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* Dipartimento di Medicina Sperimentale e Clinica ‘G. Salvatore’, Cattedra di Endocrinologia, Università degli Studi di Catanzaro ‘Magna Græcia’, Catanzaro, Italy;
Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Università degli Studi di Trieste, Trieste, Italy;
Department of Medicine, Division of Diabetes and Endocrine Research, Mount Zion Medical Center, University of California San Francisco, San Francisco, California 94120, USA; and
§ Dipartimento di Medicina Sperimentale e Clinica ‘G. Salvatore’, Cattedra di Medicina Interna, Università degli Studi di Catanzaro ‘Magna Græcia’, Catanzaro, Italy
1Correspondence: Cattedra di Endocrinologia, Policlinico ‘Mater Domini’, Via T. Campanella 115, 88100 Catanzaro, Italy. E-mail: antonio.brunetti{at}tin.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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15 kDa protein, either identical to or
highly related to the architectural transcription factor HMGI(Y), has
now been identified, suggesting an essential role for HMGI(Y) in
regulating insulin receptor gene transcription. Direct evidence of
HMGI(Y) insulin receptor promoter interactions is provided by
functional analysis with the CAT reporter gene and by hormone binding
studies in cells expressing HMGI(Y) antisense RNA. In these
experiments, antisense HMGI(Y) specifically inhibits insulin receptor
promoter function and insulin receptor protein expression, indicating
that HMGI(Y) is required for proper transcription of insulin receptor
gene. Moreover, our data consistently support the hypothesis that a
putative defect in this nuclear binding protein may cause insulin
receptor dysfunction with subsequent impairment of insulin signaling
and action.—Brunetti, A., Manfioletti, G., Chiefari, E., Goldfine,
I. D., Foti, D. Transcriptional regulation of human insulin
receptor gene by the high-mobility group protein HMGI(Y).
Key Words: insulin receptor promoter • transcription factors • CAT reporter gene
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. The insulin receptor serves a critical role both
in directing insulin to specific target tissues and in initiating the
response of these tissues to the hormone (3)
. Although
insulin receptors are present on the surface of virtually all cells,
data suggest that the insulin receptor can be regulated by a wide
variety of factors and under different environmental conditions
(4
5
6)
. For example, glucocorticoids enhance transcription
of the insulin receptor gene, whereas insulin down-regulates its own
receptor (7
, 8)
. Also, insulin receptor mRNA levels
increase after the differentiation of BC3H-1 mouse myoblasts and 3T3-L1
fatty fibroblasts (4
, 9
, 10)
. In addition, developmental
regulation of insulin receptor gene expression has been documented in
Drosophila (11)
.
The insulin receptor is usually expressed at low levels in all cell
types but at higher levels in the classical insulin target tissues
muscle, liver, and fat (2)
. However, little is known about
the regulatory mechanisms controlling insulin receptor at the level of
gene expression. The insulin receptor is of major importance in certain
states of insulin resistance in humans, in which qualitative and
quantitative abnormalities of the receptor may lead to defective
transmembrane signaling (12
13
14
15
16)
. Moreover, even though it
is an open question whether insulin receptor plays a critical role in
aging and longevity in mammals as well as in Caenorhabditis
elegans (17)
, inhibition of the neuronal insulin
receptor causes Alzheimer-like disturbances in rats (18)
.
Involvement of insulin receptor has been documented in human neoplasias
in which abnormalities of insulin receptor expression and/or function
have been reported (19
20
21
22)
. Thus, regulation of insulin
receptor gene expression seems to be important from both biological and
clinical aspects.
In eukaryotes, the binding of proteins to specific DNA sequences is
critical for the regulation of many nuclear events, such as replication
and transcription. Initiation of mRNA synthesis is a major control
point in gene expression. Gene expression in eukaryotic cells is
controlled by nuclear regulatory proteins (trans-acting
factors) that modulate the transcription of genes and gene networks
(23
24
25)
. During the last decades, unique DNA sequences
that are involved in gene regulation (cis elements) have
been identified, and this process has led to the detection and
characterization of DNA regulatory proteins (25)
. As a
step toward understanding the molecular basis of regulation of insulin
receptor gene expression, the promoter region of the human insulin
receptor gene has been identified and analyzed by several groups
(26
27
28
29
30
31
32
33
34)
. This region extends over 1800 bases 5' upstream
from the insulin receptor gene ATG codon and is extremely GC-rich,
containing a series of GGGCGG repeats that are putative binding sites
for the mammalian transcription factor Sp1 (25)
. It has
neither a TATA box nor a CAAT box, reflecting the common features for
the promoters of constitutively expressed genes (so-called housekeeping
genes).
Previously we identified two similar or identical DNA nuclear binding
proteins that specifically interacted with two unique AT-rich sequences
of the insulin receptor gene promoter, C2 and E3 (between nucleotides
-782 to -800 and -1740 to -1775, respectively), which had in
vivo transcriptional activity (32)
. These nuclear
binding proteins were readily detected in cells containing high levels
of insulin receptor, but were almost undetectable in cells with low
insulin receptor content (32)
. Thus, we have hypothesized
that these DNA binding proteins interacting with these AT-rich
sequences of the insulin receptor gene promoter may have major
importance in regulating the expression of the insulin receptor in
target tissues.
We show data on purification and molecular characterization of these
nuclear binding proteins, using a cell line that readily expresses both
insulin receptor DNA binding proteins: IM-9 human lymphocytes. Analyses
of protein–DNA interactions with the positive-acting cis
elements (C2 and E3), combined with immunoblot analyses of nuclear
protein from IM-9 cells, identified a unique insulin receptor DNA
binding protein (IR-DBP) highly related to the high-mobility group
(HMG) protein: HMGI(Y). HMGI(Y) is a distinct member of the HMG protein
family that has been demonstrated to specifically interact with AT-rich
regions of DNA and to contribute to the transcriptional regulation of
many mammalian genes by interacting with different transcription
factors (35
, 36)
. Here we demonstrate for the first time
that IR-DBP and HMGI(Y) are indistinguishable in their DNA binding
properties, immunological reactivity, and in vivo activation
of transcription. We show that inhibition of HMGI(Y) in cells naturally
expressing relatively high insulin receptor levels significantly
reduces cell surface expression of the insulin receptor. Conversely,
overexpression of HMGI(Y) in transfected cultured cells with relatively
low insulin receptor content increases insulin receptor protein
expression, indicating that HMGI(Y) may play an essential role in
regulating the transcriptional activity of the insulin receptor gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were cultivated in RPMI
1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco
Laboratories, Grand Island, N.Y.), 2 mM glutamine, penicillin (100
U/ml), and streptomycin (100 µg/ml) in a humidified 5%
CO2 atmosphere at 37°C. Nuclear extracts from
IM-9 cells were prepared, with only minor modification, according to
Dignam et al. (37)
. Briefly, cells were washed with
phosphate-buffered saline (PBS) at 4°C and harvested into buffer A
[(10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl,
0.5 mM dithiothreitol (DTT)]. After centrifugation (1200 rpm for 10
min), cells were resuspended in buffer A and kept on ice for 10 min.
Cells were then homogenized with a glass Dounce (10 strokes with a type
B pestle) and centrifuged at 2000 rpm to pellet nuclei. Nuclei were
resuspended in buffer B [(20 mM HEPES, pH 7.9, 0.55 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT,
0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml antipain, 1
µg/ml leupeptin)], and homogenized as above. The suspension was
centrifuged for 1 h at 35,000 rpm and the supernatant dialyzed
against 100 volumes of buffer C (15 mM HEPES, pH 7.9, 20% glycerol, 1
mM EDTA, 40 mM KCl, 0.5 mM DTT, 0.5 mM PMSF) for at least 12 h at
4°C. The dialysate was centrifuged at 35,000 rpm for 30 min and the
resulting supernatant, designated the nuclear extract, was frozen in
liquid nitrogen and stored at -80°C. Final protein concentration in
the extract was determined by the Bradford protein assay (Bio-Rad
Laboratories, Hercules, Calif.). Typically, 20 liters of IM-9 cells
yielded 200 mg of nuclear protein. HepG2 human hepatoma cells
(38)
and HTC rat hepatoma cells (39
, 40)
were
cultivated in DME medium H-21 with 10% FBS. Chinese hamster ovary
(CHO) cells were maintained in Ham’s F-12 medium containing 10% FBS
(40)
.
Oligonucleotides and EMSA
45-mer double-stranded oligonucleotides corresponding to
C2 (5'-TATGAACAAAATAGCAAAATGGTAGAGAAAGGATCTG-TGCCGCTG-3') and
E3 (5'-GAGAAAAACTCCATCTAAAAAAAAAAAAAAAAAAAAAAAAAAACA-3')
sequences of the 5' regulatory region of the human insulin receptor
gene were synthesized chemically (BioTeZ, Berlin, Germany), end-labeled
with (32P)dATP using DNA polymerase I
(41)
and used in electrophoresis mobility shift assay
(EMSA) as described previously (32)
. 27-mer
double-stranded oligonucleotides containing wild-type
(5'-GGGAAATTCCGTGGGAAATTCCGAGCT-3') and mutated
(5'-GGGAGATTCCGTGGGAGATTCCGAGCT-3') PRDII element
motif of the ß-interferon promoter (42)
were 5' end
labeled with (
-32P)ATP and T4 polynucleotide
kinase (Sigma-Aldrich, Milwaukee, Wis.) and used in EMSA. Unlabeled
22-mer double-stranded oligonucleotide containing consensus binding
site for Sp1 transcription factor (Santa Cruz Biotechnology, Santa
Cruz, Calif.) was used in competition studies.
Purification of IR-DBP and SDS-PAGE
Prepacked HiTrap heparin-Sepharose column (1 ml) was
equilibrated with start buffer (0.01 M sodium phosphate with a neutral
pH 7) and used under conditions suggested by the supplier
(Pharmacia, Piscataway, N.J.). IM-9 nuclear protein (2 mg) was applied,
washed with start buffer, and eluted with a 10 ml gradient from 0.1 to
1.0 M NaCl in start buffer. Eluted samples were desalted using a
prepacked HiTrap Desalting column (Pharmacia) and analyzed by EMSA for
protein–DNA binding activity. Fractions containing the protein(s) of
interest were pooled and subjected to further purification by DNA
affinity chromatography, according to Kadonaga and Tijan
(43)
. Double-stranded oligonucleotides corresponding to C2
and E3 sequences of the insulin receptor gene promoter with TCGA 5'
overhanging ends were coupled to cyanogen bromide-activated Sepharose
4B (Pharmacia) at a concentration of 1.0 nmol of DNA/ml of Sepharose
gel and chromatography was carried out in binding buffer (15 mM HEPES,
pH 7.9, 1 mM EDTA, 40 mM KCl, 0.5 mM DTT, 5% glycerol). The load was
mixed with nonspecific competitor calf thymus DNA (100 µg) (Sigma,
St. Louis, Mo.) and applied dropwise to the affinity resin. The column
was washed with binding buffer, step eluted with the same buffer
containing successively 0.1 M, 0.2 M, 0.6 M NaCl; protein–DNA
complexes were visualized by EMSA.
IM-9 affinity-purified IR-DBP was resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using the
Tricine SDS-discontinuous buffer system of Schagger and Von Jagov
(44)
, and visualized by the PLUSONE Silver-Staining Kit
Protein (Pharmacia), according to the manufacturer’s instructions.
Western transfers
Aliquots of IM-9 cell nuclear extracts and purified protein were
resolved on 16.5% SDS-PAGE, electro-transferred onto 0.2 µm PVDF
membrane (Immun-Blot, Bio-Rad Laboratories) using a mini
electrophoretic transfer cell (Trans-blot, Bio-Rad Laboratories), and
detected by DNA and antibody probes. Southwestern blotting experiments
were performed on proteins transferred onto membranes that were
denatured/renatured as described by Celenza and Carlson
(45)
. This treatment increased the recovery of active
molecules. To assay for DNA binding activity, membranes were first
immersed for 12 h in blocking solution (2.5% non-fat dry milk, 25
mM HEPES, pH 8.0, 1 mM DTT, 10% glycerol, 50 mM NaCl, 1 mM EDTA) at
4°C, then incubated in TNE-50 (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM
EDTA, 1 mM DTT) containing 32P-DNA binding site
probe (either C2 or E3) (2x106 cpm/ml) and
poly(dI-dC) (1.0 mg/ml). After 1 h at room temperature, the
filters were washed with TNE-50, patted dry and exposed to Kodak X-ray
film.
For Western blot analysis, membranes from Southwestern blots were first
immersed for 2 h in blocking solution (5% non-fat dry milk in
PBS), then probed with an antibody raised against a peptide specific
for HMGI(Y) (46)
. After 2 h at room temperature, goat
anti-rabbit IgG horseradish peroxidase conjugate (Amersham, Arlington
Heights, Ill.) was added (1 h at room temperature) and immune complexes
were visualized by enhanced chemiluminescence (ECL, Amersham).
For the immunoprecipitation studies, 10 µl of anti-HMGI(Y) antibody
were incubated with 200 ng of affinity-purified IR-DBP for 2 h
nutating at 4°C. Protein A beads (50 µl; Pharmacia) were added to
the mixture and incubated overnight nutating at 4°C. Complexes were
recovered, and the beads were washed twice with 0.2 M NaCl wash buffer
(15 mM HEPES, pH 7.9; 1 mM EDTA; 40 mM KCl; 5% glycerol; 5 mM DTT) and
once with 0.1 M NaCl wash buffer. Specifically bound material and
supernatant fractions were analyzed by SDS-PAGE and immunoblotting.
Transfection studies
For the chloramphenicol acetyltransferase (CAT) assay,
recombinant vectors containing C2 (pCAT-C2) or E3 (pCAT-E3) insulin
receptor promoter sequence, both upstream from the bacterial CAT
reporter gene, were transiently transfected into HepG2 cells and CAT
activity assayed as described previously (32)
. Briefly,
16 h before transfection, HepG2 cells were plated in 60 mm dishes
at a density of 5 x 105. Plasmid constructs
were then transfected into cells by the calcium phosphate precipitation
method (47)
. Forty-eight hours after transfection, cell
extracts were prepared by three cycles of freezing-thawing, heated to
65°C for 10 min to inactivate endogenous deacetylases, then assayed
for CAT activity with 0.25 mCi [14C]
chloramphenicol in a 125-µl reaction. Quantitation of CAT activity
was done by liquid scintillation counting (LSC) of the two major
acetylated forms of 14C-labeled chloramphenicol,
as described previously (32)
. As internal control of
transfection efficiency, ß-galactosidase activity was measured
(41)
. For antisense HMGI(Y) experiments, C2- or E3-containing vectors were cotransfected into HepG2 cells with
increasing concentrations of expression plasmid pcDNA1 containing the
HMGI(Y) cDNA either in the sense (s) or antisense (as) orientation
(48)
.
For 125I-insulin binding studies, HepG2, HTC, and
CHO cells were plated in 60 mm dishes and were 50%-60% confluent
at the time of transfection. The cells were refed with fresh media
3 h before transfection. Increasing concentrations of the
expression plasmid containing the HMGI(Y) cDNA, either in the sense (s)
or antisense (as) orientation, were then transfected into cells as
described above. Seventy-two hours after transfection, the cells were
washed three times with PBS and 125I-labeled
insulin binding was measured as described previously (4)
.
IM-9 cells (4x106 cells per plate) were
transfected by the DEAE dextran method (49)
in 60 mm
dishes using 5 µg of HMGI(Y) expression plasmid pcDNA1 or vector DNA
and 1 µg of ß-galactosidase control plasmid. The cells were
harvested 72 h after transfection and
125I-insulin binding was measured, as described
previously (50)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 8)
. We have previously demonstrated
that IR-DBP is readily expressed in these cells (32)
.
Accordingly, nuclear extracts from this cell line were prepared and
used for identification and purification of this nuclear binding
protein. Initially, IM-9 nuclear extracts were loaded onto a
heparin-Sepharose column and eluted with buffer containing increasing
concentrations of NaCl (Fig. 1A
). DNA binding activity in salt-eluted fractions was
characterized by EMSA. The bulk of DNA binding activity detectable with
E3 probe eluted between 0.5 and 0.8 M NaCl. Identical results were
obtained with the C2 probe (data not shown).
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Next, fractions from the heparin-Sepharose column containing the
protein(s) of interest were pooled and subjected to further
purification with C2 or E3 site-specific DNA affinity column (Fig. 1B
). In all preparations, sonicated calf thymus competitor
DNA was combined with the sample and the DNA–protein mixture in
solution was applied to the affinity column (43)
. The
protein–DNA mixture was eluted with steps of 0.1–0.6 M NaCl and
binding activity was monitored by EMSA, in the absence of competitor
DNA. The peak of sequence-specific DNA binding activity detected with
E3 probe eluted at 0.6 M NaCl. Identical results were obtained with the
C2 probe (data not shown).
Purification efficiency was analyzed by SDS-PAGE (Fig. 1C
).
An identical protein species with a mass of 15 kDa was detected in
silver-stained gel of active fractions from either C2- or E3-DNA
affinity chromatography.
Because of the low molecular mass of affinity-purified IR-DBP (15
kDa), in addition to its ability to specifically bind AT-rich regions
of DNA, we investigated the relationship between the IR-DBP and
HMGI(Y), a small basic protein that binds specifically to AT-rich
sequences through the minor groove of DNA (35)
. Thus, DNA
binding studies using the affinity-purified IR-DBP and the HMGI(Y)
nuclear protein were conducted.
EMSA with affinity-purified IR-DBP and pure HMGI(Y)
Analyses of protein–DNA interactions with pure IR-DBP and HMGI(Y)
with the positive-acting cis elements C2 and E3 of the
insulin receptor promoter revealed similar electrophoretic migration
characteristics (Fig. 2
, lanes 2, 3, 8, 9). EMSA was performed using the well-characterized
PRDII element of the ß-interferon promoter as probe
(42)
. It has been shown that HMGI(Y) protein binds
specifically to this element (42
, 48)
. Therefore, binding
of IR-DBP and HMGI(Y) nuclear protein to PRDII was studied. As with C2
and E3 probes, binding of IR-DBP to PRDII probe produced a protein–DNA
complex, whose mobility was identical to that of HMGI(Y) protein (Fig. 2
, lanes 14, 15). Neither HMGI(Y) nor IR-DBP showed detectable retarded
bands when a mutated PRDII element was used as probe (Fig. 2
, lanes 17,
18). Similarly, no retarded bands were observed using either mutant C2
or E3 probe (data not shown). To determine whether the IR-DBP is
related to HMGI(Y), we carried out antibody supershift experiments with
anti-HMGI(Y) antibody (46)
. As with pure HMGI(Y) protein,
protein–DNA complexes from IR-DBP were recognized and supershifted to
a slower migrating form by anti-HMGI(Y) antibody (Fig. 2
, lanes 4, 5,
10, 11), indicating an antigenic similarity between HMGI(Y) and the
IR-DBP detected with C2 and E3 site-specific DNA affinity columns.
Control (unrelated rabbit serum IgG) antibody did not alter the
mobility of the complex (Fig. 2
, lanes 6, 12). Taken together, these
results indicate that HMGI(Y) and IR-DBP are highly related proteins.
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To determine the specificity of DNA–protein binding, competition
assays were performed. A 50-fold molar excess of either unlabeled C2,
E3, or PRDII oligonucleotide significantly reduced the binding of
labeled C2, E3, or PRDII to DNA binding proteins, respectively
(Fig. 3
). Moreover, either unlabeled C2 or unlabeled E3 competitively inhibited
the binding of PRDII to nuclear proteins. In concert with this
observation, unlabeled PRDII inhibited the binding of labeled C2 or E3
to nuclear proteins (Fig. 3)
. A 50-fold molar excess of an unrelated
oligonucleotide containing the consensus binding site for the
transcription factor Sp1 did not compete for binding of nuclear
proteins to oligonucleotides C2, E3, and PRDII (Fig. 3)
.
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Western blot analysis of affinity-purified IR-DBP and pure HMGI(Y)
Direct evidence that the HMGI(Y) nuclear protein was responsible
for the sequence-specific insulin receptor DNA binding activity was
obtained by screening Western blots with DNA and antibody probes. For
Southwestern blotting analysis, two equivalent transfers were probed
with either labeled C2 or E3 element of the insulin receptor gene.
Figure 4A
shows the result obtained with the E3 probe, using crude
(lane 1) and IM-9 affinity-purified (lane 2) nuclear protein and pure
HMGI(Y) nuclear protein (lane 3). A major band labeled DNA-P was
observed in all three lanes. An identical result was obtained with C2
probe (data not shown). The same transfers were then probed with
anti-HMGI(Y) antibody. A 15 kDa protein band corresponding to the
species DNA-P detected with DNA probe was observed in each lane (Fig. 4B
, lanes 4–6), indicating again an antigenic similarity
between HMGI(Y) and the IR-DBP detected within C2 and E3 complexes.
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These observations were supported further by tests of
immunoprecipitation and immunodepletion, showing that the 15 kDa
IR-DBP detected in nuclear extracts from IM-9 cells and the HMGI(Y)
nuclear factor did exhibit identical immunological properties. Figure 4C
shows a Western blot analysis of C2 and E3
affinity-purified IR-DBP after immunoprecipitation and immunodepletion
using anti-HMGI(Y) antibody. The 15 kDa protein detected with C2
(lane 7) and E3 (lane 8) comigrated with pure recombinant HMGI(Y)
protein (lane 13). Control antibody failed to precipitate the same
protein species. In this latter case, an identical 15 kDa protein
species was detected in the supernatant of the same fractions (lanes 9
and 10), whereas it was barely detectable in the supernatant of
fractions after depletion with anti-HMGI(Y) antibody (lanes 11 and 12).
Inhibition of CAT activity by antisense HMGI(Y) RNA
The results of DNA binding studies and Western blot analyses
presented above indicate that the 15 kDa IR-DBP and the HMGI(Y)
nuclear protein are highly related, or identical, proteins. To test
whether HMGI(Y) interacted with and regulated insulin receptor promoter
function in intact cells, the ability of both C2 and E3 to drive the
expression of the CAT gene was measured in HepG2 human hepatoma cells
in which the expression of the HMGI(Y) protein was inhibited by
antisense RNA (32
, 48
, 51)
. Increasing concentrations of
the HMGI(Y) antisense expression plasmid inhibited the activity of C2
and E3 promoter elements as judged by the decrease in CAT activity in
transfected cells (Fig. 5
). Inhibition by the HMGI(Y) antisense plasmid was specific, since no
inhibition of CAT activity was observed when C2- or E3-containing
vectors were transfected into cells in the presence of expression
vector containing HMGI(Y) cDNA in the sense orientation (Fig. 5)
. To
ensure that inhibition by HMGI(Y) antisense plasmid was not related to
a nonspecific effect, we transfected the pCAT-Promoter vector, a
positive control having the simian virus 40 gene promoter upstream of
the CAT coding region, into HepG2 and exposed the cells to HMGI(Y)
antisense expression plasmid. No inhibition of this CAT reporter
plasmid was observed (data not shown). As shown by Western blot
analysis of cellular protein from HepG2 transfected cells, CAT activity
correlated with HMGI(Y) expression levels (Fig. 5)
.
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Insulin binding studies
The above results strongly suggest that HMGI(Y) plays an essential
role in the regulation of the human insulin receptor gene. We obtained
direct evidence for this conclusion by measuring
125I-insulin binding to HepG2 and IM-9 cells, two
cell lines with relatively high insulin receptor expression levels
(32)
. Exposure of these cells to the HMGI(Y) antisense
expression plasmid inhibited cell surface expression of the insulin
receptor in a dose-dependent manner (Fig. 6
). 125I-labeled insulin binding to HepG2 and IM-9
transfected cells was decreased to 30% and 35% of untransfected
control cells, respectively. The decrease in
125I-labeled insulin binding paralleled the
decrease in C2 and E3 promoter activity observed in HepG2 cells and was
specific, since no inhibition of insulin binding was observed in cells
transfected with HMGI(Y) cDNA in the sense orientation.
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Next, we carried out transfection experiments in HTC rat hepatoma cells
and CHO cells, two cell lines with relatively low insulin receptor
expression (39
, 40)
. Transfection of these cells with the
expression vector containing HMGI(Y) cDNA in the sense orientation was
followed by a six- to eightfold increase in specific
125I-insulin binding (Fig. 7
). Transfection efficiency was normalized to the expression of
ß-galactosidase from a separate reporter plasmid. In contrast,
transfection of the antisense HMGI(Y) expression plasmid had no effect
on cell surface expression of the insulin receptor (Fig. 7)
. These
experiments provide direct evidence that HMGI(Y) protein is required
for proper expression of the insulin receptor. Consistent with this,
125I-labeled insulin binding to these cells
correlated with HMGI(Y) protein expression, as shown by Western blot
analysis of cellular protein from either IM-9, HepG2, HTC, or CHO
transfected cells (Figs. 6
, 7)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 53)
. Several lines of
evidence suggest that AT-rich sequences of DNA constitute a robust
nucleation site that promotes transcription initiation in the absence
of conventional promoter elements (54
55
56
57
58)
. It has been
proposed that transcription activation at this site may be due to
structural perturbation, which may result in bending of the DNA that
promote the interaction between transcription factors and the basal
transcriptional machinery (48)
. DNA binding protein
interactions with AT-rich regions of promoters have been reported in
mammalian genes (36
, 59
, 60)
.
We have previously identified two closely related DNA nuclear binding
proteins that specifically interact with two unique functional AT-rich
sequences of the 5' regulatory region of the insulin receptor gene
(32)
. In this report, the identities of both nuclear
proteins have been determined. Analysis of protein–DNA interactions
with the positive-acting cis elements, C2 and E3, combined
with Western and Southwestern blotting analyses, identified a unique
DNA binding protein highly related to the HMG family, which is composed
of abundant and highly conserved nuclear proteins that are thought to
play important roles in the transcriptional regulation of mammalian
genes (35)
. An important member of this family is HMGI(Y),
which refers to two proteins, HMGI and HMGY, closely related splice
variants of the same gene (61)
. HMGI(Y) is an
architectural transcription factor that binds to AT-rich regions in the
minor groove of DNA via short basic domains, termed AT hooks, and
functions mainly as a specific cofactor for gene activation (35
, 48)
. HMGI(Y) by itself has no intrinsic transcriptional
activity; rather, it has been shown to trans-activate
promoters through mechanisms that facilitate the assembly and stability
of stereospecific DNA–protein complexes (enhanceosomes) that drive
gene transcription (35
, 48)
. HMGI(Y) performs this task by
modifying DNA conformation and by recruiting transcription factors to
the transcription start site, facilitating DNA–protein and
protein–protein interactions (35
, 48)
.
Our data on molecular and biochemical characterization show unequivocally that the identified IR-DBP displays many properties common to HMG protein family. First, EMSA (using IM-9 affinity-purified IR-DBP) showed the same electrophoretic migration characteristics of pure recombinant HMGI(Y) protein with either the C2 or E3 element of the insulin receptor gene. Second, a similar behavior was observed when EMSA was performed using the PRDII element of the ß-interferon promoter as probe. Third, antibody directed against HMGI(Y) significantly reduced the formation of DNA–protein complexes on the C2 and E3 oligonucleotides, suggesting that a protein antigenically related to HMGI(Y) binds to insulin receptor promoter in vitro.
The above findings were supported by Western and Southwestern
blotting analyses. Anti-HMGI(Y) antibody specifically recognized a
major protein species with mass of 15 kDa in both unpurified and
IM-9 affinity-purified nuclear protein. As in protein–DNA interaction
analysis, in Western blot analysis this immunoreactive protein
exhibited identical electrophoretic and immunological properties as
recombinant HMGI(Y) protein. In addition, Southwestern studies revealed
that the 15 kDa protein species present in IM-9 cells binds either
probe C2 or E3, as does recombinant HMGI(Y).
We have shown before that both C2 and E3 sequences of the regulatory
region of the insulin receptor gene have functional activity and act as
promoter elements driving transcription of the CAT reporter gene when
transfected into mammalian cells (32)
. This observation
agrees closely with the results reported in a previous work, in which
deletional analysis of the human insulin receptor promoter confirms the
contribution of C2- and E3-containing regions to overall promoter
activity (29)
. Here, direct evidence of HMGI(Y) insulin
receptor promoter interactions is provided by reporter gene analysis
with the CAT reporter gene and by hormone binding studies in cells in
which the expression of the HMGI(Y) protein was inhibited by antisense
RNA. We have shown that antisense HMGI(Y) RNA significantly decreased
the level of C2 and E3 promoter function in intact cells, indicating
that HMGI(Y) protein is required for proper transcription of insulin
receptor gene. This result is consistent with our previous published
data indicating that the ability of C2 and E3 to drive transcription in
intact cells was almost completely abolished by preventing the binding
of nuclear proteins to AT-rich sequences of DNA using the synthetic
polynucleotide poly(dA-dT) (32)
. In concert with these
findings, we have also observed that in HepG2 hepatocytes and IM-9
lymphocytes, two cell lines with relatively high insulin receptor
expression, transfection of antisense HMGI(Y) significantly decreases
insulin receptor binding. In contrast, in HTC and CHO cells, two cell
lines with relatively low insulin receptor content, overexpression of
HMGI(Y) significantly increases cell surface expression of the insulin
receptor.
Taken together, these observations strongly support the conclusion that
the identified nuclear binding protein for the 5' regulatory region of
the human insulin receptor gene is identical to the HMG protein,
HMGI(Y). To our knowledge, the present finding that HMGI(Y) is
specifically required for efficient expression of the insulin receptor
gene, constitutes the first demonstration revealing the potential of
this protein to modulate insulin receptor gene promoter. Studies are in
progress to better define the role of HMGI(Y) during transcriptional
regulation of the insulin receptor gene. We have preliminary evidence
suggesting that this protein may act on the insulin receptor promoter
as an architectural factor necessary for the formation of a
transcriptionally active multiprotein–DNA complex involving the Sp1
nuclear transcription factor. A similar mechanism has already been
described for the transcriptional activation of the human
interferon-ß gene (62)
. Thus, these studies add further
insight into the molecular mechanisms that control insulin receptor
gene expression. Moreover, they provide a paradigm to suggest another
type of molecule, HMGI(Y), which should be searched for in
investigations designed to evaluate the causes of impairment of insulin
receptor function and/or expression. This observation has relevance to
the pathophysiology of insulin-resistant syndromes and other
pathological states in humans.
|
ACKNOWLEDGMENTS |
|---|
Received for publication April 12, 2000. Accepted for publication July 27, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B mediated transcriptional activation is enhanced by the architectural factor HMGI-C. Nucleic Acids Res 26,1433-1439B-dependent virus induction of the human IFN-ß gene. Cell 71,777-789[Medline]
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, mock: no DNA; 
, pCAT-Basic:
vector without an insert; 
, pCAT-C2; 
