Transactivation of the Mouse Sulfonylurea Receptor I Gene by BETA2/NeuroD
Ji-Won Kim,
Victor Seghers,
Jang-Hyeon Cho,
Yup Kang,
Soyeon Kim,
Yoonseok Ryu,
Kwanghee Baek,
Lydia Aguilar-Bryan,
Young-Don Lee,
Joseph Bryan and
Haeyoung Suh-Kim
Departments of Anatomy (J.-W.K., J.-H.C., S.K., Y.-D.L., H.S.-K.) and Endocrinology (Y.K.), Brain Disease Research Center (H.S.-K.), Ajou University, School of Medicine, Suwon, 442-749, Korea; Department and Institute of Genetic Engineering (Y.R., K.B.), Kyung Hee University, Yongin City, 449701, Korea; and Departments of Molecular and Cellular Biology (V.S., L.A.-B., J.B.) and Medicine (L.A.-B.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Haeyoung Suh-Kim, Departments of Anatomy and Endocrinology, Ajou University, Paldal-Gu wonchone-Dong San 5 South Korea, South Korea 442-749. E-mail: hysuh{at}madang.ajou.ac.kr.
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ABSTRACT
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The sulfonylurea receptor 1 (SUR1) plays a key role in regulation of insulin secretion in pancreatic ß-cells. In this study we investigated the mechanism for tissue-specific expression of the SUR1 gene. A -138/-20 fragment exhibited basal promoter activity while the -660/-20 fragment contained a regulatory element for tissue-specific expression of the mouse SUR1 gene. A pancreatic ß-cell-specific transcription factor, BETA2 (ß-cell E box transcription factor)/NeuroD, enhanced the promoter activity of the -660/-20 fragment in cooperation with E47. Coexpression of a dominant negative mutant of BETA2/NeuroD, BETA2(1233), repressed the promoter activity of the -660/-20 fragment. BETA2/NeuroD bound specifically to the E3 element located at -141. The E3 sequence in a heterologous context conferred transactivation by BETA2/NeuroD in HeLa and HIT cells. Mutation of E3 eliminated the stimulatory effect of BETA2/NeuroD. Unlike BETA2/NeuroD, neurogenin 3 (ngn3) could not activate the E3 element in HeLa cells. Overexpression of ngn3 concomitantly increased expression of BETA2/NeuroD and SUR1 in HIT cells but not in HeLa cells. These results indicate that BETA2/NeuroD induces tissue-specific expression of the SUR1 gene through the E3 element. These results also suggest that E3 is specific for BETA2/NeuroD, and the stimulatory effect of ngn3 in HIT cells may require factors specifically expressed in HIT cells.
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INTRODUCTION
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ATP-SENSITIVE K+ CHANNELS (KATP) play a critical role in converting changes in the ATP:ADP ratio to differences in electrical activity of the membrane in most excitable cells. KATP is composed of two subunits, the pore-forming unit, KIR6.x, and the regulator of KIR6.x, a sulfonylurea receptor (SUR), with a 4:4 stoichiometry (1, 2). SUR is a member of the ATP-binding cassette superfamily with multiple transmembrane-spanning domains and two nucleotide-binding folds (3, 4). Several isoforms of SUR, i.e. SUR1, SUR2A, and SUR2B, have been cloned, and combination of different isoforms of SUR and KIR6.x leads to the distinct functional and pharmacological channel profiles in various tissues (4, 5). SUR2A and SUR2B with KIR6.2 constitute the KATP channels of the cardiac and vascular smooth muscle type, and SUR1 with KIR6.2 constitutes the ß-cell-specific KATP channel.
In pancreatic ß-cells, increased glucose metabolism causes an increase in the ATP:ADP ratio, which blocks KATP and leads to activation of voltage-dependent Ca2+ channels. As a result, Ca2+ ions influx into the cell and induce insulin secretion (1). SUR1 is the target of sulfonylurea drugs, e.g. tolbutamide and glibenclamide, that are widely used to promote insulin secretion in the treatment of noninsulin-dependent diabetes mellitus (3, 7). Mutations in the human SUR1 gene cause familial persistent hyperinsulinemic hypoglycemia of infancy, which is characterized by continuous insulin secretion in spite of low concentrations of blood glucose (8, 9). Compared with humans, targeted mutations of SUR1 are less effective in mice. SUR1 knockout mice lack the first-phase insulin secretion and exhibit an attenuated glucose-stimulated second-phase insulin secretion (10).
The promoters of SUR1 have been cloned from human (11) and mouse (12). Human and mouse promoters are TATA-less and GC-rich with several SP1 binding sites around transcription initiation sites. Although human and mouse promoters are relatively similar, sequence analysis does not reveal any particular region of high similarity between two promoters except the transcription initiation sites (12). Interestingly, while the 1.3 kb-long 5'-flanking sequence of the human promoter is sufficient to drive the ß-cell-specific expression (11), the corresponding region of the mouse SUR1 promoter does not seem to be tissue specific (12). Thus, it is necessary to define the sequences for ß-cell-specific expression of the SUR1 gene. In addition, transcription factors responsible for ß-cell-specific expression have not been determined, although putative binding sites for several transcription factors in the human promoter have been suggested.
Several transcription factors have been isolated from pancreatic islet cells including pancreatic duodenal homeobox-1 (PDX-1/STF/insulin promoter factor-1, hepatocyte nuclear factor 3ß, Nkx2.2, islet factor-1, paired-box transcription factor 4 and 6, ß-cell E box transcription factor (BETA2), and neurogenin 3 (ngn3) (13). BETA2/NeuroD and ngn3 are members of the basic helix-loop-helix (bHLH) transcription factor family. The bHLH transcription factors heterodimerize with E47, a ubiquitous member of the bHLH family, bind to the E box sequence (CANNTG), and activate the target genes. A cascade of bHLH transcription factors is required for proper development of pancreatic islets. ngn3 Is transiently expressed in islet progenitor cells during embryogenesis and barely detectable in the adult pancreas. The ngn3 mutant mice fail to generate pancreatic endocrine cells (14) and to express BETA2/NeuroD, indicating that ngn3 functions upstream of BETA2/NeuroD (15). Indeed, ngn3 binds to the E boxes in the BETA2/NeuroD promoter and increases the expression of BETA2/NeuroD (16). Targeted mutations of BETA2/NeuroD lead to a reduction of islet cells, resulting in early onset of diabetes (17). BETA2/NeuroD is persistently expressed after birth, suggesting an additional role of BETA2/NeuroD in maintaining the endocrine function of adult pancreas. Indeed, BETA2/NeuroD is a ß-cell-specific transactivator of the insulin gene (18).
In this study we show that the 2.4 kb-long 5'-flanking sequence of the mouse SUR1 gene is sufficient for ß-cell-specific expression. We also demonstrate that BETA2/NeuroD can confer the ß-cell-specific gene expression of SUR1 and identify the BETA2/NeuroD binding site in the SUR1 promoter.
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RESULTS
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Characterization of the 5'-Flanking Region of the SUR1 Gene
To clone the promoter region of the mouse SUR1 gene, we isolated the 5'-flanking region of the mouse SUR1 gene by screening with the first exon as a probe. An 18-kb long genomic DNA was isolated from the mouse library and subcloned into the pBluescript to obtain MG101. The 2.4 kb of the 5'-flanking region was sequenced (GenBank accession number AF037274). The upstream region is G+C rich (61%) and contains putative sites for CCAAT enhancer binding protein, SP1, and cAMP responsive element binding protein. There is no TATA-like sequence at the proximal 5'-flanking region of the first ATG codon (Fig. 1
).

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Figure 1. Characterization of the SUR1 Promoter
The most 5'-end of transcripts in MIN cells and mouse adult whole brain are marked as * (-67 bp) and # (-64 bp) upstream of the first ATG codon, respectively. Putative cAMP-responsive element (CRE) is marked with a box; CAAT box with a dashed line; four putative E-boxes with dotted lines; and five SP1 sites with solid lines. The primers used for cRACE are also shown as arrows.
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To determine the transcription initiation site, we carried out circular rapid amplification of cDNA ends (RACE) (see Materials and Methods for details) with total RNA isolated from mouse insulinoma (MIN) cells and mouse adult whole brain. The products of the second PCR fragments were subcloned into pGEM-T vector and sequenced. The most 5'-ends of the transcripts from MIN cells and mouse brain were -67 and -64 bp upstream of the first ATG codon, respectively (Fig. 1
). The sequences of the 5'-untranslated region of the SUR1 transcripts were identical between MIN and mouse brain, indicating that pancreatic ß-cells and the nervous tissue use the same promoter for SUR1 expression.
Tissue-Specific Expression of the SUR1 Gene in Insulin-Producing Cells
To determine the cis-elements required for tissue-specific expression, 5' deletion constructs were prepared (Fig. 2A
) and tested in two cell lines: insulinoma cells (HIT-T15) and cervical carcinoma cells (HeLa). The -138/-20 fragment exhibited minimum chloramphenicol acetyltransferase (CAT) activity in HIT-T15 cells as well as in HeLa cells. In HIT-T15 cells, the -660/-20 and -2,432/-20 fragments exhibited higher CAT activity by 2.3- and 3.9-fold than the -138/-20. In HeLa cells, the same fragments showed similar CAT activity to that of the -138/20 fragment (Fig. 2B
). This result suggests that the -138/-20 fragment contains a basal promoter, whereas the -660/-20 fragment contains regulatory elements for ß-cell-specific activation of the SUR1 gene.

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Figure 2. Tissue-Specific Expression of the SUR1 Gene
A, Several reporter genes were constructed by ligating the 5'-flanking regions to the CAT reporter gene, pCAT3M. B, The SUR1 reporter genes (0.5 µg) were transfected into HIT-T15 (solid bars) and HeLa cells (open bars). Experiments were carried out in duplicate. Data are presented as average ± SE with respect to pCAT3M from four independent experiments. P values were estimated from t test compared with the value of pCAT3M (**, P < 0.01).
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Transactivation of the SUR1 Gene by BETA2/NeuroD
Because it has been shown that BETA2/NeuroD mRNA is abundant in HIT-T15 cells and is expressed specifically in pancreatic islets (18), we investigated the possibility that BETA2/NeuroD is a tissue-specific regulator of the SUR1 gene in HIT-T15 cells. We cotransfected reporter genes with expression vectors for BETA2/NeuroD and E47. It has been shown that these two factors heterodimerize and transactivate the insulin promoter (18). All reporter genes were partially activated by expression of BETA2/NeuroD or E47 (Fig. 3
). Importantly, maximum transactivation was obtained with pSUR-660CAT and pSUR-2432CAT when BETA2/NeuroD and E47 were coexpressed. In contrast, although the reporter gene containing the -138/-20 fragment was minimally activated, coexpression of BETA2/NeuroD and E47 did not further increase the reporter activity. Under the same condition, coexpression of BETA2/NeuroD and E47 gave a 3.7-fold transactivation of the reporter gene containing three copies of the rat insulin promoter E box (RIPE3), RIPE3(3+) (Fig. 3
). The result indicates that the -660/-20 fragment contains the responsive elements for BETA2/NeuroD that are absent in the -138/-20 fragment.

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Figure 3. Activation of the SUR1 Promoter by BETA2/NeuroD
Reporter plasmids (0.5 µg), pCMV-BETA2 (0.5 µg), and pSVE25 (0.07 µg) were used for transfection in HIT-T15 cells. Coexpression of BETA2/NeuroD and E47 synergistically enhanced the promoter activity of -660 and -2,432. Data from three independent experiments are presented as average ± SE compared with the basal CAT activity in the absence of BETA2/NeuroD and E47. P values were estimated from t test compared with the values of reporter gene alone (*, P < 0.05; **, P < 0.01).
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To confirm whether the endogenous BETA2/NeuroD confers high activity of pSUR-660CAT in HIT-T15 cells as shown in Fig. 2
, we determined the effect of a dominant negative form of BETA/NeuroD, BETA2(1233). BETA2(1233) functions as a dominant negative mutant because it contains the bHLH domain for heterodimerization with E47 but lacks transactivation domain (19). As a positive control we used an insulin reporter gene, pINSCAT448-, which contained the RIPE3 sequence (20). Coexpression of BETA2(1233) reduced the promoter activity of the -660/-20 fragment in a dose-dependent manner (Fig. 4
). Thus, transfection of 0.3 µg pCHA-BETA2(1233) decreased the promoter activity of the -660/-20 fragment and the insulin promoter to about 20% and 35% of the reporter alone, respectively. Under the same condition, BETA2(1233) did not affect the pSV2CAT. This result indicates that repression by BETA2(1233) is specific to ß-cell-specific genes and high promoter activity of -660/-20 is due to BETA2/NeuroD-like factors in HIT-T15 cells.

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Figure 4. Repression of the SUR1 Promoter by a Dominant Negative Mutant of BETA2/NeuroD
A, Full-length BETA2/NeuroD and truncated form BETA2(1233) peptides were epitope tagged with hemagglutinin (HA) at the N terminus. AD, Activation domain. B, Reporter genes, pSUR-138CAT, pSUR-660CAT, pSV2CAT, and pINSCAT448- were cotransfected with pcHA-BETA2(1233), an expression vector for BETA2(1233). Although pSV2CAT lacking the E box was not affected by BETA2(1233), pINSCAT448- containing the E box of the rat insulin II promoter was repressed 3-fold by BETA2(1233). Data are relative values with respect to the CAT activity of pSV2CAT and presented as average ± SE of three independent experiments. *, P < 0.05; **, P < 0.01; both P values were estimated from t test compared with the value of the reporter gene alone.
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Determination of Binding Sites for BETA2/NeuroD
Four E box-like sequences (E1E4), which are potential sites for BETA2/NeuroD action, were found within the -660/-20 fragment (Fig. 1
). Because the -138/-20 fragment containing E4 was not synergistically activated by BETA2/NeuroD and E47, E4 alone is insufficient for tissue-specific regulation. To determine which of the remaining E box-like sequences was necessary for BETA2/NeuroD binding, we carried out EMSAs using RIPE3 as a probe and the E box sequences (E1E3) of the SUR1 promoter as competitors (Fig. 5
). As a source of BETA2/NeuroD, nuclear extracts were prepared from 293T cells transfected with expression vectors for BETA2(1233) and E47. BETA2(1233) has been shown to bind RIPE3 stronger than the full-length BETA2 when expressed in 293T cells (19). Several complexes were detected, and specific binding of the labeled RIPE3 probe was verified using an excess amount of unlabeled RIPE3 oligonucleotide (data not shown). Interestingly, only E3 was able to compete with RIPE3 for binding to BETA2/NeuroD (lanes 6 and 7). The binding was specific because the same complexes disappeared by addition of an anti-NeuroD antibody (lane 8). This result indicates that the E3 box is necessary for binding of BETA2/NeuroD. To confirm the specificity of binding to E3, we carried out EMSA using E3 as a probe (Fig. 6
). Like RIPE3, BETA2/NeuroD bound to E3 specifically. E3 oligonucleotide (lanes 2 and 3) and RIPE3 (lanes 6 and 7) could compete with E3 of the SUR1 gene (closed triangle). In contrast, the E3m, a mutant oligonucleotide, could not compete with E3 (lanes 4 and 5). The specific complexes disappeared in the presence of an anti-NeuroD antibody (lane 8). This result indicates that BETA2/NeuroD can bind the E3 element at -141 bp of the SUR1 gene.

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Figure 5. Determination of DNA Binding Ability of Three Putative E Boxes
Nuclear extracts were prepared from 293T cells expressing a dominant negative form of BETA2/NeuroD, BETA2(1233). EMSA was carried out with RIPE3, the E box of the rat insulin II promoter. Double-stranded oligonucleotides containing E1, E2, or E3 of the SUR1 promoter were used as competitors. Only E3 was able to compete with RIPE3 (solid triangle in lanes 6 and 7). The same complex disappeared in the presence of an anti-BETA2 antibody (lane 8).
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Figure 6. Binding of BETA2/NeuroD to E3
Nuclear extracts were prepared from 293T cells expressing a dominant negative form of BETA2/NeuroD, BETA2(1233). A double-stranded oligonucleotide containing E3 of the SUR1 promoter was used as a probe. Specific binding was confirmed by competition with an excess amount of the unlabeled E3 (lanes 2 and 3) or E3m containing mutation in E3 (lanes 4 and 5) and RIPE3 (lanes 6 and 7). The specific complexes (solid triangle) disappeared by addition of an anti-BETA2 antibody (lane 8).
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E3-Mediated Transactivation by BETA2/NeuroD
To determine whether the E3 box could confer transactivation of the SUR1 gene by BETA2/NeuroD, reporter genes pSURE3(1+) and pSURE3m(1+) were constructed by ligating one copy of E3 or E3m to a heterologous promoter driving expression of luciferase in the pGL3-promoter vector, respectively (Fig. 7A
). Coexpression of BETA2/NeuroD and E47 enhanced the luciferase expression by 3.8-fold for pSURE3(1+), whereas the pGL3-promoter was not affected. Mutation of E3 eliminated the stimulatory effect of BETA2/NeuroD (Fig. 7B
, left panel).

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Figure 7. Mutation of E3 Abolishes Transactivation by BETA2/NeuroD
A, Reporter genes, pSURE3(1+), pSURE3m(1+), pSUR(-2,432/-660), were constructed by ligating one copy of E3, E3m, or -2,432/-660 to pGL3-promoter. pSUR-660 and pSUR-660E3m were constructed using pGL2-basic (see Materials and Methods for details). B, Each of the reporter genes (0.3 µg) was cotransfected with expression vectors for BETA2 and E47 into HIT-T15 cells. Note that mutation in E3 caused a loss of transactivation by BETA2/NeuroD and E47. Data are shown as average ± SE of three independent experiments with respect to the luciferase activity of pGL3 promoter or pGL2 basic. P values were estimated from t test compared with the value of reporter genes alone (*, P < 0.05; **, P < 0.01).
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To confirm that E3 is the only functional E box element in the -660/-20 fragment, we constructed pSUR-660E3m, which contained a linker scanning mutation in E3 of the -660/-20 fragment. Mutation of E3 in the whole promoter context eliminated the stimulatory effect of BETA2/NeuroD (Fig. 7B
, right panel). Sequence analysis revealed nine more E box-like sequences upstream of -660. To determine whether they might confer higher activity of pSUR-2432CAT than pSUR-660CAT in HIT-T15 cells, as shown in Fig. 2
, we generated a reporter construct, pSUR-2,432/-660 by ligating the -2,432/-660 fragment to the pGL3-promoter. Coexpression of BETA2/NeuroD with or without E47 did not significantly enhance the -2,432/-660 fragment (Fig. 7B
, left panel). Thus, E3 is essential for transactivation by BETA2/NeuroD.
Specificity of the E Box-Mediated Transactivation by BETA2/NeuroD
In addition to BETA2/NeuroD, ngn3 is also a bHLH transcription factor expressed in pancreatic islet cells during their development (14). Thus, it is possible that ngn3 can also activate the SUR1 gene through the E3 element. We tested this possibility by coexpressing ngn3 and E47 in HIT-T15 cells. In HIT-T15 cells, the stimulatory effect of ngn3 was similar to that of BETA2/NeuroD (compare HIT-T15 cells in Fig. 8
, A and B). A reporter gene containing one copy of E3 was increased 5.7-fold by coexpression of ngn3 and E47 (Fig. 8B
). A similar result was obtained with a reporter gene containing the -660/-20 fragment. The stimulatory effect of ngn3 was abrogated when E3 was mutated as shown with E3m (Fig. 8B
) or -660E3m (data not shown). In contrast to HIT-T15 cells, transactivation by ngn3 was minimal compared with BETA2/NeuroD in HeLa cells (Fig. 8B
). Coexpression of ngn3 and E47 could activate the promoter activity of pSURE3 by only 1.4-fold. In contrast, BETA2/NeuroD enhanced it by 3.1-fold under the same conditions. Interestingly, neither BETA2/NeuroD nor ngn3 could activate E3 in the homologous context of -660/-20 in HeLa cells (see Discussion). These results indicate that the E3 element is somehow specific for BETA2/NeuroD in HeLa cells. It has been shown that ngn3 is not detectable in mature ß-cells, and forced expression of ngn3 causes an increase in the level of BETA2/NeuroD in HIT-T15 cells (16). Thus, it is possible that activation of E3 by ngn3 in HIT-T15 cells may be due to enhanced expression of BETA2/NeuroD, which is absent in HeLa cells. To test this possibility we investigated mRNA levels of SUR1 and BETA2/NeuroD before and after overexpression of ngn3. Stable cells were obtained by transfecting HIT-T15 and HeLa cells with the ngn3 expression vector. Total RNAs were prepared from pools of G418-resistant cells and subjected to RT-PCR. Overexpression of ngn3 enhanced expression of both SUR1 and BETA2/NeuroD by 2.3- and 2.4-fold, respectively, in HIT-T15 cells (Fig. 8
, C and D). In contrast, ngn3 could not induce expression of SUR1 and BETA2/NeuroD in HeLa cells. This result suggests that ngn3 might activate the SUR1 promoter indirectly by inducing the expression of BETA2/NeuroD in HIT-T15 cells but not in HeLa cells.

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Figure 8. Specificity of E3 for BETA2/NeuroD
A, Reporter genes (0.3 µg) were cotransfected with expression vectors for BETA2/NeuroD (0.1 µg) and E47 (0.05 µg) into HIT-T15 (hatched bar) or HeLa cells (solid bar). B, Reporter genes (0.3 µg) were cotransfected with expression vectors for ngn3 (0.1 µg) and E47 (0.05 µg) into HIT-T15 or HeLa cells. Data are shown as average ± SE of three independent experiments with respect to the luciferase activity of pSURE3(1+) or pSUR-660 in the absence of BETA2/NeuroD and E47. Note that the effect of ngn3 is minimal in HeLa cells. P values were estimated from t test compared with the values of pSURE3 or pSUR-660Luc (*, P < 0.05; **, P < 0.01). C, RT-PCR products of SUR1 and BETA2/NeuroD with HIT and HeLa cells stably transformed with ngn3. BETA2/NeuroD and SUR1 products were detected as 620-bp and 117-bp fragments, respectively. Data shown are the most representative of three independent experiments. D, The mRNA levels of SUR1 were normalized to the ß-actin mRNA, and the RT-PCR data from three independent experiments were summarized as average ± SE with respect to the value of untreated cells (*, P < 0.05; **, P < 0.01).
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DISCUSSION
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In this study, we showed that BETA2/NeuroD could transactivate the SUR1 promoter in a synergistic manner with E47. We also identified an E box through which BETA2/NeuroD enhances the SUR1 promoter activity.
We showed that the basal promoter is located in -138/-20, and cis-elements for ß-cell-specific expression are located in -660/-20 or -2,432/-20 (Fig. 2
). BETA2/NeuroD activates both -660/-20 and -2,432/-20. In contrast, coexpression of BETA2/NeuroD with E47 cannot enhance -2,432/-660 in the heterologous context (Fig. 7
). In addition, BETA2(1233), a dominant negative form of BETA2/NeuroD, represses -660/-20 (Fig. 3
). These results suggest that although there are 13 E box-like sequences in -2,432/-20, and nine of them are located between -2,432 and -660, they are not operative and the functional E-box(es) is located in -660/-20.
Among four putative E boxes between -660 and -20, the E3 box mediates transactivation of the SUR1 gene by BETA2/NeuroD on the basis of the following results. 1) BETA2/NeuroD binds E3 as efficiently as the insulin E box (RIPE3) (Figs. 5
and 6
). 2) E3 can confer transactivation by BETA2/NeuroD in the context of a heterologous promoter (Fig. 7
). 3) A mutation of E3 in the homologous promoter context (-660E3m) abolishes the stimulatory effect of BETA2/NeuroD (Fig. 7
).
The mouse SUR1 promoter has been cloned and characterized by Hernandez-Sanchez et al. (12). In that study, the basal promoter has been located in -84/+54 from the transcription initiation site, which is consistent with our observation. In contrast to our findings, the promoter activity of longer 5'-flanking sequences up to -4.5 kb is similar to the basal activity of -84/+54 when tested in ßTC3 and MIN-6 cells. The difference in two studies for tissue-specific expression may reflect differential expression of ß-cell-specific transcription factors in the various insulinoma cells used to assay for tissue-specific expression.
As mentioned earlier, pancreatic ß-cells express several members of the class B bHLH proteins including BETA2/NeuroD and ngn3. Although all bHLH factors can bind to E boxes, the E3 element in the SUR1 promoter is specific for BETA2/NeuroD because E3 can be activated by BETA2/NeuroD but not by ngn3 in HeLa cells (Fig. 8
). In HIT-T15 cells, like BETA2/NeuroD, ngn3 can activate the E3 element. The stimulatory effect of ngn3 in HIT-T15 cells may be indirect because overexpression of ngn3 leads to an increase in BETA2/NeuroD expression (Fig. 8C
), which may in turn enhance the SUR1 expression. In contrast, overexpression of ngn3 is not sufficient to induce expression of BETA2/NeuroD as well as SUR1 in HeLa cells. Interestingly, neither BETA2/NeuroD nor ngn3 can activate the -660/-20 in HeLa cells. The lack of stimulatory effect of BETA2/NeuroD in the homologous promoter can be explained in several ways. It may be because bHLH factors require additional factors to build up the functional transcription machinery in the homologous promoter context. It has been shown that p300/cAMP responsive element binding protein is necessary for the proper function of BETA2/NeuroD (21, 22). A homeodomain protein, PDX-1/IPF-1, interacts with E47, an ubiquitous partner of BETA2/NeuroD and ngn3, and activates the E2A3/4 minienhancer of the rat insulin gene (23). Alternatively, a negative regulatory element (NRE) may reside within -660, which may inhibit expression of the SUR1 gene in non-insulin-producing HeLa cells. An NRE is identified within the insulin promoter (24), and the negative activity of the NRE is dependent on its interaction with other regulatory sites within the insulin gene (25). The role of the NRE is unclear in the insulin promoter, although it may be involved in restricting expression of the insulin gene to pancreatic ß-cells. Further studies will be necessary to identify other elements for tissue-specific expression of the SUR1 gene.
BETA2/NeuroD was originally isolated as a transactivator of the insulin gene. Many studies have shown that BETA2/NeuroD is present in a number of cell types including pancreatic ß-cells, intestinal endocrine cells, pituitary gland, and brain (18). During development, BETA2/NeuroD is highly expressed in differentiating, postmitotic neurons and dorsal root ganglion (26) as well as in some mitotic cells of the dentate gyrus and cerebellum during postnatal stages (27). In the adult brain, localization of BETA2/NeuroD is similar to that of SUR1. Both BETA2/NeuroD and SUR1 are found in the hippocampus, dentate gyrus, piriform cortex, nucleus of the lateral olfactory tract, and cerebellum (27, 28). The overlapping distribution of SUR1 and BETA2/NeuroD is consistent with our finding that BETA2/NeuroD may be responsible for tissue-specific expression of the SUR1 gene.
There is increasing evidence that ß-cell-specific genes are regulated by common transcription factors. For example, PDX-1/IPF-1 is essential for ß-cell-specific expression of insulin, islet amyloid polypeptide (29, 30), glucose transporter type 2 (31), and glucokinase (32). Interestingly, PDX-1/IPF-1 plays a critical role in pancreatic development, and targeted mutation of PDX-1/IPF-1 results in a lack of pancreatic endocrine and exocrine cells (33). Interestingly, like PDX-1/IPF-1, BETA2/NeuroD regulates tissue-specific expression of pancreatic islet-specific genes, including insulin (18), glucagon (34), and SUR1. BETA2/NeuroD is also known to play critical roles for development of pancreatic islet cells (17). Thus, transcription factors essential for cell fate determination and specification may also regulate expression of tissue-specific genes.
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MATERIALS AND METHODS
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RACE
Circular RACE was performed as described by Maruyama et al. (35). Briefy, cDNA was synthesized from 5 µg of total RNA in the presence of a phosphorylated oligonucleotide, 9219R (5'-CGATGAAGAGGATG-3'), as a specific primer. After cDNA synthesis, ribonuclease H was added to the reaction and single-stranded cDNA was precipitated with ethanol. The cDNA was circularized in 40 µl of the reaction mixture containing 25% polyethylene glycol 6000, 10 U T4 RNA ligase at 22 C for 16 h. The first PCR was performed for 30 cycles with the annealing temperature at 55 C in the presence of the circularized cDNA as a template and the oligonucleotides 9,154R (5'-ACGAAGCAGCCGTTGTTGAGGAC-3') and 9,162 (5'-TCAACGTAGTGCCACATGTCTTC-3') as primers using the LA PCR kit (TaKaRa, Otsu, Shiga, Japan). The second PCR was carried out with 2 µl of the first PCR product (1:1,000 dilution) as a template and oligonucleotides 9,144R (5'-CGTTGTTGAGGACACCTTGGTC-3') and 9,175 (5'-CACATGTCTTCCTGCTCTTCATC-3') as primers using the LA PCR kit. The final PCR fragment was isolated on agarose gel and cloned in pGEM-T easy vector system (Promega Corp., Madison, WI). The clones containing the PCR fragments were isolated and sequenced.
Cloning of the SUR1 Promoter and DNA Constructs
MG101, an approximately 18-kb mouse genomic DNA fragment containing the 5'-end of the SUR1 gene, was isolated from a mouse genomic library made in
-phage using the SUR1 cDNA as a probe and used as the source of promoter region. The EcoRI/SmaI fragment (-1,373/+583) from MG101 was subcloned into pUC19. The 3'-end of the insert was deleted to -20 bp from the first ATG codon using exonuclease III and reinserted into the EcoRI site of pUC19 to obtain pMSUR1-d/E. The BamHI/BamHI fragment (-660/-20) was isolated from pMSUR1-d/E and linked to a CAT reporter plasmid, pCAT3M, to obtain pSUR-660CAT. To obtain pSUR-138CAT, the PvuII/BamHI fragment (-138/-20) was isolated from pMSURI-d/E and inserted into the BglII site of pCAT3M using a BglII linker. A KpnI/KpnI fragment (-2,432/-627) from MG101 was inserted to the KpnI site of pSUR-660CAT to generate pSUR-2432CAT. Reporter genes containing the E box from the SUR1 promoter were constructed by inserting a double-stranded E3 oligonucleotide into the BglII site of the pGL3-promoter luciferase vector (Promega Corp.). pSUR-2,432/-660 was made by ligating the KpnI/BamHI fragment (-2,432/-660) to the pGL3promoter vector. To introduce a linker scanning mutation at E3, PCR was carried out using MG101 as a template and the oligonucleotides 8,411 (5'-GGATCCAAGTTCCTCTTCTGGCCTCTATTGGTA-3') and 8,939R (5'-CCCCCGGGCTCTTGTGGGGC GAGGGTGGG-3') or the oligonucleotides 8,930 (5'-CCCGGGGAA GGGCGGGGGCCAGCGGCA-3') and 9,052R (5'-CTGCTCTGGCTCCGCGCGCCT-3'). Two PCR products were subcloned into pGEM-T easy vector and subsequently isolated from the vector by digestion with BamHI and XmaI. The two BamHI/XmaI fragments were inserted to the BglII site of pGL2-basic vector to obtain pSUR-660E3m.
Cell Culture and Transfection Assay
HIT-T15 and HeLa cells were maintained in DMEM with 4,500 mg/liter glucose, supplemented with 4 mM L-glutamine, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells (2.5 x 105 cells/35-mm dish) were transfected using LipofectAMINE PLUS (Life Technologies, Inc., Gaithersburg, MD). Reporter plasmids (0.5 µg), 0.5 µg each of expression vectors for BETA2/NeuroD, pCMV-BETA2, and ngn3, pCR3.1-ngn3 (19), and 0.07 µg of an expression vector for E12, pSVE-5, or pCR3.1-E47 were used. The total DNA amount was maintained with pcDNA3 (Invitrogen, Carlsbad, CA). For CAT assays, cell extracts were prepared 48 h after transfection by repeated cycling of freezing and thawing and heat inactivated at 65 C for 10 min. Protein concentration was determined by Bradford assay and 1020 µg of cell extracts were assayed for CAT activity using [3H]-chloramphenicol (NEN Life Science Products, Boston, MA) and butyryl-coenzyme A (Sigma, St. Louis, MO). Activity was normalized to ß-galactosidase activity. For luciferase assays, cell extracts were prepared according to the manufacturers protocol, and luciferase activity was determined with 520 µg of cell extracts using the Dual-Luciferase assay system (Promega Corp.). The data are presented as an average ± SE from at least three independent experiments. To obtain stably transformed cells with ngn3, pCR3.1-ngn3 was transfected into HIT-T15 and HeLa cells and selected in the presence of G418 (600 µg/ml) for 2 wk.
EMSA
293T cells were transfected with expression vectors for BETA2/NeuroD and E47 using calcium phosphate. After 36 h, nuclear extracts were prepared from transfected cells as described by Attardi and Tjian (36). Briefly, cells were lysed in 25 mM Tris-Cl (pH 8.0), 2 mM MgCl2, 0.5 mM dithiothreitol (DTT), and 0.01% phenylmethyl sulfonyl fluoride (PMSF) for 5 min at room temperature. Nonidet P-40 was then added to a final concentration of 0.05% and incubated for 2 min. After centrifugation at 1,700 x g for 5 min, the resulting pellet was suspended in 10 mM Tris-Cl (pH 8.0), 400 mM LiCl, 0.5 mM DTT, and 0.01% PMSF and kept at room temperature for 5 min. After centrifugation at 12,000 x g for 2 min, the supernatant was collected and used for EMSA. EMSA was performed using double-stranded oligonucleotides as probes containing the E box of rat insulin (RIPE3) or the E3 of SUR1 promoter. RIPE3, 5'-GATCTGGAAACTGCAGCTTCAGCCCCTCTGGCCATCTGCTGATCCA-3' (sense), and 5'-GATCCGGATCAGCAGATGGCCAGAGGGGCTGAAGCTGCAGTTTCCA-3' (antisense) were annealed and end labeled with [
-32P] dATP (NEN Life Science Products) and Klenow fragment. The sequences of putative E boxes of SUR1 are illustrated in Fig. 5
. The 32P-labeled probe (3 x 104 cpm/lane) and 1 µg of nuclear extracts were incubated in 7% glycerol, 60 mM LiCl, 0.5 mM PMSF, 5 mM MgCl2, 2 mM DTT (pH 7.4), with 2 µg of polydeoxyinosinicdeoxycytidylic acid (Sigma) for 30 min at room temperature. To confirm specific binding, the unlabeled probe of 30- to 100-fold excess or 0.6 µg of an anti-NeuroD antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the reaction mixture. Samples were loaded onto 4% polyacrylamide gels and subjected to electrophoresis at 8 V/cm. Gels were dried and exposed to x-ray film for 12 d at -70 C.
RT-PCR
Total RNA was isolated using a RNAzol B (Tel-Test, Friendswood, TX), and cDNA was synthesized using First-strand cDNA synthesis kit (Roche, Mannheim, Germany) and 1 µg of total RNA following the manufacturers instructions. The PCR was conducted with 3 µl of the first-strand cDNA; 98 C for 1 min, followed by 25 cycles for BETA2 and ß-actin, and 30 cycles for SUR1 at 94 C for 1 min, 55 C for 30 sec, and 72 C for 1 min, and finally 72 C for 7 min. Primers were designed to recognize the separate exons to eliminate possible DNA contamination. The PCR primers for SUR1 were 5'-GCTCTTCATCACCTTCCCCATCCTC-3' (forward) and 5'-CACAAC CTGCGCTGGATCCTTACC-3' (reverse); for BETA2, 5'-CTCCGGGGTTATGAGATCGTCAC-3' (forward) and 5'-GATCTCTGACAGAG CCCA-3' (reverse); and for ß-actin, 5'-CATGTTTGAGACCTTCAACACCCC-3' (forward) and 5'-GCCATCT CCTGCTCGAAGTCTAG-3' (reverse). The PCR products were analyzed on a 2% agarose gel.
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ACKNOWLEDGMENTS
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We would like to thank Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX) for helpful discussion and generous gifts of PCR3.1-ngn3 and pCR3.1-E47.
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FOOTNOTES
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This work was supported by Korea Research Foundation (Grant 1998-019-F00002) (to H.S.-K. and Y.K.); by Brain Science and Engineering Research Program, and Life Phenomena and Function Group Program of Korean Ministry of Science and Technology (to H.S.-K.); and by Korea Science & Engineering Foundation through Brain Disease Research Center at Ajou University (to H.S.-K.).
Abbreviations: BETA2, ß-Cell E box transcription factor; bHLH, basic helix-loop-helix; CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; MIN, mouse insulinoma; ngn3, neurogenin 3; NRE, negative regulatory element; PDX-1, pancreatic duodenal homeobox-1; PMSF, phenylmethyl sulfonyl fluoride; RACE, rapid amplification of cDNA ends; RIPE3, rat insulin promoter E box; SUR, sulfonylurea receptor.
Received for publication September 6, 2001.
Accepted for publication January 18, 2002.
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REFERENCES
|
---|
-
Efrat S, Tal M, Lodish HF 1994 The pancreatic ß-cell glucose sensor. Trends Biochem Sci 19:535538[CrossRef][Medline]
-
Inagaki N, Gonoi T, Clement IV JP, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, Bryan J 1995 Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270:11661170[Abstract]
-
Aguilar-Bryan L, Nichols CG, Wechsler SW, Clement IV JP, Boyd III AE, Gonzalez G, Herrera-Sosa H, Nguy K, Bryan J, Nelson DA 1995 Cloning of the ß-Cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268:423425[Medline]
-
Schwanstecher M, Siverding C, Dorschner H, Gross I, Aguilar-Bryan L, Schwanstecher C, Bryan J 1998 Potassium channel openers require ATP to bind to and act through sulfonylurea receptor. EMBO J 17:55295535[Abstract/Free Full Text]
-
Inagaki N, Gonoi T, Clement IV JP, Wang CZ, Aguilar-Bryan L, Bryan J, Seino S 1996 A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 16:10111017[Medline]
-
Deleted in proof
-
Rajan AS, Aguilar-Bryan L, Nelson DA, Yaney GC, Hsu WH, Kunze DL, Boyd III AE 1990 Ion channels and insulin secretion. Diabetes Care 13:340363[Abstract]
-
Tomas PM, Wohllk N, Huang E, Kuhnle U, Rabl W, Gagel RF, Cote GJ 1996 Inactivation of the first nucleotide-binding fold of the sulfonylurea receptor, and familial persistent hyperinsulinemic hypoglycemia of infancy. Am J Hum Genet 59:510518[Medline]
-
Tomas PM, Cote GJ, Wohllk N, Haddad B, Mathew PM, Rabl W, Aguilar-Bryan L, Gagel RF, Bryan J 1995 Mutation in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy. Science 268:426429[Medline]
-
Seghers V, Nakazaki M, DeMayo F, Aguilar-Bryan L, Bryan J 2000 Sur1 knockout mice. A model for K(ATP) channel-independent regulation of insulin secretion. J Biol Chem 275:92709277[Abstract/Free Full Text]
-
Ashfield R and Ashcroft JH 1998 Cloning of the promoters for pancreatic ß-cell ATP-sensitive K-channel subunits Kir6.2 and SUR1. Diabetes 47:12741280[Abstract]
-
Hernandez-Sanchez C, Ito Y, Ferrer J, Reitman M, and LeRoith D 1999 Characterization of the mouse sulfonylurea receptor 1 promoter and its regulation. J Biol Chem 274:1826118270[Abstract/Free Full Text]
-
Edlund H 1999 Pancreas: how to get there from the gut? Curr Opin Cell Biol 11:663668[CrossRef][Medline]
-
Gradwohl G, Dierich A, LeMeur M, Guillemot F 2000 neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci USA 97:16071611[Abstract/Free Full Text]
-
Schwitzgebel VM, Scheel DW, Conners JR, Kalamaras J, Lee JE, Anderson DJ, Sussel L, Johnson JD, German MS 2000 Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development 127:35333542[Abstract/Free Full Text]
-
Huang HP, Liu M, El-Hodiri HM, Chu K, Jamrich M, Tsai MJ 2000 Regulation of the pancreatic islet-specific gene BETA2 (neuroD) by neurogenin 3. Mol Cell Biol 20:32923307[Abstract/Free Full Text]
-
Naya FJ, PoHuang H, Qiu Y, Mutoh H, DeMayo FJ, Leiter AB, Tsai MJ 1997 Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/NeuroD-deficient mice. Genes Dev 11:23232334[Abstract/Free Full Text]
-
Naya FJ, Stellrecht Christine MM, Tsai MJ 1995 Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor. Genes Dev 9:10091019[Abstract]
-
Cho JH, Kwon IS, Kim S, Ghil SH, Tsai MJ, Kim YS, Lee YD, Suh-Kim H 2001 Overexpression of BETA2/NeuroD induces neurite outgrowth in F11 neuroblastoma cells. J Neurochem 77:103109[CrossRef][Medline]
-
Hwung YP, Gu YZ, Tsai MJ 1990 Cooperativity of sequence element mediates tissue specificity of the rat insulin II gene. Mol Cell Biol 10:17841788[Medline]
-
Sharma A, Moore M, Marcora E, Lee JE, Qiu Y, Samaras S, Stein R 1999 The NeuroD1/BETA2 sequences essential for insulin gene transcription colocalize with those necessary for neurogenesis and p300/CREB binding protein binding. Mol Cell Biol 19:704713[Abstract/Free Full Text]
-
Mutoh H, Naya FJ, Tsai MJ, Leiter AB 1998 The basic helix-loop-helix protein BETA2 interacts with p300 to coordinate differentiation of secretin-expressing enteroendocrine cells. Genes Dev 12:820830[Abstract/Free Full Text]
-
Ohneda K, Mirmira RG, Wang J, Johnson JD, German MS 2000 The homeodomain of PDX-1 mediates multiple protein-protein interactions in the formation of a transcriptional activation complex on the insulin promoter. Mol Cell Biol 20:900911[Abstract/Free Full Text]
-
Boam DS, Clark AR, Docherty K 1990 Positive and negative regulation of the human insulin gene by multiple trans-acting factors. J Biol Chem 265:82858296[Abstract/Free Full Text]
-
Clark AR, Wilson ME, Leibiger I, Scott V, Docherty K 1995 A silencer and an adjacent positive element interact to modulate the activity of the human insulin promoter. Eur J Biochem 232:627632[Abstract]
-
Lee JE, Hollenberg SM. Sinder L, Turner DL, Lipnick N, Weintraub H 1995 Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268:836844[Medline]
-
Liu M, Pleasure SJ, Collins AE, Noebels JL, Naya FJ, Tsai MJ, Lowenstein DH 2000 Loss of BETA2/NeuroD leads to malformation of the dentate gyrus and epilepsy. Proc Natl Acad Sci USA 97:865870[Abstract/Free Full Text]
-
Karschin C, Ecke C, Ashcroft FM, Karschin A 1997 Overlapping distribution of K (ATP) channel-forming Kir6.2 subunit and the sulfonylurea receptor SUR1 in rodent brain. FEBS Lett 1:5964[CrossRef]
-
Madsen OD, Jensen J, Petersen HV, Petersen EE, Oster A, Andersen FG, Jorgensen MC, Jensen PB, Larsson LI, Serup P 1997 Transcription factors contributing to the pancreatic ß-cell phenotype. Horm Metab Res 29:265270[Medline]
-
Serup P, Jensen J, Andersen FG, Jorgensen MC, Blume N, Holst JJ, Madsen OD 1996 Induction of insulin and islet amyloid polypeptide production in pancreatic islet glucagonoma cells by insulin promoter factor 1. Proc Natl Acad Sci USA 93:90159020[Abstract/Free Full Text]
-
Waeber G, Pedrazzini T, Bonny O, Bonny C, Steinmann M, Nicod P, Haefliger JA 1995 A 338-bp proximal fragment of the glucose transporter type 2 (GLUT2) promoter drives reporter gene expression in the pancreatic islets of transgenic mice. Mol Cell Endocrinol 114:205215[CrossRef][Medline]
-
Watada H, Kajimoto Y, Umayahara Y, Matsuoka T, Kaneto H, Fujitani Y, Kamada T, Kawamori R, Yamasaki Y 1996 The human glucokinase gene ß-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells. Diabetes 11:14781488
-
Marshak S, Totary H, Cerasi E, Melloul D 1996 Purification of the ß-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells. Proc Natl Acad Sci USA 93:1505715062[Abstract/Free Full Text]
-
Dumonteil E, Laser B, Constant I, Philippe J 1998 Differential regulation of the glucagon and insulin I gene promoters by the basic helix-loop-helix transcription factor E47 and BETA2. J Biol Chem 32:1994519954[CrossRef]
-
Maruyama IN, Rakow TL, Maruyama HI 1995 cRACE: a simple method for identification of the 5' end of mRNAs. Nucleic Acids Res 23:37963797[Medline]
-
Attardi LD, Tjian R 1993 Drosophila tissue-specific transcription factor NTF-1 contains a novel isoleucine-rich activation motif. Genes Dev 7:13411353[Abstract]