(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) ()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) .
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 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.
Figure 1:
Construction of DNA templates. The
template pIGF(C
AT) was constructed first (see
``Materials and Methods''). The 5`-upstream region was
extended by adding a 580-bp NcoI genomic fragment to produce
pIGF
(C
AT). The templates
pIGF
(C
AT) and
pIGF
(C
AT) were constructed by deleting NarI/NarI and BanII/NdeI fragments
from pIGF
(C
AT) and
pIGF
(C
AT), 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
(C
AT), subcloned on the SmaI
site on pUC19, and designated as pIGF
(C
AT)
and pIGF
(C
AT), 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.
Figure 2:
Expression of the IGF-I gene in
vitro. Each reaction contained 50 ng of
pML(CAT)
, 60 µg of liver nuclear extract (L, lanes 1, 3, and 4) or spleen nuclear extract (S, lane 2), and 1 µg of pUC13(C
AT) (lane
1) or pIGF
(C
AT) (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.
Figure 3:
In vitro transcription-mixing experiment.
Each reaction contained 50 ng of pML(CAT)
(panel A) or pML(C
AT)
(panel B), 1 µg of pIGF
(C
AT), 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(C
AT)
or
pML(C
AT)
.
Figure 4:
Optimization of assay conditions. In
vitro transcription assays were performed as described under
``Materials and Methods'' with 50 ng of
pML(CAT)
(panel A) or
pML(C
AT)
(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.
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, 60 µg of extract at 30
°C for 45 min, as described under ``Materials and
Methods.'' The DNA templates were
pIGF
(C
AT),
pIGF
(C
AT),
pIGF
(C
AT),
pIGF
(C
AT),
pIGF
(C
AT), and
pIGF
(C
AT). Panel B, IGF-I signals
determined by densitometric scanning were normalized to those of
pML(C
AT)
. The signal obtained from
pIGF
(C
AT) was designated as 1. Mean ±
S.E. for four different nuclear extracts is
shown.
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 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.
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 (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.
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
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.
Figure 9:
Competition analysis. A 183-bp fragment
(-240/-58) was incubated with liver nuclear extract on ice
for 20 min before pIGF(C
AT) 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.
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
(CAT), 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.
The present finding of maximal promoter activity with 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.
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 90% by the
presence of a fragment containing sites I, II, III, and IV (partial).
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.