From the Section of Islet Transplantation & Cell
Biology, Joslin Diabetes Center and the § Department of
Medicine, Harvard Medical School, Boston, Massachusetts 02215
Received for publication, September 13, 2000
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ABSTRACT |
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Transcription factors binding the insulin
enhancer region, RIPE3b, mediate In adult mammals, the insulin gene is expressed only in the
pancreatic Three conserved insulin enhancer elements, A3 ( The RIPE3b binding activity has been extensively characterized in
nuclear extracts from both insulin-producing and non-insulin-producing cell lines (14, 27-31). Two specific RIPE3b-binding complexes have
been identified: 1) a cell-specific complex, RIPE3b1, which is detected
only in pancreatic In addition to regulating cell type-specific expression, factors
binding to the enhancer elements, A3, E1, and RIPE3b, also regulate
glucose-mediated alterations in insulin gene expression (30, 31,
33-36). Our earlier studies demonstrated that the binding activity of
the cell-specific RIPE3b1 activator play a key role in regulating this
glucose responsiveness (30, 31). Although the binding activity of the
RIPE3b1 activator is induced in response to acute change in glucose
concentration, under glucotoxic conditions, as observed in HIT T-15
cells cultured chronically in the presence of high concentrations of
glucose, the RIPE3b1 and PDX-1 binding activities were inhibited
(37-39). PDX-1 protein and mRNA levels were also inhibited in rat
islets, following development of diabetes after partial pancreatectomy
(40). These observations suggest that in vitro and in
vivo glucotoxic conditions regulate the levels and activity of
insulin gene transcription factors. Interestingly, the binding activity
of the RIPE3b1 factor, but not of PDX-1, is inhibited under glucotoxic
conditions in the insulinoma cell line The cell type-specific and glucose-responsive transcription factors of
the insulin gene also play an important role in pancreatic development
and differentiation of Similar to the PDX-1 and BETA2 factors, the RIPE3b1 activator, is an
important regulator of cell type-specific and glucose-responsive expression of the insulin gene and may play an important role in
pancreatic development and/or -cell type-specific and
glucose-responsive expression of the insulin gene. Earlier studies
demonstrate that activator present in the
-cell-specific
RIPE3b1-binding complex is critical for these actions. The DNA binding
activity of the RIPE3b1 activator is induced in response to glucose
stimulation and is inhibited under glucotoxic conditions. The C1
element within the RIPE3b region has been implicated as the binding
site for RIPE3b1 activator. The RIPE3b region also contains an
additional element, A2, which shares homology with the A elements in
the insulin enhancer. Transcription factors (PDX-1 and HNF-1
)
binding to A elements are critical regulators of insulin gene
expression and/or pancreatic development. Hence, to understand the
roles of C1 and A2 elements in regulating insulin gene expression, we
have systematically mutated the RIPE3b region and analyzed the effect
of these mutations on gene expression. Our results demonstrate that
both C1 and A2 elements together constitute the binding site for the
RIPE3b1 activator. In addition to C1-A2 (RIPE3b) binding complexes,
three binding complexes that specifically recognize A2 elements are
found in nuclear extracts from insulinoma cell lines; the A2.2 complex
is detected only in insulin-producing cell lines. Furthermore, two base
pairs in the A2 element were critical for binding of both RIPE3b1 and
A2.2 activators. Transient transfection results indicate that both C1-A2 and A2-specific binding activators cooperatively activate insulin
gene expression. In addition, RIPE3b1- and A2-specific activators
respond differently to glucose, suggesting that their overlapping
binding specificity and functional cooperation may play an important
role in regulating insulin gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells in the islets of Langerhans. In studies using transgenic animals and transient transfection analysis, the proximal 5'-flanking region of the insulin promoter was shown sufficient for
directing
-cell-specific expression of the insulin gene (1-7). Further, mutational analysis of the promoter proximal region identified several cis-acting enhancer elements that are important for
insulin expression. The insulin enhancer elements, with the exception of E-box elements, have been classified based on the nucleotide sequence of the element (8). Most of these enhancer elements are well
conserved in various species, suggesting the presence of a common
regulatory mechanism(s) controlling insulin expression.
201 to
196
bp)1 (9-13), RIPE3b/C1-A2
(
126 to
101 bp) (14), and E1 (
100 to
91 bp) (4, 15, 16), play
an important role in regulating cell-specific expression of the insulin
gene. Factors binding these sites have a very limited cellular
distribution. Transcription factors that bind and activate expression
from two of the three conserved insulin enhancer elements (A3 and E1)
have been cloned. PDX-1, a member of the homeodomain family of
transcription factor expressed in cells of the pancreas and duodenum,
binds the A3 element (17-22). Heterodimers of ubiquitously distributed
basic helix-loop-helix family members (E2A and HEB (23, 24)) and the cell type enriched basic helix-loop-helix member (BETA2 (25, 26)) bind the E1 element. BETA2 is expressed in all pancreatic endocrine cell types, in some intestinal endocrine cells, and in the
brain (25-27). Limited but not identical cellular distribution of
transcription factors PDX-1 and BETA2 suggests that cell-specific insulin gene expression may result because of the presence of a unique
combination of enhancer element binding factors in the pancreatic
-cells. Interestingly, one of the RIPE3b element-binding complexes
is expressed only in insulin-producing cells (14, 28) and may play a
critical role in regulating the cell type specificity of insulin gene expression.
-cell lines, and 2) the RIPE3b2-binding complex
that is detected in nuclear extracts from all cell lines examined to
date. The importance of the factor(s) binding to the RIPE3b element in
mediating cell type-specific insulin gene expression is further
emphasized from transgenic mice studies (5). It was demonstrated that
an enhancer construct containing both RIPE3b and E1 elements, RIPE3,
could correctly regulate temporal and spatial expression of the
transgene in vivo (5), whereas a construct containing the E1
element alone was unable to induce expression in transgenic animals
(32). In all RIPE3 transgenic lines, the transgene (growth hormone)
expression was detected in
-cells, whereas in other lines,
expression of growth hormone was also noted in
-cells (5).
TC-6 (41). This suggests that
the inhibition of the RIPE3b1 activator binding may be the primary or
initial defect under glucotoxic conditions.
-cells. Mice homozygous for the null mutation
in BETA2/NeuroD have a striking reduction in the
number of
-cells and fail to develop mature islets, develop severe
diabetes, and die perinatally (26). The pdx-1 knockout mice
have a more profound phenotype, being apancreatic; these animals also
develop extreme hyperglycemia and die perinatally (20, 21, 42). During
embryonic development, PDX-1 is also expressed in exocrine and ductal
epithelial cells, whereas in the adult pancreas expression is
predominantly restricted to
-cells (20-22, 43). However, expression
of PDX-1 is induced in the ductal epithelium during pancreatic
regeneration in adult animals (44).
-cell differentiation. The C1 element
(Fig. 1) in the RIPE3b region has been
implicated as the binding site for the RIPE3b1 activator (14, 28, 31).
Factors binding the C1 element were shown to functionally co-operate
with E1 binding factors (BETA2-E12/E47) in regulating insulin gene expression (14, 25, 31, 45). In addition to C1, the RIPE3b region also
contains an A element, A2 (Fig. 1 and Refs. 8, 46, and 47). The
homeodomain family of transcription factors (PDX-1, HNF1
, and
Isl-1) that are important for pancreatic development bind insulin A
elements (48-51). Furthermore, factors binding to A elements are
implicated in synergistically interacting with other insulin gene
transcription factors and regulating gene expression (52-54).
View larger version (13K):
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Fig. 1.
Top, organization of the rat insulin II
enhancer/promoter region with new and old names of different enhancer
elements based on the simplified nomenclature.
Bottom, suggested DNA binding sequence for the A2/GG1
and the C1 element in the RIPE3b region from Ref. 8.
Hence, in this study, we have further characterized the RIPE3b region
to better understand the role of C1 and A2 elements in regulating
insulin gene expression. We demonstrate that the presence of both C1
and A2 elements are essential for binding of the RIPE3b1 activator. We
also identified factors that require the A2 element but not the C1
element for binding. Interestingly, one of the A2 element-binding
complexes, A2.2, is selectively expressed in insulin-producing cell
lines. We further demonstrate that two base pairs in the A2 element are
critical for the formation of both -cell-specific DNA-binding
complexes, RIPE3b1 and A2.2. Results from transient transfection
analysis indicate that C1-A2 (RIPE3b1) and A2-specific factors
positively regulate insulin gene expression. A mutation that prevents
binding of both activators shows greater inhibition of insulin gene
expression than a mutation that prevents binding of either of these
factors alone. Based on these results we suggest that the presence of
two
-cell-specific factors with overlapping DNA-binding sites may
provide an important means of regulating insulin gene expression in
response to various metabolic/environmental signals.
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EXPERIMENTAL PROCEDURES |
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Tissue Culture--
The HIT T-15 cell line was obtained from
American Type Culture Collection (Manassas, VA), and all experiments
were conducted with cells between passage numbers 68 and 80. TC-3
and
TC·TET cell lines have been described earlier (55, 56) and
were provided by Dr. Shimon Efrat. Non-insulin-producing cell lines
used in this study are: HeLa cells (human cervical carcinoma), baby
hamster kidney cells, and IM Duct (immorto
mouse ductal cell line,
provided by Dr. Susan Bonner-Weir, Boston, MA). Cells were maintained
in Dulbecco's modified Eagle's medium supplemented with Ham's F-12 (22 mM glucose), 10% fetal bovine serum, penicillin, and
streptomycin. For the preparation of medium with different glucose
concentration, glucose-free Dulbecco's modified Eagle's medium and
dialyzed fetal bovine serum (Life Technolgies Inc.) were used. Glucose
levels in the medium were adjusted to the desired concentration by
addition of filter sterilized glucose.
Extract Preparation-- HIT T-15 nuclear extracts, prepared using a large scale nuclear extract preparation protocol (57), were generously provided by Dr. Larry Moss (Tufts University, Boston, MA). Other insulin-producing and non-insulin-producing cell lines were cultured for 4-6 days, and nuclear extracts were prepared as described (58). To prepare extracts from HIT T-15 cells grown in the presence of different concentrations of glucose, cells were cultured for 4 days in Dulbecco's modified Eagle's medium/Ham's F-12 medium. Medium was then removed, cells were washed twice with phosphate-buffered saline, and medium containing the desired concentration of glucose was added. Cells were harvested after 48 h, and nuclear extracts were prepared (58).
Electrophoretic Mobility Shift Assays--
Oligonucleotides used
as probes and competitors (Table I) were
synthesized either by Sigma-Genosys (The Woodlands, TX) or by the DNA
core facility at the Joslin Diabetes Center. In addition to the
oligonucleotides listed in Table I, a series of two base pairs
substitution (A C and G
T) mutations in the RIPE3b (
126 to
101 bp) oligonucleotide were synthesized and named according to the
two mutated nucleotides (e.g.
126·125m oligonucleotide is mutated at positions
126 and
125 bp in the RIPE3b
oligonucleotide). Double-stranded oligonucleotides were radiolabeled
with [
-32P]dCTP and the Klenow fragment of DNA
polymerase I and used as probes. Binding reaction conditions for the
RIPE3b,
139 to
113 and thyroid transcription factor-1 probes were
identical (10 mM Tris, pH 7.8, 150 mM NaCl, 2 mM EDTA, 8 mM dithiothreitol, 0.4 mg of
poly(dI-dC), 5% glycerol, 50-100 fmol of probe, and 3-5 µg of
nuclear extracts). Binding reaction conditions for A3 (FLAT-E) and
major late transcription factor probes have been described (31, 37).
Competition experiments were performed by simultaneous addition of
radiolabeled probe and excess unlabeled competitors to the binding
reaction.
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The anti-IDX-1 (PDX-1) antibody was generously provided by Dr. Joel Habener (Massachusetts General Hospital, Boston, MA), whereas monoclonal anti-Nkx2.2 antibody was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA). For antibody supershift experiments, antibodies were preincubated with the nuclear extract for 20 min at room temperature, followed by another 20-min incubation in the presence of the radiolabeled probe. Binding reactions were then loaded onto a 6% nondenaturing polyacrylamide gel and were run in Tris-glycine-EDTA buffer (31). After completion of the run, gels were dried and scanned on a Molecular Dynamics PhosphorImager (Sunnyvale, CA), and bands were quantitated using Imagequant software.
DNA Constructs--
The insulin reporter construct-238 WT LUC
has been described earlier (31). The QuickChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA) was used to construct
additional plasmids with specific mutations in the insulin enhancer.
Double-stranded oligonucleotides containing desired mutations
(underlined nucleotides) were synthesized to construct 125·124m LUC
(
138 CA GGC AAG TGT TTT TAA ACT GCA GCT TCA GCC CCT CTG
G,
100 bp),
122·121m LUC (
135 GC AAG TGT TTG GAC
CCT GCA GCT TCA GCC CCT CTG G,
100 bp), and
110·109m LUC
(
135 GC AAG TGT TTG GAA ACT GCA GCT TCC TCC CCT CTG GCC,
98 bp). The
238 WT LUC plasmid was used as the template with these
oligonucleotides to construct reporter plasmids with mutations at the
desired positions in the insulin enhancer. Plasmids were sequenced at
the DNA core facility at Joslin Diabetes Center to confirm the
construction of each mutant plasmid. To avoid the effect of nonspecific
mutations outside the insulin enhancer region on the reporter gene
expression, the plasmids were digested with restriction enzymes to
release the mutated insulin enhancer fragments. These fragments were
then used to replace the wild type insulin enhancer from the
238 WT
LUC construct, resulting in the generation of reporter plasmids that
differed only at the specific mutations in the insulin enhancer.
Transient Transfections and Luciferase Assays--
Approximately
18 h before transfection, 5 × 105 HIT T-15 or
TC-3 cells were plated onto 60-mm2 plates. Reporter
plasmids (2.5 or 2.0 µg for HIT T-15 or
TC-3 cell line,
respectively) were co-transfected with 0.1 µg of a pRL-Null (Promega,
Madison, WI) plasmid, as an internal control. Although the pRL-Null
plasmid lacks any of the eukaryotic enhancer elements, significant
levels of constitutive Renilla luciferase expression have
been observed from this construct (59). We also observed a linear
relationship between the reporter gene expression and the amount of the
pRL-Null plasmid used (data not shown). Plasmid DNA were transfected
using LipofectAMINE (HIT T-15 cells, DNA:LipofectAMINE ratio of 1:4) or
LipofectAMINE PLUS (
TC-3 cells, DNA:LipofectAMINE PLUS ratio of 1:5)
as descried earlier (31). To determine reporter gene expression a
commercial Dual Luciferase Kit (Promega) was used. Whole cell extracts
were prepared 48 h after transfection, using passive lysis buffer.
Extracts were used to determine firefly (reporter plasmids) and
Renilla (internal control) luciferase expression using the
Moonlight 3010 Luminometer (Analytical Luminescence Laboratory, Sparks,
MD). The ratio of the firefly:Renilla luciferase activity
represents the normalized reporter gene expression. Transfection
experiments were repeated several times, with at least two or three
different plasmid preparations.
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RESULTS |
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C1 and A2 Elements Together Constitute the Binding Site for the
RIPE3b1 and RIPE3b2 Factors--
Two sequencespecific RIPE3b (126 to
101 bp) binding complexes have been identified. The RIPE3b1 complex
is detected in insulin-producing cells, whereas the RIPE3b2 complex is
detected in nuclear extracts from all cell lines examined to date (14,
27-31). Mutating two nucleotides (
112 and
111, a TC to CG
substitution) in the RIPE3b probe prevented the formation of both the
RIPE3b1 and the RIPE3b2 complexes (14, 28, 31). Methylation
interference analysis indicated that the RIPE3b1 complex exerted a
strong interference at Gs
107,
108, and
111 and weak interference
at G
114 on the bottom strand, suggesting that the binding site may
span from
114 to
107 bp (14). Based on these observations and other results, it was suggested that the RIPE3b region may contain two elements, C1 (
116 to
107) (for the binding of RIPE3b1 and RIPE3b2 factors) and A2 element (
126 to
113) (8, 46, 47). The C1 and A2
elements share a 4-bp segment (
116 to
113 bp) that contains the G
114 (bottom strand) implicated in the binding of the RIPE3b1 factor.
Hence, to define the binding site for the RIPE3b1 activator as well as
to characterize the role of the A2 element in this binding, we made a
series of 2-bp substitution mutations in the RIPE3b oligonucleotide and
studied the effect of these mutations on the RIPE3b1 binding.
Electrophoretic mobility shift assays (EMSA) were performed using
radiolabeled RIPE3b (126 to
101) probe and HIT T-15 nuclear extracts, in the presence or absence of 50-fold excess of unlabeled competitors (Fig. 2A). As
expected, a 50-fold excess of the previously described unlabeled mutant
RIPE3b oligonucleotide (having TC to CG substitution at
112 and
111
bp) did not compete for binding to either RIPE3b1 or RIPE3b2 factors,
whereas the unlabeled wild type RIPE3b oligonucleotide successfully
competed for binding of these factors. Competition between the RIPE3b
probe and a series of RIPE3b oligonucleotides with mutations in two
consecutive nucleotides showed interesting binding profiles for the
RIPE3b1 and RIPE3b2 factors (Fig. 2A). Mutations in the
nucleotides from
112 to
107 bp (C1 region) inhibited the ability of
excess unlabeled competitors to compete as effectively as the wild type
oligonucleotide, confirming the importance of this region for binding
of these factors. Interestingly, the oligonucleotides with mutations in
the A2 element (
124 to
117 bp) were also ineffective competitors
implying a role of A2 element in the binding of the RIPE3b1 and RIPE3b2
factors. Substitution mutations in the four nucleotides
116 to
113
bp (residing between these two regions) had no effect on their ability to compete as effectively as the wild type oligonucleotide. These results indicate that binding of the RIPE3b1 and the RIPE3b2 complexes require sequences present in both the C1 and A2 elements. Our results
also suggest that nucleotides at
110 to
109 bp and
122 to
121
bp are most critical for the binding of these factors (Fig.
2B).
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Because two distinct elements, C1 and A2, in the RIPE3b region are
required for binding of the RIPE3b1 and the RIPE3b2 factors, we next
asked whether the exact spacing between these elements was essential
for the binding activity. Competitor oligonucleotides were synthesized
with an insertion of two nucleotides or a deletion of two or five
nucleotides between the C1 and A2 elements. As shown in Fig.
2C, altering the distance between the two elements prevented
these oligonucleotides from competing for binding to these factors.
Results presented in Fig. 2 suggest that the RIPE3b1 and RIPE3b2
factors bind a large (107 to
124 bp) DNA-binding element of the
insulin enhancer region. The RIPE3b binding activity not only requires
the nucleotide sequence present in both the C1 and A2 elements but also
requires the exact distance between these two elements. Hence, we
suggest that RIPE3b1 and RIPE3b2 factors bind a composite C1-A2 element.
A -Cell-specific Factor Selectively Binds the A2
Element--
Because the RIPE3b1 factor binds the C1-A2 element, we
were interested in identifying factors that selectively bind either the
C1 or A2 elements. Factors that selectively bind one of these elements
could have a potential regulatory role in modulating the DNA binding
and transcriptional activation mediated by the RIPE3b1 activator. We
consistently observed only two specific RIPE3b (
126 to
101 bp)
binding complexes when nuclear extracts from insulin-producing cell
lines were used. When the C1 element-specific probe (
116 to
101 bp)
was used in EMSA with HIT T-15 nuclear extracts, only nonspecific
DNA-binding complexes were detected (data not shown). These
observations indicate that insulin-producing cells may not contain a C1
element-specific binding factor.
To identify factors that selectively bind the A2 element, we designed a
new probe, 139 to
113 bp, that included the A2 element and
additional sequences upstream of the RIPE3b region (Fig.
3A). In EMSA using
radiolabeled
139 to
113 bp probe and HIT T-15 nuclear extracts, we
detected three binding complexes with gel mobilities distinct from both
the RIPE3b1 and RIPE3b2 complexes (Fig. 3B). Competition
analysis indicated that these complexes bind specifically to the
139
to
113 bp region of the insulin enhancer. Formation of A2 complexes
was not inhibited in the presence of excess unlabeled RIPE3b, C1, A2
(
126 to
113) or sequences upstream of the RIPE3b region (
139 to
127) (Fig. 3). These observations suggest that the formation of A2
complexes requires the rat insulin II enhancer region from
139 to
127 bp, in addition to sequence spanning A2 element (
126 to
113).
Furthermore, these results demonstrate that the A2-specific complexes
selectively bind to the
139 to
113 region of the insulin enhancer,
independent of the presence of the C1 element. Results presented in
Fig. 3 also demonstrate that formation of the RIPE3b1 and RIPE3b2
complexes is not inhibited in the presence of excess unlabeled C1, A2,
or
139 to
113 regions of the insulin enhancer. Taken together, these observations demonstrate the presence of factors with overlapping C1-A2 and A2-specific DNA binding specificity in nuclear extracts from
insulin-producing cells.
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The region corresponding to the 139 to
113 bp of the rat insulin II
gene is highly conserved in the rat insulin I and the human insulin
genes (Fig. 4A). Competition
analysis was performed to confirm that the A2 factors that bind the rat
insulin II
139 to
113 probe would successfully bind the
corresponding region from rat insulin I and the human insulin gene
(Fig. 4B). EMSA were performed with the rat insulin II
139
to
113 probe and HIT T-15 nuclear extracts in the absence or presence
of increasing concentrations of unlabeled excess
139 to
113 bp
oligonucleotide or the corresponding region from the rat insulin I and
human insulin gene and the RIPE3b oligonucleotide. Formation of
A2-specific complexes was inhibited by excess unlabeled
139 to
113
oligonucleotide and its homologous regions from rat I and human insulin
gene but not by the RIPE3b oligonucleotide. These observations suggest that the DNA-binding site recognized by A2-specific factors is highly
conserved.
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Because tissue- or cell type-specific transcription factors are major
regulators of critical genes in a given cell or tissue, we next
determined whether A2-specific factors were selectively expressed in
insulin-producing cells. Nuclear extracts from insulin-producing and
non-insulin-producing cell lines were analyzed for their ability to
form A2-specific complexes. Nuclear extracts from HIT T-15, TC-3,
and
TC·TET (insulin-producing cell lines) and baby hamster kidney,
HeLa, and a pancreatic ductal cell line IM Duct (non-insulin-producing cells) were incubated with radiolabeled
139 to
113 probe from rat
insulin II gene and analyzed by EMSA. As shown in Fig.
5, the A2.1 binding activity was detected
in all cell lines, whereas the A2.2-binding complex was formed only
with the nuclear extracts from insulin-producing cell lines. These
results demonstrate that the A2.2 factor is selectively expressed in
the insulin-producing cells. In addition, the A2.2 factor binds a
conserved region in the insulin enhancer that overlaps with the binding
site for RIPE3b1. The presence of two distinct
-cell-specific
DNA-binding factors with overlapping DNA-binding sites suggest an
important role for these factors in regulating insulin expression.
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A2-specific Factors Are Distinct from PDX-1 and Nkx2.2--
The A2
element has been classified based on its homology to other A elements.
The homeodomain family of transcription factors, such as PDX-1, bind
these A elements (48-51). In addition to homology with the A elements,
the 139 to
113 bp region of rat insulin II also contains a
consensus-binding element (CAAGTG) for the Nkx2 family of transcription
factors (60-62). Nkx2.2, a member of Nkx2 family, is important for
differentiation of pancreatic
-cells (63). Hence, we performed
competition analysis and antibody supershift assay to determine whether
the PDX-1 and Nkx2.2 are present in the A2-specific complexes.
Nuclear extracts from HIT T-15 cells were incubated with radiolabeled
A3 or 139 to
113 probes, in the absence or presence of excess cold
A3 and
139 to
113 bp oligonucleotide. In addition, anti- PDX-1
antibody was added to binding reactions containing the A3 and
139 to
113 probes, and the reactions were analyzed by EMSA. As shown in Fig.
6A, formation of the
A2-binding complex is not inhibited by the presence of excess unlabeled
A3 oligonucleotide. In addition, the anti-PDX-1 antibody neither
prevented the formation of the A2-specific complexes nor altered their
mobility, suggesting that the A2-specific complex does not contain
PDX-1. These observations are consistent with the converse experiment,
where presence of the excess unlabeled
139 to
113 oligonucleotide
did not prevent formation of PDX-1 or other A3-specific complexes (Fig.
6A). We used a similar strategy to identify the presence of
Nkx2.2 in A2-specific complexes. Because the Nkx2.2 binding site is not known, we used the binding element from the rat thyroglobulin gene for
the thyroid transcription factor-1, a Nk2 family member (60). As shown
in Fig. 6B, Nkx2.2 is not part of the A2-specific complexes.
Additionally, oligonucleotides with mutation in the Nkx consensus
sequence at positions
133 to
132 (
133·132m) competed as
effectively as the wild type
139 to
113 oligonucleotide, suggesting
this sequence was not required for binding of A2-specific factors.
These results support the conclusion that the A2 binding factors are
distinct from other homeodomain proteins and the Nkx2 family of
transcription factors. These important observations suggest that the
-cell-specific A2.2 activator may represent a novel factor.
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The Binding Activity of A2-specific Factors Is Not Regulated by
Glucose--
DNA binding activity of the RIPE3b1 activator is induced
in response to an increase in glucose concentrations (28, 30, 31). This
increased binding of the RIPE3b1 activator to the insulin enhancer may
be responsible for the observed induction of insulin gene expression.
To test whether glucose can stimulate binding activity of A2-specific
factors, nuclear extracts were prepared from HIT T-15 cells grown in
the presence of different concentrations of glucose. Equal
concentrations of nuclear extracts from HIT T-15 cells grown in 0.2, 1.0, 5.0, and 22.0 mM glucose were incubated with RIPE3b
and 139 to
113 probes, and binding reactions were analyzed by EMSA
(Fig. 7). As a control, equal concentration of HIT T15 nuclear extracts were also analyzed for binding to Adenoviral major late transcription factor (data not shown).
As we showed earlier (31), increasing concentrations of glucose
significantly induced the binding of the RIPE3b1 activator (Fig. 7) but
had no effect on the binding activity of ubiquitously expressed major
late transcription factor (data not shown). However, when the same
experiment was performed with
139 to
113 probe under identical
conditions, there was no difference in the binding activity of any
A2-specific activators in response to alteration in glucose
concentrations. These results demonstrate that glucose differentially
regulates binding activity of the RIPE3b- and A2-specific activators.
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Nucleotides at Positions 122 and
121 bp Are Essential for the
Binding of Both RIPE3b (C1-A2) and A2-specific Activators--
An
oligonucleotide probe spanning the binding region of both RIPE3b- and
A2-specific factors,
139 to
101, can bind all five (RIPE3b1,
REIP3b2, A2.1, A2.2, and A2.3) DNA-binding complexes (data not shown).
To analyze the role of overlapping binding regions in the binding of
these factors, various mutations in the larger
139 to
101
oligonucleotide (LM) were constructed. To determine the role of
nucleotides in the overlapping region in the formation of C1-A2 and
A2-specific DNA-binding complexes, competition analysis was performed.
HIT T-15 nuclear extract was incubated with the RIPE3b (
126 to
101)
or
139 to
113 probes, in the absence or presence of excess wild
type (
139 to
101, RIPE3b,
116 to
101 and
139 to
113) or
mutant (
110·109LM,
114·113LM,
122·121LM,
125·124LM, and
127·126LM) oligonucleotides. These binding reactions were analyzed
by EMSA, and results are presented in Fig.
8. Presence of excess cold wild type
oligonucleotides in the binding reaction had the expected effect (see
Figs. 2 and 3) on binding of factors to either the RIPE3b or
139 to
113 probe. Mutating nucleotides
110 to
109 bp and
122 to
121
bp in the larger
139 to
101 oligonucleotide prevented these
oligonucleotides from competing with the RIPE3b probe for binding of
RIPE3b1 and RIPE3b2 factors, whereas mutations at
114 to
113 bp,
125 to
124 bp, and
127 to
126 bp positions had no effect on
binding of these factors. These observations confirm that binding of
RIPE3b1 and RIPE3b2 factors depends on nucleotides at positions
110
to
109 bp and
122 to
121 bp in the insulin enhancer, whereas the
other six nucleotides can be substituted without any significant effect on the binding activity. The results from competition analysis for
binding of A2-specific complexes to
139 to
113 probe were extremely
interesting. The formation of the A2-specific complexes, like the C1-A2
complexes, depends on nucleotides at positions
122 to
121 bp in the
insulin enhancer but not on nucleotides at positions
110 to
109 bp.
Furthermore, nucleotides at positions
125 to
124 bp were only
essential for formation of the A2-specific complexes and not for C1-A2
specific complexes. We also used these oligonucleotides as probes and
confirmed the requirement of these nucleotides for binding of the
RIPE3b- and A2-specific complexes (data not shown). These observations
suggest that the rat insulin enhancer region from
139 to
101 bp
represents the binding site for two distinct
-cell-specific factors,
and nucleotides at positions
122 and
121 bp are critical for the
binding of each factor. Furthermore, these observations suggest that
mutations at positions
110 to
109 bp or
125 to
124 bp can
selectively prevent binding of C1-A2 or A2-specific factors,
respectively.
|
Both C1-A2 and A2-specific Activators Positively Regulate Insulin
Gene Expression--
To determine the role of C1-A2 and A2-specific
factors in regulating insulin gene expression, mutations were made in
the rat insulin II enhancer (238 to +2 bp) construct driving
expression of the LUC reporter gene. Wild type (
238 WT LUC) and the
mutant insulin enhancer luciferase reporter constructs (
110·109m
LUC,
122·121m LUC, and
125·124m LUC), were transfected into two
insulin-producing cell lines, HIT T-15 and
TC-3. 48 h after
transfection, cells were harvested, and whole cell extracts was
prepared as described under "Experimental Procedures." Luciferase
activity from insulin reporter constructs was normalized for
transfection efficiency, and the results are presented relative to the
activity of
238 WT LUC construct (Fig.
9).
|
Earlier studies showed that an insulin enhancer construct with
mutations at 112 to
111 bp prevents formation of the RIPE3b1 and
RIPE3b2 complexes and inhibits insulin gene expression (14, 28, 31).
Similarly we observed that mutations at
110 to
109 bp that prevents
formation of C1-A2 specific binding complexes, significantly inhibit
(76 and 87% in HIT T-15 and
TC-3 cell lines, respectively) insulin
gene expression. Mutations at positiona
125 to
124 that selectively
prevent binding of A2-specific factors also inhibited insulin gene
expression by 43 and 37% in HIT T-15 and
TC-3 cell lines,
respectively. Interestingly, mutations that prevent binding of both
C1-A2 and A2-specific binding factors (
122-121 bp) showed
significantly more inhibition (92 and 96% in HIT T-15 and
TC-3 cell
lines, respectively) of insulin gene expression than mutations that
prevented binding of either of these factors. This inhibition of
insulin gene expression was consistently more (about 1.7-2.0-fold)
than that that can be accounted by simple additive contributions of
these factors. Hence, we suggest that C1-A2 (RIPE3b) and A2-specific
binding factors may functionally cooperate with each other and
positively regulate insulin gene expression. Mutations that prevents
binding of these factors to the insulin enhancer can drastically
inhibit insulin gene expression even when the binding site for other
key transcription factors (PDX-1 and BETA2) is unaffected, emphasizing
the importance of these factors on insulin gene expression. We are
currently investigating the mechanism by which C1-A2 and A2-specific
factors contact overlapping DNA-binding sites and cooperatively
activate insulin gene expression.
![]() |
DISCUSSION |
---|
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---|
The present study demonstrates that -cell-specific RIPE3b1 and
A2.2 factors recognize overlapping DNA-binding sites within the insulin
enhancer. These factors can each positively regulate insulin gene
expression as well as cooperatively activate insulin gene expression.
Because the binding activity of RIPE3b1 activator is regulated by
glucose (Fig. 7 and Refs. 28, 30, and 31) and phosphorylation (28),
overlapping binding specificity and functional cooperation between A2.2
and RIPE3b1 activator may play major roles in regulating insulin
expression in response to various metabolic stimulus. These
observations suggest that like RIPE3b1, the A2.2 activator is an
important regulator of insulin gene expression. Initial
characterization indicates that A2-specific binding factors are
distinct from PDX-1 (Fig. 6A). Excess unlabeled A3
oligonucleotide did not prevent formation of A2-specific complexes
(Fig. 6A), suggesting that A2 binding factors might belong
to a different family of transcription factors than the PDX-1 and other
A element binding homeodomain factors. Similarly, anti-Nkx2.2 antibody
did not recognize any of the A2-specific complexes, and mutating the
Nk2 consensus sequence did not affect the formation of A2 complexes.
These results indicate that A2-specific activators are novel and
distinct from the Nk2 and other homeodomain family of transcription factors.
The importance of the RIPE3b region in mediating -cell-specific and
glucose-responsive insulin gene expression is well established (5, 14,
27-31, 37, 41). Although two insulin enhancer elements, C1 and A2,
were described in the RIPE3b region, we were unable to detect any C1
selective binding activity in insulin-producing cells. Our results
demonstrate that C1 binding factors (RIPE3b1 and RIPE3b2) require
sequences present in the A2 element. Similarly, A2-specific factors
require additional nucleotides upstream of the RIPE3b region for their
binding (Figs. 2 and 3). These observations suggest a need to redefine
the enhancer elements in the RIPE3b region. Because we were unable to
identify any C1-specific binding factor, we suggest that designation of
the C1 region as a DNA-binding element may not be accurate. We suggest
that the binding element for RIPE3b1 and RIPE3b2 activator may
accurately be described as C1-A2 or the CA element. Similarly,
additional nucleotides must be included within the A2 element to
describe it as a binding site for A2-specific factors. These criteria
would help interpret earlier studies that describe mutations and
deletions in C1 and A2 elements and their effect on insulin gene
expression (47, 64). For example, Tomonari and co-workers (47) reported
the role of the A2 (GG1) element in regulating human insulin gene expression. Their results obtained from an A2 deletion construct would
not only inhibit activation from the A2-specific factor but also from
the RIPE3b1 activator. Similarly, other mutations in the A2 region
described in the their study would also inhibit activation mediated by
both RIPE3b1 and A2.2 factors. In addition, functional cooperation
between the C1-A2 and A2-specific factors should be considered prior to
interpreting results from earlier studies. We therefore suggest that a
clear designation of the C1-A2 and A2 elements would be helpful in
analyzing the role of this region in insulin gene expression.
Mutational analysis of the RIPE3b region has provided important
information regarding the RIPE3b1 and RIPE3b2 activators. Binding of
these factors requires at least a 16-bp-long region (123 to
107 bp;
Figs. 2 and 8) of the rat insulin II enhancer. Nucleotides critical for
binding of these factors are separated into two distinct regions,
112
to
107 bp and
123 to
117 bp. Although the nucleotides between
these regions can be mutated without any effect on binding activity and
insulin gene expression (Fig. 2A, data not shown, and Ref.
28), attempts to alter spacing between these regions by either
insertion or deletion mutations prevented DNA binding (Fig.
2C). These observations suggest that RIPE3b1 and RIPE3b2
factors not only recognize specific nucleotide sequence but also the
recognize specific structure/organization of these nucleotides. The two
critical nucleotide regions are spaced apart by about one helical turn
of DNA, suggesting that RIPE3b1 and RIPE3b2 activators may recognize
only one surface of the DNA helix. This is consistent with the results
from methylation interference analysis of RIPE3b1 and RIPE3b2 complexes
by Shieh and colleagues (14, 29), showing a strong interference at Gs
107,
108, and
111 and a weak interference at
114 bp.
Furthermore, results from the methylation interference experiment
demonstrated interference at Gs only on the bottom strand and not on
the top strand. Our mutational analysis (Fig. 2) and transient
transfection data (Fig. 9) are consistent with the importance of
nucleotides
107,
108, and
111 bp in insulin gene expression. The
role of weak interference at
114 bp is unclear, because
113 and
114 bp are not critical for binding nor gene expression (data not shown). Also, Zhao and co-workers (28) demonstrated that mutating
114
bp had no effect on insulin gene expression, suggesting that weak
interference at
114 bp is not critical for binding of the RIPE3b1
factor and insulin gene expression. Our results demonstrate the
requirement of a large binding region for the RIPE3b1 and the RIPE3b2
factors (
123 to
107 bp; Figs. 2 and 8); however, it is unclear why
no interference was observed at Gs on the bottom strand at positions
117 and
120 bp.
RIPE3b1 and RIPE3b2 factors have identical DNA binding properties, suggesting that they may belong to the same family of transcription factors. These observations are in contrast with the results described by Shieh and colleagues (14, 29) in that RIPE3b1 and RIPE3b2 complexes have distinct methylation interference profiles and recognize distinct DNA-binding sites. Rip-1, one of the components of the RIPE3b2 complex, was cloned and identified as the hamster homologue of the mouse and human gene Smbp-2, a protein that binds to the immunoglobulin m chain switch region and was also cloned as glial factor-1 (29). Glial factor-binding element (GFE) selectively competes for the formation of the RIPE3b2 complex but not for the RIPE3b1 complex. Although the nucleotide sequence in the GFE element shares some homology with the C1 element, no homology in the A2 region can be observed (29). Similarly, GFE lacks significant homology with that for Smbp-2 (Sm), and Shieh and colleagues (29) suggested that the Rip-1 may bind both sites by recognizing a structure rather than the DNA sequence. However, based on sequence requirements for the binding of RIPE3b1 and RIPE3b2 factors (Fig. 2), it is unclear how the GFE element can selectively inhibit the formation of RIPE3b2 complex. One possible explanation could be that the binding specificity of Rip-1 might be distinct from that of the multi-protein RIPE3b2 complex. Consequently, excess unlabeled GFE oligonucleotide will selectively remove Rip1 from the DNA binding reaction, thereby preventing formation of RIPE3b2 complex.
The salient finding of the present study is that two base pairs are
critical for DNA binding of two -cell-specific transcriptional activators. Several possibilities could explain how two factors can
recognize the same base pairs and not hinder the action of the other
factor. It is possible that the different factors recognize the binding
site on different alleles within a cell or in different cells in a
population. Transient transfection analysis of a cell population does
not discriminate between these possibilities and the binding of both
factors to the same allele. Nonetheless, because C1-A2 and A2-specific
activators cooperatively activate insulin gene expression, these
factors may bind the same element on an allele.
Other examples of transcription factors binding to an overlapping
binding site that result in either activation or inhibition of gene
expression exist. Overlapping binding specificity of SP1 and C/EBPs in
the C/EBP promoter has been implicated in regulating adipocyte
differentiation (65). During normal growth conditions, the SP1 factor
binds to the overlapping C/EBP binding site and prevents binding of
other factors. Differentiation signals decrease levels of the SP1
factor, which facilitates access of C/EBPs to the binding site and
results in the activation of the C/EBP
gene expression (65).
Similarly, there are several examples where both factors recognizing
overlapping binding sites positively activate gene expression (66-69).
Overlapping binding of transcription factors NF-
B and STAT6 can
positively and negatively regulate eotaxin and E-selectin gene
expression, respectively (69, 70). In the E-selectin promoter, the
STAT6 binding site shares 5 bp with the NF-
B binding site, whereas
in the eotaxin gene only 4 bp are shared. Matsukura and colleagues (69)
suggested that this difference in DNA structure may permit simultaneous
binding of these factors to overlapping binding sites and activate
eotaxin gene expression. Similarly, it is possible that the RIPE3b1
activator may bind only on one surface of the C1-A2 region (as
discussed above), which permits simultaneous occupation of overlapping
regions by the A2.2-specific activator, providing a possible mechanism by which both these factors can positively regulate insulin gene expression. Transcription factors recognizing overlapping binding sites
can integrate responses from multiple positive and negative signals by
inhibiting or cooperatively activating gene expression (65-70). Key
insulin gene transcription factors must respond to multiple signals
required to mediate
-cell-specific and glucose-responsive insulin
gene expression as well as regulate pancreatic development and
differentiation of
-cells. Because insulin gene expression is
regulated in response to metabolic and environmental signals, key
insulin gene transcription factors may integrate these responses by
functionally cooperating with each other and/or binding overlapping sites. We suggest that the RIPE3b1 and A2.2 activators may represent one such regulatory unit. Because phosphorylation and alterations in
glucose concentration regulate the activity of the RIPE3b1 factor, we
suggest that RIPE3b1 and A2.2 activators may play a major role in
regulating insulin gene expression in response to metabolic signals. In
addition, overlapping binding specificity of these factors may also
have a role in regulating pancreatic development and/or differentiation
of
-cells. Availability of cloned RIPE3b1 and A2.2 factors would
permit better understanding of the mechanisms by which these factors
bind and regulate insulin gene expression; we are currently pursuing
these objectives.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr Joel Habener (Massachusetts General Hospital, Boston, MA) for IDX-1 antibody; Dr. Larry Moss (Tufts University, Boston, MA) for providing the HIT T-15 nuclear extracts and helpful comments on the manuscript; Drs. Gordon Weir and Susan Bonner-Weir for constructive criticism of the manuscript and for their support; and Martin Olbrot for helpful suggestions. We acknowledge the services provided by the DNA and Tissue culture cores of Joslin Diabetes Center (supported by National Institutes of Health Grant DK-36836 to the Diabetes Endocrinology Research Center).
![]() |
FOOTNOTES |
---|
* This work was supported by a Juvenile Diabetes Foundation International Research Grant, a National Institutes of Health Diabetes Endocrinology Research Center Pilot and Feasibility Study Grant, and a Career Development Award from American Diabetes Association (to A. S.).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: Research Div., Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-264-2756; Fax: 617-732-2650; E-mail: arun.sharma@joslin.harvard.edu.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M008415200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: bp, base pair(s); WT, wild type; LUC, luciferase; EMSA, electrophoretic mobility shift assay(s); GFE, glial factor-binding element.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Dandoy-Dron, F., Monthioux, E., Jami, J., and Bucchini, D. (1991) Nucleic Acids Res. 19, 4925-4936[Abstract] |
2. | Edlund, T., Walker, M. D., Barr, P. J., and Rutter, W. J. (1985) Science 30, 912-916 |
3. | Hanahan, D. (1985) Nature 315, 115-122[Medline] [Order article via Infotrieve] |
4. | Crowe, D. T., and Tsai, M.-J. (1989) Mol. Cell. Biol. 9, 1784-1789[Medline] [Order article via Infotrieve] |
5. |
Stellrecht, C. M. M.,
DeMayo, F. J.,
Finegold, M. J.,
and Tsai, M.-J.
(1997)
J. Biol. Chem.
272,
3567-3572 |
6. | Stein, R. (1993) Trends Endocrinol. Metab. 4, 96-100 |
7. | Sander, M., and German, M. S. (1997) J. Mol. Med. 75, 327-340[CrossRef][Medline] [Order article via Infotrieve] |
8. | German, M., Ashcroft, S., Docherty, K., Edlund, H., Edlund, T., Goodison, S., Imura, H., Kennedy, G., Madsen, O., Melloul, D., Moss, L. G., Olson, L. K., Permutt, M. A., Philippe, J., Robertson, R. P., Rutter, W. J., Serup, P., Stein, R., Steiner, D., Tsai, M.-J., and Walker, M. D. (1995) Diabetes 44, 1002-1004[Medline] [Order article via Infotrieve] |
9. | German, M. S., Moss, L. G., Wang, J., and Rutter, W. J. (1992) Mol. Cell. Biol. 12, 1777-1788[Abstract] |
10. | Peshavaria, M., Gamer, L., Henderson, E., Teitelman, G., Wright, C. V. E., and Stein, R. (1994) Mol. Endocrinol. 8, 806-816[Abstract] |
11. |
Petersen, H. V.,
Serup, P.,
Leonard, J.,
Michelsen, B. K.,
and Madsen, O. D.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10465-10469 |
12. | Ohlsson, H., Thor, S., and Edlund, T. (1991) Mol. Endocrinol. 5, 897-904[Abstract] |
13. | Boam, D. S. W., and Docherty, K. (1989) Biochem. J. 264, 233-239[Medline] [Order article via Infotrieve] |
14. |
Shieh, S-Y.,
and Tsai, M.-J.
(1991)
J. Biol. Chem.
266,
16708-16714 |
15. | Karlsson, O., Edlund, T., Barnett Moss, J., Rutter, W. J., and Walker, M. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8819[Abstract] |
16. | Whelan, J., Cordle, S. R., Henderson, E., Weil, P. A., and Stein, R. (1990) Mol. Cell. Biol. 10, 1564-1572[Medline] [Order article via Infotrieve] |
17. | Ohlsson, H., Karlsson, K., and Edlund, T. (1993) EMBO J. 12, 4251-4259[Abstract] |
18. | Leonard, J., Peers, B., Johnson, T., Ferreri, K., Lee, S., and Montminy, M. R. (1993) Mol. Endocrinol. 1275-1283 |
19. | Miller, C. P., McGehee, R. E., and Habener, J. F. (1994) EMBO J. 13, 1145-1156[Abstract] |
20. |
Offield, M. F.,
Jetton, T. L.,
Labosky, P.,
Ray, M.,
Stein, R.,
Magnuson, M.,
Hogan, B. L. M.,
and Wright, C. V. E.
(1996)
Development
122,
983-985 |
21. |
Ahlgren, U.,
Jonsson, J.,
and Edlund, H.
(1996)
Development
122,
1409-1416 |
22. |
Guz, Y.,
Montminy, M. R.,
Stein, R.,
Leonard, J.,
Gamer, L. W.,
Wright, C. V. E.,
and Teitelman, G.
(1995)
Development
121,
11-18 |
23. | German, M. S., Blanar, M. A., Nelson, C., Moss, L. G., and Rutter, W. J. (1991) Mol. Endocrinol. 5, 292-299[Abstract] |
24. |
Peyton, M.,
Moss, L.,
and Tsai, M. J.
(1994)
J. Biol. Chem.
269,
25936-25941 |
25. | Naya, F. J., Stellrecht, C. M. M., and Tsai, M.-J. (1995) Genes Dev. 9, 1009-1019[Abstract] |
26. |
Naya, F. J.,
Huang, H. P.,
Qiu, Y.,
Mutoh, H.,
DeMayo, F. J.,
Leiter, A. B.,
and Tsai, M.-J.
(1997)
Genes Dev.
11,
2323-2334 |
27. |
Robinson, G. L. W. G.,
Peshavaria, M.,
Henderson, E.,
Shieh, S.-Y.,
Tsais, M.-J.,
Teitelman, G.,
and Stein, R.
(1994)
J. Biol. Chem.
269,
2452-2460 |
28. |
Zhao, L.,
Cissell, M. A.,
Henderson, E.,
Colbran, R.,
and Stein, R.
(2000)
J. Biol. Chem.
275,
10532-10537 |
29. |
Shieh, S. Y.,
Stellrecht, C. M. M.,
and Tsai, M. J.
(1995)
J. Biol. Chem.
270,
21503-21508 |
30. | Sharma, A., Fusco-DeMane, D., Henderson, E., Efrat, S., and Stein, R. (1995) Mol. Endocrinol. 9, 1468-1476[Abstract] |
31. | Sharma, A., and Stein, R. (1994) Mol. Cell. Biol. 14, 871-879[Abstract] |
32. | Dandoy-D, F., Monthioux, E., Jami, J., and Bucchini, D. (1991) Nucleic Acids Res. 19, 4925-4930[Abstract] |
33. | MacFarlane, W. M., Read, M. L., Gilligan, M., Bujalska, I., and Docherty, K. (1994) Biochem. J. 303, 625-631[Medline] [Order article via Infotrieve] |
34. | Melloul, D., Ben-Neriah, Y., and Cerasi, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3865-3869[Abstract] |
35. | German, M. S., and Wang, J. (1994) Mol. Cell. Biol. 14, 4067-4075[Abstract] |
36. |
Odagiri, H.,
Wang, J.,
and German, M. S.
(1996)
J. Biol. Chem.
271,
1909-1915 |
37. | Sharma, A., Olson, L. K., Robertson, R. P., and Stein, R. (1995) Mol. Endocrinol. 9, 1127-1134[Abstract] |
38. | Olson, L. K., Sharma, A., Peshavaria, M., Wright, C. V. E., Towle, H. C., Robertson, R. P., and Stein, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9127-9131[Abstract] |
39. | Olson, L. K., Redmon, J. B., Towle, H. C., and Robertson, R. P. (1993) J. Clin. Invest. 92, 514-519[Medline] [Order article via Infotrieve] |
40. | Zangen, D. H., Bonner-Weir, S., Lee, C. H., Latimer, J. B., Miller, C. P., Habener, J. F., and Weir, G. C. (1997) Diabetes 46, 258-264[Abstract] |
41. |
Poitout, V.,
Olson, L. K.,
and Robertson, R. P.
(1996)
J. Clin. Invest.
97,
1041-1046 |
42. | Jonsson, J., Carlsson, L., Edlund, T., and Edlund, H. (1994) Nature 371, 606-609[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Slack, J. M. W.
(1995)
Development
121,
1569-1580 |
44. | Sharma, A., Zangen, D. H., Reitz, P., Taneja, M., Lissauer, M. E., Miller, C. P., Weir, G. C., Habener, J. F., and Bonner-Weir, S. (1999) Diabetes 48, 507-513[Abstract] |
45. | Hwung, Y.-P., Gu, Y.-Z., and Tsai, M.-J. (1990) Mol. Cell. Biol. 10, 1784-1788[Medline] [Order article via Infotrieve] |
46. |
Boam, D. S. W.,
Clark, A. R.,
and Docherty, K.
(1990)
J. Biol. Chem.
265,
8285-8296 |
47. | Tomonari, A., Yoshimoto, K., Tanaka, M., Iwahana, H., Miyazaki, J., and Itakura, M. (1996) Diabetologia 39, 1462-1468[CrossRef][Medline] [Order article via Infotrieve] |
48. | Karlsson, O., Thor, S., Norberg, T., Ohlsson, H., and Edlund, T. (1990) Nature 344, 879-882[CrossRef][Medline] [Order article via Infotrieve] |
49. | Emens, L. A., Landers, D. W., and Moss, L. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7300-7304[Abstract] |
50. | Ahlgren, U., Pfaff, S. L., Jessell, T. M., Edlund, T., and Edlund, H. (1997) Nature 385, 257-260[CrossRef][Medline] [Order article via Infotrieve] |
51. |
Rudnick, A.,
Ling, T. Y.,
Odagiri, H.,
Rutter, W. J.,
and German, M. S.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12203-12207 |
52. | German, M. S., Wang, J., Chadwick, R. B., and Rutter, W. J. (1992) Genes Dev. 6, 2165-2176[Abstract] |
53. |
Ohneda, K.,
Mirmira, R. G.,
Wang, J.,
Johnson, J. D.,
and German, M. S.
(2000)
Mol. Cell. Biol.
20,
900-911 |
54. | Peshavaria, M., Henderson, E., Sharma, A., Wright, C. V. E., and Stein, R. (1997) Mol. Cell. Biol. 17, 3987-3996[Abstract] |
55. |
Efrat, S.,
Surana, M.,
and Fleischer, N.
(1991)
J. Biol. Chem.
266,
11141-11143 |
56. | Efrat, S., Fusco-DeMane, D., Lemberg, H., Al Emran, O., and Wang, X. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3576-3580[Abstract] |
57. | Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract] |
58. | Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve] |
59. | Behre, G., Smith, L. T., and Tenen, D. G. (1999) BioTechniques 26, 24-28[Medline] [Order article via Infotrieve] |
60. | Damante, G., Fabbro, D., Pellizzari, L., Civitareale, D., Guazzi, S., Polycarpou-Schwartz, M., Cauci, S., Quadrifoglio, F., Formisano, S., and Di Lauro, R. (1994) Nucleic Acids Res. 22, 3075-3083[Abstract] |
61. |
Weiler, S.,
Gruschus, J. M.,
Tsao, D. H. H., Yu, L.,
Wang, L.-H.,
Nirenberg, M.,
and Ferretti, J. A.
(1998)
J. Biol. Chem.
273,
10994-11000 |
62. | Pllizzari, L., Tell, G., Fabbro, D., Pucillo, C., and Damante, G. (1997) FEBS Lett. 407, 320-324[CrossRef][Medline] [Order article via Infotrieve] |
63. |
Sussel, L.,
Kalamaras, J.,
Hartigan-O'Connor, D. J.,
Meneses, J. J.,
Pedersen, R. A.,
Rubenstein, J. L. R.,
and German, M. S.
(1998)
Development
125,
2213-2221 |
64. |
Lu, M.,
Seufert, J.,
and Habner, J.
(1997)
J. Biol. Chem.
272,
28349-28359 |
65. |
Tang, Q.-Q.,
Jiang, M.-S.,
and Lane, M. D.
(1999)
Mol. Cell. Biol.
19,
4855-4865 |
66. |
Raychowdhury, R.,
Zhang, Z.,
Hocker, M.,
and Wang, T. C.
(1999)
J. Biol. Chem.
274,
20961-20969 |
67. | Xiao, S., Matsui, K., Fine, A., Zhu, B., Marshak-Rothstein, A., Widom, R. L., and Ju, S.-T. (1999) Eur. J. Immunol. 29, 3456-3465[CrossRef][Medline] [Order article via Infotrieve] |
68. |
Thomas, M. A.,
Mordvinov, V. A.,
and Sanderson, C. J.
(1999)
Eur. J. Biochem.
265,
300-307 |
69. |
Matsukura, S.,
Stellato, C.,
Plitt, J. R.,
Bickel, C.,
Miura, K.,
Georas, S. N.,
Casolaro, V.,
and Schleimer, R. P.
(1999)
J. Immunol.
163,
6876-6883 |
70. |
Benett, B. L.,
Cruz, R.,
Lacson, R. G.,
and Mannings, A. M.
(1997)
J. Biol. Chem.
272,
10212-10219 |