specific activators in response to alteration in glucose concentrations. These results demonstrate that glucose differentially regulates binding activity of the RIPE3b- and A2-specific activators.



View larger version (81K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of glucose concentrations on the A2 element binding activity. Equal concentrations of nuclear extracts prepared from HIT T-15 cells grown in the presence of 0.2, 1.0, 5.0, or 22.0 mM glucose were analyzed for RIPE3b and -139 to -113 (A2) binding activity by EMSA. Positions of the specific DNA-binding complexes, RIPE3b1, RIPE3b2, A2.1, A2.2, and A2.3, are indicated.

Nucleotides at Positions -122 and -121 bp Are Essential for the Binding of Both RIPE3b (C1-A2) and A2-specific Activators-- An oligonucleotide probe spanning the binding region of both RIPE3b- and A2-specific factors, -139 to -101, can bind all five (RIPE3b1, REIP3b2, A2.1, A2.2, and A2.3) DNA-binding complexes (data not shown). To analyze the role of overlapping binding regions in the binding of these factors, various mutations in the larger -139 to -101 oligonucleotide (LM) were constructed. To determine the role of nucleotides in the overlapping region in the formation of C1-A2 and A2-specific DNA-binding complexes, competition analysis was performed. HIT T-15 nuclear extract was incubated with the RIPE3b (-126 to -101) or -139 to -113 probes, in the absence or presence of excess wild type (-139 to -101, RIPE3b, -116 to -101 and -139 to -113) or mutant (-110·109LM, -114·113LM, -122·121LM, -125·124LM, and -127·126LM) oligonucleotides. These binding reactions were analyzed by EMSA, and results are presented in Fig. 8. Presence of excess cold wild type oligonucleotides in the binding reaction had the expected effect (see Figs. 2 and 3) on binding of factors to either the RIPE3b or -139 to -113 probe. Mutating nucleotides -110 to -109 bp and -122 to -121 bp in the larger -139 to -101 oligonucleotide prevented these oligonucleotides from competing with the RIPE3b probe for binding of RIPE3b1 and RIPE3b2 factors, whereas mutations at -114 to -113 bp, -125 to -124 bp, and -127 to -126 bp positions had no effect on binding of these factors. These observations confirm that binding of RIPE3b1 and RIPE3b2 factors depends on nucleotides at positions -110 to -109 bp and -122 to -121 bp in the insulin enhancer, whereas the other six nucleotides can be substituted without any significant effect on the binding activity. The results from competition analysis for binding of A2-specific complexes to -139 to -113 probe were extremely interesting. The formation of the A2-specific complexes, like the C1-A2 complexes, depends on nucleotides at positions -122 to -121 bp in the insulin enhancer but not on nucleotides at positions -110 to -109 bp. Furthermore, nucleotides at positions -125 to -124 bp were only essential for formation of the A2-specific complexes and not for C1-A2 specific complexes. We also used these oligonucleotides as probes and confirmed the requirement of these nucleotides for binding of the RIPE3b- and A2-specific complexes (data not shown). These observations suggest that the rat insulin enhancer region from -139 to -101 bp represents the binding site for two distinct beta -cell-specific factors, and nucleotides at positions -122 and -121 bp are critical for the binding of each factor. Furthermore, these observations suggest that mutations at positions -110 to -109 bp or -125 to -124 bp can selectively prevent binding of C1-A2 or A2-specific factors, respectively.



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 8.   Nucleotides at -122 and -121 bp in the rat insulin II enhancer are critical for RIPE3b (C1-A2) and -139 to -113 (A2-specific) binding activity. The RIPE3b and -139 to -113 bp binding activity in the HIT T-15 nuclear extract was analyzed by EMSA. Cold competitors (50×) used were either wild type rat insulin II oligonucleotides (-139 to -101 bp, RIPE3b, -116 to -101 bp, and -139 to -113 bp) or were mutant -139 to -101 bp oligonucleotide with the mutations in the indicated base pairs. Positions of specific DNA-binding complexes are indicated. An asterisk denotes mutation critical for the formation of C1-A2 and A2-specific binding complexes, whereas a pound sign denotes mutations that selectively prevents formation of either C1-A2 or A2-specific complexes.

Both C1-A2 and A2-specific Activators Positively Regulate Insulin Gene Expression-- To determine the role of C1-A2 and A2-specific factors in regulating insulin gene expression, mutations were made in the rat insulin II enhancer (-238 to +2 bp) construct driving expression of the LUC reporter gene. Wild type (-238 WT LUC) and the mutant insulin enhancer luciferase reporter constructs (-110·109m LUC, -122·121m LUC, and -125·124m LUC), were transfected into two insulin-producing cell lines, HIT T-15 and beta TC-3. 48 h after transfection, cells were harvested, and whole cell extracts was prepared as described under "Experimental Procedures." Luciferase activity from insulin reporter constructs was normalized for transfection efficiency, and the results are presented relative to the activity of -238 WT LUC construct (Fig. 9).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of mutations in the C1 and A2 regions on the rat insulin II gene expression. The structure of rat insulin II LUC reporter constructs (-238 WT LUC) denotes positions of A2 and C1 elements, the RIPE3b (C1-A2) binding factors (open box) and A2-specific binding factors (oval). Reporter construct -110·109m LUC, -122·121m LUC, -125·124m LUC with mutations (designated by ×) that prevent binding of either C1-A2 factors or A2-specific factor or both factors is shown. Equal concentration of wild type or mutant plasmids were transfected into the HIT T-15 (hatched bar) or beta TC-3 (filled bar) cell lines. Luciferase activity was determined 48 h after transfection, and the results are presented as percentages of relative luciferase activity by the -238 WT LUC plasmid ± S.E.

Earlier studies showed that an insulin enhancer construct with mutations at -112 to -111 bp prevents formation of the RIPE3b1 and RIPE3b2 complexes and inhibits insulin gene expression (14, 28, 31). Similarly we observed that mutations at -110 to -109 bp that prevents formation of C1-A2 specific binding complexes, significantly inhibit (76 and 87% in HIT T-15 and beta TC-3 cell lines, respectively) insulin gene expression. Mutations at positiona -125 to -124 that selectively prevent binding of A2-specific factors also inhibited insulin gene expression by 43 and 37% in HIT T-15 and beta TC-3 cell lines, respectively. Interestingly, mutations that prevent binding of both C1-A2 and A2-specific binding factors (-122-121 bp) showed significantly more inhibition (92 and 96% in HIT T-15 and beta TC-3 cell lines, respectively) of insulin gene expression than mutations that prevented binding of either of these factors. This inhibition of insulin gene expression was consistently more (about 1.7-2.0-fold) than that that can be accounted by simple additive contributions of these factors. Hence, we suggest that C1-A2 (RIPE3b) and A2-specific binding factors may functionally cooperate with each other and positively regulate insulin gene expression. Mutations that prevents binding of these factors to the insulin enhancer can drastically inhibit insulin gene expression even when the binding site for other key transcription factors (PDX-1 and BETA2) is unaffected, emphasizing the importance of these factors on insulin gene expression. We are currently investigating the mechanism by which C1-A2 and A2-specific factors contact overlapping DNA-binding sites and cooperatively activate insulin gene expression.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that beta -cell-specific RIPE3b1 and A2.2 factors recognize overlapping DNA-binding sites within the insulin enhancer. These factors can each positively regulate insulin gene expression as well as cooperatively activate insulin gene expression. Because the binding activity of RIPE3b1 activator is regulated by glucose (Fig. 7 and Refs. 28, 30, and 31) and phosphorylation (28), overlapping binding specificity and functional cooperation between A2.2 and RIPE3b1 activator may play major roles in regulating insulin expression in response to various metabolic stimulus. These observations suggest that like RIPE3b1, the A2.2 activator is an important regulator of insulin gene expression. Initial characterization indicates that A2-specific binding factors are distinct from PDX-1 (Fig. 6A). Excess unlabeled A3 oligonucleotide did not prevent formation of A2-specific complexes (Fig. 6A), suggesting that A2 binding factors might belong to a different family of transcription factors than the PDX-1 and other A element binding homeodomain factors. Similarly, anti-Nkx2.2 antibody did not recognize any of the A2-specific complexes, and mutating the Nk2 consensus sequence did not affect the formation of A2 complexes. These results indicate that A2-specific activators are novel and distinct from the Nk2 and other homeodomain family of transcription factors.

The importance of the RIPE3b region in mediating beta -cell-specific and glucose-responsive insulin gene expression is well established (5, 14, 27-31, 37, 41). Although two insulin enhancer elements, C1 and A2, were described in the RIPE3b region, we were unable to detect any C1 selective binding activity in insulin-producing cells. Our results demonstrate that C1 binding factors (RIPE3b1 and RIPE3b2) require sequences present in the A2 element. Similarly, A2-specific factors require additional nucleotides upstream of the RIPE3b region for their binding (Figs. 2 and 3). These observations suggest a need to redefine the enhancer elements in the RIPE3b region. Because we were unable to identify any C1-specific binding factor, we suggest that designation of the C1 region as a DNA-binding element may not be accurate. We suggest that the binding element for RIPE3b1 and RIPE3b2 activator may accurately be described as C1-A2 or the CA element. Similarly, additional nucleotides must be included within the A2 element to describe it as a binding site for A2-specific factors. These criteria would help interpret earlier studies that describe mutations and deletions in C1 and A2 elements and their effect on insulin gene expression (47, 64). For example, Tomonari and co-workers (47) reported the role of the A2 (GG1) element in regulating human insulin gene expression. Their results obtained from an A2 deletion construct would not only inhibit activation from the A2-specific factor but also from the RIPE3b1 activator. Similarly, other mutations in the A2 region described in the their study would also inhibit activation mediated by both RIPE3b1 and A2.2 factors. In addition, functional cooperation between the C1-A2 and A2-specific factors should be considered prior to interpreting results from earlier studies. We therefore suggest that a clear designation of the C1-A2 and A2 elements would be helpful in analyzing the role of this region in insulin gene expression.

Mutational analysis of the RIPE3b region has provided important information regarding the RIPE3b1 and RIPE3b2 activators. Binding of these factors requires at least a 16-bp-long region (-123 to -107 bp; Figs. 2 and 8) of the rat insulin II enhancer. Nucleotides critical for binding of these factors are separated into two distinct regions, -112 to -107 bp and -123 to -117 bp. Although the nucleotides between these regions can be mutated without any effect on binding activity and insulin gene expression (Fig. 2A, data not shown, and Ref. 28), attempts to alter spacing between these regions by either insertion or deletion mutations prevented DNA binding (Fig. 2C). These observations suggest that RIPE3b1 and RIPE3b2 factors not only recognize specific nucleotide sequence but also the recognize specific structure/organization of these nucleotides. The two critical nucleotide regions are spaced apart by about one helical turn of DNA, suggesting that RIPE3b1 and RIPE3b2 activators may recognize only one surface of the DNA helix. This is consistent with the results from methylation interference analysis of RIPE3b1 and RIPE3b2 complexes by Shieh and colleagues (14, 29), showing a strong interference at Gs -107, -108, and -111 and a weak interference at -114 bp. Furthermore, results from the methylation interference experiment demonstrated interference at Gs only on the bottom strand and not on the top strand. Our mutational analysis (Fig. 2) and transient transfection data (Fig. 9) are consistent with the importance of nucleotides -107, -108, and -111 bp in insulin gene expression. The role of weak interference at -114 bp is unclear, because -113 and -114 bp are not critical for binding nor gene expression (data not shown). Also, Zhao and co-workers (28) demonstrated that mutating -114 bp had no effect on insulin gene expression, suggesting that weak interference at -114 bp is not critical for binding of the RIPE3b1 factor and insulin gene expression. Our results demonstrate the requirement of a large binding region for the RIPE3b1 and the RIPE3b2 factors (-123 to -107 bp; Figs. 2 and 8); however, it is unclear why no interference was observed at Gs on the bottom strand at positions -117 and -120 bp.

RIPE3b1 and RIPE3b2 factors have identical DNA binding properties, suggesting that they may belong to the same family of transcription factors. These observations are in contrast with the results described by Shieh and colleagues (14, 29) in that RIPE3b1 and RIPE3b2 complexes have distinct methylation interference profiles and recognize distinct DNA-binding sites. Rip-1, one of the components of the RIPE3b2 complex, was cloned and identified as the hamster homologue of the mouse and human gene Smbp-2, a protein that binds to the immunoglobulin m chain switch region and was also cloned as glial factor-1 (29). Glial factor-binding element (GFE) selectively competes for the formation of the RIPE3b2 complex but not for the RIPE3b1 complex. Although the nucleotide sequence in the GFE element shares some homology with the C1 element, no homology in the A2 region can be observed (29). Similarly, GFE lacks significant homology with that for Smbp-2 (Sm), and Shieh and colleagues (29) suggested that the Rip-1 may bind both sites by recognizing a structure rather than the DNA sequence. However, based on sequence requirements for the binding of RIPE3b1 and RIPE3b2 factors (Fig. 2), it is unclear how the GFE element can selectively inhibit the formation of RIPE3b2 complex. One possible explanation could be that the binding specificity of Rip-1 might be distinct from that of the multi-protein RIPE3b2 complex. Consequently, excess unlabeled GFE oligonucleotide will selectively remove Rip1 from the DNA binding reaction, thereby preventing formation of RIPE3b2 complex.

The salient finding of the present study is that two base pairs are critical for DNA binding of two beta -cell-specific transcriptional activators. Several possibilities could explain how two factors can recognize the same base pairs and not hinder the action of the other factor. It is possible that the different factors recognize the binding site on different alleles within a cell or in different cells in a population. Transient transfection analysis of a cell population does not discriminate between these possibilities and the binding of both factors to the same allele. Nonetheless, because C1-A2 and A2-specific activators cooperatively activate insulin gene expression, these factors may bind the same element on an allele.

Other examples of transcription factors binding to an overlapping binding site that result in either activation or inhibition of gene expression exist. Overlapping binding specificity of SP1 and C/EBPs in the C/EBPalpha promoter has been implicated in regulating adipocyte differentiation (65). During normal growth conditions, the SP1 factor binds to the overlapping C/EBP binding site and prevents binding of other factors. Differentiation signals decrease levels of the SP1 factor, which facilitates access of C/EBPs to the binding site and results in the activation of the C/EBPalpha gene expression (65). Similarly, there are several examples where both factors recognizing overlapping binding sites positively activate gene expression (66-69). Overlapping binding of transcription factors NF-kappa B and STAT6 can positively and negatively regulate eotaxin and E-selectin gene expression, respectively (69, 70). In the E-selectin promoter, the STAT6 binding site shares 5 bp with the NF-kappa B binding site, whereas in the eotaxin gene only 4 bp are shared. Matsukura and colleagues (69) suggested that this difference in DNA structure may permit simultaneous binding of these factors to overlapping binding sites and activate eotaxin gene expression. Similarly, it is possible that the RIPE3b1 activator may bind only on one surface of the C1-A2 region (as discussed above), which permits simultaneous occupation of overlapping regions by the A2.2-specific activator, providing a possible mechanism by which both these factors can positively regulate insulin gene expression. Transcription factors recognizing overlapping binding sites can integrate responses from multiple positive and negative signals by inhibiting or cooperatively activating gene expression (65-70). Key insulin gene transcription factors must respond to multiple signals required to mediate beta -cell-specific and glucose-responsive insulin gene expression as well as regulate pancreatic development and differentiation of beta -cells. Because insulin gene expression is regulated in response to metabolic and environmental signals, key insulin gene transcription factors may integrate these responses by functionally cooperating with each other and/or binding overlapping sites. We suggest that the RIPE3b1 and A2.2 activators may represent one such regulatory unit. Because phosphorylation and alterations in glucose concentration regulate the activity of the RIPE3b1 factor, we suggest that RIPE3b1 and A2.2 activators may play a major role in regulating insulin gene expression in response to metabolic signals. In addition, overlapping binding specificity of these factors may also have a role in regulating pancreatic development and/or differentiation of beta -cells. Availability of cloned RIPE3b1 and A2.2 factors would permit better understanding of the mechanisms by which these factors bind and regulate insulin gene expression; we are currently pursuing these objectives.


    ACKNOWLEDGEMENTS

We thank Dr Joel Habener (Massachusetts General Hospital, Boston, MA) for IDX-1 antibody; Dr. Larry Moss (Tufts University, Boston, MA) for providing the HIT T-15 nuclear extracts and helpful comments on the manuscript; Drs. Gordon Weir and Susan Bonner-Weir for constructive criticism of the manuscript and for their support; and Martin Olbrot for helpful suggestions. We acknowledge the services provided by the DNA and Tissue culture cores of Joslin Diabetes Center (supported by National Institutes of Health Grant DK-36836 to the Diabetes Endocrinology Research Center).


    FOOTNOTES

* This work was supported by a Juvenile Diabetes Foundation International Research Grant, a National Institutes of Health Diabetes Endocrinology Research Center Pilot and Feasibility Study Grant, and a Career Development Award from American Diabetes Association (to A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Research Div., Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-264-2756; Fax: 617-732-2650; E-mail: arun.sharma@joslin.harvard.edu.

Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M008415200


    ABBREVIATIONS

The abbreviations used are: bp, base pair(s); WT, wild type; LUC, luciferase; EMSA, electrophoretic mobility shift assay(s); GFE, glial factor-binding element.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Dandoy-Dron, F., Monthioux, E., Jami, J., and Bucchini, D. (1991) Nucleic Acids Res. 19, 4925-4936[Abstract]
2. Edlund, T., Walker, M. D., Barr, P. J., and Rutter, W. J. (1985) Science 30, 912-916
3. Hanahan, D. (1985) Nature 315, 115-122[Medline] [Order article via Infotrieve]
4. Crowe, D. T., and Tsai, M.-J. (1989) Mol. Cell. Biol. 9, 1784-1789[Medline] [Order article via Infotrieve]
5. Stellrecht, C. M. M., DeMayo, F. J., Finegold, M. J., and Tsai, M.-J. (1997) J. Biol. Chem. 272, 3567-3572[Abstract/Free Full Text]
6. Stein, R. (1993) Trends Endocrinol. Metab. 4, 96-100
7. Sander, M., and German, M. S. (1997) J. Mol. Med. 75, 327-340[CrossRef][Medline] [Order article via Infotrieve]
8. German, M., Ashcroft, S., Docherty, K., Edlund, H., Edlund, T., Goodison, S., Imura, H., Kennedy, G., Madsen, O., Melloul, D., Moss, L. G., Olson, L. K., Permutt, M. A., Philippe, J., Robertson, R. P., Rutter, W. J., Serup, P., Stein, R., Steiner, D., Tsai, M.-J., and Walker, M. D. (1995) Diabetes 44, 1002-1004[Medline] [Order article via Infotrieve]
9. German, M. S., Moss, L. G., Wang, J., and Rutter, W. J. (1992) Mol. Cell. Biol. 12, 1777-1788[Abstract]
10. Peshavaria, M., Gamer, L., Henderson, E., Teitelman, G., Wright, C. V. E., and Stein, R. (1994) Mol. Endocrinol. 8, 806-816[Abstract]
11. Petersen, H. V., Serup, P., Leonard, J., Michelsen, B. K., and Madsen, O. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10465-10469[Abstract/Free Full Text]
12. Ohlsson, H., Thor, S., and Edlund, T. (1991) Mol. Endocrinol. 5, 897-904[Abstract]
13. Boam, D. S. W., and Docherty, K. (1989) Biochem. J. 264, 233-239[Medline] [Order article via Infotrieve]
14. Shieh, S-Y., and Tsai, M.-J. (1991) J. Biol. Chem. 266, 16708-16714[Abstract/Free Full Text]
15. Karlsson, O., Edlund, T., Barnett Moss, J., Rutter, W. J., and Walker, M. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8819[Abstract]
16. Whelan, J., Cordle, S. R., Henderson, E., Weil, P. A., and Stein, R. (1990) Mol. Cell. Biol. 10, 1564-1572[Medline] [Order article via Infotrieve]
17. Ohlsson, H., Karlsson, K., and Edlund, T. (1993) EMBO J. 12, 4251-4259[Abstract]
18. Leonard, J., Peers, B., Johnson, T., Ferreri, K., Lee, S., and Montminy, M. R. (1993) Mol. Endocrinol. 1275-1283
19. Miller, C. P., McGehee, R. E., and Habener, J. F. (1994) EMBO J. 13, 1145-1156[Abstract]
20. Offield, M. F., Jetton, T. L., Labosky, P., Ray, M., Stein, R., Magnuson, M., Hogan, B. L. M., and Wright, C. V. E. (1996) Development 122, 983-985[Abstract/Free Full Text]
21. Ahlgren, U., Jonsson, J., and Edlund, H. (1996) Development 122, 1409-1416[Abstract/Free Full Text]
22. Guz, Y., Montminy, M. R., Stein, R., Leonard, J., Gamer, L. W., Wright, C. V. E., and Teitelman, G. (1995) Development 121, 11-18[Abstract/Free Full Text]
23. German, M. S., Blanar, M. A., Nelson, C., Moss, L. G., and Rutter, W. J. (1991) Mol. Endocrinol. 5, 292-299[Abstract]
24. Peyton, M., Moss, L., and Tsai, M. J. (1994) J. Biol. Chem. 269, 25936-25941[Abstract/Free Full Text]
25. Naya, F. J., Stellrecht, C. M. M., and Tsai, M.-J. (1995) Genes Dev. 9, 1009-1019[Abstract]
26. Naya, F. J., Huang, H. P., Qiu, Y., Mutoh, H., DeMayo, F. J., Leiter, A. B., and Tsai, M.-J. (1997) Genes Dev. 11, 2323-2334[Abstract/Free Full Text]
27. Robinson, G. L. W. G., Peshavaria, M., Henderson, E., Shieh, S.-Y., Tsais, M.-J., Teitelman, G., and Stein, R. (1994) J. Biol. Chem. 269, 2452-2460[Abstract/Free Full Text]
28. Zhao, L., Cissell, M. A., Henderson, E., Colbran, R., and Stein, R. (2000) J. Biol. Chem. 275, 10532-10537[Abstract/Free Full Text]
29. Shieh, S. Y., Stellrecht, C. M. M., and Tsai, M. J. (1995) J. Biol. Chem. 270, 21503-21508[Abstract/Free Full Text]
30. Sharma, A., Fusco-DeMane, D., Henderson, E., Efrat, S., and Stein, R. (1995) Mol. Endocrinol. 9, 1468-1476[Abstract]
31. Sharma, A., and Stein, R. (1994) Mol. Cell. Biol. 14, 871-879[Abstract]
32. Dandoy-D, F., Monthioux, E., Jami, J., and Bucchini, D. (1991) Nucleic Acids Res. 19, 4925-4930[Abstract]
33. MacFarlane, W. M., Read, M. L., Gilligan, M., Bujalska, I., and Docherty, K. (1994) Biochem. J. 303, 625-631[Medline] [Order article via Infotrieve]
34. Melloul, D., Ben-Neriah, Y., and Cerasi, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3865-3869[Abstract]
35. German, M. S., and Wang, J. (1994) Mol. Cell. Biol. 14, 4067-4075[Abstract]
36. Odagiri, H., Wang, J., and German, M. S. (1996) J. Biol. Chem. 271, 1909-1915[Abstract/Free Full Text]
37. Sharma, A., Olson, L. K., Robertson, R. P., and Stein, R. (1995) Mol. Endocrinol. 9, 1127-1134[Abstract]
38. Olson, L. K., Sharma, A., Peshavaria, M., Wright, C. V. E., Towle, H. C., Robertson, R. P., and Stein, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9127-9131[Abstract]
39. Olson, L. K., Redmon, J. B., Towle, H. C., and Robertson, R. P. (1993) J. Clin. Invest. 92, 514-519[Medline] [Order article via Infotrieve]
40. Zangen, D. H., Bonner-Weir, S., Lee, C. H., Latimer, J. B., Miller, C. P., Habener, J. F., and Weir, G. C. (1997) Diabetes 46, 258-264[Abstract]
41. Poitout, V., Olson, L. K., and Robertson, R. P. (1996) J. Clin. Invest. 97, 1041-1046[Abstract/Free Full Text]
42. Jonsson, J., Carlsson, L., Edlund, T., and Edlund, H. (1994) Nature 371, 606-609[CrossRef][Medline] [Order article via Infotrieve]
43. Slack, J. M. W. (1995) Development 121, 1569-1580[Abstract/Free Full Text]
44. Sharma, A., Zangen, D. H., Reitz, P., Taneja, M., Lissauer, M. E., Miller, C. P., Weir, G. C., Habener, J. F., and Bonner-Weir, S. (1999) Diabetes 48, 507-513[Abstract]
45. Hwung, Y.-P., Gu, Y.-Z., and Tsai, M.-J. (1990) Mol. Cell. Biol. 10, 1784-1788[Medline] [Order article via Infotrieve]
46. Boam, D. S. W., Clark, A. R., and Docherty, K. (1990) J. Biol. Chem. 265, 8285-8296[Abstract/Free Full Text]
47. Tomonari, A., Yoshimoto, K., Tanaka, M., Iwahana, H., Miyazaki, J., and Itakura, M. (1996) Diabetologia 39, 1462-1468[CrossRef][Medline] [Order article via Infotrieve]
48. Karlsson, O., Thor, S., Norberg, T., Ohlsson, H., and Edlund, T. (1990) Nature 344, 879-882[CrossRef][Medline] [Order article via Infotrieve]
49. Emens, L. A., Landers, D. W., and Moss, L. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7300-7304[Abstract]
50. Ahlgren, U., Pfaff, S. L., Jessell, T. M., Edlund, T., and Edlund, H. (1997) Nature 385, 257-260[CrossRef][Medline] [Order article via Infotrieve]
51. Rudnick, A., Ling, T. Y., Odagiri, H., Rutter, W. J., and German, M. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12203-12207[Abstract/Free Full Text]
52. German, M. S., Wang, J., Chadwick, R. B., and Rutter, W. J. (1992) Genes Dev. 6, 2165-2176[Abstract]
53. Ohneda, K., Mirmira, R. G., Wang, J., Johnson, J. D., and German, M. S. (2000) Mol. Cell. Biol. 20, 900-911[Abstract/Free Full Text]
54. Peshavaria, M., Henderson, E., Sharma, A., Wright, C. V. E., and Stein, R. (1997) Mol. Cell. Biol. 17, 3987-3996[Abstract]
55. Efrat, S., Surana, M., and Fleischer, N. (1991) J. Biol. Chem. 266, 11141-11143[Abstract/Free Full Text]
56. Efrat, S., Fusco-DeMane, D., Lemberg, H., Al Emran, O., and Wang, X. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3576-3580[Abstract]
57. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract]
58. Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve]
59. Behre, G., Smith, L. T., and Tenen, D. G. (1999) BioTechniques 26, 24-28[Medline] [Order article via Infotrieve]
60. Damante, G., Fabbro, D., Pellizzari, L., Civitareale, D., Guazzi, S., Polycarpou-Schwartz, M., Cauci, S., Quadrifoglio, F., Formisano, S., and Di Lauro, R. (1994) Nucleic Acids Res. 22, 3075-3083[Abstract]
61. Weiler, S., Gruschus, J. M., Tsao, D. H. H., Yu, L., Wang, L.-H., Nirenberg, M., and Ferretti, J. A. (1998) J. Biol. Chem. 273, 10994-11000[Abstract/Free Full Text]
62. Pllizzari, L., Tell, G., Fabbro, D., Pucillo, C., and Damante, G. (1997) FEBS Lett. 407, 320-324[CrossRef][Medline] [Order article via Infotrieve]
63. Sussel, L., Kalamaras, J., Hartigan-O'Connor, D. J., Meneses, J. J., Pedersen, R. A., Rubenstein, J. L. R., and German, M. S. (1998) Development 125, 2213-2221[Abstract/Free Full Text]
64. Lu, M., Seufert, J., and Habner, J. (1997) J. Biol. Chem. 272, 28349-28359[Abstract/Free Full Text]
65. Tang, Q.-Q., Jiang, M.-S., and Lane, M. D. (1999) Mol. Cell. Biol. 19, 4855-4865[Abstract/Free Full Text]
66. Raychowdhury, R., Zhang, Z., Hocker, M., and Wang, T. C. (1999) J. Biol. Chem. 274, 20961-20969[Abstract/Free Full Text]
67. Xiao, S., Matsui, K., Fine, A., Zhu, B., Marshak-Rothstein, A., Widom, R. L., and Ju, S.-T. (1999) Eur. J. Immunol. 29, 3456-3465[CrossRef][Medline] [Order article via Infotrieve]
68. Thomas, M. A., Mordvinov, V. A., and Sanderson, C. J. (1999) Eur. J. Biochem. 265, 300-307[Abstract/Free Full Text]
69. Matsukura, S., Stellato, C., Plitt, J. R., Bickel, C., Miura, K., Georas, S. N., Casolaro, V., and Schleimer, R. P. (1999) J. Immunol. 163, 6876-6883[Abstract/Free Full Text]
70. Benett, B. L., Cruz, R., Lacson, R. G., and Mannings, A. M. (1997) J. Biol. Chem. 272, 10212-10219[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.