(Received for publication, June 29, 1994; and in revised form, September 22, 1994)
From the
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, 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.
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) ()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, 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.
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,
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.
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.
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.
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),
(C1B),
(C2),
and
(C3).
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).
,
, 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.
,
, 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, pSVApap, 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.
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,
TC1) and cells producing either insulin (lane 3,
TC1) 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.
,
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.
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,
pSVApap. Insulin (10
M) 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.
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 SGH 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
-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.