COMMUNICATION
Loss-of-function and Dominant-negative Mechanisms Associated
with Hepatocyte Nuclear Factor-1
Mutations in Familial Type 2 Diabetes Mellitus*
Hideaki
Tomura
,
Hidekazu
Nishigori
,
Kimie
Sho
,
Kazuya
Yamagata§,
Ituro
Inoue
, and
Jun
Takeda
¶
From the
Laboratory of Molecular Genetics, Department
of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma
University, Gunma 371-8512, Japan and the § Second
Department of Internal Medicine, Osaka University Medical School,
Osaka 565-0871, Japan
 |
ABSTRACT |
Hepatocyte nuclear factor (HNF)-1
, a
homeodomain-containing transcription factor, regulates gene expression
in a dimerized form in pancreas, liver, and some other tissues. Recent
genetic studies have identified two HNF-1
mutations, R177X and
A263fsinsGG, in subjects with a monogenic form of type 2 diabetes.
Despite the defects being in the same gene, diverse severities of
disease are observed in the affected subjects. To investigate the
molecular mechanism by which mutations might cause various phenotypic
features, wild type and mutant proteins were transiently expressed in
insulin-producing (MIN6) and hepatic (HepG2) cells. Luciferase reporter
assay showed that both mutations resulted in a marked reduction of
transactivation activity. Because their dimerization activity was found
to be intact by the yeast two-hybrid system, it was possible that they were dominant-negative to wild type activity. When co-expressed with
wild type, both of the mutants significantly decreased wild type
activity in HepG2 cells. In contrast, although A263fsinsGG functioned
similarly in MIN6 cells, R177X failed to affect wild type activity in
this cell line. Immunohistochemical analysis of the mutants suggests
that this functional divergence might be generated by the modification
of nuclear localization. These results suggest that HNF-1
mutations
may impair pancreatic
-cell function by loss-of-function and
dominant-negative mechanisms.
 |
INTRODUCTION |
Noninsulin-dependent (type 2) diabetes mellitus is a
genetic disorder characterized by elevated plasma glucose levels due to
an absolute or relative deficiency of insulin. Recent progress in
modern genetics research makes precise localization of disease genes
within the genome possible, and this is especially relevant in the
study of monogenic diseases.
Maturity onset diabetes of the young
(MODY)1 is a monogenic form
of type 2 diabetes characterized by onset usually under 25 years of age
and autosomal dominant inheritance (1). Genetic linkage studies first
localized three genes responsible for the development of MODY on
chromosomes 20, 7, and 12 (2-4). The third form of MODY was found to
result from mutations in the gene encoding hepatocyte nuclear factor
(HNF)-1
(5), a homeodomain-containing transcription factor (6-8).
HNF-1
forms a homodimer or heterodimer with structurally related
HNF-1
(9-11), and they function together to regulate gene
expression in liver, pancreas, and some other tissues.
In this context, the HNF-1
gene was also screened for mutations in
subjects with early onset type 2 diabetes/MODY, and two mutations of
R177X and A263fsinsGG were found in two Japanese families (12, 13).
HNF-1
is a protein of 557 amino acids comprising three functional
domains: a dimerization domain (residues 1-32), a DNA-binding domain
with a POU subdomain and a homeosubdomain (residues 88-178 and
229-299, respectively), and a transactivation domain (residues
314-557) (9-11). The R177X nonsense mutation generates a truncated
protein of 176 amino acids with the N-terminal dimerization and POU
domains. The A263fsinsGG frameshift mutation due to insertion of a GG
dinucleotide also generates a truncated mutation of 264 amino acids
that lacks a third helix structure at the C terminus of the homeodomain
and the entire transactivation domain. Interestingly, although the same
gene is mutated, differing clinical features including diverse severity
and inconsistency of onset age of diabetes are observed in these
families (12, 13). In this study, the molecular mechanisms by which
mutations in the HNF-1
gene might cause diabetes and generate the
diverse phenotypic features were addressed by functional analysis of
the mutant proteins.
 |
MATERIALS AND METHODS |
Wild Type and Mutant Plasmid Constructs--
The mutations were
generated by polymerase chain reaction-based site-directed mutagenesis
and cloned in pSP72 (Promega, Madison, WI) to generate the pSP-R177X
and pSP-A263fsinsGG and also in the expression vector pCMV-6b to
generate the pCMV-R177X and pCMV-A263fsinsGG. Wild type HNF-1
was
also cloned in pSP72 and pCMV6b to generate pSP-WT and pCMV-WT,
respectively. For luciferase reporter assay, the promoter region
(nucleotides
1296/+312) of the human gene for GLUT2 (14), a liver and
pancreatic
-cell glucose transporter, was cloned in the pGL3-Basic
Reporter vector (Promega) to generate the pGL3-GT2. The consensus
cis-element for HNF-1
/-1
binding locates at nucleotide
1030 of the promoter region of the GLUT2 gene. For
immunohistochemical analysis, the nucleotide sequence encoding the HA
epitope of YPYDVPDYA was introduced in frame at the 3' end of the
sequence for R177X, A263fsinsGG, and wild type in the expression vector
to generate the pCMV-R177X-HA, pCMV-A263fsinsGG-HA, and pCMV-WT-HA, respectively.
For verification of dimerization activity, yeast two-hybrid analysis
(15) was performed to monitor interaction of the mutant protein with
wild type. The entire coding region of the wild type and mutant
proteins were fused to the yeast GAL4 DNA-binding and transcriptional
activation domains in pAS2-1 and pACT2
(CLONTECH), respectively.
Transient Transfection Assays--
HepG2 and MIN6 cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10 and
15% fetal calf serum, respectively. Cells were transfected with the
liposomal DOTAP/nucleic acid mixture including 5 µg of DOTAP (Roche
Molecular Biochemicals), 333 ng of pGL3-GT2, 0-96 ng of pCMV-WT,
pCMV-R177X, and pCMV-A263fsinsGG, and 17 ng of pRL (Renilla
luciferase)-SV40. Luciferase reporter assay was performed using a
Dual-Luciferase Reporter Assay System (Promega) according to the
manufacturer's instructions. Renilla luciferase activity
was used to normalize transfection efficiencies among experiments.
DNA Binding Assay--
Wild type and mutant proteins were
prepared by in vitro transcription and translation with
pSP-WT, pSP-R177X, and pSP-A263fsinsGG using a TNT Coupled Reticulocyte
Lysate System (Promega) according to the manufacturer's instructions.
For electrophoretic mobility shift assay, in vitro
translated products of the mutant and wild type proteins were incubated
in a 20-µl reaction containing 10 mM Hepes, pH 7.4, 10%
glycerol, 25 mM KCl, 5 mM MgCl2, 2 mM spermidine, 0.1 mM EDTA, 0.5 mM
dithiothreitol, 2 µg of poly(dI-dC), and 0.3 pmol of
32P-labeled double-stranded oligonucleotides (10,000 cpm)
of 5'-ACCTCAGTAAAGATTAACCATCA-3'. DNA-protein complexes were resolved
on a 5% nondenaturing polyacrylamide gel in 0.25 × TBE (45 mM Tris borate, 45 mM boric acid, and 2 mM EDTA). The gel was then dried and exposed to x-ray film
for autoradiography.
Interactions of Wild Type and Mutant Proteins by Yeast Two-hybrid
System--
Verification of two-hybrid interactions of wild type and
mutant proteins was performed according to the manufacturer's
instructions. Briefly, the yeast strain YRG-2 was transformed by
electroporation with the above bait and prey constructs in pAS2-1 and
pACT2, respectively. The yeast competent cells used were purchased from
Stratagene (La Jolla, CA). Cotransformation mixtures were plated on
synthetic dropout medium (SD/-Trp/-Leu/-His) and incubated at 30 °C
for several days to select for colonies expressing interacting hybrid proteins. If the target protein interacts with a test protein, a
functional GAL4 activator is reconstituted, and the expression of the
HIS3 reporter gene is activated. For qualitative blue/white screening of the His+-cotransformants, colony lift
-galactosidase filter assay was performed.
Immunolocalization of Mutant and Wild Type Proteins--
MIN6
and HepG2 cells were transfected with the pCMV-R177X-HA,
pCMV-A263fsinsGG-HA, and pCMV-WT-HA and grown on the slide containing appropriate medium. After 48 h of culture, cells were fixed with 3% formaldehyde, washed with phosphate-buffered saline, and incubated in the primary antibody at 37 °C for 30 min. After washing with phosphate-buffered saline, the cells were incubated in the
fluorescence-labeled secondary antibody at 37 °C for 30 min.
Localization of fusion proteins within the cells was analyzed using
fluorescence microscopy. The mouse anti-HA primary antibody and
secondary antibody of fluorescein isothiocyanate-conjugated anti-mouse
IgG were purchased from Roche Molecular Biochemicals. Nuclear DNA was
stained with 4',6-diamidino-2-phenylindole.
 |
RESULTS |
Transactivation Activity of Wild Type and Mutant HNF-1
Proteins--
To investigate the molecular mechanism by which HNF-1
mutations cause impaired glucose tolerance, transactivation activities of R177X and A263fsinsGG were analyzed in liver cell and pancreatic
-cell lines by luciferase reporter assay using the promoter of the
human gene for GLUT2, which mediates facilitative glucose transport in
these tissues. Wild type HNF-1
bound to the cis-element and efficiently increased reporter gene activity directed by
transcription from the GLUT2 gene promoter in HepG2 and MIN6 cells
(Figs. 1 and
2A). When the region from
1056 to
1026 containing the cis-element for binding was
deleted from the promoter segment, the reporter activity was
significantly reduced (85 ± 4%, p < 0.001).
However, the deleted reporter construct still represented a significant increase of activity mediated by wild type HNF-1
, suggesting that
GLUT2 gene expression could also be indirectly regulated by other
transcription factors whose expression is up-regulated by HNF-1
.
When the HNF-1
mutations of R177X and A263fsinsGG were expressed, a
marked reduction of transactivation activity of the mutant proteins was
observed both in MIN6 and HepG2 cells.

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Fig. 1.
In vitro transcription and
translation and electrophoretic mobility shift assay.
A, pSP-WT, pSP-R177X, and pSP-A263fsinsGG were in
vitro transcribed and translated in the presence of
[35S]methionine. The products were separated on a 10%
SDS/polyacrylamide gel. B, in vitro translated
products were incubated with a reaction solution containing
32P-labeled DNA fragments. The binding complex was
separated on a 5% nondenaturing polyacrylamide gel. The complex
formation was inhibited by adding excess unlabeled oligonucleotides
(0.02, 0.2, and 2 pmol), whereas the complex formation was not affected
by addition of mutated oligonucleotides of
5'-ACCTCAGGTAAGAGGAACCATCA-3'. In contrast, when
R177X and A263fsinsGG were examined with the same oligonucleotides, no
shift of binding complex was observed. C, interaction of
A263fsinsGG with wild type. The DNA binding activity of wild type was
significantly reduced when mutant proteins were added in the
reaction.
|
|

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Fig. 2.
A, transactivation activity of wild type
(WT) and mutant proteins. pGL3-GT2 and each test plasmid
were cotransfected into MIN6 and HepG2 cells. The relative luciferase
activity (Firefly/Renilla luciferase) of each construct at
0, 6, 12, and 24 ng for MIN6 cells and at 0, 24, 48, and 96 ng for
HepG2 cells was measured by four independent experiments. Mean ± S.D. is shown. **, p < 0.01. B,
co-expression of wild type and each mutant protein. pGL3-GT2, wild
type, and each mutant plasmid were co-transfected into MIN6 and HepG2
cells. The effect of increasing amounts (12 and 24 ng for MIN6 cells
and 24, 48, and 96 ng for HepG2 cells) of each test plasmid on the wild
type activity was examined. The open bar on the left
side indicates the endogenous transcription activity. The other
bars indicate the activities of the WT (wild type) alone (open
bar), WT+R177X (hatched bar), and WT+A263fsinsGG
(filled bar). **, p < 0.01.
|
|
Because A263fsinsGG retains the greater part of the DNA-binding domain,
it is possible that this mutation may interfere with DNA binding of
wild type in a dominant-negative manner. To determine whether
A263fsinsGG may compete with wild type to bind the target DNA sequence
to diminish normal activity, the electrophoretic mobility shift assay
was performed, resulting in no DNA binding activity in A263fsinsGG
(Fig. 1A). Because the C terminus of the helix-turn-helix
motif of Drosophila homeodomain proteins has been shown to
be necessary to confer the DNA binding specificity (16), the third
helix structure of the DNA-binding domain of human HNF-1
, in which
21 amino acids are looped out of the turn between helix 2 and helix 3 unlike other homeodomain proteins (8), also appears to be crucial for
DNA binding.
Interaction of HNF-1
Mutants with Wild Type
Protein--
Because the dimerization domain at the N terminus is
retained in both mutations, the nonfunctional truncated proteins may form a heterodimer with wild type HNF-1
and so interfere with the
normal function. To test this possibility, dimerization activity of the
mutants was examined using the yeast two-hybrid system (15). The bait
and prey DNA constructs were introduced into the same host cells to
verify mutual interaction. When either pAS2-WT (bait) or pACT2-WT
(prey) for wild type was introduced into the cells with a counter
vector, no reporter activity was observed, suggesting that HNF-1
alone does not have an autonomous function that activates GAL4 gene
expression. When both pAS2-1-WT and pACT2-WT were introduced together,
the efficient growth of transformants in the selection medium and blue
color formation by
-galactosidase assay were observed, indicating
that the specific HNF-1
dimerization should be readily detected by
the two-hybrid system. Using this method, the dimerizing activity of
A263fsinsGG and R177X was examined in relation to wild type, resulting
in efficient growth of cotransformants in the selection medium and generation of
-galactosidase activity (Table
I). The positive interaction of each
mutation with wild type was confirmed by switching the test molecules
in bait and prey constructs.
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Table I
Verification of dimerization activity of wild type and mutant proteins
Dimerization activity of the HNF-1 mutants was examined using yeast
two-hybrid system. indicates no growth of cotransformants in the
selection media (SD/ Trp/ Leu/ His). + indicates the efficient
growth of the cotransformants in the selection media and the
generation of positive -galactosidase activity. ± indicates the
efficient growth of the cotransformants in the selection medium but
extremely weak generation of -galactosidase activity. pAS2-1 and
pACT2 are bait and prey plasmid vectors, respectively. ND, not done.
|
|
Loss-of-function and Dominant-negative Mechanisms of Reduction of
Transactivation Activity--
To determine whether A263fsinsGG or
R177X acts as a dominant-negative regulator, varied amounts (molar
ratio, 1:1 to 1:2 in MIN6 cells and 1:0.5 to 1:2 in HepG2 cells) of the
mutant constructs were expressed together with wild type, and the
luciferase reporter activity driven by the GLUT2 gene promoter was
measured (Fig. 2B). When equimolar amounts of DNA for the
wild type and each mutant were transfected into HepG2 cells, both of
the mutations significantly reduced wild type activity. With increasing
amounts of mutant DNA, both of the mutations were also found to reduce normal activity, suggesting that A263fsinsGG and R177X both function as
a dominant-negative regulator in hepatic cells. Interestingly, however,
when the mutations were expressed in MIN6 cells, although A263fsinsGG
also similarly reduced wild type activity in this cell line, R177X
failed to affect wild type activity, suggesting that A263fsinsGG and
R177X function differently in pancreatic
-cells.
Intracellular Localization of Wild Type and Mutant HNF-1
Proteins--
To determine whether a difference in intracellular
localization of mutant proteins could affect transcription activity,
localization of HA-tagged proteins within the HepG2 and MIN6 cells was
examined by fluorescence microscopy (Fig.
3). Strong signals for A263fsinsGG and
wild type were similarly observed only in the nucleus in both cells. In
contrast, the distribution of R177X within the cells was found to be
differently patterned and markedly modified among the cells. In MIN6
cells, 90% of the cells transfected with R177X showed strong signals
only in the cytoplasm, whereas the other 6 and 4% of the cells showed
relatively weak signals in both nucleus and cytoplasm or only in the
nucleus, respectively; 50-100 cells in two experiments were counted to
estimate the frequency of each pattern. On the other hand, 92% of the
transfected HepG2 cells showed strong staining in the nucleus (nucleus
only, 19%; nucleus and cytoplasm, 73%). The other 8% of the cells
showed only cytoplasmic staining. Although the frequency of nuclear or
cytoplasmic staining varied to some extent in two experiments, the
general patterns of intracellular localization were similar. The
modified localization of R177X within the cells might be due to lack of
a short stretch of basic residues (residues 229-237) in the POU
domain, which has been suggested as a nuclear localization signal (16,
17).

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Fig. 3.
Intracellular localization of the wild type
and mutant proteins in MIN6 and HepG2 cells. The cells were
transiently transfected with pCMV-R177X-HA, pCMV-A263fsinsGG-HA, and
pCMV-WT-HA. Immunohistochemical analysis was performed using anti-HA
antibody. A typical staining pattern from each experiment is shown. Two
different patterns of R177X staining in HepG2 cells are shown in the
two bottom panels (R177X(a) and
R177X(b)).
|
|
 |
DISCUSSION |
Liver and pancreatic
-cells play a central role in regulation
of glucose homeostasis by glucose uptake and disposal and insulin secretion, respectively, in response to the levels of plasma glucose. Because expression of the genes involved in such liver- and
-cell-specific function is regulated by a tissue-specific subset of
transcription factors, molecular defects of these factors could lead to
the development of impaired glucose tolerance.
In this study, the HNF-1
mutations of R177X and A263fsinsGG were
found to have markedly reduced activity of transactivation of the human
GLUT2 gene in liver or pancreatic
-cell lines. These results are
consistent with the contribution of the mutations in the development of
diabetes in patients with the mutation. Because a functional loss of
GLUT2 has been suggested to be responsible for hyperglycemia and
relative hypoinsulinemia in the fed state in patients with
Fanconi-Bickel syndrome and early onset diabetes in mice lacking GLUT2
(18, 19), the decreased expression of GLUT2, which could be generated
by HNF-1
mutations, might be involved in the pathogenesis of
hyperglycemia in patients in concert with other target gene defects. In
this regard, further identification of the target genes of HNF-1
,
which are expressed in pancreatic
-cells and liver, is important to
better understand the pathogenesis of HNF-1
-deficient diabetes. The
present study also shows that the two mutations function differently in
pancreatic
-cells, possibly due to the difference of intracellular
localization of the protein. Because the DNA binding activity of wild
type is found to be reduced in the presence of A263fsinsGG (Fig.
1C), mutation in pancreatic
cells could interfere with
the wild type regulation of target gene expression. Accordingly, the
differing functional properties of the mutations in pancreatic
-cells may explain, at least in part, the differing severity of
insulin secretion deficiency in the affected subjects. However, because
only two mutations have so far been reported, further identification of families with the mutation will be necessary to understand to what
extent these distinct molecular behaviors could account for differing
clinical features in patients.
Because the primary structures of HNF-1
and HNF-1
, which
recognize the same DNA sequence, are highly related and the tissues that express these proteins are mostly overlapped except lung and ovary
(6-11), the structure and function relationships in HNF-1
may also
apply to HNF-1
. The possible nuclear localization sequence of
HNF-1
, which is absent in R177X, is also found in HNF-1
(residues
197-205), so the modification of nuclear targeting could be observed
in HNF-1
mutations involved in this region (20). Accordingly,
functional analysis of the HNF-1
mutations should be helpful to
clarify the molecular mechanism of impaired insulin secretion not only
in HNF-1
-deficient diabetes but also in the related form of
HNF-1
-deficient diabetes.
 |
ACKNOWLEDGEMENT |
We thank Dr. J. Miyazaki (Osaka University,
Osaka, Japan) for providing MIN6 cells.
 |
FOOTNOTES |
*
This study was supported by a Grant-in-Aid for Creative
Basic Research from the Japanese Ministry of Science, Education, Sports and Culture and by the Uehara Memorial Foundation.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.
¶
To whom correspondence should be addressed: Dept. of Cell
Biology, Inst. for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan. Tel.:
81-27-220-8830; Fax: 81-27-220-8889; E-mail:
jtakeda{at}akagi.sb.gunma-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
MODY, maturity onset
diabetes of the young;
GLUT2, glucose transporter type 2;
HNF, hepatocyte nuclear factor;
HA, hemagglutinin;
DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
methylsulfate.
 |
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