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From the
Received for publication, February 15, 2002, and in revised form, May 1, 2002
Maturity onset diabetes of the young, subtype 1 (MODY1), is associated with defective glucose-dependent
insulin secretion from pancreatic beta cells. MODY1 is caused by
mutation in the transcription factor hepatocyte nuclear factor 4 In adult mammals, the insulin gene is expressed
exclusively in the beta cells of the endocrine pancreas (1-3). This
selectivity is controlled primarily at the transcriptional level
through well defined promoter elements (4-6). Although a number of
transcription factors have been defined that interact with the insulin
promoter and activate it in synergistic fashion (7-10), the action of
these factors alone is not sufficient to explain the remarkable degree of cell specificity observed in vivo (9). Most likely,
additional transcription factors participate in achieving specificity.
MODY1 is a monogenic form of
diabetes characterized by impaired glucose-dependent
insulin secretion in the absence of insulin resistance in peripheral
tissues (11, 12). This indicates that a beta cell defect is the primary
cause of MODY and implies that MODY genes have a role in insulin
production and/or secretion. Consistent with this is the fact that four
of the five known MODY subtypes are caused by mutations in distinct
transcription factors expressed in pancreatic beta cells (13). The
striking MODY phenotype caused by such single gene disorders provides
clear evidence of the important role of each of these transcription
factors in beta cell function.
MODY1 is caused by mutation in HNF4 MODY3 is caused by mutation in the HNF1 Recently, however, strong evidence has been obtained indicating that
this hypothesis may be an over-simplification and that the actual
relationship between HNF4 In this study, we have examined this idea through a detailed analysis
of the insulin gene promoter. In addition to confirming that HNF4 Cell Culture
293T, HeLa S3 Tet-Off (CLONTECH PT3001-1),
and COS7 cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, penicillin (200 IU/ml), and
streptomycin (100 µg/ml). INS-1 DN-HNF4 Plasmids
Reporter Plasmids--
pGL3.Synt.LUC, a plasmid containing the
luciferase (LUC) gene controlled by the rat insulin I gene promoter,
was generated by replacement of the SV40 promoter of pGL3-promoter
vector (Promega) with the insulin promoter fragment ( Expression Plasmids--
pSG5.HNF4 Transfection
Transfections of 293T, HeLa, and COS7 cells were carried out by
the calcium phosphate coprecipitation technique (32). 4-7 h after
addition of DNA, cells were exposed to 20% glycerol in Dulbecco's
modified Eagle's medium for 2 min. Cells were harvested 40-48 h after
transfection, and cell extracts were assayed for reporter enzyme
activity. INS-1 DN-HNF4 Reporter Gene Assay
Following transfection, cell extracts were prepared by 3 cycles
of freeze-thawing, followed by centrifugation to remove cell debris.
The firefly and Renilla luciferase assays were carried out
as follows: extracts containing 5-50 µg (2-5 µl) of protein were
added to 50 µl of firefly luciferase assay buffer (Promega E1501) or
100 µl of Renilla luciferase assay buffer (33). The samples were placed in a luminometer (LUMAC Biocounter M2500), and
light output was determined over a 10-s interval. Statistical significance was evaluated by t test using InStat software.
Oligonucleotide Probes
Oligonucleotides were synthesized corresponding to the
HNF4 Electrophoretic Mobility Shift Assay (EMSA)
Cells were transfected with 10 µg of expression plasmids as
described above. Whole cell extracts (10-15 µg of protein) were incubated for 10 min on ice in binding buffer (10 mM HEPES,
pH 7.8, 50 mM KCl, 10% glycerol, 1 mM
dithiothreitol, 2.5 mM MgCl2) containing 600 ng
of poly(dI·dC) and 600 ng of poly(dA·dT) (Sigma) in a final assay
volume of 14 µl. Subsequently, 1 µl of 32P-labeled
probe was added (~10 fmol), and incubation was allowed to continue on
ice for an additional 25 min. Samples were subsequently resolved on 6%
polyacrylamide gels (37.5:1 acrylamide/bisacrylamide) in 45 mM Tris borate, 1 mM EDTA (0.5× TBE) at 184 V
for 2 h at 4 °C. The gel was dried, and the labeled DNA-protein
complexes were visualized by autoradiography. In supershift
experiments, 1 µl of anti-HNF4 HNF4 Identification of the Region in the Insulin Gene Promoter Important
for HNF4 HNF4 HNF4
To investigate further this augmented activation generated by HNF4 Mechanism of Action of HNF4
To assess the significance of the HNF4
To confirm the role of HNF4 Identification of HNF1 In this report, we have examined the above hypothesis. Expression of
DN-HNF4 These results are consistent with recent reports (29, 30) indicating
that the epistatic relationship between HNF4 We thank D. Gerber for assistance with
plasmid construction and Dr. R. Dikstein for gifts of plasmids.
*
This work was supported in part by grants from the Roy and
Ellen Rosenthal Family Foundation, the Israel Academy of Sciences and
Humanities (to M. W.), and Swiss National Science Foundation Grant
32-49755.96 (to C. W.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, May 6, 2002, DOI 10.1074/jbc.M201582200
The abbreviations used are:
MODY, maturity-onset
diabetes of the young;
HNF4
Activation of the Insulin Gene Promoter through a Direct Effect
of Hepatocyte Nuclear Factor 4
*
,
Department of Biological Chemistry, Weizmann
Institute of Science, Rehovot 76100, Israel, the
§ Department of Human Nutrition and Metabolism, Faculty of
Medicine, Hebrew University, Jerusalem 91120, Israel, and the
¶ Division of Clinical Biochemistry, Department of Internal
Medicine, University Medical Center,
CH-1211 Geneva 4, Switzerland
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(HNF4). To understand better the MODY1 phenotype, we tested
whether HNF4 was able to modulate directly the insulin gene
promoter. Transfection of cultured 293T cells with an HNF4
expression vector led to 10-fold activation of a cotransfected reporter
plasmid containing the rat insulin I gene promoter. Computer analysis
revealed a potential HNF4-binding site between nucleotides 
57 and
69 of the promoter; mutation of this sequence led to reduced ability of HNF4 to activate the promoter. The ability of HNF4 to bind this sequence was confirmed using gel shift analysis. In transfected INS-1 beta cells, mutation of either the HNF1 site or the HNF4 site in the insulin gene promoter led to 50-75% reduction in reporter gene activity; expression of dominant negative HNF4 led to
significant reduction in the activity of wild type and both mutated
promoters. Thus, in addition to the previously described indirect
action of HNF4 on insulin gene expression mediated through elevated HNF1 levels, HNF4 also activates the insulin gene directly, through a previously unrecognized cis element.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
, a transcription factor of the
nuclear receptor subfamily (14). At least eight different mutations
have been identified in MODY1 patients (15). Four mutations result in
truncated HNF4 proteins, of which two are nonsense mutations (14,
16), and two are frameshift mutations (17, 18). The other four
mutations are missense mutations that affect various domains of the
HNF4 protein (19-22).
gene encoding a
transcription factor of the homeodomain family (23). Like HNF4, HNF1 is expressed in liver and beta cells. In fact, HNF1
was shown to be a direct transcriptional target of HNF4 in
liver (24). Furthermore, a mutation in the HNF4-binding site in the HNF1 gene promoter produces a MODY phenotype (25).
Consistent with these observations, MODY1 and MODY3 show a similar
clinical phenotype. Recent studies (26) have shown that induction of dominant negative (DN) HNF1 in beta cells leads to alterations in
expression of a number of beta cell genes including the insulin gene.
Because DN-HNF4 led to reduced activity of the Hnf1
promoter and reduced insulin mRNA levels (27), it was hypothesized
that HNF4 increases insulin mRNA levels indirectly by increasing
HNF1 levels, which in turn activate the promoter through binding to an HNF1 consensus sequence (28).
and HNF1 may be more complex; it has
been shown (29, 30) that P2, the major promoter of the
Hnf4 gene in beta cells, is regulated by HNF1.
Therefore, activation of beta cell genes by HNF4 may involve more
than a simple transcriptional regulatory cascade mediated through
HNF1, as previously envisaged.
activates insulin gene expression in an indirect fashion, through an
HNF1-binding site, we have identified a novel cis element in the
insulin gene promoter, which mediates direct activation of the promoter
by HNF4 in beta cells.
EXPERIMENTAL PROCEDURES
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DISCUSSION
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26 cells (27) were
cultured in RPMI 1640 medium containing 10% heat-inactivated fetal
calf serum, 25 mM HEPES, 2 mM
L-glutamine, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, penicillin (200 IU/ml), and
streptomycin (100 µg/ml) supplemented with 150 µg/ml G418 and 100 µg/ml hygromycin.
410 to 1).
pGL3.SV40.LUC is the plasmid pGL3-promoter vector (Promega), containing
the luciferase gene controlled by the SV40 early promoter. RSV.LUC contains the luciferase gene controlled by the RSV promoter (31). 
-Actin.LUC contains the luciferase gene controlled by -actin promoter. CMV.TK.LUC contains the luciferase gene controlled by CMV
enhancer and herpes simplex virus-thymidine kinase (TK) promoter. The
reporter plasmids Synt 5 LUC, Synt 6 LUC, Synt 7 LUC, and Synt 8 LUC
contain the luciferase gene controlled by Synt 5-8 block replacement
mutants of the rat insulin I promoter (5). pGL3.
HNF4Synt.LUC was
generated by PCR-mediated site-directed mutagenesis of pGL3.Synt.LUC.
The nucleotides mutated (57 to 69) were ACGGCAAAGTCCA to
CATTACCCTGAAC. pGL3.HNF1Synt.LUC was generated by subcloning of
the mutated insulin promoter from 410MFInsLUC to pGL3-promoter vector
(Promega). The nucleotides mutated (222 to 218) were TTAAT to
GTCCG. The Renilla luciferase internal control plasmid
pRL.RSV contains the Renilla luciferase coding sequence from
pRL null vector (Promega) controlled by the RSV promoter.
encodes rat HNF4
controlled by the SV40 early promoter. pcDNA3.HNF4 encodes rat
HNF4 controlled by the CMV promoter. pCMV.PDX1 is a PDX1 expression
vector controlled by the CMV promoter. pCMV.E2A is a mouse E2A (E47)
expression vector (9) controlled by the CMV promoter. pCGN.HA.BETA is a
BETA2 expression vector encoding hemagglutinin epitope-tagged BETA2
controlled by the CMV promoter (9).
26 cells (27) were transfected using
electroporation as follows. Cells were suspended in 450 µl of cold
phosphate-buffered saline (calcium/magnesium-free), and plasmid
DNA dissolved in 50 µl of H2O was added. Cells were incubated on ice for 10 min, then transferred to 4-mm gap cuvettes (BTX), and pulsed for 6 ms at 320 V/cm, 975 microfarads using an ECM
830 pulse generator. For induction of DN-HNF4, cells were incubated
with 1 µg/ml doxycycline for 24 h prior to transfection.
binding element (HBE) region
(5'-gatccGCCCTTAATGGGCCAAACGGCAAAGTCCAGGGa-3') from the rat insulin I
promoter (5) (positions 86 to 53) and C3P region
(5'-agctGCAGGTGACCTTTGACCAGCTc-3') from the rat ApoCIII
promoter (34). Radioactive probes were generated by incubation of the
appropriate annealed oligonucleotides (5 pmol) in the presence of 50 µCi of [-32P]dATP (3,000 Ci/mmol), 0.25 mM dCTP, dGTP, and dTTP, and 5 units of DNA polymerase I
(New England Biolabs, Klenow fragment) for 40 min at room temperature.
The radiolabeled oligonucleotides were separated from free nucleotide
by spin dialysis using Sephadex G-50. Specific activity obtained was
typically ~2,000 cpm/fmol.
antiserum or preimmune serum was
added after the first 10 min of incubation, and the mixture was
incubated for an additional 10 min before addition of the
32P-labeled probe.
RESULTS
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ABSTRACT
INTRODUCTION
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DISCUSSION
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Can Activate the Insulin Gene Promoter--
Because
HNF4 was previously shown to affect insulin mRNA levels in beta
cells (27), its involvement in the direct transcriptional regulation of
the insulin gene was examined. 293T cells were transfected with a
reporter plasmid encoding the luciferase gene controlled by the rat
insulin I gene promoter (410 to 1) in the presence or absence of an
HNF4 expression plasmid. For comparison, cells were also transfected
with luciferase reporter plasmids controlled by no promoter or by other
promoters as follows: RSV, -actin, CMV.TK, and SV40 in the presence
or absence of HNF4 expression plasmid. HNF4 activated
transcription of the insulin gene promoter 9.5-fold (Fig.
1) but did not activate the other
promoters tested, indicating that this activation is specific. Similar
results were obtained upon transfection of HeLa cells (not shown).
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Fig. 1.
Activation of the insulin gene promoter by
HNF4 protein. 293T cells were transfected
with 1 µg of luciferase reporter plasmid containing either no
promoter (pGL3.basic), the rat insulin I gene promoter (pGL3.Synt), or
one of the following promoters: SV40, RSV, -actin, or CMV.TK in the
presence of 1 µg of HNF4 expression plasmid (pcDNA3.HNF4)
or empty expression plasmid control. Results are expressed as the ratio
of luciferase activity observed in the presence as compared to the
absence of HNF4. Each data point represents the mean ± S.E. of
at least three independent transfection experiments for each reporter
plasmid. The asterisk indicates statistical significance
(p < 0.0001) in the presence compared to the absence
of HNF4.
Activation--
In order to characterize further this
HNF4-dependent activation, the insulin gene promoter was
scanned for the presence of an HNF4 consensus binding sequence using
TESS software (www.cbil.upenn.edu/tess). Such a consensus site was
found between nucleotides 57 and 69 (Fig.
2), suggesting the presence of an
HNF4-binding site in the promoter of the insulin gene. To determine
whether this putative HNF4-binding site is necessary for HNF4
activation of the insulin gene promoter, we examined the ability of
HNF4 to activate the insulin gene promoter mutated at this site.
Four block replacement mutants of the insulin promoter, spanning the
consensus binding sequence and designated Synt 5-8, were used (5)
(Fig. 2). As before, 293T cells were transfected with reporter plasmids
encoding the luciferase gene controlled by the wild type rat insulin I promoter or by one of its mutated variants, Synt 5-8, in the presence or absence of HNF4 expression plasmid. HNF4 activation of mutants Synt 5 and Synt 8, in which mutations are located outside the putative
HNF4-binding site, was not reduced compared with wild type promoter
activation. In contrast, mutation Synt 6 and Synt 7, each abolishing a
part of the HNF4 consensus site, showed significantly attenuated
activation (Fig. 3). These results
indicate that the nucleotides mutated in mutants Synt 6 and Synt 7 are important for HNF4 activation of the insulin gene promoter.
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Fig. 2.
A consensus binding site for
HNF4 in the insulin gene promoter. An
HNF4 consensus site (boldface letters) is located between
nucleotides 57 and 69 and adjacent to PDX1-binding site
(boldface letters). Block replacement mutants of the insulin
promoter, designated Synt 5-8 (5), were generated by mutation of all
nucleotides of the relevant portion of the wild type sequence as
follows: A 
C and G T. The location of the HBE probe is also
shown.
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Fig. 3.
Block replacement mutations in the
HNF4 consensus binding site in the insulin
gene promoter reduce HNF4 activation of the
promoter. 293T cells were transfected with 1 µg of luciferase
(LUC) reporter plasmid containing the wild type rat insulin
I promoter or each of the block replacement mutants Synt 5-8 in the
presence of 1 µg of HNF4 expression plasmid (pcDNA3.HNF4)
or empty expression plasmid control. The nucleotides mutated in Synt
5-8 are 43 to 53, 54 to 63, 64 to 68, and 79 to 88,
respectively (5). Results are expressed as the ratio of luciferase
activity observed in the presence as compared to the absence of
HNF4. Each data point represents the mean ± S.E. of at least
three independent transfection experiments for each reporter plasmid.
Asterisks indicate statistical significance
(p = 0.024 for Synt 6 and p = 0.0006 for Synt 7) compared with the values obtained for wild type
promoter.
Can Bind the Insulin Gene Promoter--
We next tested the
ability of HNF4 to bind to the putative binding sequence in the
insulin gene promoter. An EMSA was performed using a probe containing
the putative HNF4-binding site, designated the HBE element (Fig. 2).
Whole cell extracts were made from HeLa cells transfected with HNF4
expression plasmid and from untransfected HeLa cells. Incubation of
transfected extracts with the radiolabeled HBE probe resulted in a
strong band that was absent when extracts from mock-transfected cells
were used (Fig. 4A,
lanes 1 and 2). Competition with unlabeled HBE
oligonucleotide abolished this band (lane 3). Addition of
anti-HNF4 serum generated a supershifted band that was not observed
with preimmune serum, confirming the presence of HNF4 in the complex
(lanes 4 and 5). Apolipoprotein C III
(ApoCIII) is a well characterized target gene of HNF4
(35, 36). Competition with unlabeled oligonucleotide containing the C3P
element, an HNF4-binding site in the ApoCIII gene
promoter (37), also abolished the observed band (lane 6).
Competition with increasing amounts of unlabeled HBE and C3P
probes indicates that HNF4 affinity to its binding site in
ApoCIII gene is higher than its affinity to the binding site
in the insulin gene (Fig. 4B).
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Fig. 4.
HNF4 can bind the
insulin gene promoter, as demonstrated in EMSA. A,
whole cell extracts from mock-transfected HeLa cells (lane
1) or HeLa cells transfected with HNF4 expression vector
(lanes 2-6) were incubated with 32P-labeled HBE
probe and separated on a non-denaturing polyacrylamide gel. Specificity
of binding was analyzed by adding 100- or 60-fold molar excess of
unlabeled HBE probe (lane 3) or C3P probe (lane
6), respectively. The formation of an immune complex is shown with
the addition of anti-HNF4 serum (lane 4) and compared
with the addition of preimmune serum (lane 5). B,
affinity of HNF4 to HBE probe is lower than its affinity to C3P
probe. Whole cell extracts made from untransfected COS7 cells
(lane 1) or COS7 cells transfected with HNF4 expression
vector (lanes 2-10) were incubated with
32P-labeled HBE probe and separated on a non-denaturing
polyacrylamide gel. Competition was performed using 10-, 20-, 50-, and
100-fold molar excess of unlabeled HBE oligonucleotide (lanes
3-6, respectively) or 3-, 10-, 20-, and 50-fold molar
excess of unlabeled C3P oligonucleotide (lanes 7-10,
respectively).
and Other Insulin Gene Transcription Factors--
E2A
(38-40), BETA2 (NeuroD) (41, 42), and PDX1 (also known as IPF1, STF1,
and IDX1) (43-45) are well characterized insulin gene transcription
factors that can synergistically activate the insulin gene promoter
when cotransfected to HeLa cells with a reporter gene controlled by the
promoter; however, when the insulin promoter construct is transfected
to beta cells, it is ~4 times more active (9). This implies that
additional factor(s), present in beta cells, may be important for
insulin gene regulation. In light of our data showing that HNF4 can
bind and activate the insulin gene promoter, its possible cooperation
with the other three known insulin gene transcription factors was
examined. 293T cells were transfected with a luciferase reporter
plasmid controlled by the rat insulin I gene promoter and with
expression plasmids encoding the four transcription factors E2A, BETA2,
PDX1, and HNF4. Expression of HNF4 with E2A, BETA2, and PDX1
produced a synergistic activation of the insulin gene promoter that was ~2-fold higher than the activation generated by the three factors alone (Fig. 5A). Similar
results were obtained upon transfecting HeLa cells (not shown).
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Fig. 5.
A, HNF4 enhances the insulin
gene promoter activation generated by E2A, BETA2, and PDX1. 293T cells
were cotransfected with 1 µg of luciferase reporter plasmid
containing the rat insulin I promoter in the presence or absence of 0.1 µg of expression vectors encoding HNF4, E2A, BETA2, and PDX1 as
indicated. Results are expressed relative to the activity observed in
the presence of E2A + BETA2 + PDX1. Each bar represents the
mean ± S.E. of 8 independent experiments. The asterisk
indicates statistical significance (p = 0.005) compared
with the value obtained upon transfection with E2A + BETA2 + PDX1.
B, HNF4 enhances PDX1 activation of the promoter in
a synergistic manner. 293T cells were cotransfected with 1 µg of
luciferase reporter plasmid containing the rat insulin I gene promoter
in the presence or absence of 0.1 µg of expression plasmids encoding
the indicated transcription factors. Results are expressed relative to
the activity observed in the presence of HNF4 + PDX1. Each data
point represents the mean ± S.E. of 8 independent transfection
experiments. Asterisks indicate statistical significance
(p < 0.0001 for HNF4 and for PDX1) compared with
HNF4 + PDX1.
,
the effect of HNF4 and PDX1 was tested in the absence of E2A and
BETA2. 293T cells were cotransfected with a reporter plasmid encoding
the luciferase gene controlled by the rat insulin I promoter and with
expression vectors encoding PDX1 and HNF4. Expression of HNF4 and
PDX1 generated synergistic activation of the promoter (Fig.
5B). Synergistic activation was also observed in HeLa cells
(not shown). This cooperativity of HNF4 with PDX1 is probably
responsible for the synergistic activation observed previously upon
coexpression of HNF4 with E2A, BETA2, and PDX1.
in Beta Cells--
Expression of
dominant negative HNF1 in the beta cell line INS-1 reduced the
expression of several beta cell genes, including the insulin gene (26),
and has similar effects to that of dominant negative HNF4 (27).
Furthermore, HNF4 is a transcription activator of HNF1 (24, 27),
and mutations in HNF1 are responsible for MODY3 (23). This raises
the possibility that HNF4 may activate the insulin gene indirectly,
through Hnf1 gene function. In non-beta cells, the
HNF4-dependent activation we observed (Fig. 1) may be
mediated in part through HNF1, but the substantial reduction in the
activation of promoter mutated in the HNF4-binding site (Fig. 3)
indicates that a significant portion of the effect is mediated directly.
site in the insulin gene
promoter in beta cells, and to test whether in beta cells HNF4
activates the insulin gene promoter directly or indirectly through
HNF1, we used INS-1 DN-HNF4 26 cells, a derivative of INS-1 cells
that contains a plasmid encoding doxycycline-inducible dominant
negative HNF4 (27). This dominant negative protein possesses a
functional dimerization domain but lacks a DNA binding domain and
therefore suppresses activity of the endogenous wild type HNF4.
Luciferase reporter plasmids controlled by wild type rat insulin I gene
promoter or by promoter mutated either in HNF1- or HNF4-binding
sites were electroporated into these cells. In the absence of
doxycycline (i.e. no expression of DN-HNF4), mutations in
either the HNF4 site or the HNF1 site led to reduced activity of
the insulin gene promoter (Fig.
6A). Thus both sites are
important for full transcriptional activation in beta cells.
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Fig. 6.
Effect of DN-HNF4 on
wild type and mutated insulin gene promoter in beta cells.
A, luciferase reporter genes containing wild type insulin
gene promoter (wild type, WT) and insulin gene promoter
containing mutations in the HNF1 site (HNF1) or
HNF4 site (HNF4) were electroporated into INS-1
DN-HNF4 26 cells in the presence (light bars) and absence
(dark bars) of doxycycline (Dox., to induce
DN-HNF4). Cells were harvested 48 h following electroporation,
and normalized luciferase activity was determined. Results are
expressed relative to the activity obtained for wild type promoter in
the absence of doxycycline. Each data point represents the mean ± S.E. of at least three independent electroporation experiments.
Asterisks indicate statistical significance
(p = 0.04 for wild type (WT),
p = 0.02 for HNF4, and p = 0.02 for
HNF1) in the presence compared to the absence of doxycycline.
B, model depicting possible effects of DN-HNF4 on insulin
gene expression.
and to distinguish between a direct
action of HNF4 as compared with an indirect action mediated through
HNF1, we compared the activity of wild type and mutated promoters in
the presence and absence of doxycycline. If HNF4 acts exclusively in
an indirect manner, through HNF1, induction of DN-HNF4 should not
affect the activity of promoter mutated at the HNF1 site. In fact,
induction of DN-HNF4 significantly reduced the activity of both the
wild type promoter and promoter mutated in the HNF1-binding site
(Fig. 6A), confirming the involvement of HNF4 in insulin
promoter activation, and showing that at least part of this effect does
not involve HNF1. Furthermore, expression of DN-HNF4 led to
significant reduction in activity of a promoter fragment mutated in the
HNF4 site (Fig. 6A), confirming that HNF4 also
increases insulin gene transcription through an indirect effect via
HNF1 (Fig. 6B). Although this experimental system does
not permit quantitative comparison of the relative contribution of the
direct effects as compared with the indirect effects of HNF4, the
significant DN-HNF4-dependent reduction in observed activity for
both the HNF1 and HNF4 promoter clearly indicates that both
effects are substantial.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
and HNF4 as the
mutated genes responsible for MODY3 and MODY1, respectively, has
prompted efforts to establish the precise role of these factors in
health and disease. Because the MODY phenotype involves a primary
defect in beta cells, the defective function of these transcription
factors must be manifested in these cells. Indeed, expression of
DN-HNF1 and DN-HNF4 leads to clear alterations in expression of
key beta cell genes, including the insulin gene (26, 27). Because
previous studies have shown that HNF4 is an activator of HNF1
(24) and that MODY3 can be caused by mutation in an HNF4-binding
site in the HNF1 promoter (25), it was proposed that the effect of
HNF4 on the insulin gene promoter is indirect, mediated through activation of HNF1 (27).
in beta cells led to reduced activity of both wild type
insulin gene promoter and promoter mutated in the HNF1-binding site,
strongly suggesting the presence of an HNF4-binding site elsewhere
in the promoter region. Indeed, we identified such a sequence between
nucleotides 57 and 69 of the promoter, and we were able to show
that it is capable of binding HNF4 in vitro. We further
showed that this site is essential for full activation of the promoter
in beta cells and for efficient HNF4-dependent activation in non-beta cells. Expression of DN-HNF4 in beta cells also led to reduced activity of a promoter construct bearing mutations in the HNF4 site. The data therefore indicate that in addition to
the previously recognized indirect action of HNF4 on insulin gene
expression mediated through HNF1, there is also a direct action
mediated through binding to the novel HNF4 consensus sequence in the promoter.
and HNF1 in beta
cells is more complex than previously appreciated and may differ from
that in liver. It has been shown that the Hnf4 promoter
contains an upstream beta cell-specific promoter whose activity is
regulated by HNF1. This promoter also contains a binding site for
PDX1, mutations in which strongly correlate with the diabetic phenotype
in a MODY family (30). Taken together, the data are consistent with the
idea that HNF1, HNF4, and PDX1 participate in a complex
autoregulatory transcriptional network that controls many of the
functional properties of developing and mature beta cells. Of central
importance in this regard is the insulin gene, whose expression is
modulated by all three factors. Interestingly, we have observed
synergistic activation of the insulin gene promoter by HNF4 and
PDX1, which have adjacent binding sites in this promoter, thus adding
an additional level of complexity to the transcriptional control
network. The current results indicating that HNF4 activates the
insulin gene through both direct and indirect actions will permit a
more detailed understanding of the MODY phenotype.
ACKNOWLEDGEMENTS
FOOTNOTES
Holds the Marvin Meyer and Jenny Cyker Chair of Diabetes
Research. To whom correspondence should be addressed. Tel.: 972- 8 934 3597; Fax: 972- 8 934 4118; E-mail: m.walker@weizmann.ac.il.
ABBREVIATIONS
, hepatocyte nuclear factor 4;
DN, dominant negative;
HNF1, hepatocyte nuclear factor 1;
EMSA, electrophoretic mobility shift assay;
RSV, Rous sarcoma virus;
CMV, cytomegalovirus;
TK, thymidine kinase;
LUC, luciferase;
HBE, HNF4
binding element.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Steiner, D. F.,
Chan, S. J.,
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