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Unité de Diabétologie Clinique, Centre Médical Universitaire, 1211 Genève 4, Switzerland
Address all correspondence and requests for reprints to: Benoit Gauthier, Ph.D., Division de Biochimie Clinique, Centre Médical Universitaire, 1211 Genève 4, Switzerland. E-mail: benoit.gauthier{at}medecine.unige.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-cell-specific element G1. Two proteins interacting with G1 have
previously been identified as Pax6 and Cdx2/3. We identify here the
third yet uncharacterized complex on G1 as hepatocyte nuclear factor 3
(HNF-3)ß, a member of the HNF-3/forkhead transcription family, which
plays an important role in the development of endoderm-related organs.
HNF-3 has been previously demonstrated to interact with the G2 element
and to be crucial for glucagon gene expression; we thus define a second
binding site for this transcription on the glucagon gene promoter. We
demonstrate that both HNF-3 and -ß produced in heterologous cells
can interact with similar affinities to either the G1 or G2 element.
Pax6, which binds to an overlapping site on G1, exhibited a greater
affinity as compared with HNF-3 or -ß. We show that both HNF-3ß
and - can transactivate glucagon gene transcription through the G2
and G1 elements. However, HNF-3 via its transactivating domains
specifically impaired Pax6-mediated transactivation of the glucagon
promoter but had no effect on transactivation by Cdx2/3. We suggest
that HNF-3 may play a dual role on glucagon gene transcription by 1)
inhibiting the transactivation potential of Pax6 on the G1 and G3
elements and 2) direct activation through G1 and G2. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-cells of the endocrine pancreas,
the L cells of the intestine, and certain areas of the brain (2, 3). Previous studies have shown that tissue-specific expression
of the glucagon gene is conferred by four cis-acting
elements (G1, G2, G3, and G4) located within the proximal promoter
region of the gene (4, 5). Elements G2, G3, and G4 confer
islet-specific expression while G1 restricts glucagon gene
transcription to the -cells. Some of the transcription factors
regulating the activity of the glucagon gene have been identified.
Cdx2/3, Brn-4, hepatocyte nuclear factor-3ß (HNF-3ß), Pax2,
NeuroD/Beta2, and E47 interact with the G1, G2, G3, and G4 element,
while Pax6 binds both the G1 and G3 elements (6, 7, 8, 9, 10, 11, 12, 13).
Recently, the heterodimeric Pbx-Prep1 homeodomain protein was also
shown to interact with G3 and with a novel binding element identified
as G5 (14). Optimal and regulated expression of the
glucagon gene in -cells of the pancreas results from the
combinatorial interactions of these factors binding to their cognate
site(s).
Initial studies characterized G1 as a 41-bp element harboring two
A/T-rich sequences that form a nearly perfect repeat and are putative
binding sites for homeodomain-containing DNA binding proteins (4, 15). We further demonstrated that the direct repeat was critical
for -cell-specific expression of the glucagon gene (5).
A third proximal A/T-rich sequence was shown to interact with the
homeobox protein Isl-1, which is expressed in all four principal cell
types of the endocrine pancreas (16). In the presence of
nuclear extracts derived from a glucagon-producing cell line, InR1G9,
at least three protein complexes interact with an oligonucleotide
comprising the A/T-rich direct repeat (G1-56 element). The
paired-homeodomain transcription factor, Pax6, can bind as a monomer to
the distal AT-rich site or form an heterodimer with the caudal
related protein Cdx2/3 that will interact with both AT-rich sites
(6, 8, 9, 10, 12). The third complex forming on the G1
element remains to be characterized.
In this study, the nuclear protein forming the third protein(s) complex
on the distal AT-rich site of G1 is molecularly identified as HNF-3ß,
a member of the HNF-3/forkhead transcription factor family, which plays
an important role in the development of endoderm-related organs
(17, 18, 19, 20, 21, 22). We previously demonstrated that the G2 element
interacts with HNF-3 (23); we now delineate G1 as a second
HNF-3 binding site on the glucagon gene promoter. We demonstrate that
HNF-3 and -ß are able to interact with G2 and the distal AT-rich
site of G1 with similar affinities. However, in InR1G9 cells, HNF-3ß,
as opposed to HNF-3, appears to be the predominant binding activity
detected on the G1 element. Transactivation experiments demonstrate
that HNF-3ß and - can activate glucagon gene transcription through
the G2 and G1 elements. Pax6, which also binds the distal AT-rich site
of G1, exhibits a greater affinity as compared with HNF-3 or -ß.
However, HNF-3 specifically impairs Pax6-mediated transactivation of
the glucagon promoter through G1 and G3 but has no effect on
transactivation by Cdx2/3. We show that this inhibition is conferred by
the two transactivation domains of HNF-3. We suggest that HNF-3 may
play a dual role on glucagon gene transcription by 1) inhibiting the
transactivation potential of Pax6 on the G1 and G3 elements and 2)
direct activation through G1 and G2.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-cell-specific expression of the
glucagon gene. At least three protein complexes can be resolved by EMSA
when an oligonucleotide corresponding to G1 (G1-56) is incubated with
nuclear protein extracts derived from the hamster glucagon-producing
cell line InR1G9 (Fig. 1B
and Ref.
5). The fastest migrating complex, B1, was identified as
the paired homeodomain transcription factor Pax6, while the slowest
migrating complex, B3, was shown to be an heterodimer between Pax6 and
the caudal related factor Cdx2/3 (Fig. 1B). As shown in Fig. 1A, binding of Pax6 to G1 requires the distal A/T-rich region while Cdx2/3
binds to the proximal A/T-rich site of G1 (6, 10, 12).
Several lines of evidence suggested that the third, yet
uncharacterized, complex, B2, which as Pax6 forms on the distal
A/T-rich site of the G1 element belongs to the HNF-3 family of
transcription factors: 1) the G2 element, which binds HNF-3 ß, can
effectively compete for factors interacting with G1 (4);
2) nuclear protein extracts derived from HNF-3expressing but not
from other cell lines produced in EMSA a complex of similar
electrophoretic mobility as B2 in the presence of G1 (5);
and 3) sequence comparison analysis revealed that the distal A/T-rich
region of G1 harbors an inverted DNA-binding site for the family of
HNF-3 transcription factors. This family of nuclear activators consists
of three genes (, ß, and 
) encoding proteins that bind to DNA
via a highly conserved winged helix domain (24). To
determine whether the B2 complex detected in nuclear protein extracts
derived from the glucagon-producing cell line, InR1G9, corresponded to
an HNF-3 family member, we performed EMSA using specific antisera
against each of the members (Fig. 1B). Whereas incubation of binding
reactions with preimmune serum or antibodies raised against HNF-3
and - had little or no effect on the formation of the various
complexes (Fig. 1B, lanes 1, 2, 5 and 6), the addition of HNF-3ß
antibodies completely abolished B2 (Fig. 1B, lane 4). Antibodies
against HNF-3ß also abolished the Pax6/Cdx2/3 heterodimer and altered
the migration pattern of Pax6 (10), indicating a
cross-reactivity of the Ig to Pax6. Complexes containing either Pax6 or
HNF-3ß were recognized by HNF-3ß antisera while Cdx2/3 or HNF-3
were unaffected by the antibody, confirming a nonspecific
cross-reactivity of the antibody to Pax6 (data not shown). We conclude
that B2 represents essentially HNF-3ß. Similar results were obtained
with the mouse -cell line TC-1 (data not shown). Glucagon gene
expression has also been reported in enteroendocrine cells of the
intestine (3). We therefore investigated whether or not
HNF-3 (, ß, or ) binding activity on G1 could be detected using
nuclear protein extracts isolated from the intestinal cell line GLUTag
(25). For this purpose, we used an oligonucleotide
harboring only the distal A/T-rich site of G1 (G1-54) to which B2 but
not Pax6 or the heterodimer Pax6/Cdx2/3 can interact (Fig. 1, A and C,
lane 1). This oligonucleotide produced one predominant complex in the
presence of InR1G9 nuclear protein extract that was mostly eliminated
by HNF-3ß antibodies, while only a slight decrease in band intensity
was observed with the HNF-3 antibody (Fig. 1C, lanes 5 and 6).
Nuclear protein extracts from GLUTag cells generated two retarded
complexes on G1-54 (Fig. 1C, lane 8). The first complex was abolished
when antibodies to HNF-3 were added to the binding reaction (Fig. 1C, lane 9) while the second complex, of weaker intensity, was
supershifted in the presence of HNF-3ß antibodies (Fig. 1C, lane 10).
A similar binding pattern was generated using nuclear protein extracts
from HepG2 that express both HNF-3 and -ß (Fig. 1C, lanes 12–15).
Of note, the mobility of mouse and human HNF-3 and -ß complexes on
G1 was slightly slower as compared with the hamster factors.
Interestingly, the addition of HNF-3 antibodies to InR1G9, GluTag,
or HepG2 nuclear extracts caused the appearance of new faster migrating
complexes that was not observed with G1-56 (Fig. 1C, lanes 5, 9, and
13). Potentially, sequestration of HNF-3 may allow a yet unknown
factor to interact with G1 in the absence of Pax6. Taken together,
these results indicate that HNF-3 and -ß, but not -, can
interact with the G1 element of the glucagon gene promoter. However,
the binding activity of HNF-3ß is favored in pancreatic endocrine
cells while the reverse is observed in enteroendocrine cells. Thus, we
define G1 as a second HNF-3 binding site on the glucagon gene
promoter.
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-cell lines. However, recently HNF-3 was demonstrated
to interact with the G2 element of the glucagon gene promoter, and the
HNF-3 null mutation in mice leads to a marked decrease in glucagon
content and hypoglycemia (26, 27). We therefore determined
whether or not expression levels, compartmentalization of HNF-3 and
-ß proteins, or differences in affinity for the G1 and G2 binding
sites could account for these discrepancies. Western blot analysis
revealed that both factors are expressed and properly localized to the
nucleus in InR1G9 cells (Fig. 2A). The
same results were obtained with nuclear extracts derived from the
hepatoma cell line HepG2, which exhibits HNF-3 and -ß binding
activity to G1 in EMSA. To determine the relative binding activities of
HNF-3 and -ß from normal pancreatic endocrine cells, nuclear
protein extracts were prepared from human islets, and binding assays
were performed with the G2 element. Three complexes were detected, one
of which was recognized by antibodies raised against HNF-3ß, but not
- (Fig. 2B). The remaining complexes have yet to be characterized in
these human pancreatic islet nuclear protein extracts. Of note, the
addition of the HNF-3, but not -ß, antibody resulted in the
formation of a nonspecific low mobility complex in the presence of G2
(Fig. 2B, lanes 2, 4, and 5). To evaluate the relative binding affinity
of HNF-3 and -ß for either G1 or G2, gel shift competition
experiments were performed with nuclear protein extracts derived from
BHK-21 cells overexpressing HNF-3 or -ß. The HNF-3 complex
formed on labeled G1-54 was effectively competed off by a 50-fold molar
excess of cold G1-54 and G2 oligonucleotides, respectively (Fig. 3A, left panel). In the
presence of a labeled G2 element, the HNF-3 complex was partially
competed by a 100-fold molar excess of either G2 or G1-54 (Fig. 3A, right panel). Similar results were obtained in the presence
of HNF-3ß (Fig. 3B), indicating that both HNF-3 and -ß display
very similar binding characteristics to either the G1 or G2 element of
the glucagon gene promoter. Taken together, these results indicate that
HNF-3 is expressed in InR1G9 cells but that HNF-3ß is the
predominant EMSA binding activity on the G1 and G2 elements from both
InR1G9 cells and normal human islets. The fact that the marked decrease
in glucagon content observed in HNF-3 homozygous mutant mice cannot
be corrected by the substitution of HNF-3ß cannot be explained by the
relative affinities of HNF-3 and -ß for the G1 and G2 sites.
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or
ß) and Pax6, we compared the relative binding affinity of the three
factors for this element. Pax6 and HNF-3 ( or ß)-containing
nuclear protein extracts from BHK-21 cells were incubated
simultaneously with labeled G1-56 before the addition of cold
competitor. As shown in Fig. 4, Pax6 was
effectively competed off by a 50- to 100-fold molar excess of unlabeled
G1-56 while a 100-fold molar excess of the same oligonucleotide
slightly competed for HNF-3 or -ß binding. G1 is therefore a
better target site for Pax6 than for either HNF-3 or -ß.
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and -ß for G1. The G1-56,
as well as the proximal (G1-52) and the distal (G1-54) A/T-rich sites
of G1, were used as competitors. As expected, G1-54, but not G1-52,
competed effectively for the binding of HNF-3 and -ß to labeled
G1-56 at a 100-fold molar excess (Fig. 5, A and B). Inversely, Cdx2/3 was completely competed off by a 100- to
500-fold molar excess of unlabeled G1-52, whereas G1-54 appeared to
compete only at higher molar fold excess (500-fold). Unlabeled G1-56
appeared to compete equally well for both HNF-3 and Cdx2/3 (Fig. 5, A
and B). We conclude that the distal A/T-rich region of G1 displays a
greater binding affinity for HNF-3 than for Cdx2/3 while the opposite
is observed for the proximal A/T-rich site.
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and ß), we performed transient transfection studies in the
nonislet cell line BHK-21. HNF-3 increased chloramphenicol
acetyltransferase (CAT) activity of reporter constructs containing
either G1 (G1–31Glu) or G2 (G2–31Glu) in a dose-dependent manner up
to 4-and 5-fold higher than control levels, respectively (Fig. 6A). An additive effect on CAT activity,
as compared with individual sites, was observed when reporter
constructs harboring both elements (G2–138Glu, 16-fold;
-292Glu, 16-fold; and -350Glu, 10-fold) were cotransfected
with increasing amounts of HNF-3. Similar quantitative results
were also obtained for HNF-3ß (Fig. 6B). We then assessed the
consequences of a point mutation (A to C; at position -84) within the
first 350 bp of the glucagon promoter which abrogate HNF-3 binding to
G1 in vitro but leaves binding to G2 intact (G1M4–350Glu)
(Fig. 1A
and Ref. 5). This mutation resulted in an
impaired induction of CAT activity at levels quantitatively similar to
those observed with constructs containing either G1 or G2 (Fig. 6, A
and B). Taken together, these results indicate that both G1 and G2
binding sites are required for maximal activation of the glucagon gene
promoter by HNF-3 ( or ß) in BHK-21 cells and that each element
contributes to half of the full activation.
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and ß) in
the glucagon-producing cell line InR1G9, we cotransfected expression
vectors for either HNF-3 or -ß along with CAT reporter gene
constructs driven by the G1, G2, or both G1 and G2 elements of the
glucagon gene promoter. Overexpression of HNF-3 ( or ß) had no
significant effect on basal activity of the various constructs (Fig. 7, A and B). Interestingly, similar
results have been obtained in InR1G9 cells with Pax6 and Cdx2/3, which
are key transcription factors involved in the regulation of glucagon
gene transcription (12). These results suggest that in
pancreatic -cells, glucagon gene expression is governed by a complex
regulatory mechanism involving multiple proteins that may tether the
effect of individual factors such as HNF-3, Pax6, or Cdx2/3. We
therefore pursued our study in an heterologous system in which the
functional impact of individual components can be assessed.
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and ß) were capable of
functionally interacting on the G1 element, combinations of HNF-3 or
-ß and Cdx2/3 expression vectors were cotransfected along with the
reporter construct G1–31Glu; no significant increase in CAT activity,
as compared with Cdx2/3 alone, was observed in BHK-21 cells (Fig. 8A). Higher amounts of HNF-3 or -ß
(up to 2 µg) produced similar results (data not shown). We conclude
that HNF-3 ( and ß) and Cdx2/3 do not have additive or synergistic
activity on glucagon gene transcription. To assess the functional
relevance of HNF-3 ( and ß) and Pax6 binding to the same A/T-rich
site of the G1 element, BHK-21 cells were cotransfected with fixed
amounts of either G1–31Glu or -138Glu and Pax6 expression vector along
with increasing amounts of either HNF-3 or -ß. As previously
reported, transfection of 0.25 µg of Pax6 induced a 15- to 30-fold
increase in CAT activity as compared with the control sample (Fig. 8B
and Ref. 12). The addition of 30 ng of either HNF-3 or
-ß to transfectants containing 0.25 µg of Pax6 resulted in a
drastic decrease in Pax6-induced activity (Fig. 8B). These data suggest
that even though Pax6 displays a better affinity for G1 (Fig. 4),
HNF-3 and -ß can hinder Pax6-mediated transactivation through the
G1 element of the glucagon promoter. Similar results were also obtained
with the reporter construct G2–138Glu, which harbors both HNF-3
binding sites (Fig. 8B). However, the presence of G2 resulted in a
small increase in the CAT activity of G2–138Glu at higher amounts of
HNF-3 or -ß (0.5 µg), indicating that these transcription
factors have the potential to increase glucagon gene expression, albeit
to lower levels, once they have suppressed Pax6 transactivation.
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and ß) interaction with the G1
element is necessary for the inhibition of Pax6-mediated activation, we
conducted cotransfection experiments with a reporter construct
harboring the G3 element (G3–31Glu), which binds Pax6 but not HNF-3.
Cotransfection with Pax6 resulted in a 14-fold induction in CAT
activity of the reporter construct, which was inhibited by the presence
of either HNF-3 or -ß (Fig. 9A).
Similar results were obtained with the mutant reporter construct
G1M4–350Glu (Fig. 9B). In an attempt to investigate a possible
mechanism for HNF-3-mediated repression of Pax6 transactivation, we
tested for direct protein-protein interaction between these two
transcription factors (Fig. 9C). In a
glutathione-S-transferase (GST) pull-down experiment,
HNF-3 and -ß were found to bind efficiently to
glutathione-Sepharose beads containing GST-Pax6 but not GST alone (Fig. 8B). We thus suggest that HNF-3 ( or ß) can directly interact with
Pax6 and attenuate its transactivation potential on the glucagon gene
promoter.
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) (28). Although a
significant decrease in CAT activity was observed at 0.5 µg of
DN-HNF-3ß, the truncated protein was unable to effectively inhibit
Pax6 transactivation of the -138Glu reporter construct as compared
with the wild-type HNF-3ß for similar amounts of transfected cDNA
(compare Fig. 8B to Fig. 10 B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-cells. We show that two members of the
HNF-3 family, HNF-3 and -ß, can interact with similar affinities
to either the G1 or G2 element. These two sites contain an identical
core sequence (5'-GTAAATAA-3'), albeit on opposite strands, which is
reminiscent of the HNF-3 consensus DNA binding element (WTRTTKRYTY,
where W = A or T; K = G or T; Y = pyrimidine; and r
= purine) (29). However, it appears that HNF-3ß, as
opposed to HNF-3, is the predominant binding activity detected on
both elements in the glucagon-producing cell lines InR1G9 and -TC-1
and in normal human islets, even though both factors are expressed in
these cells. Two independent studies have recently demonstrated that,
in HNF-3-deficient mice, glucagon-producing pancreatic -cells
developed normally, but glucagon mRNA steady state levels were reduced
by 50–70%, implying a direct role of HNF-3 in the regulation of
this gene, which cannot be substituted by HNF-3ß (26, 27). Discrepancies observed between our results and transgenic
animals indicate that although HNF-3 and -ß may be concomitantly
expressed in similar cells and share identical consensus DNA binding
sites, their binding activities may be regulated by cellular
constraints in vivo. Such constraints may be imposed by
chromatin, which is intimately related to the expression of eukaryotic
genes in vivo. HNF-3 is of particular interest as it has
been shown to induce chromatin modifications on several genes such as
the albumin, ER, and the -fetoprotein gene (30, 31, 32, 33). A
similar situation could occur for the glucagon gene in which HNF-3
may act directly or indirectly as a cellular determinant that
establishes a promoter environment favorable for transcriptional
activation by HNF-3ß or other critical transcription factors. In mice
lacking HNF-3, chromatin restructuring may be less efficient through
HNF-3ß, thus resulting in lower levels of glucagon gene expression.
Alternatively, HNF-3ß, which plays a determinant function during
embryonic endoderm development, may be down-regulated and replaced by
HNF-3 in glucagon-producing cells after terminal differentiation in
mice, as recently suggested by Kaestner and co-workers for liver gene
expression (34). In contrast, HNF-3ß may be reactivated
in immortalized cell lines becoming the predominant HNF-3 activity as
observed in InR1G9 and -TC-1 cells. The analysis of the functional
impact of HNF-3ß on glucagon gene expression in vivo
awaits new experimental strategies since homozygous mice bearing a
targeted null mutation for this transcription factor die early in
embryogenesis before the differentiation of pancreatic endoderm
(17, 18, 21).
The proximal promoter element G1 is a well conserved regulatory element
that confers -cell-specific expression of the glucagon gene in the
pancreas. Characterization of HNF-3 as the last major complex forming
on G1 in InR1G9 cells will now permit a detailed analysis of the
molecular mechanism governing cell-specific expression of glucagon in
endocrine cells. Interestingly, neither HNF-3, Pax-6, nor Cdx2/3 is
found exclusively in pancreatic endocrine cells; rather, they are all
expressed at varying degrees in different tissues. Hussain and
co-workers (35) have previously demonstrated that Brn-4,
which is predominantly expressed in neuronal cells and -cells of the
endocrine pancreas, could interact with the distal G1 element and
potentially confer -cell-specific expression of the glucagon gene.
The respective roles of Brn-4, Pax6, Cdx2/3, and HNF-3 in the
cell-specific expression of the glucagon gene thus remain to be
established.
HNF-3 and -ß were equally capable of activating the glucagon gene
promoter in a heterologous assay system, confirming that there are no
differences in the ability of these factors to bind the G1 or G2
element. Both elements appear of similar functional importance, and
each contributes to half of the full activation of the glucagon
promoter in BHK-21 cells. However, in pancreatic -cells, G1 by
itself confers only weak transcriptional activation and is dependent on
the upstream enhancer element G2 (or G3) for high levels of expression.
Conversely, G1 is required for G2 to enhance transcription indicating a
potential interaction between these two sites (4).
According to a current view of enhancer function, specific interactions
between enhancer-binding proteins and factors that bind proximal
promoter elements are important to achieve enhancer-promoter
selectivity (36). HNF-3 may thus function as an accessory
factor between the G1 and G2 elements to orchestrate enhanced
transcription of the glucagon gene promoter in -cells (37, 39). Interestingly, HNF-3-mediated induction of glucagon gene
expression through the G1 element was much lower than that observed
with either Pax6 or Cdx2/3. This binding site, which is located
adjacent to the TATA box and thus in close proximity to the basal
transcription machinery, may stimulate transcription of the glucagon
gene by recruiting components of the basal transcription complex via
HNF-3, Pax6, and Cdx2/3. However, it has been proposed that
heterologous cells, such as BHK-21, are deprived of coactivator
proteins that are required to interact with HNF-3 activation domains
and allow proper stimulation of the basal transcription complex
(40). Lower levels of glucagon gene promoter expression
and the lack of synergism between G1 and G2 in the presence of HNF-3
may be partly explained by the absence of these coactivators in BHK-21
cells.
The concept that HNF-3 suppresses Pax6-mediated activation of the
glucagon gene through the G1 and G3 elements of the promoter by
physical protein interaction, rather than by competition for a common
binding site, defines a novel function for this transcription factor.
Pdx1 as been ascribed to physically interact with HNF-3 to up-regulate
its own gene transcription. However, this interaction was shown to be
dependent on DNA binding at two different promoter elements
(41). Consistent with this inhibitory effect, we have
previously demonstrated that a mutation abrogating binding of HNF-3,
but not of Pax6, to G1 resulted in an increase in glucagon gene
transcription in the pancreatic -cell line InR1G9 (11).
Interestingly, a recent study concluded that repression of glucagon
gene transcription by insulin implicated the transcription factor Pax6
and a complex interaction between the proximal promoter elements G1 and
G4 and the more distal enhancer-like elements G2 and G3
(42). It may thus be possible that the insulin-induced
pathway utilizes HNF-3 as target protein to modulate Pax6 activity on
glucagon gene transcription. In this regard, coincidence of insulin
target sequences with HNF-3 binding sites has been reported for several
genes such as PECK, IGF binding protein, tyrosine aminotransferase, and
cholesterol 7-hydroxylase (43, 44, 45, 46). We have delineated
the two transactivating domains of HNF-3 as the regions involved in
conferring Pax6-mediated transactivation of the glucagon gene.
Potentially, these regions may interact with either the paired or homeo
DNA-binding domain of Pax6 to destabilize the interaction of this
protein to the G1 or G3 element. GST pull-down experiments, performed
in the absence of DNA, suggest that HNF-3-Pax6 protein interactions are
probably DNA independent. Furthermore, heterodimers between Pax6 and
HNF-3 were never observed in EMSA on either G1 or G3.
Full expression of the glucagon gene in pancreatic -cells is
dictated by the combinatorial effect of various transcription factors
assembling on different cis-acting elements. We demonstrate
that HNF-3 and -ß bind independently to at least two DNA control
elements, G1 and G2, and transactivate glucagon gene expression.
Furthermore, HNF-3 ( and ß) physically interacts with Pax6 to
down-regulate glucagon gene transcription. HNF-3 or -ß may mediate
-cell- specific expression by permitting access of both Pax6 and
Cdx2/3 to a chromatin-free G1 element while conferring optimal
expression by interacting with the enhancer-like G2 element. The
significance of HNF-3 functional dichotomy on glucagon gene regulation
remains to be further defined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and -ß (kindly provided by Robert Costa,
University of Illinois, Chicago, IL) were subcloned into the
EcoRI site of pSG5 (Stratagene, Amsterdam, The
Netherlands). Expression vectors harboring the hamster
Cdx-2/3 and quail pax6 cDNAs were obtained from
Michael S. German (University of California, San Francisco, CA) and
Simon Saule (Institut Curie, Orsay Cedex, France) respectively.
pEBOTd harboring the cDNA of DN-HNF-3ß was kindly provided by Axel
Kahn (INSERM, Paris, France). Plasmid G2–31GluCAT was constructed by
ligation of a double strand oligonucleotide corresponding to the G2
cis-acting element of the glucagon gene promoter
(5'-GATCCAGGCACAAGAGTAAATAAAAAG- TTTCCGGGCCTCTGC-3') (11)
into the -31GLUCAT vector (47), which had been cut with
BamHI and blunt ended using the Klenow fragment of DNA
polymerase I (Roche Diagnostics, Rotkreuz,
Switzerland).
Cell Culturing
The Syrian baby hamster kidney BHK-21, the human hepatoma HepG2,
the enteroendocrine GLUTag, and the glucagon-producing hamster InR1G9
(48) cell lines were grown and maintained in RPMI 1640
(Seromed, Basel, Switzerland) supplemented with 5% FCS, 5% newborn
calf serum (Life Technologies, Inc.; Basel, Switzerland),
100 U/ml penicillin (Seromed), 100 µg/ml streptomycin (Seromed), and
2 mM glutamine (Life Technologies, Inc.;
Basel, Switzerland). Human pancreatic islets were isolated from whole
pancreata obtained from multiorgan cadaveric donors (20–65 yr) as
described previously (49).
Transient Transfection and CAT Assay
The BHK-21 cell line was transiently transfected using the
calcium phosphate precipitation technique (50). Each 10-cm
Petri dish received a precipitate containing 10 µg of cat
gene reporter construct, 0.5 µg of pSV2PAP
(internal control), and variable amounts of expression vectors for the
various transcription factors (all cloned into pSG5). The final amount
of DNA in each transfection was maintained constant by adding the
expression vector pSG5 without an insert. Cells were harvested 48
h after transfection in 250 mM Tris-HCl and
disrupted by three consecutive freeze-thaw cycles. CAT and alkaline
phosphatase (PAP) activities were determined as previously
described (5). PAP activity was used to standardize for
transfection efficiency. The CAT/PAP activity values presented for each
set of experiments correspond to the mean and SD
of at least three individual transfections performed in duplicate. The
values calculated were normalized as fold induction of the control
sample obtained from cells transfected with the empty pSG5 expression
vector. InR1G9 cells were transfected in suspension by the
diethylaminoethyl-dextran method as described previously
(4).
Nuclear protein extracts enriched for Pax6, Cdx2/3, and HNF-3 and
-ß were obtained by transfecting BHK-21 cells with 10 µg of various
transcription factor cDNAs. Cells were harvested 48 h after
transfection, and nuclear protein extracts were prepared as described
by Schreiber et al. (51).
EMSA
Oligonucleotides used in EMSA are described in Fig. 1A.
Double-strand forms were radioactively labeled by filling in the ends
using the Klenow fragment of DNA polymerase I in the presence of
[32P]-dCTP and purified using the QIAquick
nucleotide removal kit (QIAGEN AG, Basel, Switzerland).
DNA binding assays were performed as described previously
(52) using nuclear extracts prepared by the method of
Schreiber et al. (51).
Western Blot Assay for HNF-3 Proteins
Cytoplasmic and nuclear fractions were isolated from InR1G9 and
HepG2 cells according to the protocol of Schreiber et al.
(51). Approximately 25 µg of each protein extract were
resolved on a 10% SDS-polyacrylamide gel and transferred
electrophoretically to polyvinylidene difluoride membranes.
Immunoblotting was performed with polyclonal antibodies to HNF-3 and
-ß (1:5,000) (R. H. Costa, University of Illinois, Chicago, IL)
and TFIIE- (1:1,000) (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) and goat antirabbit IgG antisera conjugated with
horseradish peroxidase (1:7,500) (Amersham Pharmacia Biotech, Lagerstrasse, Switzerland). Immunoreactive products
were detected on x-ray films using enhanced chemiluminescence
(SuperSignal West Pico), as directed by the manufacturer (Pierce Chemical Co., Rockford, IL).
GST Pull-Down Assay
L-[35S]methionine-labeled
HNF-3 and -ß polypeptides were produced using the TNT rabbit
reticulocyte lysate-coupled transcription-translation system
(Promega Corp., Madison, WI) according to the
manufacturer’s protocol. The labeled proteins were incubated with
either GST or GST-Pax6, and the binding reactions were treated as
outlined by Ritz-Laser et al. (12).
Statistical Analysis
Results are expressed as mean ± SE. Where
indicated, the statistical significance of the differences between
groups was estimated by t test. * and ** indicate
statistical significance with P < 0.05 and
P < 0.01, respectively.
|
ACKNOWLEDGMENTS |
|---|
and
-ß. |
FOOTNOTES |
|---|
Abbreviations: CAT, Chloramphenicol acetyltransferase; DN-HNF-3ß, dominant negative form of HNF-3ß; GST, glutathione-S-transferase; HNF, hepatocyte nuclear factor; PAP, alkaline phosphatase.
Received for publication July 19, 2001. Accepted for publication September 7, 2001.
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