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(Received for publication, July
31, 1995; and in revised form, December 6, 1995) From the
Although the liver is the major source of circulating
insulin-like growth factor-I (IGF-I), relatively little is known about
the regulation of IGF-I gene transcription in this tissue. Since
transcripts are initiated largely in exon 1, we established an in
vitro transcription system to evaluate activation of transcription
via the major exon 1 initiation site. Transcription of a G-free
cassette reporter was directed by rat IGF-I genomic fragments, and the
adenovirus major late promoter was used as an internal control. Tissue
specificity was demonstrated by a 60-90% decrease in transcripts
with spleen extracts as compared with liver. 54 base pairs (bp) of
upstream sequence were sufficient to direct IGF-I gene transcription,
and activity increased 5-fold with 300 bp of upstream sequence. DNase I
footprinting revealed four protected regions between -300 and
-60 bp; binding was confirmed by gel shift analysis, and tissue
specificity was demonstrated by reduced shifts with spleen extracts.
The necessity of transcription factor binding to such sites was
established by competition analysis, which revealed a specific decrease
in IGF-I transcription in the presence of a competing fragment. Use of
this in vitro transcription system should permit analysis of
the function of individual transcription factors involved in regulation
of IGF-I gene expression.
Insulin-like growth factor-I (IGF-I) ( The
rat IGF-I gene contains at least six exons with total length over 80
kb(5) . A single copy of the IGF-I gene gives rise to four
major mRNA species, with size differences due primarily to multiple
polyadenylation sites(6) . Transcription is initiated at
multiple loci in exons 1 and 2, but exon 1 transcripts predominate in
all tissues (7) . While IGF-I production appears to be
regulated mainly at the level of gene transcription, underlying
mechanisms are not well understood. The 5`-flanking sequences for IGF-I
genes from several species exhibit common features, including lack of a
TATA box, the presence of ``initiator'' elements, and binding
sites for recognized transcription factors such as Sp1, C/EBP, HNF-1,
and AP-1. However, although studies in neuroblastoma SK-N-MC
cells(8) , rat fibroblasts, and rat C6 glioma cells (9, 10) indicate the presence of functioning promoter
regions in exon 1, it has been more difficult to characterize IGF-I
transcription in the liver, the dominant source of IGF-I in
vivo(4) . While the regulation of many genes has been
examined by transient transfection in cultured cell models, this
approach is less well suited to the liver, because IGF-I expression in
immortal cell lines tends to be low (11) and cultured primary
hepatocytes are difficult to transfect(12) . Accordingly, we
have utilized nuclear extracts of normal rat liver in an in vitro transcription system to examine the effect of 5`-flanking
sequences on IGF-I gene expression. We demonstrate that maximal
promoter activity requires
Figure 1:
Construction of DNA templates. The
template pIGF
Figure 2:
Expression of the IGF-I gene in
vitro. Each reaction contained 50 ng of
pML(C
Figure 3:
In vitro transcription-mixing experiment.
Each reaction contained 50 ng of pML(C
Figure 4:
Optimization of assay conditions. In
vitro transcription assays were performed as described under
``Materials and Methods'' with 50 ng of
pML(C
Figure 5:
The
effect of 5`-deletions on IGF-I gene expression. Panel A,
assays here and below were performed in the presence of 60 mM KCl, 6 mM MgCl
Figure 6:
DNase
I protection assay. The probe was incubated with liver nuclear extract
for 20 min at 25 °C and then digested with DNase I at 20 units/ml
for 2 min as described under ``Materials and Methods.'' The
DNA was electrophoresed on an 8 M urea, 6% polyacrylamide gel.
The DNA size marker (M) was pBR322-digested by HpaII.
Protected regions were determined with Maxam-Gilbert sequencing (A
+ G) using naked DNA digested with DNase I as a negative
control (lane 1). Panel A, an NcoI/AccI (-471/-54) fragment was labeled
on the coding strand. The amount of protein used was 4, 8, 16, and 24
µg in lanes 2-5, respectively. Panel B, a DdeI/PvuII (-350/-66) fragment was
labeled on the noncoding strand. The amount of protein used was 8, 16,
24, and 32 µg in lanes 2-5,
respectively.
Figure 7:
DNase I-protected regions are indicated.
Protected regions on coding and noncoding strands are shown by lines above and beneath sequences, respectively. The major
exon I transcription initiation site is indicated by an arrow.
The restriction sites used to generate probes are also
indicated.
The tissue specificity of
DNA-protein interactions within the -300-bp promoter was
evaluated by gel mobility shift analysis (Fig. 8). Using a
247-bp DNA fragment (-300/-54) as a probe, DNA-protein
complexes were readily apparent with a liver nuclear extract but barely
detectable with a spleen nuclear extract (panel A). The
formation of DNA-protein complexes was specific, because binding with
liver extracts could be competed with a 100
Figure 8:
Gel mobility shift analysis. Panel
A, a 247-bp (-300/-54) DNA fragment was incubated with
1, 2, and 4 µg of nuclear protein from liver (lanes
2-4) or spleen (lanes 7-9) as described under
``Materials and Methods.'' In competition assays, 4 µg of
protein was incubated with a 50
To determine whether DNA-protein interactions within the
-300-bp region might be essential for IGF-I gene expression, in vitro transcription was performed with and without addition
of a 183-bp DNA fragment (-240/-58 bp), which included
DNase I-protected regions I-III and IV (partial). Although a 5
Figure 9:
Competition analysis. A 183-bp fragment
(-240/-58) was incubated with liver nuclear extract on ice
for 20 min before pIGF
The present studies demonstrate that the molecular regulation
of rIGF-I gene expression can be evaluated by in vitro transcription with nuclear extracts and genomic templates.
Specific IGF-I transcripts from the major exon 1 transcription
initiation site were detected by expression of a 373-bp G-free cassette
(C The present finding of maximal promoter activity with Variation among these findings may have several causes. Our studies
utilized extracts from normal liver, while other
workers(8, 9, 10, 18) utilized
fibroblasts or immortal cell lines, in which the concentration and/or
activity of transcription factors are recognized to be different from
that of liver (20, 21, 22) and could
contribute to the observed discrepancies in promoter activity. Because
of the constraints of the G-free cassette system, our constructs
contained little downstream sequence as compared with the chimeric
rIGF-I genes used in transfection studies. However, both our laboratory (23) and other workers (8, 9, 10) have noted that downstream
sequences may be important for expression and conceivably could also
modify the interactions of transcription factors with upstream
sequences. In addition, our system provided evaluation of transcripts
initiated only at a defined site, whereas transient transfection
studies included transcripts potentially initiated at multiple sites.
Finally, formation of the transcription machinery to assemble
initiation complexes in vitro also differs from that in
vivo since chromatin structure is disrupted with the use of
purified DNA as a template(24) . Taken together, these
possibilities may underlie our finding of maximal promoter activity
with a -300-bp construct using normal liver extracts, as opposed
to a -1-kb construct in SK-N-MC neuroblastoma cells(8) ,
and -156-bp constructs in rat fibroblasts and C6 glioma
cells(9, 10) . For many genes, the dominant control
of liver-specific expression is at the level of transcription (25, 26) and depends on binding of trans-acting factors to cis-regulatory DNA sequences,
often located in the 5`-flanking region. For the rat IGF-I gene,
developmental activation is associated with the progressive appearance
of DNase I-hypersensitive sites, consistent with the impact of trans-acting factors(27) . In the present study, the
four protected regions identified by DNase I footprinting are
compatible with the location of DNase I cleavage sites described by
Kikuchi et al.(27) . While regions I and II are
similar to locations HS3A and HS3B described by Thomas et
al.(28) , we are not aware of binding of nuclear factors
to regions III and IV in previous reports on the rat IGF-I gene. The
combined functional importance of these sites is illustrated by our
competition study, in which expression was decreased The regulation of hepatic gene transcription depends in part on the
coordinated contributions of basal transcription factors,
tissue-specific transcription factors, and transcription factors that
are modulated by hormonal and metabolic status(26) . Thus,
relatively liver-specific genes such as albumin contain binding sites
for basal transcription factors such as TATA box binding protein (TBP),
AP1, and NF-Y(29, 30, 31) , as well as more
liver-specific factors such as hepatic nuclear factors I, II, III, and
V (HNF-1, -2, -3, and -5), CCAAT/enhancer binding protein (C/EBP), and
D-site binding protein
(DBP)(30, 31, 32, 33, 34) .
Based on comparisons with consensus sequences for binding sites of such
factors(35) , it seems likely that both liver-specific and
general transcription factors will be found to interact with the
proximal IGF-I promoter region(36, 37) . In
summary, the present studies constitute the first use of an in
vitro transcription system to examine the regulation of IGF-I gene
expression in normal liver, the major source of circulating
IGF-I(4) . In the future, this system should permit functional
assays of promoter activity assessment to be combined with
transcription factor binding in order to identify critical regulatory
elements on the IGF-I gene and elucidate mechanisms by which both
circulating hormones and locally expressed effectors modulate the
synthesis of IGF-I.
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 8667-8674
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is a 70-amino
acid peptide that is similar to proinsulin in structure (1) and has major anabolic effects on growth, development, and
metabolism during both fetal and postnatal
life(2, 3) . The liver is the major origin of IGF-I
acting in an endocrine mode(4) , but IGF-I is also synthesized
in many other tissues, with local autocrine/paracrine
actions(2, 3, 4) . Although IGF-I was first
thought to be regulated mainly by growth hormone, it is now recognized
that nutrition, local cellular factors, and other hormones also
modulate IGF-I production(2, 3, 4) .
300 bp upstream from the major exon 1
transcription initiation site and that binding of nuclear factors to
this region is essential for IGF-I gene expression.
Chemicals
Restriction endonucleases, T4
polynucleotide kinase, Klenow fragment of DNA polymerase I, and mung
bean nuclease were obtained from New England BioLabs (Beverly, MA); a
24-bp oligonucleotide containing consensus Sp1 binding sites was from
Stratagene (La Jolla, CA); nucleotides and DNase I were ordered from
Pharmacia Biotech Inc.; Taq DNA and Pfu DNA
polymerases were from Perkin-Elmer and Stratagene, respectively;
[
-
P]ATP,
[-P]deoxy-ATP, -GTP, -TTP, and -CTP (800
Ci/mmol), and [-P]UTP (400 Ci/mmol) were
from Amersham Corp.; oligonucleotides were from Operon Technology
(Alameda, CA); and other molecular biology grade reagents were from
Sigma.Animals
Male Sprague-Dawley rats (Charles River,
Lexington, MA), weighing 120-160 g, were fed ad libitum.
Animals were sacrificed by cervical dislocation, and livers or spleens
were used for nuclear extract preparation immediately.Liver/Spleen Nuclear Extract Preparation
Nuclear
extracts were prepared according to modifications of the methods of
Gorski et al.(13) and Triezenberg et
al.(14) . Briefly, 10-12 g of tissue were
homogenized in 85 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 protease inhibitors leupeptin, aprotonin, and
pepstatin A (all at 1 µg/ml), 0.5 mM PMSF, and 1 mM benzamidine were added just before use. The homogenate was layered
onto a 2 M sucrose cushion and centrifuged at 27,000 rpm in an
SW 28 rotor for 1 h at 2 °C. Nuclei were resuspended in lysing
buffer containing 10 mM Hepes, pH 7.6, 100 mM KCl,
0.1 mM EDTA, 1 mM DTT, 3 mM MgCl
, 10% glycerol, leupeptin, aprotonin, pepstatin A
(all at 1 µg/ml), and 0.1 mM PMSF. Nuclei were lysed by
adding 0.1 volume of 4 M ammonium sulfate, and lysate was left
on ice for 30 min with gentle mixing. Chromatin was removed by
centrifugation at 39,000 rpm in an SW 40 rotor for 1.5 h at 2 °C.
Nuclear protein was concentrated by
(NH
)SO precipitation (0.33 g/ml)
and dialyzed against buffer containing 25 mM Hepes, pH 7.6,
100 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 10%
glycerol for 4 h at 4 °C. Nuclear protein was frozen and stored at
-70 °C.Template Construction
The plasmids
pUC13(CAT)
,
pML(CAT)
, and pML(CAT)
were kindly provided by Dr. Lee E. Babiss. Although there are
four transcription initiation sites in exon 1, the G-free cassette was
placed downstream from site 3 because this is the major initiation site in vivo(15) , and there are no G residues immediately
downstream from this site. Two oligonucleotides 5`-
GTTATAGAATTCACCATGGTCATTTCAGGG-3` (-471/-454) and
5`-GCTGCATACGTAAGAAGAGGGATTTAGAG-3` (-13/+4) were used as
primers for PCR amplification(16) . After digestion with EcoRI and SnaBI, the gel-purified fragment was
subcloned into pUC13(CAT), and the construct,
pIGF
(CAT), was sequenced. The 5`-upstream
sequence was extended to 1 kb by insertion of a 581-bp NcoI
IGF-I genomic fragment, and the recombinant was designated as
pIGF
(CAT). A series of 5`-deletion mutants
were then prepared by conventional subcloning, as summarized in Fig. 1. Plasmid DNAs were purified by standard CsCl gradient
methods, linearized, deproteinized, and used as templates in
transcription assays.
(CAT) was constructed first (see
``Materials and Methods''). The 5`-upstream region was
extended by adding a 580-bp NcoI genomic fragment to produce
pIGF(CAT). The templates
pIGF
(CAT) and
pIGF
(CAT) were constructed by deleting NarI/NarI and BanII/NdeI fragments
from pIGF(CAT) and
pIGF(CAT), respectively. The BanI
and AccI fragments, containing 136 and 54 bp of 5`-upstream
sequence together with the 373-bp G-free cassette, were excised from
pIGF(CAT), subcloned on the SmaI
site on pUC19, and designated as pIGF
(CAT)
and pIGF
(CAT), respectively. The exon 1
transcription initiation site designated as +1 is indicated above
the constructs; for convenience, the initiation sites identified by
Adamo et al.(15) are shown
below.
In Vitro Transcription Assay
The optimal assay
conditions were defined in preliminary studies. In general, reactions
(30 µl) contained 1.0 µg of IGF-I template DNA, 50 ng of
pML(CAT) or
pML(CAT), 60 µg of extract, 60 mM KCl, 6 mM MgCl, 0.5 mM ATP and CTP,
35 µM UTP, 10 µCi of
[-P]UTP, 0.1 mM 3`-O-methyl-GTP, 10% glycerol, 1 unit/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
addition of nucleotides, and reaction mixtures were incubated for 45
min at 30 °C. Reactions were terminated by adding 300 µl of
stop buffer (20 mM Tris, pH 7.5, 5 mM EDTA, 1% SDS,
0.25 M NaCl) containing 5 µg of tRNA and 60 µg of
proteinase K and incubating for 30 min at 37 °C. The RNA was
purified by phenol/chloroform extraction, ethanol-precipitated, and
then applied onto an 8 M urea, 6% polyacrylamide gel, and
visualized by autoradiography. For competition studies, the
oligonucleotides 5`-AACGTCTGCTAACCCTGTCA-3` and
5`-AAACAGCTGGGGGAACATTCG-3` were used as primers to synthesize a 183-bp
(-240/-58) fragment by polymerase chain
reaction(16) . The 183-bp fragment including footprint sites
I-IV (partial) was used as a competitor in transcription assays
in order to ensure that competition was specific.DNase I Protection Assay
The end-labeled DNA
fragment was incubated with 4-24 µg of nuclear extract in 20
µl of binding buffer containing 25 mM Hepes, pH 7.6, 0.1
mM EDTA, 50 mM KCl, 1 mM DTT, 1 µg of
poly(dI-dC), and 10% glycerol for 20 min at 25 °C. An equal volume
of binding buffer containing 10 mM MgCl, 2 mM CaCl, and 40 units/ml DNase I was added, and reactions
were carried out for 2 min at 25 °C. Reactions were stopped by
buffer containing 10 mM Tris, 40 mM EDTA, and 0.25 M NaCl, 200 µg/ml proteinase K, and 20 µg/ml tRNA.
After incubation for 30 min at 37 °C, the purified DNA was resolved
on an 8 M urea, 6% polyacrylamide gel.Gel Mobility Shift Assay
An end-labeled 247-bp DNA
(-300/-54) was incubated with 1-4 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, 1%
Nonidet P-40, 20 µg of bovine serum albumin, 4 µg of
poly(dI-dC), and 10% glycerol for 25 min on ice. Protein-DNA complexes
were separated at 4 °C on a 5% polyacrylamide gel in 1 
TBE
(89 mM Tris, 89 mM borate, 2 mM EDTA) at 11
volts/cm for 2-3 h and visualized by autoradiography.Statistics
To analyze the effect of 5`-deletions
on IGF-I gene expression, differences between means were examined by
analysis of variance with significance established as p <
0.05.
In Vitro Transcription of the Rat IGF-I Gene
In
initial experiments, the pIGF(CAT) construct
was used to examine rIGF-I gene expression in vitro, as shown
in Fig. 2. With the IGF-I template, the G-free cassette
(CAT) provided a 373-bp signal. A second template from the
adenovirus major late gene promoter (AdMLP) was included in all
reactions as an internal control, with a shorter CAT
cassette providing a 190- or 270-bp signal. In the presence of a
nuclear extract from normal rat liver, a strong IGF-I signal was
observed (lane 3). The IGF-I signal was abolished by use of
vector (pUC13(CAT)) as template (lane 1), and both
the IGF-I and AdMLP signals were abolished by addition of
-amanitin (lane 4). These findings indicate that the
IGF-I transcripts were directed by RNA polymerase II and originated
from an authentic promoter on the rIGF-I gene and that 471 bp of
5`-flanking sequence contain cis-elements sufficient to
activate the IGF-I promoter in vitro. A nuclear extract from
rat spleen was transcriptionally active as shown by a strong AdMLP
signal but provided an IGF-I signal only 10% as strong as that with
extracts from liver (lane 2 versus 3), similar to liver
specificity found in vivo(4) . In mixing experiments,
progressive substitution of spleen nuclear extracts for liver nuclear
extracts led to a proportionate decrease in the IGF-I transcripts, with
little change in the AdMLP transcripts (Fig. 3A).
Moreover, in a separate mixing experiment (Fig. 3B),
addition of 40 µg of spleen extract did not diminish transcription
driven by 40 µg of liver extract (lanes 7 and 8 versus
lanes 1 and 2). In combination, these data indicate that
the reduction in IGF-I transcripts observed with spleen nuclear
extracts likely reflects the absence of tissue-specific activators
rather than the presence of inhibitory factors.
AT), 60 µg of liver nuclear extract (L, lanes 1, 3, and 4) or spleen nuclear extract (S, lane 2), and 1 µg of pUC13(CAT) (lane
1) or pIGF(CAT) (lanes
2-4). In lane 4, the reaction contained 3 µg/ml
-amanitin. The RNA was electrophoresed on an 8 M urea, 6%
polyacrylamide gel. A pBR322 plasmid DNA digested by HpaII was
used as a size marker.
AT) (panel A) or pML(CAT) (panel B), 1 µg of pIGF (CAT), and nuclear extracts from liver and/or spleen
as indicated. Bovine serum albumin (BSA) was used to balance
total protein to 80 µg for each reaction. The IGF-I transcripts
were quantitated by densitometric scanning, and expression was
normalized to those of pML(CAT) or
pML(CAT).
Optimization of in Vitro Transcription
In order to
obtain strong and consistent signals, in vitro transcription
conditions were examined in detail, as shown in Fig. 4. Both
AdMLP and IGF-I transcripts were increased with increasing nuclear
protein from 20 to 50 µg, and IGF-I transcripts were increased
further with 60 µg; the latter concentration was used in subsequent
studies (Fig. 4A). Assays were carried out for 45 min
at 30 °C, since signal strength appeared to plateau at that time (Fig. 4B). The optimal KCl concentration was 60 mM (Fig. 4C). As shown in Fig. 4D,
AdMLP transcripts were increased when the MgCl concentration was raised from 2 to 6 mM, whereas IGF-I
transcripts were decreased with MgCl concentrations from 8
to 12 mM. To maximize the activities of both general and
tissue-specific transcription factors, 6 mM was considered to
be optimal concentration for MgCl. In separate studies, we
found that 50 ng of pAdMLP (CAT) or pAdMLP
(CAT) used as an internal control did not
compete with the IGF-I signal provided by 1.0 µg of IGF-I template
(data not shown).
AT) (panel A) or
pML(CAT) (panels B-D) as an
internal control. Panel A, assays were performed with 60
mM KCl, 6 mM MgCl, and various amounts of
extract, as indicated. Panel B, assays were carried out in the
presence of 60 µg of extract, 6 mM MgCl, and
60 mM KCl at 30 °C for 15 min to 1 h. Panel C,
assays were performed in the presence of 60 µg of extract, 6 mM MgCl, and KCl concentrations at 45-120
mM, at 30 °C for 45 min. Panel D, assays were
carried out with 60 µg of extract, 60 mM KCl, and various
concentrations of MgCl at 30 °C for 45
min.
5`-Flanking Sequences Essential for rIGF-I Gene
Expression
To identify promoter regions, we examined in
vitro transcription supported by IGF-I templates with different
lengths of 5`-flanking sequence: -1050, -701, -471,
-300, -136, and -54bp with respect to the major exon
1 transcription initiation site, as shown in Fig. 5. In four
separate experiments, core promoter activity was consistently detected
with the pIGF(CAT) construct. Transcriptional
activity was increased 2.2-fold with -136 bp of 5`-flanking
sequence and was 5-fold with -300 bp of 5`-flanking sequence
(both p < 0.05 versus -54 bp).
Transcriptional activity was decreased 50% with another 171 bp of
5`-flanking sequence, and there was no significant change in promoter
activity with further addition of upstream sequence to -1050 bp.
Similar results were obtained when circular plasmid DNA was used a
template, indicating that changes in promoter activity were not due to
restraint effects (data not shown).
, 60 µg of extract at 30
°C for 45 min, as described under ``Materials and
Methods.'' The DNA templates were
pIGF(CAT),
pIGF(CAT),
pIGF(CAT),
pIGF(CAT),
pIGF(CAT), and
pIGF(CAT). Panel B, IGF-I signals
determined by densitometric scanning were normalized to those of
pML(CAT). The signal obtained from
pIGF(CAT) was designated as 1. Mean ±
S.E. for four different nuclear extracts is
shown.
DNA-Protein Interactions in rIGF-I 5`-Flanking
Regions
Potential transcription factor binding within the
critical region from -300 to -54 bp was examined with DNase
I protection analysis, as shown in Fig. 6. Three major protected
regions were identified on the coding strand at -119/-100
(I), -149/-126 (II), and -213/-193 bp (III),
with weaker binding at -248/-238 bp (IV). Protection on the
noncoding strand was generally comparable, except that incomplete
protection was observed with region III from -202/-186 bp,
and protection remained weak with region IV. Comparisons with
transcription factor consensus sequences indicate homology with binding
sites for both ubiquitous and liver-specific factors, as summarized in Fig. 7and Table 1.
excess of unlabeled
probe (lane 6) and with a 50 molar excess with spleen
extracts (lane 10); binding was not competed with a 300
excess (0.35 µg) of SspI- and PvuII-digested pBR322 plasmid DNA (not shown). The reduced
binding observed with spleen nuclear extracts was due primarily to
absence of tissue-specific IGF-I-related factors, because the spleen
nuclear extract provided stronger binding activity than the liver
nuclear extract when an Sp1 probe was used (panel B, lanes 2 and 3 versus 4 and 5). The formation of
DNA-protein complexes with Sp1 was also found to be specific via
competition studies (data not shown). Taken together, our findings
suggest that the majority of transcription factor binding within the
300-bp IGF-I promoter region is likely to be tissue-specific.
(lanes 5 and 10) or 100 (lanes 6 and 11) excess
of unlabeled fragment on ice for 20 min before addition of probe. Panel B, a 24-bp double-stranded oligonucleotide containing
Sp1 binding sites was incubated with nuclear extracts from liver (lanes 2 and 3) and spleen (lanes 4 and 5) for 20 min on ice as described. DNA-protein complexes were
resolved on a 5% polyacrylamide gel and visualized by
autoradiography.
molar excess of the competing fragment decreased IGF-I
transcription 15%, AdMLP transcription was unaffected. With a 25
molar excess, IGF-I transcription was decreased by 70% (Fig. 9, lane 3 versus lane 1), but AdMLP transcription
was minimally affected, showing that competition was specific under
these conditions. With a 100 molar excess of the competing
fragment, IGF-I transcription was decreased by 90% (lane 4 versus
lane 1), while 45% of AdMLP activity was retained. In contrast, a
35 excess of a 190-bp (-178/+10) AdMLP fragment
decreased AdMLP expression by 45% but did not interfere with IGF-I gene
expression (not shown). With a 70 molar excess of the AdMLP
competing fragment, expression of both IGF-I and AdMLP was decreased
(not shown), consistent with competition for general transcription
factors. In combination, these findings suggest that binding of
transcription factors to the -240/-58-bp region is
necessary to direct IGF-I gene transcription from the major exon 1
initiation site.
(CAT) template DNA was
added. The in vitro transcription assay was performed as
described under ``Materials and Methods.'' 
174 DNA
digested by HinfI was used as a size marker (M); lane 1, control; lane 2, 5 excess; lane
3, 25 excess; lane 4, 100
excess.
AT), while shorter 190- or 270-bp G-free cassettes
reflected transcription of the adenovirus major late promoter, as an
internal control. Using nuclear extracts shown to be transcriptionally
competent on the basis of AdMLP activity, we found that IGF-I
transcriptional activation was liver-specific, because transcription
was decreased 90% with spleen extracts. Mixing experiments
indicated that reduced transcriptional activation with spleen extracts
may be attributable to the lack of putative tissue-specific activators,
a finding similar to previous observations with the L-type pyruvate
kinase and albumin genes(13, 17) . Transcription was
polymerase II-dependent, as shown by inhibition with -amanitin.
While basal promoter activity could be detected with 54 bp of IGF-I
5`-flanking sequence, a 5-fold stronger signal was detected with 300 bp
of 5`-flanking sequence, with a decrease in signal strength with
addition of further 5`-sequence. Within the -300/-54-bp
region, four DNase I footprints were identified, and competition
studies indicated that binding of putative transcription factors to
such regions is necessary for IGF-I gene expression in vitro. 300 bp of
sequence 5` to the major exon 1 transcription initiation site, in an in vitro transcription system driven by normal liver extracts,
may be compared with observations by other workers who evaluated
promoter activity by transient transfection in extrahepatic immortal
cell lines. Hall et al.(8) examined rIGF-I gene
expression in SK-N-MC neuroblastoma cells and found very limited
promoter activity with a construct extending from -533 to
+190 bp (see Fig. 1), and maximal promoter activity with a
construct with the 5` terminus at -1 kb. Similar findings in
SK-N-MC cells transfected with human IGF-I constructs were reported by
Kim et al.(18) , who found greatest activity with a
construct extending from -1.8 kb to +181 bp. However, Jansen et al.(19) also reported greatest activity in SK-N-MC
cells with a human IGF-I construct extending from -690 to
+55 bp. In contrast, Lowe and Teasdale (9) found maximal
promoter activity in rat dermal fibroblasts and C6 glioma cells with a
rat IGF-I construct extending from -550 to +224 bp and
observed that addition of 700 bp of further 5`-flanking sequence
led to reduced expression. More recently, Lowe (10) used a
similar model to examine rIGF-I gene expression with constructs having
the 3` terminus at +40 bp and reported little fall off in
expression when the 5`-flanking sequence was reduced to -156 bp. 90% by the
presence of a fragment containing sites I, II, III, and IV (partial).
)
We thank Drs. Jeremy Boss, Daniel Reines, and Shouting
Huang for helpful discussion, Liang Zhu for assistance in preparation
of the figures, and Mary Lou Mojonnier and Sharon DePeaza for
assistance in preparation of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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  T. A. Ahmed, M. D. Buzzelli, C. H. Lang, J. B. Capen, M. L. Shumate, M. Navaratnarajah, M. Nagarajan, and R. N. Cooney Interleukin-6 inhibits growth hormone-mediated gene expression in hepatocytes Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1793 - G1803. [Abstract] [Full Text] [PDF]
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 M. Yaekashiwa and L.-H. Wang Transcriptional Control of the Human Thromboxane Synthase Gene in Vivo and in Vitro J. Biol. Chem., June 14, 2002; 277(25): 22497 - 22508. [Abstract] [Full Text] [PDF]
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 S. Harish, T. Khanam, S. Mani, and P. Rangarajan Transcriptional activation by hepatocyte nuclear factor-4 in a cell-free system derived from rat liver nuclei Nucleic Acids Res., March 1, 2001; 29(5): 1047 - 1053. [Abstract] [Full Text] [PDF]
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 J.-L. Zhu, C.-I Pao, E. Hunter Jr., K.-w. M. Lin, G.-j. Wu, and L. S. Phillips Identification of Core Sequences Involved in Metabolism-Dependent Nuclear Protein Binding to the Rat Insulin-Like Growth Factor I Gene Endocrinology, October 1, 1999; 140(10): 4761 - 4771. [Abstract] [Full Text]
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