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(Received for publication, July 11, 1995 ) From the
Insulin-like growth factor-I (IGF-I) gene transcription is
mediated largely via exon 1. In an initial search for regulatory
regions, rat hepatocytes were transfected with IGF-I constructs. Since
omission of downstream sequences led to reduced expression, we then
used in vitro transcription to evaluate potential metabolic
regulation via downstream regions. With templates including 219 base
pairs of downstream sequence, transcriptional activity was reduced
70-90% with hepatic nuclear extracts from diabetic versus normal rats. However, activity was comparable with templates
lacking downstream sequences. The downstream region contained six DNase
I footprints, and templates with deletion of either region III or V no
longer provided reduced transcriptional activity with nuclear extracts
from diabetic rats. Nuclear protein binding to regions III and V
appeared to be metabolically regulated, as shown by reduced DNase I
protection and activity in gel mobility shift assays with nuclear
extracts from diabetic rats. Southwestern blotting probes corresponding
to regions III and V recognized a
The insulin-like growth factors (IGFs) ( The single rIGF-I gene
gives rise to a complex family of mRNAs with both size and coding
sequence heterogeneity (9, 10, 11) and
polypeptides which are products of multiple translational initiation
sites. Multiple in-frame initiator codons within 5` sequences specify
different amino-terminal signal peptides, and preprolGFs with signal
peptides containing 22, 32, or 48 amino acids are synthesized depending
on utilization of different AUGs(9, 12) . Initiation
of transcription is also complex, as several laboratories have
identified multiple transcription initiation sites in exons 1 and 2 of
the rat, sheep, and human
genes(12, 13, 14, 15, 16) .
In the rIGF-I gene, initiation sites extend over 140 bp in exon 1, and
770 bp in exon 2(13) . However, Adamo et al.(13) found that two initiation sites in exon 1 could
account for 70-80% of IGF-I gene transcription in adult rat
liver. Thus, although rat IGF-I gene transcription is regulated by two
distinct promoters, the exon 1 promoter appears to be dominant. Since relatively little is known about molecular regulation of IGF-I
gene transcription, we have focused on the liver; while several
laboratories have begun to study the basis of IGF-I gene transcription
in different immortal cell
lines(14, 17, 18, 19) , there has
been little evaluation of underlying mechanisms in the dominant source
of IGF-I production(1, 3, 4) . Analysis of
findings from several laboratories suggests that downstream regions may
play a role in IGF-I gene
expression(17, 18, 19, 20) , but
such an hypothesis has not been tested in the liver. In the present
study, we demonstrate that sequences downstream from the exon 1 major
transcription initiation site are important both for hepatic IGF-I
expression and metabolic regulation, we characterize nuclear protein
binding to downstream sequences, and we identify two regions that may
be involved in the decreased IGF-I gene transcription associated with
diabetes mellitus.
Figure 1:
Luciferase activity in cultured normal
rat hepatocytes transfected with constructs containing rIGF-I sequences
in a reporter vector (see ``Materials and Methods'').
Sequences are shown relative to the exon 1 major transcription
initiation site. Promoter activity was expressed relative to activity
with p0Luc. Results shown are representative of three experiments.
Separate studies indicated that transfection efficiency (measured
according to expression of CMV-
Because of relatively low expression in transient transfection
studies (possibly due to the difficulty in maintaining IGF-I gene
expression in hepatocytes (30) as well as the difficulty of
transfecting cells in primary culture(31) ), a different model
was used to test the hypothesis that downstream sequences contribute to
metabolic regulation of IGF-I gene transcription. A genomic IGF-I
template containing 471 bp of upstream sequence and 219 bp of
downstream sequence was incubated with nuclear extracts from the livers
of normal and diabetic rats, and in vitro transcriptional
activity was evaluated by primer extension. As shown in Fig. 2A, the dominant transcription initiation site in vitro was identical to that used in vivo. The signal originated
from RNA polymerase II transcripts, since it was sensitive to
Figure 2:
In
vitro transcriptional activity of nuclear extracts from the livers
of normal and diabetic rats. Panel A, genomic IGF-I templates
containing 471 bp of upstream sequence and 219 bp of downstream
sequence were incubated with separate batches of hepatic nuclear
extracts from normal (lanes 4 and 6) and diabetic (lanes 5 and 7) rats, and the in vitro transcripts were quantitated by primer extension; 30 µg of
total liver RNA from normal (lane 2) and diabetic (lane
3) rats were used as positive controls, and yeast tRNA (lane
8) was used as a negative control. f174 DNA digested by HinfI was used as a size marker (lane 1). Panel
B, a genomic IGF-I fragment from -471 to +3 bp was
subcloned to pUC13(C
Figure 3:
Binding of nuclear proteins to the 272-bp AccI/BglII (-54/+219) fragment.
End-labeled probe (5000 cpm) was incubated with liver extract at 25
°C for 25 min. Protein-DNA complexes I and II were visualized on a
5% polyacrylamide gel as described under ``Materials and
Methods.''
Figure 4:
DNase I protection assay. An end-labeled
272-bp AccI/BglII (-53/+219 bp) fragment
was incubated with normal or diabetic liver extract at 25 °C for 20
min and then digested with DNase I as described. Protected regions
determined according to Maxam-Gilbert sequencing are shown as boxes, with naked DNA digested with DNase I as a negative
control (C). pBR322 plasmid DNA digested with HpaII
was used as a size marker (M). Panel A, probe labeled
on coding strand; Panel B, probe labeled on noncoding
strand.
Figure 5:
Nucleotide sequence of 272 hp AccI/BglII fragment. Sequence originally determined
by Shimatsu and Rotwein (32) was confirmed in our laboratory.
Protected regions are underlined. The exon 1 major
transcription initiation site is indicated by an arrow and
designated +1. The transcription initiation sites
identified by Adamo et al.(12, 13) are also
indicated.
Figure 6:
In vitro transcription assay with
deletion mutants as templates. Panel A, summary of deletion
mutant constructs. Regions of DNase I protection are indicated, and
deleted regions are shown by a thin line. The right side of
the panel shows the relative transcriptional activities of diabetic rat
liver nuclear extracts as compared with those from normal animals for
each template. Panel B, relative transcriptional activities of
normal (lanes 3 and 5) and diabetic (lanes 4 and 6) nuclear extracts with both wild type DNA (lanes 3 and 4) and a deletion mutant (lanes 5 and 6) as template. Lane 1 contained 20 pg of
liver RNA; lane 2 contained tRNA. In vitro transcripts were quantitated by primer extension as described
under ``Materials and Methods.'' Sequencing reactions
utilized an oligonucleotide complementary to +141 to 161 bp
electrophoresed along with primer extension products on a 6%
polyacrylamide-8 M urea gel. Panel C, transcriptional
activities of normal (lanes 1, 3, and 5) and
diabetic (lanes 2, 4, and 6) nuclear
extracts with both wild type DNA (lanes 1 and 2) and
mutants lacking region III (lanes 3 and 4) or region
V (lanes 5 and 6) as templates. Lane 7 contained 10 µg of liver RNA. pBR322 DNA digested with HpaII was used as a size marker (M). A 23-bp
oligonucleotide complementary to +79/+101 (region IV) was
used as a primer to quantitate in vitro transcripts.
Sequencing reactions were performed using the same primer, but with a
construct lacking region III d(42-68) as a template. Both
sequencing reactions and primer extension products were electrophoresed
on a 10% polyacrylamide-8 M urea gel. IGF-I cDNA is indicated
with an arrow. Panel D, transcriptional activities of normal (lanes 3 and 5) and diabetic (lanes 4 and 6) nuclear extracts with both wild type DNA (lanes 3 and 4) and a mutant lacking region IV (lanes 5 and 6) as templates. Liver RNA (10 µg, lane
2) and yeast tRNA (lane 7) were used as positive and
negative controls for primer extension. pBR322 plasmid DNA digested
with HpaII was used as a size marker (lane 1). A
20-bp oligonucleotide complementary to +42/+61 was used as a
primer to quantitate in vitro transcripts. IGF-I cDNA is
indicated with an arrow.
Figure 7:
Binding of nuclear proteins to
oligonucleotides III and V. Each binding reaction contained 6 µg of
nuclear protein from normal rat liver, 5000 cpm probe, pBR322 plasmid
DNA or/and cold double stranded oligonucleotides as indicated.
Protein/DNA complexes were separated on a 6% polyacrylamide
gel.
Figure 8:
Specific interactions of nuclear proteins
with oligonucleotides III, IV, and V. Each binding reaction contained
5,000 cpm probe, and different amounts of nuclear proteins as
indicated. Protein-DNA complexes were visualized on a 6% polyacrylamide
gel.
Figure 9:
Identification of proteins associated with
oligonucleotides III and V. Proteins (20 µg/lane) from
hepatic nuclear extracts from normal and diabetic rats were
electrophoresed, blotted onto nitrocellulose paper, and then hybridized
with oligonucleotides III and V as indicated. After washing with
binding buffer at 25 °C for 2 h with 2 changes of solution, the
filter paper was air dried and subjected to autoradiography. Molecular
markers in kDa are shown at left.
The IGF-I promoters analyzed to date have several common
features, such as lack of a ``TATA'' box, presence of
transcription ``initiator''
sequences(13, 32) , and binding sites for well
recognized transcription factors such as Sp1, C/EBP, and HNF-1 located
upstream from the major transcription initiation sites(32) .
The present studies demonstrate that sequences downstream from the
major transcription initiation site in exon 1 are important for both
IGF-I gene expression and metabolic regulation. Within the
-54/+219 bp region of exon 1, we found six loci of binding
with hepatic nuclear factors; protected regions were similar to those
described by Thomas et al.(33) . With our model, DNase
I footprinting and gel mobility shift assays revealed that nuclear
factors in the livers of diabetic rats have reduced interactions with
region III (+42/+68) and region V (+129/+152).
Transfection studies revealed a 230% increase in expression with a
construct containing 180 bp of downstream sequence (including both
regions III and V). Our findings are consistent with those of Hall et al.(14) , who observed that the presence of
downstream sequence increased IGF-I gene expression when the same
constructs were transfected into SK-N-MC cells and Lowe et al.(18) and Adamo and co-workers (19) who found that
downstream sequence increased IGF expression when constructs were
transfected into C6 glioma cells. Further evidence of biological
significance was provided by in vitro studies; specific
differences in IGF-I transcriptional activity between normal and
diabetic rat liver extracts could be detected only in the presence of
downstream sequences. The two protein-DNA complexes observed in gel
mobility shift assays with oligonucleotides III and V likely result
from binding of multiple nuclear factors rather than formation of a
dimer, since only complex I could be cross-competed with both
oligonucleotides III and V. A putative common factor could interact
with motifs such as CCTGC(G/C)CA found within both regions III and V.
In both gel mobility shift assays and Southwestern blotting studies,
the formation of protein-DNA complexes could be competed with unlabeled
oligonucleotides but was not blocked with a great excess of pBR322 DNA,
indicating that binding was specific. The DNA-binding protein(s)
identified by Southwestern blotting appears to be metabolically
regulated, as reduced binding was provided by hepatic nuclear extracts
from diabetic as compared with normal rats. While gel mobility shift
assays point to the presence of at least two DNA-binding proteins, we
do not yet know if other putative factors are metabolically regulated
as well. Lack of identification of a second DNA-binding factor by
Southwestern blotting may also be attributed to the denaturing
conditions used in this procedure, which could interfere with
protein-protein interactions that may be stabilized by the caging
effect in gel mobility shift analysis. A number of viral and
cellular transcriptional units contain essential sequences which are
downstream from transcription initiation sites (34, 35, 36, 37) . Such downstream
elements may influence RNA elongation, processing, and translation, in
addition to transcription initiation. Promoters commonly associated
with housekeeping and growth control genes often require downstream
elements to achieve full gene expression (38) . Moreover,
intragenic enhancers or activators have been described for numerous
extrahepatic genes such as immunoglobulins(39) , adenosine
deaminase(40) , and muscle creatine kinase(41) . Thus,
the requirement for both upstream and downstream elements to achieve
full gene expression is not unique to the IGF-I gene. There has been
relatively little characterization of tissue-specific and hormone
response elements of the IGF-I gene. Since this manuscript was
submitted, Nolten et al.(42) recently found that
C/EBP and HNF-1 can stimulate hIGF-I gene expression in Hep3B cells
through binding The present studies add to understanding of the regulation of
IGF-I biosynthesis. Hepatic IGF-I gene transcription is decreased under
conditions of reduced provision of essential amino acids or regulatory
hormones, due presumably to differences in nuclear factors that either
bind directly to the IGF-I gene or interact with other transcription
factors involved in the formation of transcription initiation
complexes. Our results suggest that a change in the concentration or
activity of factors bound to downstream sequences may lower IGF-I gene
transcription in conditions of diabetes mellitus. Additional studies
are now aimed at characterization of transcription factors involved in
modulation of IGF-I promoter activity.
Volume 270,
Number 42,
Issue of October 20, 1995 pp. 24917-24923
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
65-kDa nuclear factor present at
reduced levels in diabetic rats. These findings indicate that a
downstream region in exon 1 may be important for both IGF-I expression
and metabolic regulation. Altered concentration or activity of a
transcription factor(s) binding to this region may contribute to
reduced IGF-I gene transcription associated with diabetes mellitus.
)are
polypeptides with sequence, structure, and biological actions similar
to those of insulin (1) . Since circulating levels of IGF-I are
more responsive to changes in metabolic status than are levels of
IGF-II(2, 3) , IGF-I is thought to be a more important
regulatory factor during postnatal life. While IGF-I is expressed in
many organs and tissues, consistent with paracrine regulation and a
role as a local growth factor, its expression is 50-100 times
higher in the liver than in other tissues, consistent with hepatic
origin of circulating IGF-I, and a role as an endocrine regulator of
growth(1, 3, 4) . In the liver, IGF-I
expression appears to be regulated
pretranslationally(5, 6, 7) . Modulation at
the level of gene transcription is indicated by findings such as
decreased IGF-I gene transcription in streptozotocin-diabetic animals (7) and the ability of insulin to stimulate IGF-I gene
transcription in hepatocyte primary culture(8) . However,
underlying mechanisms are poorly understood.
Chemicals
Restriction endonucleases and DNA
modifying enzymes were obtained from New England BioLabs (Beverly, MA);
streptozotocin from Pfanstiehl (Waukegan, IL);
[-
P]ATP (6000 Ci/mmol),
[
-P]dATP, and
[
-P]dGTP (800 Ci/mmol) were from Amersham
Corp. Oligonucleotides were from Operon Technology (Alameda, CA);
luciferase and luciferin were from Boehringer Mannheim. Superscript
RNase H
reverse transcriptase was from Life
Technologies, Inc., and DNase I was from Pharmacia Biotech Inc. All
other chemicals (of molecular biology grade) were purchased from Sigma.
Animals
Male Sprague-Dawley rats (Charles River,
Lexington, MA), weighing approximately 120-160 g, were fed ad
libitum. Chronic diabetes was produced through tail vein injection
of streptozotocin 140 mg/kg. Animals were sacrificed by cervical
dislocation 5-7 days later, and livers were used for nuclear
extract preparation immediately. Streptozotocin at 250 mg/kg was used
to produce acute diabetes, with sacrifice of animals two days after
injection.Transfection
Constructs with IGF-I sequences
cloned into a luciferase reporter (p0Luc) were generously provided by
Dr. Peter Rotwein from Washington University, as reported
previously(14) . Relative to the rIGF-I exon 1 major
transcription initiation site (181 bp upstream from the leader
sequence(7, 13) ), constructs used for transfection
extended from -4398 to -32 bp; -1859 to -32 bp;
-1262 to -32 bp; -1859 to +55 bp; and
-1859 to +180 bp. Hepatocytes were isolated from
150-200-g male Sprague-Dawley rats using a modification of the
collagenase perfusion method of Seglen(21) , as reported
previously(5, 8) . Cells were transfected with 6
µg of supercoiled DNA calcium-phosphate precipitate in a volume of
0.3 ml/plate 5 h after plating(22) . Following overnight
exposure to the DNA, the cells were rinsed twice with 4 ml of
phosphate-buffered saline and placed in 3 ml of serum-free defined
medium containing 10M insulin and 500
ng/ml human growth hormone, since both insulin and growth hormone
increase IGF-I secretion in primary cultures of
hepatocytes(23) . After 24 h, the cells were rinsed twice with
4 ml of phosphate-buffered saline and scraped into 0.7 ml of 50 mM Tris-MES, pH 7.8, containing 1 mM DTT and 1% Triton
X-100. Following centrifugation, 50 µl of the supernatant was
assayed for luciferase activity in a 200-µl reaction containing 50
mM Tris-MES, pH 7.8, 10 mM magnesium acetate, 2
mM ATP, and 0.5 mM luciferin. Standard curves were
performed with purified luciferase to ensure linearity. In all
experiments, transfections were performed in triplicate plates for each
condition.
Rat Liver Nuclear Extract Preparation
Liver
nuclear extracts were prepared as described by Gorski et al.(24) and Triezenberg et al.(25) . Briefly, 15
g of tissue were homogenized in 120 ml of buffer containing 10
mM Hepes, pH 7.6, 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 10%
glycerol, and 2.0 M sucrose. The homogenate was layered onto a
2 M sucrose cushion and centrifuged at 27,000 rpm in an SW28
rotor at 4 °C for 1 h. The nuclear pellet was resuspended in lysing
buffer containing 10 mM Hepes, pH 7.6, 100 mM KCl, 3
mM MgCl
, 0.1 mM EDTA, and 10% glycerol at
a concentration of 10 A/ml. Nuclei were lysed by
adding one-tenth of a volume of 4 M
(NH
)SO, and chromatin was removed
by centrifugation at 39,000 rpm in an SW40 rotor for 2 h. Nuclear
proteins were concentrated by (NH)2SO precipitation (0.33 g/ml) and dialyzed against buffer containing
25 mM Hepes, pH 7.6, 100 mM KCl, 0.1 mM EDTA, and 10% glycerol for 4 h. Proteinase inhibitors (0.1
mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine,
1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 µg/ml
aprotinin) were added to buffers just before use. Extracts prepared
from animals with either acute or chronic diabetes had comparable
activity with in vitro transcription assays.Construction of 3` Deletion Mutants
For mutants
with deletion of +84 to +152 bp, the wild type template was
linearized with BstXI, treated with Bal 31 nuclease,
and then religated. Other mutants were constructed by polymerase chain
reaction, similar to methods described by Higuchi et
al.(26) . Primers used are in Table 1. In each case,
a -309/+373-bp polymerase chain reaction product with
specific internal deletions was digested with BanII and BglII, gel purified, and then subcloned into a wild type
template. All mutants were sequenced.
In Vitro Transcription Assay
Transcription
reactions (30 µl) contained 1.0 µg of linear DNA template, 50
ng of pAML(CAT), 60 µg of liver extract,
50 mM KC1, 6 mM MgCl
, 0.5 mM ATP
and CTP, 35 µM UTP, 10 µCi of
[-P]UTP (400 Ci/mmol), 0.1 mM 3`-O-methyl GTP, 10% glycerol, I u/µl RNasin, 0.05
mM EDTA, and 1 mM DTT. The DNA template and extract
were incubated on ice for 20 min. Transcription was then initiated by
the addition of nucleotides and carried out for at 30 °C for 45
min. The transcripts were purified by phenol/chloroform extraction,
ethanol precipitated, separated on an 8 M urea, 6%
polyacrylamide gel, and visualized by autoradiography. When
transcription assays were performed in the presence of 0.5 mM of ATP, UTP, GTP, and CTP, the DNA template was degraded by 50
units of RNase-free DNase I as described previously (7) , and
the RNA synthesized in vitro was quantitated by primer
extension.
Primer Extension
Oligonucleotides complementary to
the sequence from +42 to +61 or +79 to +101 were
end-labeled by polynucleotide kinase to a specific activity of 10
cpm/µg. Probe (50 
10 cpm) was annealed to
RNA synthesized in vitro, to 10 µg of yeast tRNA, or to
10-30 µg of liver RNA at 42 °C for 3 h. The cDNA was
synthesized as described by Boorstein and Craig (27) at 42
°C for 1 h, ethanol precipitated, and separated on an 8 M urea, 6% polyacrylamide gel.DNA Probes
Plasmid DNA containing an 0.9-kilobase
pair SacI/PstI rIGF-I genomic DNA fragment was
linearized with BglII or AccI, and end-labeled. After
digestion with AccI or BglII, the 272-bp fragment,
-54 to +219 bp relative to the exon 1 major transcription
initiation site(7, 13) , was purified from a
polyacrylamide gel as described previously(28) . Pairs of
oligonucleotides, corresponding to +42/+68 bp (oligo III),
+79/+101 bp (oligo IV), and +129/+152 bp (oligo V)
downstream from the exon 1 major transcription initiation site, were
annealed, labeled, and gel purified.Gel Mobility Shift Assay
End-labeled 272-bp AccI/BglII fragments were incubated with 0.5-15
µg of nuclear extract in 25 µl of binding buffer containing 10
mM Tris, pH 7.5, 50 mM KCl, 1 mM EDTA, 0.5
mM DTT, 0.2% Nonidet P-40, 20 µg of bovine serum albumin,
4 µg of poly(dI-dC), and 10% glycerol at 25 °C for 25 min.
Protein-DNA complexes were separated from free probe at 4 °C on a
5% polyacrylamide gel in 0.5 TBE (45 mM Tris, pH 8.0,
45 mM boric acid, 1 mM EDTA) at 11 V/cm for 2-3
h, and visualized by autoradiography. For double-stranded
oligonucleotides, the probe was incubated with 5-15 µg of
extract in same buffer, except that 200 µg/ml of salmon sperm DNA
and 24 µg/ml of pBR322 were included to reduce nonspecific binding. DNase I Protection Assay
End-labeled 272 bp AccI/BglII fragments were incubated with 4-24
µg of nuclear extract in 25 µl of binding buffer containing 10
mM Hepes, pH 7.9, 50 mM KCl, 1 mM EDTA, 0.5
mM DTT, 1 µg of poly(dI-dC), and 10% glycerol at 25 °C
for 25 min. An equal volume of binding buffer containing 10 mM
MgCl, 2 mM CaCl, and 10 units/ml of
DNase I was added, and the sample was incubated at 25 °C for 2 min.
The reaction was stopped with buffer containing 40 mM EDTA and
10 µg/ml tRNA and deproteinized with plenol. DNA was precipitated
with ethanol, electrophoresed on an 8 M urea, 6%
polyacrylamide gel, and visualized by autoradiography.Southwestern Blotting
20 µg of extract were
mixed with an equal volume of loading buffer containing 5 mM
Tris, pH 6.8, 200 mM DTT, 5% SDS, 20% glycerol, and 0.05%
pyromin Y. Proteins were denatured at 100 °C for 3 min, separated
on a 10% SDS-polyacrylamide gel, and then blotted onto nitrocellulose
paper(29) . The blots were incubated with buffer containing 10
mM Hepes, and 5% nonfat dry milk at 25 °C for 1 h, and
then incubated with binding buffer containing 10 mM Hepes, pH
8.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.25%
nonfat dry milk, 5 µg/ml salmon sperm DNA, and 1 10
cpm/ml probe at 25 °C for 1 h. After washing with 2 changes
of binding buffer containing 300 mM NaCl at 25 °C for 2 h,
the filter paper was air dried and subjected to autoradiography.
Sequences Downstream from Exon 1 Transcription
Initiation Sites Are Important for IGF-I Gene Expression and Metabolic
Regulation
Constructs with IGF-I 5`-flanking sequences were
transfected into rat hepatocytes in primary culture, as summarized in Fig. 1. Relative to expression of a promoterless luciferase
reporter vector (p0LUC), expression was decreased 30% by the presence
of IGF-I sequence extending from -4 kilobase pairs to
-32 bp relative to the exon 1 major transcription initiation
site(7, 13, 14) . Expression was increased 40
and 113% with constructs containing 1.86 and 1.26 kilobase pairs of 5`
sequence, respectively, and the same 3` terminus. Compared with
expression of p(-1859/-32)LUC (40% above that of p0LUC),
increased expression was obtained with constructs containing additional
downstream sequences. Expression was increased 126% above p0LUC with a
construct containing 1.86 kilobase pairs of 5` sequence and 3` sequence
terminating at +55 bp, and maximum expression, 230% above p0LUC,
was obtained with a construct containing the same 5` sequence and 3`
sequence extending to +180 bp; expression was significantly
greater than that of the construct with similar 5` sequence but lacking
downstream sequence (p < 0.005). Thus, these findings
suggested that downstream sequences enhanced IGF-I gene expression.
-galactosidase) was unaffected by
the presence of IGF-I elements. Mean ± S.E., n =
3.
-amanitin (not shown). Moreover, no signal was detected when
extracts were incubated in the absence of a DNA template, indicating
that signals originated from transcripts generated in vitro (not shown). Since in vitro transcriptional activity for
the adenovirus major late promoter template driven by nuclear extracts
from the livers of diabetic rats was comparable or greater to that with
extracts from normal rats (Fig. 2B), we concluded that
nuclear extracts from the livers of diabetic rats contained adequate
transcriptional machinery and that changes in IGF-I gene transcription
were likely to be specific. Using templates containing downstream
sequence, in vitro transcriptional activity of nuclear
extracts from the livers of diabetic rats was reduced 90%, compared
with nuclear extracts from the livers of normal rats, (p <
0.05), but transcriptional activity with an IGF-I template lacking
downstream sequence was not significantly decreased with extracts from
diabetic versus normal rats (p > 0.1, Fig. 2C). Since this observation is consistent with our
previous finding(7) , that IGF-I gene transcription rates were
reduced 97% in the livers of diabetic rats as compared with normal
rats, downstream sequences may be important for both IGF-I expression
and metabolic regulation.
AT) and used as a template in in
vitro transcription assays, and a template for the adenovirus
major late promoter (pML(CAT), AMLP)
was used as an internal control. Panel C, relative
transcriptional activities of normal and diabetic liver nuclear
extracts. Data were obtained from three different pairs of normal and
diabetic extracts; for each pair of extracts, IGF-I transcriptional
activity of normal liver extracts (determined by densitometric scanning
and expressed relative to adenovirus major late promoter activity) was
designated as 100%. Mean ± S.E., * indicates p <
0.05.
Nuclear Protein(s) Binding to Downstream
Sequences
To search for regions that might be involved in gene
regulation, the binding of hepatic nuclear factors to the 272-bp AccI/BglII fragment (-54/+219 bp) was
first studied by gel mobility shift analysis, as shown in Fig. 3. Densitometric scanning revealed that the intensity of
shifted protein-DNA complexes was reduced 30-60% with extracts
from streptozotocin-diabetic as compared with normal rats. Binding was
specific, since formation of DNA-protein complexes could be competed
with an unlabeled 272-bp fragment but not with pBR322 DNA (not shown).
All protein-DNA binding studies were repeated with at least three
different batches of extracts, and extract activity was monitored by in vitro transcription assays.
Protein Binding Sites Assessed by DNase I
Footprinting
Protein binding sites were determined on both
coding and noncoding strands, and results are shown in Fig. 4.
Region I corresponded to the major transcription initiation site in
exon 1. Five additional protected regions were observed consistently,
with footprints at +17/+25 (region II), +42/+68
(region III), +79/+101 (region IV), +129/+152
(region V), and +155/+169 (region VI). Binding of factors in
nuclear extracts from the livers of diabetic as compared with normal
rats was reduced in both region III (especially with the noncoding
strand) and region V (especially with the coding strand) (lanes
4, 5, and 6 versus lanes 1, 2, and 3 in panels A and B; differences in binding
were not consistent in other regions. Nucleotide sequences and
protected regions are summarized in Fig. 5.
Regions III and V Are Necessary for the
Diabetes-associated Reduction in IGF-I Gene Transcription
The
importance of downstream regions in metabolic regulation was evaluated
with in vitro transcription assays using deletion mutants as
templates, as shown in Fig. 6A. A template lacking regions
IV and V (+84/+152) no longer provided reduced
transcriptional activity with nuclear extracts from diabetic versus normal rats (panel B). Similar results were also obtained
with templates lacking regions III (+42/+68) or V
(+129/+152) (panel C). In contrast, a template with
deletion of region IV continued to exhibit decreased transcriptional
activity with nuclear extracts from diabetic versus normal
rats (panel D). While the potential involvement of other
regions is still being investigated in our laboratory, these data were
reproducible with different batches of extracts, and the
transcriptional activities of normal and diabetic extracts remained
comparable as determined with the adenovirus major late promoter as
template.
Nuclear Factors Associated with Regions III and V Are
Reduced by Streptozotocin-induced Diabetes
Double-stranded
oligonucleotides corresponding to regions III (+42/+68 bp)
and V (+129/+152 bp) were used in gel mobility shift analyses
to examine DNA-protein interactions, as shown in Fig. 7.
Addition of pBR322 DNA was necessary to decrease nonspecific binding (lanes 2 and 10 versus lanes 4 and 12). The
association of nuclear factors with region V was stronger than that
with region III, especially in formation of complex II (lanes 3 and 4 versus lanes 11 and 12). While formation
of both complexes I and II could be competed with unlabeled
oligonucleotides (lane 3 versus lanes 5 and 6 and lane 11 versus lanes 13 and 14), cross-competition
was incomplete (lane 3 versus lanes 7 and 8 and lane 11 versus lanes 15 and 16). Thus, binding of
nuclear factors to regions III and V was relatively specific,
particularly for complex II. Activities of nuclear extracts from the
livers of normal and diabetic rats are shown in Fig. 8. Nuclear
proteins associated with region IV showed similar affinity with normal
and diabetic rat liver extracts. In contrast, nuclear protein binding
to regions III and V was reduced with extracts from diabetic animals,
typically 30-50% of that of extracts from normal rats.
Identification of Protein Factors Associated with Regions
III and V
To characterize the size and relative abundance of
proteins associated with regions III and V, hepatic nuclear extracts
from normal and diabetic rats were subjected to polyacrylamide gel
electrophoresis and then blotted to nitrocellulose and probed with
corresponding oligonucleotides, as shown in Fig. 9. Proteins
with apparent molecular weight of 65 kDa were associated with both
regions III and V and were present in extracts from both normal and
diabetic animals. However, apparent abundance of the
65-kDa
protein in extracts from diabetic animals was
75% of normal with
region III and
50% of normal with region V, as determined by
densitometric scanning.
120 bp upstream from the major exon 1
transcription initiation site. Within regions of interest identified in
the present studies, region III includes the AAATAAA silencer motif
identified in the rat prolactin gene(43) , and the
(T/A)GATA(A/G) binding motif found in the promoters or enhancers of
erythroid-expressed genes(44) , the histone H-5
gene(37) , and immunoglobulin genes(45, 46) .
The nontranscribed strand sequence GGNGCAGGA in region V is similar to
the silencer binding protein motif GGAGCAGGA found in the rat
glutathione transferase P gene(47) . A GenBank/EMBL search
indicates that there is substantial homology between region III and
region V sequences and elements of over 50 eukaryotic and prokaryotic
genes.
)
We thank Sharon DePeaza and Mary Lou Mojonnier for
assistance in the preparation of this manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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