©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Upstream Promoter Element of the Glucagon Gene, G1, Confers Pancreatic Alpha Cell-specific Expression (*)

(Received for publication, June 29, 1994; and in revised form, September 22, 1994)

Corinne Morel Martine Cordier-Bussat Jacques Philippe (§)

From the Departments of Genetics and Microbiology and Medicine, Centre Médical Universitaire, 9 avenue de Champel, 1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The glucagon gene is expressed in the endocrine pancreas, the intestine, and the brain. In the endocrine pancreas, expression of the glucagon gene is restricted to the alpha cells of the islets of Langerhans. We previously showed that 168 base pairs of the promoter was critical for this restricted expression. To further characterize the mechanisms involved in alpha cell specificity, we analyzed the responsible DNA sequences by transient transfection studies into glucagon- and insulin-producing cell lines. We localized alpha cell-specific sequences between nt 100 and 52, a region that corresponds to the upstream promoter element G1. Four protein complexes, B1, B2, B3, and B6 interact with G1; B6 requires most of G1 to be formed. B1, B2, and B3, by contrast, bind on closely overlapping sequences, display similar methylation interference patterns, and appear to be related complexes. Point mutations of G1 indicate, however, that their binding specificities are different. All four complexes are islet-specific, and impairment of their binding results in decreased transcription. We conclude that G1 interacts with islet cell-specific proteins to restrict glucagon gene expression to the alpha cells.


INTRODUCTION

Developmental regulation of gene expression and the events leading to cell differentiation remain poorly characterized. Identification of nuclear proteins and their cis-acting cognate DNA control elements constitute a preliminary step to approach the molecular mechanisms of gene expression during embryogenesis and in the differentiated cell.

Glucagon is a 29-amino acid peptide involved in glucose homeostasis. This is the first hormone to be produced by the embryonic pancreas(1, 2) . Characterization of the factors involved in the regulated expression of the glucagon gene should help to understand the events leading to islet cell differentiation. Studies with transgenic mice and glucagon-producing cells transiently transfected with DNA constructs containing various lengths of the rat glucagon gene 5`-flanking sequence linked to the chloramphenicol acetyl transferase (CAT) (^1)reporter gene have shown that the most proximal 300 bp were sufficient to direct alpha cell-specific expression(3, 4) . Three DNA control elements have been further defined within these 300 bp, G1, G2, and G3 G2 and G3 function as enhancer-like sequences, and G1 functions as an upstream promoter element with little intrinsic activation potential.

We previously showed that 168 bp of the rat glucagon gene promoter were critical for alpha cell specificity(5) . To further understand the molecular mechanisms leading to cell-specific expression of the glucagon gene, we first localized within this region the responsible sequences by 5`- and 3`-deletional analyses of the promoter and studied their interactions with nuclear proteins by binding and functional assays. We report here that G1 is the critical DNA control element of the rat glucagon gene promoter, which confers alpha cell-specific expression. At least four protein complexes (B1, B2, B3, and B6) bind to G1 B6 requires most of G1 for its formation, whereas B1, B2, and B3 bind to overlapping sequences, display similar DNA interactions as assessed by dimethyl sulfate (DMS) interference assays, and may be related complexes. Specific mutations of G1, however, can selectively prevent or impair the binding of B1, B2, or B3. The four complexes are islet cell-specific, and mutations that affect their binding result in decreased transcriptional activity. Our data indicate that glucagon gene expression depends on the interactions of islet cell-specific DNA-binding proteins with sequences of the upstream promoter element G1.


MATERIALS AND METHODS

Cell Culture and Nuclear Extracts

alphaTC1 and betaTC1 cells were generously provided by Dr. D. Hanahan (University of California, San Francisco). Glucagon (InR1G9 (6) and alphaTC1(7) ) and insulin (HIT-15 and betaTC1(8) ) as well as non-islet cells, Syrian baby hamster kidney (BHK-21), and rat adrenal pheochromocytoma (PC-12) were grown in RPMI 1640 (Seromed; Basel, Switzerland) supplemented with 5% fetal calf serum and 5% newborn calf serum, 100 units of penicillin, and 100 µg of streptomycin/ml.

The human cell lines THP-1 (human monocyte), Hep-2 (human epidermoid carcinoma), HepG2 (human hepatocellular carcinoma), ME-43 (melanoma), JAR (choriocarcinoma), and the Epstein-Barr virus-transformed B lymphocyte line, Mann, were grown in the same conditions as described above.

Nuclear extracts were prepared according to Shapiro et al.(9) or Schreiber et al.(10) . Protein concentrations were determined by the Bio-Rad protein assay kit (Bio-Rad).

Electrophoretic Mobility Shift and DNase I Footprinting Assays

Electrophoretic mobility shift assays (EMSAs) were performed as previously described(11) . Antibodies against the helix-loop-helix proteins E12/E47 were generously provided by Drs. C. Murre (University of California, San Diego) and M. Walker (Weitzmann Institute of Science; Rehovot, Israel).

A 408-bp fragment (nt -350 to +58) subcloned into Bluescript (Stratagene, La Jolla, CA) was 5`-end-labeled on the upper strand and used for the DNase I footprinting assay essentially as described previously (5) except that no polyvinyl alcohol was added in the incubation buffer.

Methylation Interference Assays

The G1-56 oligonucleotide was individually end-labeled on the upper or lower strands with [P]ATP by T4 polynucleotide kinase and partially methylated by dimethyl sulfate for 150 s as described by Maxam and Gilbert(12) . Oligonucleotides were then used in EMSAs; the binding reactions with nuclear extracts were scaled up 8-fold. Transfer and elution of methylated DNA was performed as described by Kobr et al.(13) . Piperidine cleavage followed by sequencing gel analysis was done according to Maxam and Gilbert(12) .

Transfections and CAT/Placental Alkaline Phosphatase Assays

DNA transfection was performed by the DEAE-dextran method, essentially as described(5) . 2 times 10^5 InR1-G9 cells were trypsinized and incubated in 1 ml of TD (25 mM Tris-Cl (pH 7.4), 140 mM NaCl, 5 mM KCl, 0.7 mM K(2)HPO(4)) containing 6 µg of indicator plasmid and 300 µg/ml DEAE-dextran for 15 min at room temperature. To normalize for transfection efficiency, a plasmid containing the human placental alkaline phosphatase gene linked to the SV40 enhancer (pSV2Apap) was included in each transfection(14) . Forty-eight hours following transfection, cells were harvested, and CAT assays were performed essentially as described(5) ; 2.5 times 10^6 cells were resuspended in 100 µl of 0.25 M Tris-HCl (pH 7.4); 20 µl were incubated at 30 °C for 2-3 h to carry out alkaline phosphatase assays(14) .

Plasmids and Oligonucleotides

A 408-bp fragment (nt -350 to +58) of the 5`-flanking sequence of the glucagon gene, subcloned into Bluescript (Stratagene), was used for site-directed mutagenesis of G1, performed essentially as described previously(15) . The wild type and the mutated fragments were then subcloned into the XhoI-PstI sites of pBLCAT3(16) . All constructs were sequenced to confirm identity by the enzymatic method(17) .

Oligonucleotides used for EMSAs (see Table 1for sequences) and site-directed mutagenesis were synthesized on a gene assembler (Pharmacia Biotech Inc.) by using the phosphoramidite method and purified on 20% sequencing gels. Site-directed mutagenesis of G1 was performed on G1-56 subcloned into Bluescript. The wild-type G1-56 and the resulting mutated G1 (M1 to M12) were then isolated and used as binding sites in EMSAs. Relative localization within G1 of the mutated nucleotides is indicated in Table 1. 5` and internally deleted mutants were constructed from the appropriate 5`- and 3`-truncated DNA fragments of the rat glucagon 5`-flank, as previously described(5, 18) .




RESULTS

Alpha Cell-specific Expression of the Glucagon Gene Is Dependent on the G1 Control Element

We previously demonstrated that the promoter element of the rat glucagon gene (nt -136 to +51) was critical for alpha cell-specific expression(5) . In the present study, we attempted to more precisely localize the DNA sequences responsible for this specificity. In a first set of experiments we transfected DNA constructs containing 5`-deleted rat glucagon promoter sequences linked to the bacterial reporter gene encoding CAT into glucagon- or insulin-producing cells (Fig. 1, A and B). Transient transfection were performed in hamster (InR1G9 and HIT-15, producing glucagon and insulin, respectively) and mouse islet cells (alphaTC1 secreting glucagon and betaTC1 secreting insulin).


Figure 1: Cell-specific expression of the rat glucagon gene 5`-flanking sequences. CAT plasmids containing fragments of the 5`-flanking region of the glucagon gene were transfected along with a control plasmid, pSV2Apap, into glucagon-producing (InR1G9 and alphaTC1) and insulin-producing (HIT-15 and betaTC1) cells. CAT activities represent CAT/placental alkaline phosphatase enzymatic activity ratios and are expressed relative to that obtained with the positive control, RSVCAT; values are indicated along with the standard error of the mean (n = 4). poCAT (the CAT gene without the promoter) was used as a negative control. A and C, DNA transfection into InR1G9 and HIT-15 cells. B and D, DNA transfection into alphaTC1 and betaTC1 cells. G2 refers to sequences nt -200 to -165, and G3 refers to nt -270 to -230. *, #, and + indicate p values of <0.01, <0.02, and <0.05, respectively.



Selective expression of DNA constructs nt -1600CAT, nt -1100CAT, nt -350CAT, nt -213CAT, and nt -168CAT was observed in the glucagon-producing cell lines, InR1G9 and alphaTC1. By contrast, little or no activity above that obtained with the promoterless construct poCAT was measured in the insulin-producing cells, HIT-15 and betaTC1. The fact that nt -168CAT was still specifically expressed in InR1G9 and alphaTC1 cells indicates that a critical determinant for alpha cell-specific expression is present within the promoter of the rat glucagon gene or the 51 bp of the first exon, a result in agreement with our previous analyses(5) .

Additional DNA constructs were made in order to further localize the alpha-specific DNA element. We thus linked the rat glucagon gene enhancer sequences G2 or G3 to different lengths of the 5`-deleted promoter (nt -136 to +51). G2 and G3 were chosen for their capability to drive transcription in both glucagon- and insulin-producing cells with similar efficiency when linked to a heterologous promoter(5) . With constructs containing either 31 or 60 bp of the glucagon promoter driven by G2 or G3, no clear specificity of expression between glucagon- and insulin-producing cells could be demonstrated (Fig. 1, C and D). Specific expression into InR1G9 and alphaTC1 cells became evident, however, when at least 75 bp of the promoter were present. A further increase in specificity was observed when 136 bp were included. Replacement of G2 or G3 by a heterologous enhancer, represented by the hepatocyte nuclear factor 3 (HNF3) binding site of the transthyretin gene (HNF3-136CAT), which is active in both beta and alpha cell phenotypes(19) , gave similar results, indicating that alpha cell-specific expression is not critically dependent on G2 or G3. In addition, activities measured after transient transfection of the 3`-deleted mutant nt -200 to -118 linked to nt -31CAT, which lacks G1, were comparable in both glucagon- and insulin-producing cells. Our results indicate that critical determinants of alpha cell-specific expression are present between nt -118 and -60 of the glucagon promoter. This region corresponds to the previously identified upstream promoter element G1(5) . G1 thus serves as the alpha cell-specific element of the glucagon gene.

G1 Interacts with Four Protein Complexes

To better characterize the upstream promoter element G1, we first precisely defined its 5`- and 3`-boundaries by the DNase 1 footprinting assay. Although G1 was previously mapped by this assay, protection by InR1G9 nuclear extracts was rather weak, resulting in a poor definition of the boundaries. A 408-bp fragment of the 5`-flanking region of the rat glucagon gene (nt -350 to +58) was labeled on the upper strand and incubated with increasing amounts of InR1G9 nuclear extracts (Fig. 2). The same protected regions corresponding to the previously characterized enhancer elements G2 and G3 were found. The most proximal protected area, G1, was localized between nt -100 and -52, differing at the 3`-boundary by 11 bp from previous mapping. G1 thus spread over 49 bp. Of note, a DNase I hypersensitive site was observed at nt -96. To test whether different protein complexes specifically bind G1, EMSAs were performed by incubating oligonucleotides of different sizes corresponding to progressively shortened G1 elements ( Fig. 3and Table 1). Sequence specificity of the formed complexes was determined by adding increasing amounts of the corresponding wild type or mutated unlabeled oligonucleotides or of nonspecific DNA fragments. With the G1-121 oligonucleotide (nt -30 to -150) containing the entire G1 element, five retarded bands were detected, B1, B2, B4, B5, and B6 (Fig. 4A). All bands were competed for by an excess of unlabeled G1-121; however, bands B4 and B5 were also competed by nonspecific DNA fragments (data not shown). Three specific complexes, B1, B2, and B6, thus bind G1-121; competition for B1 and B6 was achieved by relatively lower amounts of G1-121 as compared with B2.


Figure 2: DNase I footprint analysis of the rat glucagon gene promoter. The upper strand of the nt -350 to +58 fragment of the 5`-flanking sequence of the rat glucagon gene was 5`-end-labeled as described under ``Materials and Methods.'' Lanes F, 1, 2, and 3 represent DNase I-digested free DNA (F) and DNA to which 25 (lane 1), 50 (lane 2), and 75 (lane 3) µg of nuclear extracts prepared from InR1-G9 cells were added. Protected regions are indicated by brackets, and their approximate borders are numbered according to the transcription start site. Enhanced DNase I cutting site is indicated by an arrow.




Figure 3: Schematic representation of the first 300 bp of rat glucagon gene 5`-flanking sequence. G1, G2, and G3 boxes represent regulatory elements(7) . T, TATA box. Nucleotides are numbered relative to the transcriptional start site (indicated by an arrow). The DNA sequence of G1 is indicated above the map. Fragments used for electrophoretic mobility shift assays and methylation interference experiments are represented below the diagram. The relative positions of the mutated nucleotides within G1 are represented by black boxes. See ``Materials and Methods'' for the sequences of the mutated oligonucleotides.




Figure 4: Binding of nuclear proteins from InR1G9 cells to the G1 control element. Electrophoretic mobility shift assays were performed by incubating P-end-labeled G1-121 in the absence(-) or presence of the indicated molar excess of unlabeled competitor oligonucleotides (indicated above each lane) with 6 µg of InR1-G9 extracts. B1, B2, and B6 indicate the positions of specific protein-bound complexes, whereas B4 and B5 point to nonspecific complexes. NS, nonspecific oligonucleotide. The asterisk represents migration of free oligonucleotides.



To examine the minimal sequences needed for binding of B1, B2, and B6, we first competed for these three complexes by progressively shortened oligonucleotides (Fig. 3). As shown in Fig. 4, B and C, oligonucleotides G1-56 (nt -115 to -60) and G1-33 (nt -95 to -63) were both able to compete for B1, B2, and B6, although relatively more G1-33 was necessary to displace B2 and B6 as compared with G1-56 and G1-121. By contrast, a mutant G1-56 (G1r-56, mutated from nt -89 to -68) or a nonspecific oligonucleotide were unable to displace any of the three complexes (Fig. 4B). To further localize the binding sites of B1, B2, and B6, we used two different G1-33 mutants as competitors, G1-33r3 and G1-33r5 (Table 1). As shown in Fig. 4D, only modest competition of B1 was observed with the 5`-mutant G1-33r5, whereas both B1 and B2 were efficiently displaced by the 3`-mutant G1-33r3. Neither G1-33 mutant affected B6. These results indicate that the core of G1 (nt -89 to -80) is critical for the interactions with B1 and B2 and that B6 requires at least the 33 bp of G1-33 to be formed. We then performed EMSAs with the various shortened oligonucleotides. With G1-56, we observed not only B1 and B2 but a new complex, B3, in addition to two nonspecific complexes, B4 and B5 (Fig. 5A). B3 was specifi-cally competed for by G1-56 but to a lower extent by G1r-56 or nonspecific DNA. Nonspecific competitor DNA always somewhat affected B3 formation, indicating that B3 has a lower binding specificity to G1 as compared with B1, and B2. B3 was also efficiently competed for by the mutant G1-33r3 but not by G1-33r5 (data not shown). Of note, binding of B1 and B3 to G1-56 was maximal at 5 mM MgCl(2) and at 20 °C, whereas B2 binding was optimal at 3.5 mM MgCl(2) and not influenced by temperature (4 versus 20 °C) (data not shown). B6 was never observed to form with G1-56, suggesting that sequences between nt -60 and -52, which are present in G1-121 but not in G1-56 are important for its formation. The reason for not observing B3 with labeled G1-121 is unclear, although shorter DNA fragments might be preferable binding sites in EMSAs; alternatively, B3 may be a component of B6. In this hypothesis, B3 might interact with other proteins to form the slow migrating B6 complex. When the 3`-end of G1 is deleted (as with G1-56 and G1-33), B6 may not be able to form, allowing for the appearance of the faster migrating (and thus probably smaller) B3 complex.


Figure 5: Binding of nuclear proteins from InR1G9 cells to shortened G1 oligonucleotides. Gel retardation assays were performed with 6 µg of nuclear extracts as in Fig. 4. A and C, binding of nuclear extracts to G1-56. B, binding of InR1G9 nuclear extracts to G1-33. The molar excess of the competitor oligonucleotide is indicated above each lane. B1, B2, and B3 indicate the positions of specific complexes, whereas B4, B5, and dots point to nonspecific complexes. The asterisk represents free oligonucleotides.



B1, B2, and B3 were also able to bind G1-33, although, with this shorter binding site, B2 migrated close to B1 (Fig. 5B). Addition of mutant G1-56 competitors ( Table 1and Fig. 9), which either bind B1 and not B2 (M4) or B2 and not B1 (M9), clearly showed, however, that both B1 and B2 are binding to G1-33. G1-33 is thus sufficient to bind B1, B2, and B3.


Figure 9: Effect of G1 mutants on protein binding. A, EMSA performed by incubating P-end-labeled G1-56 and 11 G1 mutant oligonucleotides (see Table 1) with 6 µg of InR1-G9 nuclear extracts. B, G1 mutant oligonucleotide-specific binding was investigated by competition experiments. The binding reactions were carried out by incubating labeled G1-56 in the absence(-) or presence of a 100-fold molar excess of mutated DNA competitor (M1 to M12) as indicated above each lane. Positions of the specific DNA-protein complexes (B1 to B3), nonspecific complexes (B4, B5, and dots), and free DNA (asterisk or F) are indicated with arrowheads.



The E Box of G1 Is Not Sufficient for the Binding of B1, B2, and B3

The core of G1 contains an E box (CAGATG) between nt -83 and -78. E boxes have been shown to be important for the cell-specific expression of the insulin gene and for other genes expressed in the endocrine pancreas, such as the gastrin and secretin genes(20, 21, 22) . Since we showed that nt -89 to -80 were critical for the binding of B1, B2, and B3, we investigated whether these complexes were specifically interacting with the E box present in G1. An oligonucleotide containing the E box sequence flanked by four additional nucleotides on each side (G1-HLH) was used to compete for the B1, B2, and B3 complexes binding to labeled G1-56. B1 and B2 were not affected, whereas B3 was only partially competed (Fig. 5C); these results indicate that the E box of G1 is not sufficient for the binding of any of the three complexes. In addition, B1, B2, and B3 did not bind to the oligonucleotide HLH-G1 or to an oligonucleotide that contains the FAR box of the rat insulin gene (data not shown). Furthermore, addition of antibodies against the ubiquitous helix-loop-helix proteins E12/E47 (23, 24) to the EMSA binding reactions did not result in any alteration in B1, B2, or B3 formation, and overexpression of E12/E47 or of the transcriptional helix-loop-helix inhibitor 1d (25) in glucagon-producing cells did not modify transcriptional activity of constructs containing G1 linked to G2 or G3 (data not shown).

Transient expression of DNA constructs containing 5`deleted fragments of the glucagon gene promoter driven by either G2 or G3 revealed that alpha cell-specific expression was partially restored with 75 bp of the promoter compared with 60 bp. Since the binding sites of B1, B2, and B3 are located 5` of nt -75, we tested whether we could detect the interaction of protein complexes with G1-3`, an oligonucleotide spanning sequences of the promoter between nt -77 and -47. However, no specific complexes were observed (data not shown).

B1, B2, and B3 are Related Complexes

To investigate a possible relationship between B1, B2, and B3, we progressively increased the amount of nuclear proteins incubated with labeled G1-56 (Fig. 6A). At high protein concentrations, we did not observe any new complex but rather a marked increase in B3 formation with a proportionate decrease in B1 and B2. These data suggest that B3 may contain components of B1 and B2. To further explore this possibility, we competed for B3 after adjusting for a protein concentration that maximized the formation of B3 while minimizing that of B2 with a mutant G1-56 oligonucleotide, M4, which binds B1 and B3 but not B2 (see Fig. 9). Addition of M4 eliminated B1 and B3, whereas it favored B2 formation (Fig. 6B, compare lanes 1 and 3), supporting the hypothesis that components of B3 may also be present in B2. We cannot exclude, however, that B2 reappearance is caused by the availability of more G1-56 binding site. To identify precisely the contact points that are involved in the formation of B1, B2, and B3 within the core of G1, we performed methylation interference assays. Comparison of the cleavage profiles obtained with free and bound DNA revealed that methylation of 2Gs (nt -76 and -83 on the upper and lower strands, respectively) interfered with the formation of B1 and B2 (Fig. 7, A and B). Methylation interference with B3 resulted in the same profile (data not shown). Interference of methylation was also reproducibly observed at nt -78 (B1, B2, and B3) and -81 (B2) on the upper strand, although this was mild and thus of unclear significance.


Figure 6: Binding of increasing amounts of InR1G9 nuclear extracts to G1-56. Gel retardation assays were performed as in Fig. 4. A, the amount of nuclear extracts incubated with labeled G1-56 was increased from 6 (lane 1) to 9 (lane 2), 12 (lane 3), 15 (lane 4), 18 (lane 5), and 21 µg (lane 6). B, the amount of nuclear extracts was 6 (first lane) and 12 µg (second and third lanes). The unlabeled competitor oligonucleotide is indicated above lane 3. B1, B2, and B3 indicate specific complexes, whereas B4 and B5 indicate nonspecific complexes. The asterisk represents free labeled G1-56.




Figure 7: Methylation interference analysis of B1 and B2. G1-56 coding (C) and noncoding (NC) strands were individually P-endlabeled and incubated with 48 µg of InR1-G9 extracts as described under ``Materials and Methods.'' A, methylation interference pattern of B1 and B2. F represents the cleavage pattern of free DNA; B1 and B2 correspond to the cleavage products obtained from B1 and B2 complexes. DNA sequences of modified methylation are indicated along the ladder. B, schematic representation of the methylation interference profiles of B1 and B2. Filled arrowheads indicate G residues in B1 and B2 complexes at which methylation specifically interferes with protein binding. Open arrowheads represent enhanced DNA cleavage. Intensity of each band was evaluated by laser densitometry.



Enhanced DNA cleavage was noted 5` and 3` of the contact points for all three complexes (at nt -109 and -74 on the upper and lower strands, respectively), probably indicating more exposed sites either by protein binding-induced structural alteration of the DNA or by the formation of hydrophobic pockets at the boundaries of the protein binding sites.

B1, B2, B3, and B6 Are Islet Cell-specific

Since G1 is critical for cell-specific expression of the rat glucagon gene, we evaluated the cell type-specific distribution of B1, B2, B3, and B6. Nuclear extracts prepared from several different cell lines were incubated with either labeled G1-121 or G1-56 (Fig. 8). The binding pattern obtained from glucagon- (InR1G9 and alphaTC1; lanes 1 of the left and right panels, respectively, of Fig. 8A) or insulin-producing cells (HIT-T15 and betaTC1, lanes 2 of the left and right panels, respectively) were similar on G1-121, as well as on G1-56 (data not shown). B1, B2, and B3 were absent from non-islet cells (Fig. 8, B, C, and D). A complex migrating with the same mobility as B2 and present in all of the non-islet cells was observed with G1-121 (Fig. 8, B and C) but not G1-56 (Fig. 8D); this complex was not competed for by an excess of unlabeled G1-121 (data not shown). We also detected a complex migrating close to B6 in HepG2 extracts; this complex, however, was shown to be nonspecific (data not shown). Additional bands were observed with labeled G1-56 and not G1-121; their migration pattern differed from that of B1, B2, and B3. The quality of the nuclear extracts was verified by their capacity to bind the ubiquitous factor NFY with an oligonucleotide containing an NFY binding site (13) (data not shown). Overall, our data indicate that the four complexes that interact with G1 are islet-specific.


Figure 8: Cell type distribution of B1, B2, B3, and B6. Equal amounts of nuclear extracts (6 µg) from cell lines indicated below were assayed for the presence of B1, B2, B3, and B6, using the G1-121 and G1-56 probes. EMSAs were performed as described in the legend to Fig. 4. A, cellular specificity of DNA-protein complexes B1, B2, and B6 in extracts from phenotypically different islet cells: InR1G9 and HIT-15 (left panel, lanes 1 and 2, respectively) and alphaTC1 and betaTC1 (right panel, lanes 1 and 2, respectively). B and D, cellular specificity from non-islet cells. B, comparison of the EMSA profiles obtained with nuclear extracts from InR1-G9 (lane 1) and the non-pancreatic rodent cell lines PC12 (lane 2) and BHK-21 (lane 3) using labeled G1-121. C, comparison of the EMSA profiles obtained with nuclear extracts from InR1G9 cells (lane 1), ME43 (lane 2), HepG2 (lane 3), Hep-2 (lane 4), JAR (lane 5), and Mann (lane 6) using labeled G1-121. D, patterns obtained with InR1G9 (lane 1) and the non-pancreatic human cell lines Mann (lane 2), THP-1 (lane 3), Hep-2 (lane 4), HepG2 (lane 5), ME-43 (lane 6), and JAR (lane 7) using labeled G1-56. Asterisk indicates unbound oligonucleotides. B1, B2, B3, and B6 indicate the positions of bound specific complexes. For descriptions of the different cell lines, see ``Materials and Methods.''



B1 and B2 Recognize Different Sequences

To dissect the sequence requirements of B1, B2, and B3, EMSAs were performed with G1 oligonucleotides containing point mutations (Table 1). Mutants were randomly chosen between nt -91 and -72. B1 and B3 formation was affected by all mutations except for M4 (nt -85) and M10 (nt -76); the absence of major effects on complex binding by M10 was unexpected since methylation at nt -76 interfered with B1, B2, and B3 binding. M11 (nt -72/73) had moderate effects on B3 (Fig. 9, A and B). M11 is particularly interesting because it has destabilizing effects on B3 although nt -72 and -73 do not appear to be absolutely required for its binding inasmuch as G1-33r3 can bind and compete for B3 (although with lower efficiency than G1-56). B2 was differently affected; mutants at nt -79 to -72 (M8 to M11) were still able to bind B2 although with somewhat decreased affinity (especially M9) in contrast to mutants M1 to M7 (nt -91 to -82). These data, together with the fact that G1-33 is sufficient to bind all three complexes, indicate that B1, B2, and B3 interact on overlapping sequences between nt -95 and -76. Our mutational analyses reveal, however, different binding specificities for B1 and B2. When the different G1 mutants were used as competitors for B1, B2, and B3, a good correlation for each mutant was observed between its capability to bind and to compete, except for B3, which was competed by all mutants (Fig. 9B).

Transcriptional Activity Is Affected by Point Mutations of G1

We previously showed that the transcriptional activity of the glucagon gene 5`-flank containing a linker scanning mutant of G1 involving nt -75 to -84 was abolished(5) . To examine more systematically the functional consequences of G1 mutations, we transfected DNA constructs consisting of 350 bp of the rat glucagon gene 5`-flank linked to the CAT gene into InR1G9 cells; mutations within G1 corresponded to those of the G1 oligonucleotide mutants used in EMSAs (Table 1). Most of the mutations affected transcription, although some had little effect (Fig. 10). Two of them (M4 and M10), however, increased activity as compared with the wild-type construct. Interestingly, these two mutations were the only ones that did not impair B1 or B3 formation. M11, by contrast, resulted in a marked decrease in transcription, whereas only B3 binding was significantly affected; nt -72 and -73 thus appear critical for G1 function, suggesting that protein complexes, other than B1, B2, or B3, interact with this region of G1.


Figure 10: Functional analysis of mutations within G1 by transient expression into InR1G9 and HIT-15 cells. Plasmid constructs containing the CAT gene (CAT) under the control of sequence nt -350 to +58 of the rat glucagon gene were tested for CAT activity by transient transfections into InR1G9 () and HIT-15 (box) cells as described under ``Materials and Methods.'' The regulatory elements are represented by hatched boxes, and the wild-type (WT) and mutated G1 sequences are indicated below the map. The E box motif is underlined. The site of transcription initiation is indicated by the arrow. Each construct was cotransfected with the plasmid pSV2Apap to correct for differences in transfection efficiencies, and relative CAT activity values represent CAT/PAP enzymatic activity ratios relative to that of construct WT with the standard error of the mean (n = 4). pBLCAT3 (the CAT gene without the promoter) was used as a negative control. #, +, and * indicate p values of <0.01, <0.02, and <0.05, respectively.



To investigate the possibility that G1 might function as silencer in non-alpha cell lines rather than as an alpha cell-specific element, we transfected the mutant G1 constructs into the insulin-producing cell line, HIT-15. None of the constructs was capable of significantly activating transcription above base line (Fig. 10). These results strengthen the proposed alpha cell-specific role of G1.


DISCUSSION

In the studies presented here, we have attempted to better define the factors that contribute to alpha cell-specific expression of the glucagon gene. We have previously shown that the glucagon gene promoter (nt -168 to +51) was critical for cell-specific expression, whereas the enhancers G2 and G3 were capable of activating transcription in phenotypically different islet cells(5) . We show here that the DNA sequences of the promoter that determine alpha cell specificity are localized between nt -118 and -60 and correspond to G1, a DNA control element previously identified by DNase I footprint assays. Additional elements are present within the rat glucagon gene promoter, but their contribution to differential expression between islet cell phenotypes is probably small. (^2)G1 is a large 49-bp element that binds at least four protein complexes, B1, B2, B3, and B6. Surprisingly, B1, B2, and B3 interact with a limited part of G1, between nt -95 and -75. No complex binding to the proximal half of G1 (G1-3`), between nt -75 and -52 was detected by EMSAs, although many different conditions were attempted both for running gels (Tris-glycine and TBE (0.045 M Tris-borate, 0.001 M EDTA) buffers) and performing the binding reactions of the EMSAs (range of KCl and MgCl(2) concentrations). Several indications suggest, however, that the proximal half of G1 plays a significant role in both transcriptional activity and cell specificity. Transcriptional activities of G2/G3-60CAT in alpha and beta cells are comparable, whereas a clear difference in favor of alpha cells is apparent for G2/G3-75CAT, indicating the presence of an alpha cell-specific determinant in the proximal 75 bp. Furthermore, mutations at nt -72 to -73 (M11) result in the most dramatic decrease in transcription. It is thus likely that the proximal part of G1 is functionally important and consequently interacts with transcription factors. The B6 complex actually requires the proximal region of G1 to be formed. The facts that B6 needs most of G1 for binding and that it is a slow migrating complex suggest that it may contain several proteins, one or more of which may interact with the proximal part of G1. Since we cannot detect specific complexes binding to G1-3`, it may be hypothesized that protein interactions with the proximal part of G1 require the cooperativity of proteins binding to its distal half and among them, potentially, B1, B2, and B3. In that regard, M11 (nt -72 to -73) decreases B3 binding, whereas B3 may not interact with these nucleotides. The facts that B6 can only bind when an intact proximal half of G1 is present and that B3 only forms in the absence of this proximal half suggest that B3 may be contained in B6.

B1, B2, and B3 probably share common subunits as suggested in the EMSAs by the preferential formation of B3 at the expense of B1 and B2 at high protein concentrations. These complexes may thus represent combinations of homo- and heterodimers belonging to a family of transcription factors. They bind on overlapping sequences between nt -95 and -75 and display very similar DMS interference patterns. Mutational analyses clearly reveal, however, different specificities at least for B1 and B2. The overlapping binding sites of B1, B2, and B3 suggest that only one of the complexes, exclusively of the others, may interact with G1 at a particular time; if this is indeed the case, it will be important to determine whether all three complexes can interact with G1 in vivo, what favors the binding of one complex over the others, and what are their respective effects on transcription. In that regard, it may be of interest to note that B1 may have a higher affinity for G1 than B2.

Can the interactions of G1 with B1, B2, B3, or B6 explain alpha cell-specific expression of the glucagon gene? All four complexes are found in both insulin- and glucagon-producing cells. The nature of the four beta cell complexes may differ from those found in alpha cells. Combinations of different subunits contained in B1, B2, B3, or B6 between alpha and beta cells might then explain cell-specific expression of the glucagon gene. Alternatively, additional complexes that we have not detected in our binding assays or proteins not directly contacting DNA and present only in alpha cells may be required to confer specificity. In any case, multiple proteins are likely to act in concert to insure specificity. This hypothesis is strengthened by our transfection studies indicating that 75 bp of the promoter impart cell specificity as compared with 60 bp but that the entire G1 is necessary for optimal alpha cell-specific expression. An additional consideration may be that complexes from non-glucagon-producing islet cells bind to G1 and prevent transcription; transfections of the mutant G1 constructs in HIT-15 cells do not support this possibility.

Search by computer analysis did not reveal any sequence homology between G1 and other known DNA control elements, except for an E box (CAGATG from nt -83 to -78) and two TAAT sequences (from nt -57 to -54 and nt -91 to -88).

We noted previously that disruption of the E box by linker scanning mutation resulted in a complete loss of transcriptional activity(5) . We thus hypothesized that the E box could serve as a main determinant of cell-specific expression of the glucagon gene. Helix-loop-helix proteins, which bind E boxes, play a central role in the differentiation process of a wide variety of cell types (26, 27, 28, 29) and have been suggested to be involved in the cell-specific expression of the insulin gene as well as other genes, such as those encoding gastrin and secretin expressed at some stages in islet cells(20, 21, 22) . We show, however, that B1, B2, and B3 do not show binding specificities for the E box of G1, suggesting that the E box motif of G1 has no relevance by itself to the cell-specific expression of the glucagon gene.

The two TAAT sequences present in G1 suggest that homeobox-containing transacting factors may play a role in alpha cell-specific expression. Such sites have already been shown to be critical for the cell-specific expression of two other islet hormone genes encoding insulin and somatostatin(30, 31, 32) . Further characterization of B1, B2, B3, and B6 should help us understand the development and differentiation of alpha cells.


FOOTNOTES

*
This work was supported by Swiss National Fund Grant 32-031327.91 and the Jules Thorn Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Genetics and Microbiology, Centre Médical Universitaire, 9 avenue de Champel, 1211 Geneva 4, Switzerland. Tel.: 41-22-702-56-66; Fax: 41-22-34672-37.

(^1)
The abbreviations used are: CAT, chloramphenicol acetyl transferase; nt, nucleotide(s); bp, base pair(s); EMSA, electrophoretic mobility shift assay.

(^2)
M. Cordier-Bussat, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. W. Reith for critically reading the manuscript and making helpful suggestions.


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