©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Islet-specific Proteins Interact with the Insulin-response Element of the Glucagon Gene (*)

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

Jacques Philippe (§) Corinne Morel Martine Cordier-Bussat

From the Departments of Genetics and Microbiology and Medicine, Centre Médical Universitaire, University of Geneva Medical School, CH-1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glucagon gene expression is negatively regulated by insulin at the transcriptional level. G3, a DNA control element located in the 5`-flanking sequence of the rat glucagon gene mediates the inhibition of transcription, which occurs in response to insulin. We show here that two islet-specific protein complexes C1A and C1B, bind to the A domain of G3, which is critical for the insulin response. These two complexes bind to overlapping sequences of the A domain and display very similar binding specificities. Point mutations in the A domain that affect binding of C1A and C1B result in both decreased G3 enhancer activity and insulin-mediated inhibitory effects with a close correlation between diminution of binding and function. One of the two complexes, C1A, is similar or identical to B1, a protein complex interacting with the upstream promoter element of the glucagon gene, G(1), implicated in the A cell-specific expression of the glucagon gene. Our data indicate that islet-specific proteins are involved in glucagon gene regulation by insulin.


INTRODUCTION

Insulin plays an important role in promoting cell growth and energy storage. One of its most important physiological effects in the adult is the maintenance of glucose homeostasis, which is realized by the control of glucose production in the liver and glucose utilization by muscle and adipose tissues. To control glucose production, insulin directly inhibits hepatic glycogenolysis and gluconeogenesis but also acts on the A cell of the islets of Langerhans by inhibiting the secretion and biosynthesis of glucagon, a polypeptide hormone that favors hepatic glucose production(1, 2, 3) . We have previously shown, using the glucagon-producing cell line, InR1G9, that the effects of insulin on glucagon biosynthesis are the result of changes in glucagon gene expression occurring at the level of transcription(3) . We recently characterized an insulin-response element (IRE) (^1)within the 5`-region of the rat glucagon gene, G3, which interacts with at least 2 protein complexes (4) . G3 is an islet-specific control element, located in tandem 5` of the major enhancer of the glucagon gene, G2; it contains two apparently distinct domains, A and B(5) . The A domain is sufficient for the enhancer activity of G3 and the mediation of the inhibitory effects caused by insulin(4, 5) . Both the A domain and the factors interacting with it remain poorly characterized.

In this study, we have attempted to precisely define the IRE of the rat glucagon gene and to better characterize the cellular factors that interact with it. We find that two islet-specific protein complexes whose respective binding sites are overlapping interact with the A domain. Mutations that severely impair their binding result in both decreased enhancer activity and insulin-mediated inhibitory effects; one of the two complexes, C1A, is very similar or identical to B1, a complex interacting with the upstream promoter element, G(1), implicated in the A cell-specific expression of the glucagon gene(7) . Our results suggest that insulin regulation of glucagon gene expression is mediated by islet-specific proteins.


EXPERIMENTAL PROCEDURES

Plasmids

Oligonucleotides containing the wild-type and mutated G3 sequence (nucleotides (nt) -274 to -234 relative to the transcriptional start site) with BamH1-compatible ends were inserted into a BamH1 site of Bluescript (Stratagene, La Jolla, CA) or 5` of the glucagon (nt -136 to +51) or of the herpesvirus thymidine kinase promoters linked to the chloramphenicol acetyltransferase (CAT) gene(4) . The oligonucleotides were synthesized by the phosphoramidite method (Pharmacia Biotech Inc.) and are listed in Fig. 1. All constructs were sequenced to confirm identity and orientation by the enzymatic method. Oligonucleotides corresponding to different CCAAT binding sites, C/EBP, CTF/NF1, NFY, and alpha-CBF were generously provided by Dr. Mario Vallejo (Harvard University), and mutants G3M3 and G3M6 were provided by Dr. W. Knepel (University of Goettingen).


Figure 1: Binding of nuclear proteins from InR1G9 cells to the G3 control element. Gel retardation assays were performed by incubating P-labeled G3 with nuclear extracts. A, effect of different amounts of proteins and competitor DNA on binding to G3. Nuclear extracts were incubated with P-labeled G3. Lane 0, free DNA; lanes 1-3, 2, 4, and 6 µg of nuclear extracts. Competitor DNA was added in a 10- (lanes 4 and 7), 25- (lanes 5 and 8), and 50-fold excess (lanes 6, 9, and 10). B, separation of C1A from the C1B complex. 4 µg of nuclear extracts were incubated with P-labeled G3. Polyacrylamide gels were run for a longer time period than in A. Lane 1, free DNA; lane 2, InR1G9 nuclear extracts. points to C1A, up triangle to C1B, and right arrowhead to the C2 and C3 complexes. Dots indicate either nonspecific or non-reproducibly observed protein-DNA complexes. comp, competitor DNA.



Cell Culture and Transfection Studies

Islet cells InR1G9 (8) , alphaTC1(9) , betaTC1(10) , and RIN B2 (11) and non-islet cells (rat pheochromocytoma PC12, mouse fibroblast Ltk, Syrian baby hamster kidney BHK-21, human hepatoma Hep G2, mouse hepatoma H4, human epidermoid laryngeal carcinoma Hep-2, rat epithelial ileum IEC, Epstein-Barr virus-transformed B lymphocyte Mann and chronic lymphocytic leukemia Cohen) were cultured in RPMI 1640 containing 5% fetal calf serum and 5% newborn calf serum. InR1G9 cells were transfected in suspension by the DEAE-dextran method (12) with 3 µg of reporter plasmid and 1 µg of the plasmid pSV(2)Apap to monitor transfection efficiency. pSV(2)Apap is a plasmid containing the human placental alkaline phosphatase gene driven by the simian virus 40 long terminal repeat(13) . Insulin was added to the culture medium at a concentration of 10M. Cell transfection studies with Bluescript alone or Bluescript-containing binding sites were done with 1 µg of reporter plasmid and either 2.5 or 5 µg of competitor plasmid. Cell extracts were prepared 48 h after transfection and analyzed for CAT and alkaline phosphatase activities as described(4) . Protein concentrations were determined with a Bio-Rad protein assay kit.

Cell Extracts and Gel Retardation Assays

Nuclear extracts were prepared by the method of Dignam et al.(14) and Schreiber et al.(15) . Gel retardation assays were performed as described(16) . Oligonucleotides were labeled by blunt-ending the BamH1 ends with the Klenow enzyme and both P-labeled and cold nucleotides.

Methylation Interference Assays

The G3 oligonucleotide was individually end-labeled on the coding or noncoding strand with P-ATP by T(4) polynucleotide kinase and partially methylated by dimethyl sulfate for 150 s as described(17) . Labeled oligonucleotides were then used in gel retardation assays, the binding reactions being scaled up 8-fold. Transfer and elution of free and protein-retarded methylated DNA were performed (18) before piperidine cleavage and analysis on 15% sequencing gels.


RESULTS

Four Protein Complexes Interact with G3

To better characterize the factors involved in the insulin-mediated negative effects on glucagon gene transcription, we performed gel retardation assays. Nuclear extracts from the glucagon-producing cells, InR1G9, were obtained within 1 h after the addition of 10M insulin. We first found three specific protein complexes interacting with G3 (C1, C2 and C3) (Fig. 1A); the complex that displayed the lowest intensity, C2, was not previously recognized(4) . An identical pattern was observed whether nuclear extracts were obtained from cells incubated with or without 10M insulin. All three complexes were competed for by an excess of unlabeled G3 but not by unrelated sequences, except for G1.

We and others (5, 7) reported previously that the upstream promoter element, G1, was capable of competing for complexes binding to G3 in the DNase I footprint and gel retardation assays. However, the amount of G1 necessary to compete was 10- to 30-fold higher compared with G3 itself, suggesting that proteins binding to G3 had a lower affinity for G1. To compare the relative affinities of G3-binding proteins for G3 and G1, we used these two binding sites as competitors for labeled G3 at increasing concentrations. Whereas G3 competed for all three complexes at all concentrations studied, only the lowest concentrations of G1 selectively displaced the fastest migrating component of C1 (C1A), indicating that C1 actually contains two different complexes, which display different affinities to G1. With increasing amounts of G1, however, the upper component of C1 (C1B) was also partially displaced (Fig. 1A), indicating that G1 can also compete for C1B at higher concentrations. The two components of the C1 complex were further separated when the gel was run for a longer time (Fig. 1B). Four protein complexes thus interact with G3, but C1A displays a similar affinity for G1 and G3.

C1 Interacts with the A Domain of G3

G3 has been proposed to represent a DNA element composed of two independent domains, A and B, located at the 5`- and 3`-half sides of G3, respectively(5) . These domains, however, have not been characterized precisely, and the nature of the proteins interacting with them remains elusive.

To localize the binding site of C1A and C1B, we performed methylation interference assays on the whole C1 complex and on C1B (after competition of C1A by G1). For the C1 complex, we observed a marked interference in methylation at nt -249 and a weaker effect at nt -252, -253, and -259 on the coding strand and nt -258 and -260 on the noncoding strand (Fig. 2, A and B). Binding of complex C1B interfered with methylation at nt -249 on the coding strand and -258 and -260 on the noncoding strand (Fig. 2C). The difference in methylation interference pattern between C1 and the C1B component lies at nt -252, -253 and -259 on the coding strand, suggesting that C1A is specifically interacting with these nucleotides. Interference with methylation at these nucleotides is weak, however. It is thus possible that C1A and C1B share a very similar or identical methylation interference pattern. The sequence that includes the protected nucleotides extends the previously defined A domain (5) on both its 5`- and 3`-ends.


Figure 2: Methylation interference assay of the C1, C2, and C3 complexes. The P-end-labeled coding and noncoding strands of the G3 oligonucleotide were partially methylated by dimethyl sulfate. DNA-protein complexes were separated from the free probe by gel retardation assay. A and B, C1 complex. A, upper strand of G3. The sequence is indicated alongside the lanes. F, free DNA; C1, C1 complex. B, lower strand of G3. The sequence is indicated alongside the lanes. Right arrowhead indicates guanosines and adenosines whose methylation was partially decreased by C1. C, C1B, C2, and C3 complexes. The upper strand of G3 is shown on the right, the lower strand on the left. The sequence is indicated alongside the lanes. F, free DNA. up triangle points to nucleotides whose methylation was partially prevented by C2, whereas indicates decreased methylation by C1B. D, summary of the methylation changes found on the coding and noncoding strands of G3. Interference of methylation is indicated by (C1), box (C1B), bullet (C2), and circle (C3).



C2 and C3 Interact with the B Domain and May Represent CCAAT Binding Factors

The B domain of G3 contains an imperfect and inverted CCAAT box (TCAATC), also present in the promoter of two other genes encoding somatostatin and the alpha subunit glycoprotein hormone (alphaSGH)(19, 20) . In all three genes, the sequence is highly conserved throughout evolution (Table 1), and the TCAATC box present in the somatostatin and the alphaSGH gene promoters has been shown to interact with a unique CCAAT-box binding factor, alpha-CBF, present in various cell types. We thus attempted to compete for complexes binding to G3 with an excess of unlabeled oligonucleotides containing the sequences of different CCAAT boxes interacting with the nuclear factors C/EBP(21) , CTF/NF1(22) , NFY(23) , and alpha-CBF. The alpha-CBF binding site was able to compete for the C2 and C3 complexes at a 10-fold excess like G3 (Fig. 1, lane 10), whereas we observed moderate competition for C2 and C3 with a 100-fold excess of the NFY site and no competition with the C/EBP and CTF/NF1 sites (data not shown). To more precisely localize the binding sites of C2 and C3, we performed methylation interference assays. We observed an identical pattern of decreased methylated guanosines at nt -243 and -247 on the coding strand for both C2 and C3 (Fig. 2). These nucleotides are part of the inverted TCAATC box. Our results indicate that C2 and C3 bind to the B domain of G3 and may represent factors that belong to the family of CCAAT-box binding proteins, which also interact with the somatostatin and the alphaSGH gene promoters. It is not entirely clear why two complexes form on the TCAATC box, although it is possible that the lower intensity complex C2 represents a monomer and that C3 represents a dimer, since CCAAT-box binding proteins bind DNA as dimers (24) ; alternatively C2 and C3 might represent different heterodimeric complexes.



C1A Is Similar or Identical to the B1 Complex of the Upstream Promoter Element G1

We recently identified three specific complexes (B1, B2, and B3) interacting with the upstream promoter element of the rat glucagon gene, G1 (Fig. 3)(6) . Since C1B was competed equally well by the G3 and G1 binding sites, we investigated whether one of the complexes that bind G1 also binds G3. We performed competition studies in gel retardation assays using labeled G1. G3 competed for B1 and B3 specifically and with a similar affinity as G1 (Fig. 3), whereas a G3 oligonucleotide mutated in the A domain (G3M6) was unable to compete any of the three complexes. In addition, the migration pattern of B1 was identical to that observed for C1A (Fig. 4, lanes 1 and 6). To further strengthen the hypothesis that C1A and B1 are the same complexes, we competed C1A with two single nucleotide mutants of G1 (Fig. 4B). Mutant 4 binds B1 and B3 but not B2, whereas mutant 9 binds B2 but not B1 or B3(6) . Mutant 4 was able to compete for C1A (Fig. 4A, lane 5), whereas mutant 9 was without effect (lane 4). Twelve additional G1 mutants were tested, and a perfect correlation between their capability to compete B1 and C1A was observed (data not shown). These data indicate that B1 and C1A are probably identical complexes. Competition for B3 by the G3 binding site was not totally unexpected since components of B1 are probably contained in B3 as demonstrated by gel retardation and methylation interference assays(6) . However, we did not unequivocally detect an equivalent of the B3 complex binding to G3, suggesting that the affinity of B3 for G3 might be lower than B1.


Figure 3: The B1 complex of the upstream promoter element, G1, is competed by the A domain of G3. Gel retardation assays were performed by incubating P-labeled G1 with nuclear extracts form InR1G9 cells in the presence of different competitors. Four µg of nuclear extract were used in each lane. Lane 1, no competitor; lane 2, 100-fold excess of nonspecific competitor DNA (N) represented by the G2 oligonucleotide; lanes 3-8, 10-, 25-, and 50-fold excess of G1 and G3 oligonucleotides, respectively; lane 9, 100-fold excess of G3M6, a G3 oligonucleotide mutated within the A domain (see Fig. 7A for sequence). , thick right arrow, and thin right arrow indicate the B1, B2, and B3 complexes. Dots indicate either nonspecific or non-reproducibly observed protein-DNA complexes. comp, competitor DNA.




Figure 4: C1A and B1 share similar binding characteristics. A, gel retardation assays were performed with 4 µg of nuclear extracts from InR1G9 cells and P-labeled G3 (lanes 1-5) or G1 (lane 6) oligonucleotides. Lane 1, no competitor; lane 2, 50-fold excess of G3; lane 3, 50-fold excess of G1; lanes 4 and 5, 50-fold excess of G1 mutants, which either do not bind (M9, lane 4) or bind very efficiently B1 (M4, lane 5). , up triangle, and right arrowhead, indicate the C1A, C1B, and C2/C3 complexes, respectively. The thin left arrow and thick left arrow point to the B1 and B2 complexes binding to labeled G1. B, coding strands of the wild-type and mutant G1 oligonucleotides used as competitors. Mutated nucleotides are in italic type and underlined.




Figure 7: Effect of G3 mutants on C1A and C1B binding. A, coding strands of the wild-type and mutant G3 oligonucleotides used in the gel retardation or in functional assays. Not shown are BamH1 ends on both sides. Mutated nucleotides are in italic type and underlined. B, summary of the six different mutations on the A domain of G3. C-E, binding of C1A, C1B, and C2/C3 to mutant G3 oligonucleotides. Gel retardation assays were performed with 4 µg of nuclear extracts from InR1G9 cells and P-labeled with wild-type or mutant G3. C, wild-type G3 (lanes 1 and 2); mutant G3 (lanes 3-6); lane 1, free DNA. D, wild-type G3 and mutant M12. E, competition of the C1A, C1B, and C2/C3 complexes bound to P-labeled G3 by wild-type (G3) mutant G3 (M7 to M12). Competitors were used in a 100-fold molar excess except for G3 (20-fold molar excess). comp, competition. , up triangle, and right arrowhead indicate C1A, C1B, and C2/C3 complexes. Dots indicate either nonspecific or non-reproducibly observed DNA-protein complexes.



If C1A and B1 are identical, introduction of an excess of G1 oligonucleotide into glucagon-producing cells should compete for C1A and affect the transcriptional activity of a reporter gene driven by a G3-containing promoter. We co-transfected into InR1G9 cells a plasmid containing G3 located 5` of the herpesvirus thymidine kinase gene promoter and the CAT gene (G3 put CAT) along with G3, G1, or the glucagon enhancer G2 sequences, inserted into Bluescript. Co-transfection of Bluescript alone or of Bluescript containing G2 did not significantly affect the activity of G3 put CAT, whereas co-transfection of Bluescript containing G1 or G3 decreased the activity of the reporter gene with similar potency (Fig. 5). These results indicate that factors necessary for G3 activity can be competed by G1. We propose that these factors are contained in B1 and C1A. Our data also suggest that C1A plays a crucial role in the positive effects of G3 on transcription.


Figure 5: Functional competition between G3 and G1. InR1G9 cells were co-transfected with an indicator plasmid containing G3 and the thymidine kinase promoter linked to the CAT gene (G3 put CAT), a control plasmid, pSV(2)Apap, and the vector Bluescript alone or containing G3, G1, or the major enhancer of the glucagon gene, G2 at two different concentrations. CAT activities were measured 48 h later and expressed as a percentage of the activity obtained in cells co-transfected with the indicator and control plasmids and Bluescript at 1.5 µg. Results represent the mean ± S.E. of four experiments.



C1A and C1B Are Islet-specific Complexes, whereas C2 and C3 Are Ubiquitous

To investigate the cell type distribution of the protein complexes binding to G3, we tested phenotypically different islet cell lines producing glucagon (InR1G9 and alphaTC1 cells), insulin (betaTC1), or somatostatin (RIN B2). All four complexes were found in the nuclear extracts of these cells (Fig. 6, lanes 1-4) (the presence of C1A and C1B was confirmed by differential competition with G1). We also looked for the presence of these complexes in non-islet cells. The C1 complex was not detected in any of these cells. A complex migrating with a similar mobility as C1 was recognized in the mouse fibroblast Ltk cell line (Fig. 6, lane 8). This complex, however, contained three different components when analyzed with different incubation conditions (concentrations of KCl and glycerol were reduced to 20 mM and 5%, respectively) (Fig. 6B). The migration pattern of the complexes present in Ltk cells and differential competitions with G3 and G1 indicated that C1A and C1B were not present in Ltk cells. The two lowest complexes migrated faster than C1A, whereas the upper complex was slower than C1B. Other complexes migrating slightly faster than C1 were also detected in other cell lines; these complexes were specifically competed by G3, but their nature remains unknown.


Figure 6: Cell type distribution of the C1A, C1B, C2, and C3 complexes. Gel retardation assays were performed as in Fig. 1with P-labeled G3 oligonucleotide and 4 µg of nuclear extracts from different cell lines. A, lanes 1-4, phenotypically different islet cell lines: glucagon-producing cells (lane 1, InR1G9; lane 2, alphaTC1) and cells producing either insulin (lane 3, betaTC1) or somatostatin (lane 4, RIN B2 cells). Lanes 5-13, non-islet cells. Lane 5, rat pheochromocytoma, PC12; lane 6, Syrian baby hamster kidney, BHK-21; lane 7, rat epithelial ileum, IEC; lane 8, mouse fibroblast (Ltk); lane 9, human hepatoma, HepG2; lane 10, human epidermoid laryngeal carcinoma, Hep-2; lane 11, mouse hepatoma, H4; lane 12, Epstein-Barr virus-transformed human B lymphocyte, Mann; lane 13, human chronic lymphocytic leukemia, Cohen. B, nuclear extracts from InR1G9 (lanes 1-3) and from Ltk cells (lanes 4-6). Competitor (comp) DNA was added at 20-fold excess. , up triangle and right arrowhead indicate the C1A, C1B, and C2/C3 complexes, respectively. Dots indicate either nonspecific or non-reproducibly observed protein-DNA complexes.



By contrast, the C2 and C3 complexes were found ubiquitously (Fig. 6) (both complexes were detected in all cell lines tested after prolonged exposure); the relative abundance of C2 and C3 was comparable in most cell lines, except in mouse H4 cells in which C2 was more abundant than C3.

Mutational Analysis of the A Domain Reveals That C1A and C1B Display Very Similar DNA Binding Characteristics

To further analyze the binding characteristics of C1A and C1B to the A domain, we examined their ability to bind mutant G3 sequences (Fig. 7, A and B). Mutants located within the core of the A domain (M9, M10, and M11) had the most dramatic effects on both C1A and C1B binding (Fig. 7C), whereas nucleotide changes at both extremities resulted in less pronounced consequences (M8 and M12) (Fig. 7, C and D). C1B appeared more affected than C1A by the latter mutants. Mutant 7 markedly decreased C1A and C1B binding but resulted in the formation of a new complex, which was specifically competed for by an excess of unlabeled mutant 7 and not by wild-type G3 (data not shown). We then attempted to compete for C1A and C1B with G3 mutants (Fig. 7E). To compete for these two complexes and illustrate their respective affinities for the mutants, we used a large excess of competitor oligonucleotides. Competition for C1A and C1B with the different G3 mutants correlated well with their ability to bind the two complexes (Fig. 7E). Our results indicate that C1A and C1B interact on overlapping sequences of the A domain with very similar binding specificities.

Transcriptional Inhibition of the Glucagon Gene by Insulin Requires That the Core of the A Domain Be Intact

To assess the functional consequences of mutations within the A domain of G3, we studied the transcriptional activities of plasmid constructs containing wild-type or mutant G3 located 5` of the glucagon gene promoter (nt -136 to +51) and the CAT gene. These plasmids were transiently transfected into InR1G9 cells, which were then incubated with or without 10M insulin. Mutants affecting the core of the A domain (M9, M10, and M11) and which affected most C1A and C1B binding resulted in the most severe effects on both basal transcription and insulin-mediated inhibition of transcription, whereas M8 and M12 did not affect either one despite moderate effects on binding of both complexes (Fig. 8). A good correlation between G3 function and C1A and C1B binding was observed though. These results indicate that mutations that affect the basal transcriptional activity conferred by G3 also decrease the insulin-mediated inhibition of transcription.


Figure 8: Consequences of G3 mutants on transcription. InR1G9 cells were transfected with an indicator plasmid containing wild-type or mutant G3 and 136 bp of the rat glucagon gene promoter linked to the CAT gene along with a control plasmid, pSV(2)Apap. Insulin (10M) was added to the culture medium 24 h after transfection. CAT activities were assessed 48 h after transfection as in Fig. 5and are expressed as a percentage of the activity obtained with the wild-type G3. Results represent the mean ± S.E. of six experiments. White bars, cells not treated by insulin; black bars, cells treated by insulin.



We next investigated whether mutations within the G1 control element, which binds B1/C1A, would affect insulin-dependent regulation of glucagon gene expression. Twelve different mutations of G1 were tested (6) . We observed a correlation between the transcriptional activity observed in the absence of insulin and the capacity of insulin to inhibit transcription. With mutants M4 and M10(6) , insulin decreased transcription by 76 and 70%, respectively; with all of the other mutants, except for M9 and M11, transcription was inhibited by 35-46%. The extent of inhibition with M9 and M11 could not be assessed because of a very low basal activity. There was no close correlation between C1A binding and the insulin-mediated effects.


DISCUSSION

The mechanisms by which insulin regulates gene transcription remain poorly defined. Several IREs have recently been identified(25) , but no unique sequence appears to mediate the effects of insulin. It is thus likely that different transcription factors are responsible for these effects.

G3 is an islet cell-specific enhancer-like element that contains two domains, A and B(5) . The A domain has been shown, by itself, to enhance transcriptional activity (4, 5) and to mediate the insulin response of the glucagon gene(4) . The function of the B domain is unknown. These domains, however, have only been defined by 3-4-nucleotide block mutations(5) , and thus the recognition sequence for the cognate transacting factors interacting with both domains have not been characterized.

Mutational analyses allowed us to identify the A domain as TCACGCCTGACTG between nt -249 and -261. Mutations of the most 5` and 3` nucleotides of the A domain resulted, however, in only moderate decreases in binding of C1A and C1B and no change in function. By contrast mutations within the core of the A domain drastically affected both binding and function. Interestingly, the A domain contains an imperfect palindrome, TCACGCCTGA, which may represent a possible binding site for dimeric proteins. Despite their different affinities for the G1 binding site, we have not been able to clearly identify differences in the specific interactions of C1A and C1B with nucleotides of the A domain. Although subtle changes with both mutational analysis and methylation interference assays were observed, these cannot firmly and definitely establish specificities in the respective binding sites of C1A and C1B. At present, our data cannot differentiate whether C1A and C1B represent different proteins or dimers/multimers sharing common subunits. It thus remains unclear whether the palindromic sequence has any relevance in C1A and/or C1B binding; definitive conclusions await the isolation of the proteins present in these two complexes. Although most IREs so far identified are not palindromes, Schütz and co-workers (26) have recently reported that the tyrosine aminotransferase gene may be regulated by insulin through the interactions of cyclic AMP-responsive element (CRE) binding protein and a CRE. The CRE of the tyrosine aminotransferase gene is recognized by CRE binding protein in a phosphorylation-dependent manner; insulin lowers the binding activity of CRE binding protein and inhibits transcription. It is becoming clear that a variety of protein-DNA complexes will be shown to mediate insulin effects on transcription, and dimeric proteins binding to palindromes may only be one example.

The functional relevance of C1A and/or C1B in the regulation of glucagon gene expression by insulin is indicated by two major observations. First, C1A and C1B interact with the A domain of G3, which is necessary and sufficient to confer insulin responsiveness to its own and to heterologous promoters(4) . Second, impairment of C1A and C1B binding to mutant G3 in gel retardation assays corresponds to a decrease in the ability of insulin to mediate its inhibitory effects, and the degree of binding impairment closely parallels the decrease in insulin action on transcription. The islet-specific distribution of C1A and C1B is rather unexpected for complexes mediating the effects of a hormone known to act on a great number of genes expressed in a wide variety of tissues. Although few data are available on the cell type distribution of IRE binding proteins, in two instances insulin has been reported to modulate the activity of transacting factors involved in the cell-specific expression of the amylase and thyroglobulin gene promoters(7, 28) . These examples may apply to G3 since G3 only functions in islet cell lines(7) .

Our data show that C1A and the B1 complex of the upstream promoter element, G1, are likely to be very similar or identical complexes; this is indicated by their identical response to wild-type and mutant G3 and G1 competitors, their identical migration pattern, and cell type distribution. Furthermore, G1 can functionally compete the transcriptional activity conferred by G3 in InR1G9 cells. Based on the sequence homology between the C1A and B1 binding sites, we propose a consensus sequence (Fig. 9) with the limitation that mutations of the flanking nucleotides of the respective sites also slightly affect binding.


Figure 9: Binding sites of the C1A and B1 complexes. Rat and human DNA sequence of the recognition site and three flanking nucleotides on each side for the C1A (G3) and B1 (G1) complexes.



Previous data already suggested that a 45-kDa DNA-binding protein might interact with both G1 and G3(5) . This protein was part of a complex that was not, however, islet cell-specific since it was also present in HeLa cells. In addition, this complex was poorly competed with a 500-fold excess of G1 in the gel retardation assay. Furthermore, we do not obtain a protection on G3 with the DNase I footprint analysis using HeLa cell extracts(7) . More detailed characterization of the C1A and C1B complexes is required to resolve these discrepancies.

What are the implications of the binding of C1A to both G3 and G1? C1A is likely to be involved in the islet cell-specific expression of the glucagon gene, probably in concert with other cell-specific factors; its transactivating properties may be modulated by other proteins depending on the context of its binding site, since G1 has low intrinsic activity as compared with G3. With regard to insulin regulation, G1 does not behave as an IRE(4) , suggesting again that the context of the C1A binding site may be crucial for that response. Alternatively, we cannot exclude the possibility that C1B and not C1A mediate the insulin effects on glucagon gene expression; in this hypothesis binding of C1B could be favored by insulin treatment, resulting in a repression of transcription. The fact that there is no close correlation between the binding of B1/C1A to mutant G1 and the degree of insulin-mediated inhibition of glucagon gene expression does not exclude either possibility.

The functional relevance of the B domain of G3 on both basal and insulin-regulated transcriptional activity of the glucagon gene is not evident from our transfection studies. Mutations of the B domain do not significantly impair either of these activities. In the case of the somatostatin and alphaSGH gene, the CCAAT box exerts positive effects on transcription(19, 20) . The CCAAT box of the somatostatin gene promoter is part of a bipartite D cell-specific enhancer; the two functional domains are interdependent, and mutations of either domains result in a more than 80% decrease in transcription. The functional role of the alpha-CBF-like factor of the B domain of G3 appears clearly different. It has been proposed that protein binding to domains A and B may occur independently, and it appears to be mutually exclusive(5) . This was based on three observations: 1) no protein interaction occurs between the A and B domains since no complex is competed for by both an A and a B domain; 2) no complex binds to both domains since no band is sensitive to mutations in the A and B domains; 3) more importantly, the mutant G3M6 (which has an intact B domain) is unable to affect the extent of G3 protection in the DNase I footprint analysis, suggesting that the complex formed over domain A protects the entire G3 sequence, including the B domain, and that the A domain-binding proteins have a higher affinity for G3 or are more abundant than the B domain-binding factors. The possibly mutually exclusive binding of A and B domain proteins to G3 may have relevance in the regulation of the glucagon gene by insulin. Competition between transcription factors for the same binding site may be important for modulating gene expression in response to developmental or environmental changes(29, 30, 31, 32, 33, 34) . A similar mechanism has been proposed for the regulation of the amylase gene by insulin(27) .

Proteins binding to the A domain of G3 have been proposed to also interact with the distal enhancer E1 of the rat insulin I gene (-332 to -291 base pair, relative to the transcriptional start site) and the rat somatostatin gene upstream element (SMS-UE, -118 to -72 base pair)(35) . It is interesting to note that both genes may be regulated negatively by insulin(36, 37) . The implication of C1A and C1B in the regulation of the insulin and somatostatin genes will be examined in future experiments.


FOOTNOTES

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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: 41-22-346-72-37.

(^1)
The abbreviations used are: IRE, insulin-response element; CAT, chloramphenicol acetyltransferase; CRE, cyclic AMP-responsive element; nt, nucleotide(s).


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