From the Department of Anatomy and Neurosciences, The
University of Texas Medical Branch, Galveston, Texas 77555-1043 and
§ Laboratory for Physiological Chemistry, Utrecht
University, Universiteitsweg 100, 3586 CG Utrecht, The
Netherlands
Received for publication, August 25, 2000, and in revised form, November 8, 2000
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ABSTRACT |
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The activity of the exogenous, full-length
insulin-like growth factor-2 (IGF-2) P3 promoter is significantly
up-regulated during the logarithmic growth phase but rapidly declines
in confluent CaCo2 cells undergoing differentiation. Nuclear run-on
assays confirmed cell density-dependent regulation of
endogenous P3 promoter. To identify regulatory elements in the P3
promoter that may be required for regulating cell
density-dependent transcriptional activity, we used the
methods of promoter truncation, electrophoretic mobility shift assay,
DNase footprinting, and mutation analysis. The relative activity of the
full-length ( A large percent of colon cancers overexpress
IGF-21 (1-5) and contain
significant concentrations of IGF-I receptor (5-7). Colon
cancer-derived cell lines secrete unprocessed IGF-2, which is even more
potent than the mature form (8) and is believed to contribute to
autocrine growth of cancer cells (9). Since a significant percent of
primary/metastatic human colon cancers express IGF-2 and IGF-I receptor
(1, 3, 5, 10) and surrounding stromal/normal epithelial cells may also
express IGFs (11, 12), IGFs are likely to be potent mitogenic factors
for colon cancers in situ by autocrine, endocrine, and
paracrine mechanisms. We and others have shown that IGF-2 is a potent
autocrine growth factor for a significant percent of human colon cancer
cells (4, 13). Evidence in literature strongly supports the contention that IGFs (via IGF-I receptor) play an important role not only in the
proliferation/tumorigenicity of colon cancers but also in inhibiting
differentiation of the cells (13-16). It is therefore important to
understand the mechanisms that contribute to up-regulation of IGF-2
levels in colon cancers.
The IGF-2 gene consists of 9 exons, and the peptide is encoded by exons
7, 8, and 9 (17, 18). The gene is transcribed from four different
promoters (P1-P4) (18). Multiple transcripts are synthesized as a
result of alternative promoter usage and the splicing of the unique
5'-untranslated region to common coding exons (18). Enhanced levels of
P3- and P4-driven IGF-2 mRNAs have been detected in many human
tumors including colorectal cancers (1-4, 9, 10, 19). Increased levels
of IGF-2 mRNA in neoplastic cells can be potentially accomplished
by loss of imprinting, loss of heterozygosity, transcriptional
activation, and/or loss of transcriptional suppression (reviewed in
Ref. 20). With loss of imprinting or loss of heterozygosity (involving
duplication of the active paternal allele), IGF transcripts are made
from two copies instead of one, leading to ~two times higher mRNA
levels. However, the expression of IGF-2 mRNA is 10-40-fold higher
in colon cancer cells compared with that in normal colonocytes (1-5). Although loss of imprinting can contribute to overexpression of IGF-2,
the majority of overexpression is now believed to be due to
transcriptional up-regulation in several cancers (4, 13, 21, 22).
Factors involved in transcriptional regulation of the IGF-2 gene in
colon cancers are as yet unknown.
In previous studies we and others have used CaCo2 cells (a human colon
cancer cell line) for investigating the role of the IGF system in
growth and differentiation of colon cancer cells (10, 13, 15, 16, 19,
23, 24). CaCo2 cells spontaneously differentiate in culture at
confluency (25) and are an ideal in vitro model for
investigating cellular mechanisms involved in proliferation and
differentiation of colonic cells. IGF-2 mRNA levels are
significantly up-regulated during logarithmic growth of CaCo2 cells
(days 3-6) in culture followed by a steep decline in confluent cells
undergoing rapid differentiation (days 7-10) (13, 23). We recently
reported that P3- and P4-derived transcripts were significantly
up-regulated during the proliferative phase of the cells (days 3-6 in
culture) and declined rapidly in cells undergoing differentiation (days
7-10); P1- and P2-derived transcripts were not detected (19).
Similarly, transcriptional activity of transiently transfected P3 and
P4 promoters reached peak levels by days 4-6 and declined rapidly
thereafter (19). At the present time, the regulatory element(s) within
the P3 and P4 promoters, which is involved in the observed cell
density-dependent regulation of the promoter activity, is
unknown. Since a majority of colon cancers mainly express P3-derived
transcripts, we further investigated the role of the endogenous P3
promoter in regulating IGF-2 mRNA levels, in a cell
density-dependent/differentiation manner using the method
of nuclear run-on assays. Additionally, for the first time, we have
defined the promoter segment(s) and a putative novel cis element that
may play a critical role in the cell density-dependent regulation of P3 promoter activity in CaCo2 cells using the methods of
transient transfection with truncated promoter-reporter constructs, electrophoretic mobility shift assay (EMSA), and DNase footprinting.
Cell Culture--
The CaCo2 human colon cancer cell line was
obtained from Dr. Jing Yu, Tufts School of Medicine, and cells were
grown as monolayer cultures as previously described (13, 19). For all
experimental purposes, exponentially growing cells were seeded at
~150,000 cells/35-mm cell culture dishes (Falcon, Fisher), and the
cell culture medium was changed every second day with fresh growth medium containing 10% FCS.
Nuclear Run-on Assay--
Nuclei were obtained, and assays were
performed by a modification of previously described procedures (26).
Briefly, 7-10 × 107 CaCo2 cells, at the indicated
time points, were washed 2 times in phosphate-buffered saline
(4 °C). Cells were scraped and lysed in 2 ml of Nonidet P-40 buffer
(10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40), and nuclei
were harvested and resuspended in 100 µl (~1-2 × 107 nuclei/100 µl) of glycerol storage buffer (40%
glycerol, 10 mM Tris, pH 7.5, 5 mM
MgCl2, 80 mM KCl, and 0.1 mM EDTA)
and stored at Human IGF-2 P3 Expression Vectors Used for
Transfections--
The IGF-2 promoter-luc constructs used for
transfection are presented diagrammatically in Fig.
1. The full-length human promoter P3 DNA
fragment was subcloned into the BamHI and SaII
sites of the luciferase reporter gene expression vector, PSla3 (18), resulting in HUP3. Additional P3 clones containing the indicated truncated P3 fragments (Fig. 1) were derived from HUP3 either by
restriction enzyme digestions with the appropriate enzymes and filling
in of the protruding ends by Klenow DNA polymerase (Fig. 1) or
by PCR amplification of the indicated fragments (Fig. 1). For PCR
amplification, six separate 5' sense primers were used:
(a1) 5'-GCGCGCGGAGGGCGAAGTGATTGAT-3' ( DNA Transfection of CaCo2 Cells--
CaCo2 cells in culture in
35-mm dishes (as described above under cell culture) were transfected
with 5-10 µg of the indicated vector DNA on days 2-8 of cell
culture by the calcium phosphate precipitation method, as described
previously (19, 28, 29). Cells were cotransfected with the SV40
Luciferase and EMSA--
Initially we conducted EMSA with double-stranded (ds)
DNA fragments that were obtained by restriction digestion of the full length HUP3-luc vector, as shown in Table I. The DNA fragments were
purified by agarose gel electrophoresis and labeled with [ DNase I Footprinting Assay--
DNase I footprinting assays were
performed with the help of Core Footprinting System (Promega) by our
published procedures (31). The ds DNA fragment ( Construction of a Homologous P3 Core-luc Construct Ligated to
Either WT or Mutant Distal P3 Promoter--
To examine the
functionality of the identified regulatory DNA segments within the
distal P3 promoter, we used HUP3-A6-luc vector (Fig. 1) that contained
a representative P3 core promoter ( Relative Levels of Endogenous P3-derived IGF-2 Transcripts in CaCo2
Cells on Days 3-11 of Cell Culture--
To determine the levels of
expression of endogenous P3-derived IGF-2 mRNAs in proliferating
and differentiating CaCo2 cells, nuclear run-on transcription assays
were conducted in the nuclei of CaCo2 cells on consecutive days of cell
culture. In four separate experiments, RNA prepared from nuclei on the
indicated days of cell culture were hybridized with various probes as
described under "Experimental Procedures."
Hybridization of nuclear RNA with the 18 S ribosomal DNA demonstrated a
similar intensity in the signal on different days of cell culture,
implying that equal amounts of RNA were used for the experiment (Fig.
2A). No specific signal other
than background noise was obtained with the negative control DNA
(Bluescript SK+ vector without insert). However, IGF-2
transcription was highly regulated and dependent on the differentiation
status of the CaCo2 cells (Fig. 2A). A significant
up-regulation in the relative abundance of IGF-2 transcripts was
measured on day 5 (compared with day 3 and 4 values) when cells are in
the proliferative phase. This was followed by a steep decline in the
relative abundance of IGF-2 transcripts on days 6-7, when cells enter
differentiation, and IGF-2 mRNA levels were minimal on days 8-11,
the exponential phase of differentiation (Fig. 2A).
Four independent experiments with nuclei from CaCo2 cells samples were
performed using the IGF-2 and 18 S probes. Densitometric analysis of
the autoradiographic data with the IGF-2 probe, normalized with the 18 S probe, is shown in bar graphs in Fig. 2B.
Interestingly, the observed pattern of transcriptional activity for the
endogenous IGF-2 gene is almost identical to the pattern of
transcriptional activity measured with exogenous P3 promoter-reporter
constructs as recently reported (19). The results thus confirm our
earlier findings that reduction of IGF-2 mRNA levels in CaCo2 cells
(13) reflect changes in the transcriptional activity of the exogenous
(19) and endogenous (present study) IGF-2 gene caused by gradual
progression of differentiation on consecutive days of cell culture.
Relative Contribution of the Distal, Middle, and Proximal Segments
of the P3 Promoter in Cell Density-dependent Regulation of
the P3 Promoter Activity--
To determine if the promoter region
responsible for the cell density-dependent regulation of
transcription was located within the distal, middle, or proximal
segment(s) of the P3 promoter, we used three IGF-2 P3 promoter
constructs in transient transfection assays. HUP3-LUC, the maximal
promoter (
The relative activity of HAVP3 (
To further narrow down the location of the putative regulatory
element(s) in the distal segment of P3 promoter, we initially measured
transcriptional activity of three additional truncated constructs
(HUP3-A1, HUP3-A5, and HUP3-A6) (Fig. 1) and compared the activity with
that of HBSP3-luc and HAVP3-luc vectors. The results of a
representative experiment (of a total of two similar experiments) are
presented in Fig. 4. The transcriptional
activity of HUP3-A1-luc construct was almost identical to that of the
full-length HUP3-luc vector (Fig. 4), indicating that the extreme
distal end of the P3 promoter ( The Distal P3 Promoter Fragment Contains a Specific DNA-Protein
Complex--
We initially conducted EMSA with relatively large
(100-400 bp) P3 promoter DNA fragments covering the entire distal to
middle promoter (Table I). Using
fragments 1, 3, and 4 as probes, similar patterns were observed for
extracts from proliferating and post-confluent CaCo2 cells (data not
shown). Only fragment 2 (
Based on the above truncation and EMSA results, a DNA fragment
(
As a final confirmatory step, we used a 25-bp ds oligonucleotide
sequence (
Since DNA footprinting studies also suggested the presence of a
putative binding site within the
To confirm a regulatory function of the identified regulatory element
within the distal DNA segment of P3 promoter, we used either the WT or
mutant P3-D HUP3-A6-luc vector in transfection studies, as described
under "Experimental Procedures." The results with the WT and
mutant P3-D HUP3-A6-luc vectors is presented in Table
II; the activity of the full-length
HUP3-luc vector is presented for comparison. As can be seen from the
table, the presence of the distal P3 promoter segment, containing the
WT P3-D element, significantly reduced the transcriptional activity of
the core HUP3-A6 promoter by ~80-90%, and the activity was similar
to that of the full-length HUP3 promoter. On the other hand,
transcriptional activity of mutant P3-D HUP3-A6-luc vector, which was
mutated at the P3-D element, was reduced only slightly compared with
the activity of the parent HUP3-A6-luc vector. Importantly,
transcriptional activity of the WT P3-D HUP3-A6 promoter mimicked the
activity of the full-length HUP3 vector and was similarly
down-regulated in day-7 samples. These results confirm the functional
significance of the P3-D element and once again suggest a negative role
of the P3-D element in regulating the transcriptional activity of the
P3 promoter in a cell density-dependent manner.
In a previous study, we could show by transient transfection
studies that transcription of the P3 promoter of the IGF-2 gene is
dependent on the growth phase of CaCo2 cells. Transcription was
significantly up-regulated during logarithmic growth of the cells and
rapidly declined in confluent cells undergoing differentiation (19). In
the present study, we show that the transcriptional activity of the
endogenous IGF-2 gene is identical to that of the exogenously
transfected IGF-2 P3 promoter. By applying nuclear run-on assays at
different stages of CaCo2 cell growth it was confirmed that the
transcriptional activity of the endogenous IGF-2 P3 promoter was
significantly up-regulated during the proliferative phase of cell
growth (days 4-6) followed by a steep decline in transcriptional
activity during the differentiation phase (days 7-11) (Fig. 2). This
confirms our hypothesis that the endogenous IGF-2 levels in CaCo2 cells
are regulated mainly at the transcriptional level in a cell density and
differentiation-dependent manner.
Several functional regulatory elements are present in the proximal
segment ( Transcription factors and cis elements that may be involved in the
transcriptional regulation of the P3 promoter in human colon cancer
cells have yet to be identified. In the current study we observed that
the distal segment of the P3 promoter, upstream of nucleotide The promoter segments between Using the methods of EMSA and DNase footprinting, we have identified a
putative cis element (CGAGGGC, arbitrarily named P3-D) in the distal
segment of the IGF-2 P3 promoter that specifically binds nuclear
proteins from proliferating CaCo2 cells. In addition, the promoter
truncation studies indicated the presence of physiologically relevant
regulatory element(s) in this distal segment of the P3 promoter,
encompassing the sequences indicated in the EMSA and DNase footprinting
studies. Transcriptional activity of core P3 promoter-reporter vector
was significantly reduced on ligation with distal P3 promoter sequences
containing the WT-P3-D element; mutation of P3-D element in the ligated
sequence did not reduce the activity of the core promoter
significantly. These results further confirmed our findings that the
distal segment encompassing the putative P3-D regulatory element was
indeed functional and down-regulated the activity of the core promoter
in a cell density-dependent manner. The sequence identified
by DNase footprint ( In summary, our studies suggest the presence of a 7-bp regulatory
element in the distal portion of the P3 promoter that is involved in
silencing IGF-2 gene expression in colonic cells undergoing differentiation. We hypothesize that native proteins (that are perhaps
expressed in a cell differentiation-dependent manner) bind
to the putative P3-D regulatory element in the distal segment of the P3
promoter to either suppress the down-regulation of IGF-2 expression
(during proliferation) or to silence IGF-2 expression (during
differentiation); our studies so far support the former model. The
cytokines interleukin-4 and transforming growth factor 1229/+140) and truncated (
1090/+140) promoter was
identical, being ~19, 27, 7, and 3% of pSV-luc activity on days 3, 5, 7, and 9 of cell culture, respectively. However, truncation to
1048 resulted in complete loss of cell density-dependent
down-regulation of P3 promoter activity on days 7 and 9, suggesting the
presence of regulatory elements between
1091 and
1048
sequence. Further stepwise truncation to
515 did not change promoter
activity. Truncation to
138/+140 resulted in complete loss of
promoter activity, suggesting that the core promoter was within the
515/
138 segment. A 14-base pair footprint (
1084/
1070) was
identified by DNase footprinting within the distal
1091/
1048
segment. Electrophoretic mobility shift assay with wild type and mutant
probes confirmed the presence of a novel 7-base pair (CGAGGGC)
(
1084/
1078) cis element (P3-D); its mutation abolished binding.
Functionality of P3-D cis element was confirmed by measuring the
activity of core P3 promoter ligated to distal P3 segment containing
either the mutant or wild type P3-D element. We have, therefore,
identified a novel cis element, P3-D, that appears to play a critical
role in regulating IGF-2 P3 promoter activity in a cell
density/differentiation-dependent manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. Thirty µl of nuclear run-on buffer (25 mM Tris, 12.5 mM MgCl2, 750 mM KCl, 1.25 mM ATP, 1.25 mM
CTP, 1.25 mM GTP) and 20 µl of [32P]UTP
(3000 Ci/mmol, ICN, Costa Mesa, CA) were added to 100 µl of nuclear
suspension. The transcription runoff was allowed to proceed at 30 °C
for 30 min and terminated with DNase I (300 units), and
CaCl2 (final concentration 1 mM) was allowed to
proceed at 30 °C for 10 min. One µl of proteinase K (20 mg/ml) and
25 µl of SET buffer (5% SDS, 50 mM EDTA, 100 mM Tris, pH 7.5) were added and incubated for 30 min at
37 °C. Subsequently, 550 µl of 4 M guanidinium
isothiocyanate and 90 µl of 3 M sodium acetate were added, and the mixture was extracted with phenol/chloroform/isoamyl alcohol (25:24:1). The aqueous phase was removed, and RNA was precipitated with 1 volume of isopropyl alcohol. The pellet was resuspended in 300 µl of guanidinium isothiocyanate, and RNA was reprecipitated with isopropyl alcohol. The pellet was washed with 70%
ethanol, dissolved in 100 µl of TES (10 mM Tris, pH 7.2, 1 mM EDTA, 0.1% SDS), and used for hybridization. A DNA
slot or dot blot apparatus was used to blot equal concentrations of
linearized plasmids (10 µg) onto nitrocellulose filters (Schleicher
and Schuell). The DNA plasmids used included the negative control
(Bluescript II SK+ vector without insert), the Bluescript
II SK+ vector (containing human 18S cDNA used as a
positive control for normalizing the hybridization signals), and a
plasmid containing the human IGF-2 exon 5 DNA (to measure P3
promoter-driven transcriptional activity). The plasmid DNA were
linearized by appropriate restriction endonucleases and denatured with
0.2 M NaOH. The pre-hybridization and hybridization
solutions consisted of 10 mM TES, 0.2% SDS, 10 mM EDTA, 0.3 M NaCl, 1× Denhardt's, 0.25 mg/ml, Escherichia coli DNA. DNA blots were pre-hybridized
in hybridization buffer for 3 h at 65 °C for 36 h.
Hybridization was carried out at 65 °C for 36 h. Filters were
washed in 2× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate) with 0.1% SDS for 10 min twice
and then in 0.01× SSC with 0.1% SDS for 30 min at 60 °C before
autoradiographic analysis. The nuclear run-on assay results were
quantitated by densitometric analysis of the autoradiographic results
using the UltroScan LX laser densitometer (Amersham Pharmacia Biotech). Relative levels of RNA on different days of cell culture were determined with the help of GelScan XL software (Amersham Pharmacia Biotech). RNA levels in each experimental blot are expressed as a ratio
of the corresponding 18 S RNA blot.
1090/
1070);
(a2) 5'-GCGCGCGGAGGGCGAAGTGATTGAT-3' (
1048/
1012);
(a3) 5'-AGGGGGGCAGGGGGGCGCGGGATTC-3' (
1006/
982);
(a4) 5'-TCCCCTTGGCTAGGCTTAGGCGGC-3' (
946/
923); (a5) 5'-ACCCCCAAATTATCGTGGTGG-3' (
884/
864); primer
(a6) 5'-TCCCGCCCTGATCCTCTCTCC-3' (
716/
696) along
with a common 3'-antisense primer 5'-GTCGACGCGGGGCCGCCTTGCCCGA-3' (+139/+115). The full-length HUP3-luc vector (linearized) was used as
template for the PCR amplifications. The PCR reaction was performed in
the presence of AmpliTaq DNA polymerase (PerkinElmer Life Sciences) as
described previously (27). The PCR products were subcloned into PCR 2.1 cloning vector using a TA cloning kit (Invitrogen, San Diego, CA) as
per protocols provided by the supplier. Positive clones (that were
determined to have the appropriate orientation of the P3 promoter DNA
fragment in relation to the KpnI site on the PCR 2.1 vector)
were selected and checked by DNA sequencing. The inserts were released
from the PCR 2.1 vectors by XhoI and KpnI
endonuclease digestion and religated into the same sites in
promoterless luciferase reporter vector, pGL2-Basic (Promega, Madison,
WI). The six new luciferase expression vectors, thus constructed, were
termed HUP A1-HUP A6 luc vectors as shown in Fig. 1. The orientation
and the sequence of the inserts were confirmed by DNA sequencing using
pGL primers one, 5'-TCTATCTTATCCTACTGTAACTG-3' (~10 bp upstream from
multiple cloning site of pGL-2 basic vector), and two,
5'-CTTTATGTTTTTGGCGTCTTCC-3' (~20 bp downstream from multiple cloning
site of pGL-2 basic vector).
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Fig. 1.
Schematic representation of the IGF-2
promoter-luc constructs used in transient transfection assays. The
restriction sites available in the P3 promoter and exon 5 were used for
constructing truncated constructs HBSP3 and HAVP3 in luciferase
expression vector, pSla3. Six truncated constructs are also shown
(HUP3-A1-luc to HUP3-A6-luc) that were PCR-amplified from the HUP3
vector using primers a1-a6, as described under
"Experimental Procedures."
-galactosidase expression vector (pSV
-Gal vector, Promega) to
normalize for transfection efficiency (19, 29). Duplicate dishes were
transfected with 5 µg of luciferase expression vector (pGL2, Promega)
under the transcriptional control of the SV40 promoter as a positive
control (19, 29). Cells were grown in regular growth medium containing 10% FCS until 22 h before transfection. Fresh growth medium
containing 10% FCS was added on alternate days until the day of
transfection. Additionally, before transfection, fresh growth medium
plus 10% FCS was added. Post-transfection, the cells were washed with
fresh medium plus 10% FCS and cultured for 24 h followed by cell
lysis for purposes of measuring luciferase and
-Gal activities.
-Gal Assays--
Luciferase and
-Gal were
measured in the cell lysates as described by us (29). Briefly, cells
were washed twice with phosphate-buffered saline and lysed using the
reporter lysis buffer (Promega) as per the protocols provided by the
company. Luciferase activity was measured with the luciferase assay
system (Promega) using 50 µl of cell extract and 100 µl of
luciferase buffer at room temperature. Luciferase activity was measured
within 15 s of adding the substrate, luciferin, with Berthold
AutoLumaat LB953 (Wallac-Berthold, Gaithersburg, MD). The
-Gal assay
was performed with the
-galactosidase enzyme assay system (Promega)
as per the protocols provided by the company, as described by us (29).
At the end of the reaction, the absorbance of the samples was read at
420 nm in a Umax kinetic microplate reader (Molecular Devices, Menlo
Park, CA).
-32P]ATP by T4 polynucleotide kinase (Promega). The
labeled DNA fragments were separated from unincorporated nucleotides by
chromatography through a G50 spin column (Amersham Pharmacia Biotech).
Nuclear extracts were prepared from CaCo2 cells on the indicated days of cell culture by methods described by Abmayr et al. (30). Protein concentrations were measured, and nuclear extracts were separated into aliquots and frozen at
70 °C. EMSA was performed essentially as described by Rietveld et al. (31). Briefly,
EMSA was performed in a 20-µl binding reaction containing 5-10 µg
of crude nuclear extract prepared from CaCo2 cells on different days of
cell culture, 5 µl of DNA probe containing ~200,000 cpm, 1 µg of
poly(dI-dC), 10 mM Tris-HCl, pH 7.5, 50 mM
NaCl, 0.05 mM EDTA, 1 mM MgCl2, 4%
glycerol, and 1 mM dithiothreitol and incubated for 15 min
at room temperature. For competition, a 100-200-fold excess of the
unlabeled DNA probe was mixed with the radiolabeled probe before the
addition of the protein extract. After incubating the samples for 15 min at room temperature, the DNA protein complexes were separated from
the free DNA by electrophoresis in 5% nondenaturing polyacrylamide
gels in 0.5× Tris-boric acid-EDTA buffer. The gels were dried
and subjected to autoradiography in the presence of intensifying
screens at
80 °C. We additionally conducted EMSA with the
indicated ds oligonucleotide probes using methods that were essentially
similar to that described previously (31). The oligonucleotide probes
used are detailed under "Results."
1164/
871) was
amplified with sense primer, 5'-AGGGACGCGGAGGAGAGGCGC TCC-3', and
antisense primer, 5'-AGGATCCATTTGGGGGTCTGGGGAAACCAT-3', by PCR using
HUP3-luc vector as the template. PCR products were cloned into PCR 2.1 vector by using TOPO cloning kit (Invitrogen, CA). The DNA fragment was released from the new vector by EcoRI digestion and isolated
from agarose gel by using QIAEX II gel extraction kit (Qiagen, Inc.). The resulting 293-bp template was labeled with
[
-32P]ATP by T4 DNA kinase (New England Biolabs). The
labeled DNA was digested with BamHI to get a single-stranded
template for the DNase footprinting assay. Unincorporated nucleotides
were removed from the labeled sample by using NucTrap® probe
purification columns (Stratagene). The binding reaction was set up as
per the protocol provided by the manufacturer. The binding reaction (50 µl) contained 100,000 cpm of labeled DNA probe, 20 µl of nuclear protein (prepared as described above), 25 mM Tris-HCI, pH
8.0, 50 mM KCl, 6.125 mM MgCl2, 0.5 mM EDTA, 10% glycerol, and 1 mM dithiothreitol. After incubation for 20 min at 4 °C, 50 µl of 10 mM MgCl2, and 5 mM
CaCl2 were added followed by the addition of 0.14 units of
DNase I, and incubation was continued at room temperature for 3 min.
DNase I digestion was quenched by the addition of 90 µl of stop
solution (200 mM NaCl, 30 mM EDTA, 1% SDS),
and the DNA template was purified by phenol/chloroform (1:1) extraction and ethanol precipitation. Samples were analyzed by electrophoresis through denaturing 6% polyacrylamide/urea sequencing gels. Gels were
dried on Whatman No. 3MM paper and visualized by autoradiography. To
identify the nt sequence of the footprint, we simultaneously ran a
20-bp DNA ladder (Sigma) labeled with [
-32P]ATP using
T4 DNA kinase. The labeled DNA ladder was run along with the
DNase-treated samples as a size marker by the Maxam and Gilbert G+A
track method, as published previously (31).
712/+140). Two pairs of ~100-bp
oligonucleotides were synthesized (Genemed Syn Inc.)
for ligation to the homologous core P3 promoter. The WT sense and
antisense oligonucleotide sequences represented
1091/
991
nucleotides of the P3 promoter. The mutant ~100-bp oligonucleotides
were mutated at the
1087/
1074 nucleotide sequence by AT repeats in
the sense oligo and by TA repeats in the antisense oligo. The
ds-synthesized oligos contained KpnI (5') BamHI
(3') overhangs for ligating to the core promoter. The HUP3-A6-luc
vector was pre-digested with KpnI and BamHI. The
pre-digested vector was ligated with the ds-synthesized oligos using T4
DNA ligase. The constructs were confirmed by DNA sequencing using the
pGL primers as indicated above and were arbitrarily named P3-D WT- and
P3-D mut-HUP3-A6-luc vectors. The WT and mutant vectors thus constructed were transfected into CaCo2 cells on days 5 and 7 along
with the
-Gal vector, and the luciferase and
-Gal activities were
measured in the cells 24 h post-transfection as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Nuclear run-on analysis of endogenous IGF-2
gene transcription in CaCo2 cells on days 3-11 of cell culture.
A, representative autoradiographs from experiments I and II
from a total of four experiments are shown. The probe used for
measuring IGF-2 P3-derived transcripts is described under
"Experimental Procedures." As controls, 18 S ribosomal transcripts
were measured simultaneously. B, the ratios of IGF-2/18 S
mRNAs measured for all four experiments on different days of cell
culture are shown as bar graphs. The data in the bar
graphs from days 3-11 are presented as the mean ± S.D. of
duplicate measurements from 2-4 separate experiments.
1229/+140), and two truncated promoter fragments, HBSP3-luc
(
515/+140) and HAVP3-luc (
138/+140) (Fig. 1), were used in these
studies. CaCo2 cells were transfected on days 3, 5, 7, and 9 of cell
culture with one of the P3 promoter constructs and with a
-Gal
expression vector to correct for transfection efficiency. To normalize
the activities of the IGF-2 promoter constructs, pSV-luc was included
as a positive control, and its value was arbitrarily assigned to 100%.
Data from a representative experiment (from a total of three similar
experiments) are presented in Fig.
3A as a percent of the
pSV-luc/
-Gal activity on different days of cell culture.
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Fig. 3.
Transcriptional activity of the full-length
and truncated IGF-2 P3-luc vectors in CaCo2 cells. A,
CaCo2 cells were cotransfected with DNA from the full-length (HUP3-luc)
and truncated (HBSP3-luc and HAVP3-luc) vectors (Fig. 1) and with a
-Gal expression vector to normalize for transfection efficiency as
described under "Experimental Procedures." Duplicate dishes were
transiently transfected with DNA from the pSV-luc vector. The
luc/
-Gal activity for each sample was determined, and the relative
activity of the P3-luc vectors is presented as a percent of the pSV-luc
activity (arbitrarily assigned a 100% value) on the indicated days of
cell culture. Each bar graph represents the mean ± S.E. of data obtained from three separate experiments. B,
the fold increase in the activity of the HBSP3-luc vector compared with
that of the full-length HUP3-luc vector (based on data presented in
A) is presented as a ratio of HBSP3-luc/HUP3-luc activity on
the indicated days of cell culture. a, b, and
c indicate data from three separate experiments. The
numbers on top of each bar graph represent the
fold increase of the luc activity of construct HBSP3 compared with that
of the HUP3-luc activity.
138/+140) was minimal on all days of
cell culture, suggesting that even for basal P3 promoter activity, some
core elements critical for transcription must be present upstream of
position
138. On the other hand, HBSP3 (
515/+140) appeared to
contain all the critical basal promoter elements and demonstrated
maximal promoter activity. Surprisingly, the activity of HBSP3 was
significantly higher than that of the full-length HUP3 (
1229/+140) on
all days of cell culture, suggesting that specific inhibitory elements
may be located between
1229 and
515. The fold increase in the
activity of HBSP3 compared with that of HUP3 (as measured in 3 separate
experiments, a-c, on days 3, 5, and 7 of cell culture) was
determined (Fig. 3B). Although the relative activity of
HBSP3 decreased 2-3-fold from days 4-5 to 7-8 of cell culture (Fig.
3A), the relative activity of HUP3 was almost abolished on
days 7-10 of cell culture. The strong down-regulation of the
transcriptional activity of HUP3, but not of HBSP3, suggested that the
distal segment of the P3 promoter (
1229 to
515) contained a potent
inhibitory element that is regulated in a cell
density-dependent manner.
1229 to
1091) does not contain the
regulatory element(s). In contrast, further truncation to
889
(HUP3-A5-luc) resulted in a strong increase (up to 10-fold) of promoter
activity, suggesting that the cell density-dependent
regulation of P3 was lost on truncation of the segment between
1091
to
889. Further truncation of the distal segment to
712
(HUP3-A6-luc) showed no additional effects on transcriptional activity.
These results suggested that the P3 promoter contained regulatory
element(s) between
1091 and
889 (relative to the CAP site) that may
be involved in silencing of the promoter by many fold. More
importantly, the cell density-dependent regulation of the
P3 promoter was lost on removal of the distal segment between
1091
and
889 (as in HUP3-A5 vector). To further narrow down the location
of the regulatory element, additional truncated constructs between
1091 and
889 were generated as shown in Fig. 1. Transient
transfection studies with HUP3-A2-luc (
1048/+140), HUP3-A3-luc
(
1006/+140), or HUP3-A4-luc (
946/+140) demonstrated that the
luciferase activity of all these truncated constructs was identical to
that shown in Fig. 4 for HUP3-A5-luc vector (
889/+140) on days 3-9
of cell culture, indicating that the putative distal regulatory element
was between
1090 and
1048 sequence of the P3 promoter (data not
shown). To further confirm the presence of putative DNA binding sites
in the distal P3 promoter, we conducted DNase footprinting and EMSA
studies as described below.
View larger version (37K):
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Fig. 4.
Transcriptional activity of the truncated
constructs of the IGF-2 P3 promoter in CaCo2 cells on days 3-9 of cell
culture. Five truncated constructs of the distal segment of the P3
promoter were generated as described under Fig. 1 and used in transient
transfection studies as per methods described under "Experimental
Procedures." CaCo2 cells on different days of culture were
cotransfected with the -Gal expression vector, and the ratio of
luc/
-Gal was determined at each time point for the various vectors.
The luc/
-Gal values on day 5 with the HBS-luc vector were
arbitrarily assigned a 100% value. All other values are presented as a
percent of that measured with the HBS-luc vector on day 5. Data from
two separate experiments are presented as bar graphs. Data
represent the mean of 4-5 measurements from two separate experiments,
wherein individual values varied by <10%.
1090/
656) demonstrated significant
differences in the binding pattern of nuclear proteins from
proliferating (day 5) versus post-confluent (day 10) CaCo2 cells (Fig. 5A). A unique and
specific DNA-protein complex (a) was present in
proliferating cells (day 5) that was absent in post-confluent (day 10)
cells.
Restriction fragments of P3 promoter used as EMSA probes
View larger version (51K):
[in a new window]
Fig. 5.
Localization of a
differentiation-dependent DNA-protein complex.
A, EMSA of 32P-labeled IGF-2 P3 DNA fragment 2 ( 1090/
656) (Table I) with nuclear proteins prepared from CaCo2
cells on either day 5 (lane 1) or day 10 (lane 2)
of culture. Complex a is seen only in day-5 samples. All
other complexes are common to day-5 and day-10 samples. Band
e is free 32P-labeled probe. B, EMSA
with 32P-labeled ds oligonucleotide probe (
1090/
1065)
in the presence of nuclear proteins from CaCo2 cells on different days
of culture in the presence or absence of excess unlabeled probe.
Autoradiograph of EMSA with nuclear protein samples from days 5-14
(lanes 2-4) using the radiolabeled 25-bp oligonucleotide
probe in the presence (lane 1) or absence (lanes
2-4) of a 100-fold excess of unlabeled homologous probe is shown.
Lane 1, D5 sample in the presence of 100-fold excess of
unlabeled ds 25-bp probe; lane 2, D5 (50 µg) nuclear
protein; lane 3, D10 (50 µg) nuclear protein; lane
4, D14 (50 µg) nuclear protein. Complex a was
prominent in D5 samples (lane 2), whereas it was
significantly reduced in D10 samples (lane 3). Complex
a was minimally observed in day 14 samples (lane
4). The binding of proteins in day-5 samples was specific and
completely displaced by excess unlabeled probe, as shown in lane
1.
1164/
871) containing the putative regulatory element was used as a
radiolabeled probe for DNase1 footprinting experiments to confirm the
presence of the binding site in this distal segment. A specific DNA
footprint was obtained with nuclear proteins from proliferating day-5
samples but not from day-10 samples. Positive data with day-5 results
are shown in Fig. 6. The 14-bp-nt
sequence of the footprint was identified with the help of a 20-bp DNA
ladder co-run with the samples as described under
"Experimental Procedures" and is shown in Fig. 6.
View larger version (26K):
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Fig. 6.
DNA footprint of a distal fragment of
IGF-2-P3 promoter. A ds DNA fragment ( 1164/
871) was generated
and used in the DNase 1 footprinting assay, as described under
"Experimental Procedures," in the presence of nuclear proteins from
day-5 and day-10 CaCo2 cells. No footprint was visible with day-10
samples, whereas a specific footprint was visible with day-5 samples in
two separate experiments. A representative autoradiograph of DNA
footprint of a total of 2 autoradiographs from 2 experiments with day-5
samples is shown. A labeled DNA ladder was co-run with the
DNase-treated samples as a size marker, and the nt sequence of the
DNase-protected site was determined by Maxam and Gilbert G+A track
method, as published previously (31, 35). A 14-bp region
(
1084/
1070) with the indicated sequence was specifically protected
from DNase 1 digestion (containing the putative P3-D binding site) in
the presence of day-5 samples (lanes 1 and 2).
Lanes 3 and 4, DNA samples run in the absence of
nuclear proteins. The size of the DNA markers that were co-run with the
samples is indicated on the right-hand side with arrows. The
5'/3' direction of the DNA probe, used in the footprint assays, is
indicated by the arrows facing up and down. On the left, the
nt sequence of the DNA footprint is shown.
1090/
1065) encompassing the 14-bp DNA footprint (Fig. 6)
for EMSA analysis with CaCo2 samples, prepared from proliferating versus post-confluent cells (Fig. 5B). Using the
25-bp ds oligonucleotide as a probe, one DNA-protein complex
(a) was prominently present in day-5 samples from
proliferating cells (lane 2), and this band was
significantly reduced in confluent day-10 cells (lane 3) and almost absent in day-14 cells (lane 4). In the presence of
excess unlabeled probe, no complex was observed (lane 1),
indicating that the binding was specific. To confirm that promoter
sequences 5' to
1090 and 3' to
1065 did not contain any other
putative DNA binding sites, we also conducted EMSA with several other
20-25-bp oligonucleotide probes that contained sequences between
1229 to
1093 at the 5' end and between
1063 to
999 at the 3'
end of the putative regulatory element; no binding was observed with sequences that were either 5' of
1091 or 3' of
1063 (data not shown).
1090/
1065 segment of the P3
promoter, we focused on identifying the core binding element within
this region by mutation analysis of the
1090/
1065 EMSA probe.
Mutation of the 25-bp
1090/
1065 EMSA probe at the 5' end (between
1090 and
1086) or at the 3' end (between
1075 and
1065) had no
effect on the binding of the mutant probes to day-5 nuclear proteins
(data not shown). However, mutations between
1086 and
1075 had
significant effects on the binding to day-5 samples (Fig.
7). Binding of day-5 samples to mutants
M-1 and M-4 was slightly reduced by ~20%. Binding was significantly
decreased by ~40 to >60% to mutants M2, M3, and M5. Since the
mutations in M2, M3, and M5 overlapped within the region of
1084 and
1078, we mutated all 6 base pairs between this segment in mutant M6, which resulted in complete loss of binding (Fig. 7). The results with
the mutant probes suggest that the core of the binding element is
present within
1084/
1078 of the P3 promoter. Computer analysis of
the sequence (GAGGGC) using the GeneTool software program (LifeScience Software Resource, Long Lake, MN) did not demonstrate any homology with
the known cis elements for various transcriptional factors. The GAGGGC
sequence may thus represent a novel cis element (arbitrarily named
P3-D) that specifically and differentially binds nuclear proteins from
proliferating CaCo2 cells.
View larger version (52K):
[in a new window]
Fig. 7.
EMSA with WT and mutant (M1-M6) probes.
EMSA was conducted with a 25-bp ds oligonucleotide probe
( 1090/
1065) that had either a WT sequence or had mutated sequences
(underlined and bold in M1-M6 probes) in the
presence of nuclear samples from day-5 CaCo2 cells. An autoradiograph
of the resulting EMSAs with WT or M1-M6 probes is shown in the
top panel. A single band of oligos bound to day-5 nuclear
proteins was seen with WT and mutant probes other than with the
M6 probe; the pattern of binding was identical to that seen in
Fig. 5B with the WT probe. The relative density of the WT
band was arbitrarily assigned a 100% value, and the relative density
of the bands in the presence of M1-M6 probes is presented as a percent
of that measured with the WT probe and is shown as bar
graphs. Lanes 1-7, relative density of binding to WT,
M1, M2, M3, M4, M5, and M6 probes, respectively. The data are from a
representative experiment of two similar experiments. That the binding
to the probe was completely abolished on mutation of six nt (M6 probe)
(lane 7) suggests the core of the binding element is present
between
1084 and
1078 as shown by the black bar above
the WT sequence. The hatched bar below the WT sequence
represents nt sequence of the DNA footprint, as shown in Fig.
6.
Percent change in the ratio of luc/-Gal activity of core P3 promoter
ligated to distal P3 segment containing either wild type or mutated
P3-D element
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
238/+140) of the human IGF-2 P3 promoter (32). P3 contains a
TATA-box and a CAAT-box sequence, and both are bound by their cognate
factors. Furthermore, several Sp1 sites are present in the proximal
promoter, which is very GC-rich. Other zinc finger transcription
factors such as the early growth response proteins Egr-1 and Egr-2 can
also bind to multiple sites in the proximal promoter, thereby
activating transcription (20, 32). Interestingly, the Wilms' tumor
WT-1 gene product, a tumor suppressor protein, also recognizes the Egr
binding sites in the proximal promoter and suppresses the
transcriptional activity of the IGF-2 P3 promoter by binding to
Egr-l/WT-1 consensus sequences (33). The absence of WT-1 is associated
with overexpression of IGF-2 in Wilms' tumor (20). Other transcription
factors that bind the proximal segment of the P3 promoter downstream of
position
288 include p53 (34), the P3-4 cis element-binding proteins
(35, 36), and AP2 (22, 31) (reviewed in Ref. 37).
1048,
was required for the cell density and
differentiation-dependent regulation of IGF-2
transcription. Truncated promoter-luc constructs that lacked the distal
segment 5' of
1048 did not reveal any differentiation-dependent down-regulation of the P3
promoter in CaCo2 cells. Additionally, down-regulation cannot be caused
by the known tumor suppressor gene products because CaCo2 cells lack both wtp53 (38) and WT-1
proteins.2 Still the
transcriptional activity of the P3 promoter was significantly down-regulated in a cell density-dependent manner. It thus
appears unlikely that potential transcription factors identified to
date that bind the proximal segment (
288/+140) of the P3 promoter play a role in the density/differentiation-dependent
down-regulation of the transcriptional activity of IGF-2 P3 promoter in
CaCo2 cells. Instead, the results of the present study with transient transfection assays of truncated IGF-2 P3 promoter-luc vectors suggest
the novel possibility that the distal segment of the P3 promoter
contains cis elements that are involved in suppressing the
transcriptional activity of IGF-2 P3 promoter in a cell
density-dependent manner. An important finding was that the
transcriptional activity of the truncated P3 promoter that lacks the
distal segment (
1091 to
1048) was enhanced by severalfold at all
phases of cell growth. It thus appears likely that promoter sequences
between
1091 and
1048 contain cis elements, which in the presence
of specific transcription factors, confer regulated
differentiation-dependent transcription activity to the promoter.
1048 and
515 appeared to lack any
significant regulatory elements, since further truncation of the P3
promoter was not accompanied by any additional changes in the promoter
activity compared with the activity of the HUP3B-luc vector. The
activity of the HAVP3-luc vector (
138/+140) was significantly reduced
to almost negligible levels on all days of cell culture. The segment
between
515 and
138 nucleotide thus appears to contain a strong
regulatory element(s), in the absence of which the transcriptional activity of the promoter was completely abolished in CaCo2 cells. The
middle and proximal segments of the P3 promoter together (
515 to
+140) may therefore represent the core promoter. As stated above,
several cis elements have already been identified in the proximal
segment of the IGF-2 P3 promoter (32). It is likely that some of these
factors, such as Sp1 and Egr, also play an important role in the
unregulated transcriptional activity of the IGF-2 gene in CaCo2 cells.
The distal segment of the P3 promoter, however, appears to be
critically required for observing down-regulation of P3 promoter
activity in a cell density/differentiation-dependent manner
in CaCo2 cells.
1084/
1070) and by mutational analysis with EMSA
probes (
1084/
1078) did not demonstrate any homology with the
consensus sequences of known transcriptional factors as determined with
the help of the gene-runner program (Hastings Software, Inc.) and the
Gene-Tool program. It is therefore possible that the identified
sequence represents a novel cis element (P3-D element) that is
importantly involved in the differentiation-dependent
down-regulation of the IGF-2-P3 promoter in CaCo2 cells.
-1
significantly reduce IGF-2 mRNA levels in IGF-dependent
colon cancer cells (39), but mechanisms mediating the inhibitory
effects have yet to be investigated. These studies, however, suggest
that specific cytokines can down-regulate the expression of the
functional IGF-2 promoters in colon cancer cells, lending support to
the possible presence of native transcriptional suppressor proteins that perhaps mediate the inhibitory effects of certain cytokines and
that are perhaps endogenously regulated in a
differentiation-dependent manner in CaCo2 cells. Lack
and/or overexpression of native transcription factors in colon cancer
cells is speculated to result in continued expression of IGF-2,
associated with loss of differentiation.
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ACKNOWLEDGEMENTS |
---|
The secretarial help of Jean Rice is gratefully acknowledged and the technical support of Azar Owlia is gratefully acknowledged.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA60087 and CA72992 (to P. S.) and John Sealy Memorial Foundation Grant 454690 (to B. D.). Part of this study was published in its preliminary form as an abstract (Dai, B., Wu, P. H., Holthuizen, E., and Singh, P. (1999) in Proceedings of The Digestive Diseases Week, Abstr. 3777, American Gerontological Association, Orlando, FL).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom all reprint requests should be addressed: Dept. of Anatomy and Neurosciences, 10.138 Medical Research Bldg. 1043, University of Texas Medical Branch, Galveston, TX 77555-1043. Tel.: 409-772-4842; Fax: 409-772-1861; E-mail: posingh@utmb.edu.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M007789200
2 B. Dai, P. H. Wu, E. Holthuizen, and P. Singh, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
IGF-2, insulin-like
growth factor-2;
-Gal,
-galactosidase;
bp, base pair(s);
ds, double-stranded;
EMSA, electrophoretic mobility shift assay;
luc, luciferase;
nt, nucleotide(s);
WT, wild type;
TES, N-tris(hydroxymethyl)-2-aminoethanesulfonic acid;
FCS, fetal
calf serum;
PCR, polymerase chain reaction.
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