From the Department of Pharmacology and the
§ Experimental and Clinical Therapeutics Program, Barbara
Ann Karmanos Cancer Institute, Wayne State University School of
Medicine, Detroit, Michigan 48201
Received for publication, September 4, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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Our laboratory previously identified two
functional promoters (designated A and B) for the human reduced folate
carrier (hRFC) gene that result in hRFC transcripts with differing
5'-untranslated regions. By transiently transfecting HT1080 and HepG2
cells with a series of 5' and 3' deletions in the hRFC-B and -A
promoters, the minimal promoters were localized within 46 and 47 base
pairs, respectively. Gel mobility shift assays with the hRFC-B basal promoter region revealed specific DNA-protein complexes involving a
highly conserved GC-box and Sp1 or Sp3. In Drosophila SL2
cells, both Sp1 and the long Sp3 isoform potently transactivated the hRFC-B basal promoter; however, the short Sp3 isoforms were
transcriptionally inert and resulted in a potent inhibition of Sp1
transactivation. For the hRFC-A basal promoter, a CRE/AP-1-like element
was bound by the bZip superfamily of DNA-binding proteins.
Cell-specific DNA-protein complexes were identified for hRFC-A (CREB-1
and c-Jun in HT1080 cells; CREB-1 and ATF-1 in HepG2 cells). When the
GC-box and CRE/AP-1-like elements were mutated, a 60-90% decrease in promoter activity was observed in both cell lines. These results identify the critical regulatory regions for the hRFC basal promoters and stress the functional importance of the Sp and bZip families of
transcription factors in regulating hRFC expression.
Reduced folate cofactors are involved in one-carbon transfer
reactions in anabolic pathways leading to the purines, thymidylate, serine, and methionine, essential precursors for the biosynthesis of
DNA, RNA, and proteins (1). Mammalian cells lack the capability of
synthesizing these essential cofactors. Furthermore, chemically, folates are hydrophilic anionic molecules with limited capacities to
traverse biological membranes by diffusion alone. Accordingly, mammals
have evolved highly sophisticated uptake processes for transporting
folates into cells. The primary route for entry of reduced folates is
the reduced folate carrier
(RFC)1 (2-5). The biological
role of RFC is to provide adequate rates of intracellular delivery of
tetrahydrofolates to sustain DNA synthesis and cell proliferation.
In addition to the important role of RFC in tissue folate homeostasis,
membrane transport via RFC is a critical determinant of the antitumor
activity of antifolate therapeutics used in cancer chemotherapy
(i.e. methotrexate (Mtx), Tomudex; 2-4) and
alterations in RFC contribute to Mtx resistance (2-4). In cultured
cells, high levels of Mtx transport are essential for complete
inhibition of dihydrofolate reductase and for Mtx polyglutamate
synthesis (2). Transport is frequently impaired in cultured cells after prolonged Mtx exposures in vitro (6-10) and in murine tumor
cells in vivo after chemotherapy with Mtx (11). Similarly,
in leukemic lymphoblasts from patients, a wide range of RFC was
observed, and low levels of RFC transcripts were associated with small
amounts of Mtx uptake (12, 13). Most recently, Guo et al.
(14) report that osteosarcomas with decreased human RFC (hRFC)
expression were associated with a poor response to chemotherapy,
including Mtx. Taken together, these studies argue that the levels of
RFC gene expression can potentially have a major impact on clinical outcome after chemotherapy with Mtx. RFC gene expression can also influence normal tissue development and could conceivably contribute to
disease states associated with decreased accumulations of folate cofactors, exacerbating the effects of folate deficiencies on the
development of cardiovascular disease (15) and fetal abnormalities (16)
and on the carcinogenic process (17-19).
In recent years, cDNAs encoding RFC from various species have been
isolated and found to restore many of the transport characteristics typically associated with this system (20-25). Furthermore, the structures of the RFC gene from hamster (26), mouse (27, 28), and human
(29-31) cells have been established. Despite the close homologies
between the rodent and human RFCs, the murine and hamster genes are
notably smaller than the hRFC gene (23 and 15.3 kilobases, versus ~29 kilobases, respectively) (26, 27, 29). Although there is little similarity between the intron sizes for the human and
rodent RFC genes, the intron-exon junctions are highly conserved (29-31).
Multiple cDNA isoforms were described for both the rodent (26-28,
32) and human (23, 30, 31, 33, 34) RFCs, differing in their
5'-untranslated regions (UTRs). For the mouse RFC gene, the multiple
5'-UTRs arise from alternate noncoding exons that are regulated by two
distinct promoters (27, 28), each of which is regulated by multiple Sp1
binding sites (32). Our recent studies of the 5' upstream regulation of
the hRFC gene confirmed the presence of 5' transcript heterogeneity
resulting from multiple transcriptional start sites and variable
splicing of alternate noncoding exons (designated exons 1 and 2 for the
KS43 and KS32 5'-UTRs, respectively), each transcribed from separate
GC-rich, TATA-less promoters (designated A and B or, previously, Pro32 and Pro43, respectively) (33). Similar results were described for hRFC
by Tolner et al. (30) and Williams and Flintoff (31). In
this report, we significantly extend this initial characterization of
promoter structure and function by identifying the critical transcriptional elements involved in regulating basal levels of hRFC
expression. Our results suggest a remarkable complexity of basal
transcription that portends possibilities for tissue-specific hRFC
transcriptional controls, in response to requirements for folate
cofactors or other cell- or tissue-specific signals.
Chemicals and Reagents--
[ Cell Culture--
The HT1080 human fibrosarcoma and the HepG2
human hepatoma cell lines were obtained from the American Type Culture
Collection (Manassas, VA). Schneider's Drosophila SL2 cells
were provided by Dr. Bonnie Sloane (Wayne State University, Detroit,
MI). HT1080 cells were grown in RPMI 1640 medium with 10%
heat-inactivated iron-supplemented calf serum (Hyclone Labs), 2 mM L-glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin in an atmosphere of 5% CO2 and
95% air at 37 °C. HepG2 cells were cultured in minimum essential
medium unmodified with 10% fetal bovine serum and antibiotics at
37 °C. SL2 cells were grown at room temperature in Schneider's Drosophila medium (Sigma-Aldrich) containing 10%
heat-inactivated fetal bovine serum, 2 mM
L-glutamine, and antibiotics.
hRFC-Luciferase Reporter Constructs--
The full-length
hRFC-B/-2016 (positions Transient Transfections and Reporter Gene Activity--
HT1080
and HepG2 cells were seeded at 1 × 105 cells/well in
6-well dishes 24 h before transfection. The plates used for HepG2 cells were coated with 4 µg/ml poly-L-lysine.
RFC-promoter constructs or empty pGL3-basic (4 µg for HT1080, 475 ng
for HepG2 cells) were cotransfected with 25 ng of pRLSV40 plasmid using
Lipofectin® (10 µg for HT1080, 3 µg for HepG2 cells) according to
the manufacturer's protocol. After 24 h, the cells were washed,
and then the appropriate medium was added. After an additional 24-48
h, lysates were prepared, and firefly luciferase activity was assayed
using the dual luciferase kit (Promega) in a Turner 20/20 luminometer.
Firefly luciferase activity was normalized with Renilla
luciferase. Drosophila SL2 cells (5 × 105
cells/T12.5 cm2) were transfected with hRFC promoter
constructs (1 µg) and 100 ng of pPacSp1, pPacO, pPacSp3, or pPacUSp3
using Fugene 6 (Roche Molecular Biochemicals; 3 µl) in complete
media. Transfections were allowed to continue for 48 h before the
cells were lysed, and luciferase activity was assayed with the single
luciferase reporter assay system (Promega). Relative luciferase
activities were normalized to protein concentrations of all the cell
lysates. For all transfections, three or more experiments were
performed in duplicate.
Gel Mobility Shift Assays--
Nuclear extracts from HT1080 and
HepG2 cells were prepared by standard methods (35). 5-10 µg of
nuclear extract was incubated for 15 min at room temperature with
[ Western Blot Analysis--
Nuclear extract proteins from HT1080
and HepG2 cells were electrophoresed on 7.5% polyacrylamide gel in the
presence of SDS and electroblotted onto polyvinylidene difluoride
membranes (DuPont) (36). Detection was with ATF-1, CREB-1, and c-Jun
antibodies and enhanced chemiluminescence (Pierce). Light emission was
recorded on x-ray film with various exposure times.
Identification of the Basal Promoter Regions for the hRFC-B and -A
Promoters--
Previously, our laboratory identified two unique
promoters (designated A and B or Pro32 and Pro43) involved in
regulating hRFC gene expression (33). Although regions of possible
regulatory significance were suggested by 5' deletion analysis, neither
the minimal promoters nor the major transcription factors that regulate basal transcriptional activity were elucidated. Accordingly, we prepared a refined series of deletion reporter constructs to better localize the minimal hRFC-A and -B promoters. These constructs were
transiently transfected in HT1080 human fibrosarcoma and HepG2 human
hepatoma cells, which express moderate to high levels of hRFC
transcripts, with a 2-fold higher level of hRFC expression in HepG2
cells (data not shown). By using 5'-rapid amplification of cDNA
ends for both cell types, we found that each cell line uses both hRFC
promoters,2 making them a
good in vitro model for studying hRFC gene expression.
For both cell lines, up to 928 bp could be deleted from the full-length
construct (hRFC-B/-2016) with only a slight decrease (30%) of
luciferase activity (Fig. 1, panel
A). However, upon deletion of an additional 46 bp (to position
For promoter A, the levels of luciferase activity were reduced by
~65% for both cell lines upon removal of 457 bp (hRFC-A/-501) from
the full-length hRFC-A/-958 construct (Fig. 1, panel B). Luciferase activity was completely abolished by further deletion to
position
These data strongly suggest that the basal promoter for hRFC-B resides
within the 46-bp region from Identification of Transcription Factors Binding to the Basal hRFC-B
Promoters--
The 46-bp basal promoter B is GC-rich (87%), lacks a
TATA box, and contains a highly conserved GC-box (position
When incubated with the 46-bp basal promoter B oligonucleotide, both
nuclear extracts resulted in three complexes (labeled 1,
2, and 3; Fig.
3A, lanes 2 and
9). All three complexes were competed with a 50-fold molar
excess of unlabeled hRFC-B oligonucleotide (Self;
lanes 3 and 10), establishing binding
specificity. Likewise, the complexes were competed with a 100-fold
molar excess of Sp1 consensus oligonucleotide (Fig. 3A,
lanes 4 and 11). However, there was no
competition with a 200-fold molar excess mutated Sp1 consensus
oligonucleotide (Fig. 3A; lanes 5 and
12) or with hRFC-B oligonucleotides in which the GC-box
(hRFC-B/-1088 M1; Fig. 2C) or both the GC- and GT-boxes
(hRFC-B/-1088 M3; Fig. 2C) were mutated (Fig.
3B, lanes 4, 6, 10, and
12, respectively). However, when the flanking GT-box alone
was mutated (hRFC-B/-1088 M2; Fig. 2C), competition was
unaffected (Fig. 3B, lanes 5 and 11).
Thus, the highly conserved GC-box (CCCGCCC) at position
Based on our competition studies with the promoter B oligonucleotide,
supershift analyses were performed. Antibodies to Sp1 and Sp3 were
added to the nuclear extracts before the binding reactions. The Sp1
antibody resulted in a supershift in both cell lines (complex
A; Fig. 3A, lane 6 and 13).
In the presence of the Sp3 antibody, both lower complexes (2 and 3) were supershifted (complex B; Fig.
3A, lanes 7 and 14). When both
antibodies were added simultaneously to each nuclear extract, all three
complexes were supershifted (complex C; Fig. 3A,
lanes 8 and 15). In separate experiments (not
shown), we found that purified Sp1 also bound the basal promoter B
oligonucleotide. Collectively, these results demonstrate that both Sp1
and Sp3 can bind to the 46-bp minimal promoter B through the highly
conserved GC-box at positions Identification of Transcription Factors Binding to the Basal hRFC-A
Promoter--
The 47-bp hRFC-A basal promoter is 76% GC-rich, also
TATA-less, and has a CRE/AP-1-like element at positions
Based on our competition results with hRFC-A consensus sequences,
supershifts were conducted with antibodies to various members of the
CREB/ATF and AP-1 families of transcription factors. On a 6%
nondenaturing polyacrylamide gel, CREB-1 antibody supershifted complexes in both HT1080 and HepG2 cells (complex A; Fig.
5A, lanes 3 and
11, respectively). A supershifted complex was detected in
HepG2 cells with an ATF-1 antibody (complex B; Fig.
5A, lane 12); however this shift was not detected
in HT1080 cells (Fig. 5A, lane 4). Antibodies to
c-Jun or c-Jun/AP-1 (recognizes all Jun family members), JunD, JunB,
c-Fos (recognizes all Fos family members), or Nrf1 did not result in
detectable supershifts for either HT1080 or HepG2 cell lines on a 6%
gel (Fig. 5A, lanes 5-9, 13, and
14; not shown for JunD, JunB, or Nrf1 for HepG2 cells). However, a supershift with c-Jun and c-Jun/AP-1 antibodies could be
detected on a 4% gel in HT1080 cells (complex C; Fig.
5B, lane 1; data not shown for c-Jun/AP-1).
Neither c-Jun nor c-Jun/AP-1 antibodies resulted in a supershift with
the HepG2 nuclear extract (Fig. 5B, lane 4, shows
data for c-Jun antibody). For the other antibodies included on the 6%
gel, shown in Fig. 5A, identical results were obtained on a
4% gel (data not shown).
Collectively, these results identify a multiplicity of transfactor
binding associations with the hRFC-A basal promoter involving members
of the superfamily of bZip DNA-binding proteins and the conserved
CRE/AP-1-like motif at positions
To assess this possibility, Western analysis was used. As shown in Fig.
5C, CREB-1 was detected at high levels in both cell lines,
thus explaining the presence of CREB-1/hRFC-A complexes on our
supershifts. However, c-Jun was detected only in HT1080 cells, and
ATF-1 was identified only in HepG2 cells (Fig. 5C). These
striking differences between c-Jun and ATF-1 are in further agreement
with our supershift data. Taken together, they suggest that different
cellular levels of c-Jun, CREB-1, and ATF-1 determine the types of
DNA-protein complexes that are observed.
Confirmation of Transcription Elements and Promoter Transactivation
by Mutagenesis and Transient Transfections--
From our gel shift
experiments (Figs. 3-5), the GC-box (position
To confirm important transactivating roles for these regions, reporter
gene constructs for hRFC-B/-1088 and hRFC-A/-501 containing these point
mutations were transiently expressed in HT1080 and HepG2 cells (Fig.
6, A and B,
respectively). In HT1080 cells (Fig. 6A, upper
panel), the hRFC-B/-1088 M1 and M3 constructs were only 10 and
7%, respectively, as active as wild-type, whereas only slightly
greater levels (45 and 35%, respectively) were detected in HepG2 cells
(Fig. 6A, lower panel). In both HT1080 and HepG2 cells (Fig. 6, panel B), the hRFC-A promoter construct
containing a mutated CRE/AP-1-like element (hRFC-A/-501 M1) resulted in
a 60% decrease in luciferase activity compared with the hRFC-A/-501 wild-type construct. These data clearly demonstrate the critical transactivating roles for the GC-box in the hRFC-B minimal promoter and
for the CRE/AP-1-like element in the hRFC-A basal region.
Sp1 and Sp3 Transactivation of the hRFC-B Basal Promoter in
Drosophila SL2 Cells--
Sp1 has been primarily shown to act as a
positive transactivating factor (37, 38). Sp3 exists as three isoforms,
generated by different translation starts (39), all of which can act
(to varying degrees) as activators or repressors of transcription, depending on the cell and promoter context (37-43). To compare the
effects of Sp1 and the long and short isoforms of Sp3 on the hRFC-B
promoter activity, transient transfection experiments were performed
with Drosophila SL2 cells, which provide a negative background for the Sp family of transcription factors (44-46). hRFC-B/-2016 and hRFC-B/-1088 reporter constructs were transfected with
expression vectors for Sp1 (pPacSp1) (44) or the short (pPacSp3) or
long (pPacUSp3) isoforms of Sp3 (41) under the control of a SL2-
specific promoter. Relative luciferase activities for the hRFC reporter
constructs were compared with that in cotransfections with an empty
expression vector (pPacO). As shown in Fig.
7A, cotransfection with
pPacSp1 induced an ~500-fold increase in relative luciferase activity
for hRFC-B/-1088 over cotransfection with pPacO. An ~240-fold
stimulation by Sp1 was observed for hRFC-B/-2016. Conversely, there was
no stimulation of hRFC-A/-501 basal promoter by Sp1. Thus, both the
full-length and basal hRFC-B constructs are exquisitely sensitive to
transactivation by Sp1. The greater stimulation of the basal construct
in this assay implies that negative Sp1 elements or other upstream
regions in the full-length promoter are able to interfere with Sp1
transactivation of hRFC-B basal region.
Fig. 7B shows the effects of transfecting SL2 cells with
increasing amounts of pPacSp1, pPacSp3, and pPacUSp3 on hRFC-B/-1088 luciferase activity. Both pPacSp1 and pPacUSp3 exhibited a
dose-dependent transactivation of hRFC-B/-1088, differing
in potency by up to 4-fold. However, the short Sp3 isoforms were
completely inert (Fig. 7B). In combination experiments
(panel C), the stimulatory effects of Sp1 and the long Sp3
isoform on hRFC-B transcription were completely additive. Conversely,
the short Sp3 isoforms caused a striking decrease (70%) in the ability
of Sp1 to transactivate basal promoter B. Thus, Sp1, in combination
with the short and long isoforms of Sp3, can exert positive or negative
effects on the transactivation of the hRFC-B minimal promoter,
apparently by competing for the same DNA consensus sequence. This could
represent another level of cell- or tissue-specific regulation for the
hRFC gene.
The goal of this study was to identify the basal promoters for the
hRFC gene and the transcriptional elements that are responsible for
regulating hRFC gene expression. By 5' and 3' deletion analysis, the
minimal A and B promoters were localized to positions Since promoter B is GC-rich, TATA-less, Sp1-regulated, and
constitutively expressed, it resembles those for other housekeeping genes, such as dihydrofolate reductase (47) and thymidylate synthase
(48), involved in one-carbon transfer pathways that result in purines,
pyrimidines, and amino acids. The transcobalamin II receptor (vitamin
B12 receptor) is also regulated by Sp1 binding to a GC-box element with
an identical consensus sequence to that in basal promoter B (49). The
common basal regulatory regions of these assorted genes could
potentially explain their ubiquitous expression and may reflect the
absolute requirements for these biosynthetically interrelated gene
products for cell proliferation.
Sp1 is typically an activator of transcription; however, Sp3 has been
found to exert both agonistic and antagonistic effects on gene
expression (37-43). Both factors are ubiquitously expressed (37, 38),
perhaps explaining why transcripts transcribed from promoter B have
predominated in virtually every cell line examined to
date.3 When the effects of
Sp1 and Sp3 on basal promoter B were examined in Drosophila
SL2 cells, both Sp1 and the long Sp3 isoform were able to stimulate
transcription; however, the short isoforms of Sp3 were
transcriptionally inactive. In cotransfection experiments, the effects
of Sp1 and the long Sp3 isoform were completely additive, yet
transactivation by Sp1 was potently repressed in the presence of the
short Sp3 isoforms. These results strongly suggest that Sp1 and the Sp3
isoforms bind to the same GC-box in basal promoter B and may have
different effects on hRFC expression if the stoichiometric ratios
between these factors are varied. Changes in cellular levels of Sp1 and
Sp3 isoforms could conceivably exert profound effects on the overall
expression of hRFC-B in different cell and tissue types. This adds
another level of complexity and cell specificity to the already
intricate regulation of hRFC gene expression resulting from the use of
dual promoters with different transcription elements.
Likewise, the activity of promoter A could be regulated in a
tissue-specific manner by different members of the bZip superfamily of
DNA binding factors, which bind to the hRFC-A CRE/AP-1-like element. On
our gel shifts, CREB-1 was able to form a DNA-protein complex with
basal promoter A in both HepG2 and HT1080 cells. Conversely, an ATF-1
promoter complex was only identified in HepG2 nuclear extracts, and a
c-Jun complex was only detected in HT1080 cells. Interestingly, these
cell-specific differences were closely paralleled by differences in the
levels of ATF-1 (detectable in HepG2 cells, virtually undetectable in
HT1080 cells) and c-Jun (extremely low levels in HepG2 cells and high
levels in HT1080 cells) in nuclear extracts prepared from both cell
lines. High levels of CREB-1 were detected in both cell lines. Of
course, these findings could possibly be extended to other cell or
tissue types for which other members of the AP-1 and CREB/ATF families may be present and result in additional homo-/heterodimeric complexes that bind at the CRE/AP-1-like element in promoter A. It is the complexity of the bZip superfamily and the intricate interrelationships between these factors (50) that underscore the regulation of the hRFC-A
minimal promoter and may explain previous reports of the effects of
antagonists and agonists of second messenger pathways on hRFC function
(51, 52). Clearly the mechanisms that determine differential promoter
activities and alternate promoter usage from one cell or tissue type to
another is an area that needs to be further explored.
In our studies, the involvement of the GC-box and the CRE/AP-1-like
elements on transactivation of the hRFC basal promoters was confirmed
by mutating the consensus sequences and transfecting the reporter
constructs into HT1080 and HepG2 cells. The mutated GC-box resulted in
~90% or more reduction in promoter activity in HT1080 cells but only
55% or more reduction in HepG2 cells. Similarly, when the
CRE/AP-1-like site in hRFC-A promoter was mutated by altering one
nucleotide, relative luciferase activity decreased by 60% in both cell
lines. Our inability to uniformly abolish promoter activity in these
experiments may reflect partial factor binding to the mutated regions
(for CRE/AP-1) or conformational interactions between the basal
promoters and further downstream regions in the hRFC-A or -B promoter
constructs. Alternately, this could reflect important roles of
accessory proteins that coordinate transcription factor binding or,
perhaps, reflect contributions from elements in the downstream regions
of the hRFC promoter constructs. Clearly, the level of transcription
ultimately achieved depends on a large number of factors, including the
levels of the individual transcription factors and the presence of
upstream and downstream inhibitory and activating elements, and the
overall promoter architecture.
In conclusion, our results demonstrate that the hRFC-A and -B minimal
promoters can be localized to within 47 and 46 bp, respectively. We
demonstrate that the GC-box and the CRE/AP-1-like consensus sequences
are the major sites of regulation for the basal promoters; however, our
findings do not eliminate the possibility that other accessory proteins
or downstream transcription elements may also contribute to basal
transcriptional activity. Our results suggest a remarkable and
surprising complexity of basal hRFC transcription, which portends many
possibilities for tissue-specific control of hRFC transcription, in
response to cellular requirements for folate cofactors, nucleotides, or
other exogenous signals. Future studies will focus on the
interrelationships between the basal transcription for each promoter
and upstream and downstream regulatory regions. The results of these
studies should provide insights into the roles of changes in hRFC gene
expression in nutritional folate deficiency and human disease,
including possible therapeutic strategies for increasing hRFC gene
expression, and also in relation to response and resistance of cancer
in patients undergoing chemotherapy with antifolates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP
(3000Ci/mmol) was purchased from PerkinElmer Life Sciences.
Synthetic oligonucleotides were purchased from Genosys Biotechnologies,
Inc. (The Woodlands, TX). Lipofectin® was purchased from Life
Technologies, Inc. Restriction and modifying enzymes, reporter gene
vectors (pGL3-Basic, pGL3-Pro, pRLSV40), and other molecular biology
reagents were obtained from Promega (Madison, WI). pPacSp1 and pPacO
plasmid constructs were provided by Dr. Robert Tjian (University of
California, Berkeley, CA), and the pPacSp3 and pPacUSp3 constructs
were provided by Dr. Guntram Suske (Philipps-Universität,
Marburg, Germany).
2016 to
959) and hRFC-A/-958 (positions
958 to
277) promoters were prepared from a hRFC genomic clone
(RFCg1-31) as described by Zhang et al. (33). 5'-Deleted hRFC-B (hRFC-B/-1088 and hRFC-B/-1043) and -A (hRFC-A/-501 and hRFC-A/-455) constructs were prepared by successive exonuclease III
and S1 nuclease digestions (34), whereas 3' deletions (designated hRFC-B/MluI and hRFC-B/BsrBI) were introduced by restriction
endonuclease digestions with MluI and BsrBI,
respectively. hRFC-B/-1088 M1 and hRFC-B/-1088 M3, containing a mutated
GC-box, and hRFC-A/-501 M1, containing a mutated CRE/AP-1-like element,
were prepared by polymerase chain reaction using the following primers:
hRFC-B/-1088 M1, 5'-GGACGGACCCGAACACCCCGCAGCCGCG-3'; hRFC-B/-1088 M3,
5'-GGACCCGAACAAACCGCAGCCGCGCGCCCGCCGCGCCGCCTT-3'; hRFC-A/-501 M1,
5'-CGGCCCCCAGCCTGCCCTCCGCGTCCTCCTGGGGCGCCAAGTCCCA-3'. The
polymerase chain reaction conditions were as follows: 95 °C/30 s,
68 °C/45 s, 72 °C/45 s for 35 cycles. The amplicons were isolated from 2% LE-agarose and then subcloned into pGemT Easy plasmid (Promega). The inserts were subsequently subcloned into pGL3-Basic in
the sense orientation. All mutations were verified by automated DNA sequencing.
-32P]ATP end-labeled hRFC-B (
1088 to
1043) and -A
(
501 to
455) double-stranded oligonucleotides in a binding buffer
consisting of 20 mM Tris-HCl, pH 7.5, 2 mM
MgCl2, 1 mM EDTA, 50 mM NaCl, 0.1%
Triton X, 6 µg of bovine serum albumin, 2 µg of poly[dI-dC], 0.5 mM dithiothreitol, and 10% glycerol. Oligonucleotide
competition analysis was conducted with a 100-200-fold molar excess of
commercial competitor oligonucleotides (CREB, AP-1, Sp1, mutant Sp1,
and mutant CREB; Promega) or a 50-fold molar excess of unlabeled
wild-type or mutant hRFC-B and hRFC-A oligonucleotides. For the
supershift assays, the DNA-protein complexes were pretreated with
anti-Sp1 (Geneka) and Sp3, c-Jun, c-Jun/AP-1, JunB, JunD, c-Fos, Nrf1, CREB-1, ATF-1, and ATF-2 antibodies (Santa Cruz Biotechnology, Inc.).
DNA-protein complexes were separated on 4 or 6% nondenaturing polyacrylamide gels in 0.5× Tris borate-EDTA (pH 8.4) at 4 °C and
at 35 mA. The gels were dried, and the complexes were visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1043), promoter activity was completely abolished. When 141 bp were
removed from the 3'-end of hRFC-B/-2016 (to position
1099) with
MluI (hRFC-B/MluI), activity was similarly abolished.
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Fig. 1.
Deletion analysis of hRFC-B and -A promoter
activity of hRFC gene. 5' and 3' deletions were introduced into
the hRFC-B (panel A) and -A (panel B) promoters
as described under "Experimental Procedures." Promoter constructs
in pGL3-Basic were transiently expressed in HT1080 (4 µg of plasmid)
and HepG2 (475 ng of plasmid) cells for luciferase assays. Data are
reported as relative firefly luciferase activity, normalized to
Renilla luciferase activity. S.E. are shown by the
error bars.
455 (hRFC-A/-455) (Fig. 1, panel B). Removal of
261 bp from the 3'-end of the full-length hRFC-A/-958 construct (to position
541) with BsrBI (hRFC-A/BsrBI) abolished
luciferase activity for promoter A.
1088 to
1043, and the minimal promoter
for hRFC-A can be localized to within the 47 bp between positions
501
and
455.
1081 to
1075), flanked by a potential GT-box (
1077 to
1071), both of
which are recognized by the family of Sp transcription factors (Fig. 2A). To assess transcription
factor binding to these conserved elements, a double-stranded
oligonucleotide (
1088 to
1043), including these elements
(hRFC-B/-1088 Wt) was used in gel mobility shift assays. The
32P-labeled oligonucleotide was incubated with 5-10 µg
of HT1080 or HepG2 nuclear extracts and analyzed on a nondenaturing
polyacrylamide gel.
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Fig. 2.
Schematic of the hRFC-B and hRFC-A basal
promoter constructs. Schematics are shown depicting the
full-length hRFC-B (panel A) and hRFC-A (panel B)
basal reporter constructs from Fig. 1, including the minimal promoters
(capital letters) and the 5'-ends of exons 1 and 2, respectively (italicized letters). The GC/GT-box in hRFC-B
and CRE/AP-1-like element in hRFC-A are also noted. In panel
C, the wild-type and the mutated GC/GT-box and the CRE/AP-1-like
elements in the oligonucleotides used in the gel mobility shift
experiments and in the promoter constructs in the reporter gene assays
are shown.
1081 to
1075 is the major binding element in the hRFC-B basal region.
View larger version (50K):
[in a new window]
Fig. 3.
Binding of Sp1 and Sp3 transcription factors
to the 1081 to
1075 bp GC-box in the hRFC-B minimal promoter.
Gel shift assays were conducted with HT1080 and HepG2 nuclear extracts
as described under "Materials and Methods." Panel A, The
1088 to
1043-bp hRFC-B oligonucleotide was used as a probe in the
absence and presence of competitor oligonucleotides or in the presence
of anti-Sp polyclonal antibodies. Complexes were resolved on a 4%
nondenaturing gel. Lane 1, probe alone (without nuclear
extract); lanes 2 and 9, probe plus 5 µg of
total nuclear extract for each cell line; lanes 3 and
10, 50-fold molar excess of the unlabeled hRFC-B/-1088 Wt
oligonucleotide (Self); lanes 4 and
11, 100-fold molar excess of unlabeled Sp1 consensus
oligonucleotide; lanes 5 and 12, 200-fold molar
excess of mutated Sp1 consensus oligonucleotide; lanes 6-8
and 13-15, antibodies were added to the binding reactions,
including anti-SP1 (lanes 6, 8, 13,
and 15), anti-SP3 (lanes 7, 8,
14, and 15). The DNA-protein complexes are
numbered, and the supershifted complexes are noted with
letters. Panel B, lane 1, probe alone;
lanes 2 and 8, probe plus 5 µg of total nuclear
extract for each cell line; lanes 3 and 9,
50-fold molar excess of unlabeled hRFC-B/-1088 wild-type
oligonucleotide; lanes 4 and 10, 50-fold molar
excess of unlabeled hRFC-B/-1088 M1; lanes 5 and
11, 50-fold molar excess of hRFC-B/-1088 M2; lanes
6 and 12, 50-fold molar excess of hRFC-B/-1088 M3; lanes
7 and 13, 100-fold molar excess of Sp1 consensus oligonucleotide.
The DNA-protein complexes are numbered.
1081 to
1075.
485 to
471 (Fig. 2B). In gel mobility shift assays with the 47-bp
hRFC-A oligonucleotide (
501 to
455), a single DNA-protein complex
was detected on a nondenaturing 6% polyacrylamide gel for both HepG2 and HT1080 cells (Fig. 4, lanes
1 and 7). The complex was competed with a 50-fold molar
excess of unlabeled basal promoter A oligonucleotide (Self;
Fig. 4, lanes 2 and 8) and with excess (100-fold)
commercial AP-1 and CREB consensus oligonucleotides (Fig. 4,
lanes 4 and 10, 5 and 11,
respectively). However, competition was significantly diminished with a
commercial mutant CREB oligonucleotide (Fig. 4, lanes 6 and
12). When a single point mutation was introduced into the
CRE/AP-1-like binding site in the hRFC-A oligonucleotide (hRFC-A/-501
M1, Fig. 2C), competition was almost completely inhibited (Fig. 4, lanes 3 and 9). In data not shown, the
bZip DNA binding domain from CREB-1 (Santa Cruz Biotechnology) also
formed a DNA-protein complex with the 47-bp hRFC-A
oligonucleotide.
View larger version (65K):
[in a new window]
Fig. 4.
Binding of CREB/ATF- and AP-1-like proteins
to the CRE/AP-1-like element in the hRFC-A minimal promoter. Gel
shift assays were conducted with HT1080 and HepG2 nuclear extracts as
described under "Materials and Methods." Panel A, The
501 to
455-bp oligonucleotide was used as a probe in the absence
and presence of competitor sequences. Complexes were resolved on a 6%
nondenaturing gel. Lanes 1 and 7, probe plus 10 µg of total nuclear extract for each cell line; lanes 2 and 8, 50-fold molar excess of unlabeled hRFC-A/-501
wild-type oligonucleotide (Self); lanes 3 and
9, 50-fold molar excess of unlabeled hRFC-A/-501 M1
oligonucleotide; lanes 4 and 10, 100-fold molar
excess of AP-1 consensus oligonucleotide; lanes 5 and
11, 100-fold molar excess of CREB consensus oligonucleotide;
lanes 6 and 12, 200-fold molar excess of mutated
CREB consensus oligonucleotide. The specific DNA-protein complex is
designated with an arrow.
View larger version (77K):
[in a new window]
Fig. 5.
Supershift analysis of CREB/ATF and AP-1
transcription factor binding to the hRFC-A minimal promoter and Western
analysis of cell-specific factors. Gel shift assays were conducted
with HT1080 and HepG2 nuclear extracts, as described under "Materials
and Methods." Panel A, The 501 to
455-bp fragment was
used as a probe in the absence and presence of anti-CREB/ATF and
anti-AP-1 antibodies and resolved on a 4% nondenaturing polyacrylamide
gel. Lane 1, probe alone; lanes 2 and
10, probe plus 10 µg of total nuclear extract for each
cell line; lanes 3 and 11, anti-CREB-1 antibody;
lanes 4 and 12, anti-ATF-1 antibody; lanes
5-7 and 13, various anti-Jun antibodies (c-Jun, JunD,
JunB, and c-Jun/AP-1, respectively); lanes 8 and
14, anti-c-Fos antibody; lane 9, anti-Nrf-1
antibody. Panel B, supershift complexes were resolved
on a 6% nondenaturing polyacrylamide gel. Lanes 1 and
3, probe plus 10 µg of total nuclear extract for each cell
line; lanes 2 and 4, anti-c-Jun antibody.
Panel C, nuclear extracts (5, 10, 15 µg of protein) were
electrophoresed and transferred to polyvinylidene difluoride as
described under "Materials and Methods." The membranes were probed
with antibodies specific to CREB-1, ATF-1, and c-Jun proteins.
Detection was by enhanced chemiluminescence.
485 to
471. The identification of
complexes involving different members of the bZip superfamily with the
hRFC-A minimal promoter suggests that the nature of the DNA-protein
complexes formed may be cell-specific. This may reflect differences in
the levels of the individual transcription factors between the cell lines.
1081 to
1075) in
hRFC-B and the CRE/AP-1-like sequence (
485 to
471) in hRFC-A were
implicated in transactivation of the minimal promoters since the
DNA-protein complexes were selectively abolished by competitive
oligonucleotides (Sp1, and CREB and AP-1, respectively) and were
supershifted with specific antibodies to the individual transcription
factors (Sp1, Sp3, CREB-1, ATF-1, and c-Jun). However, commercial
mutant oligonucleotides (Sp1 and CREB) or hRFC oligonucleotides with
mutations in the highly conserved GC-box (hRFC-B/-1088 M1 and
hRFC-B/-1088 M3) and the CRE/AP-1-like (hRFC-A/-501 M1) elements were
no longer effective competitors (Figs. 3 and 4).
View larger version (19K):
[in a new window]
Fig. 6.
Mutational analysis of the GC-box and
CRE/AP-1-like consensus sequences in the hRFC-B and the hRFC-A minimal
promoters. HT1080 and HepG2 cells were transfected with wild-type
and mutant hRFC reporter constructs (depicted in Fig. 2C) as
described under "Material and Methods." Panel A, the
relative luciferase activities for the wild-type and mutated hRFC-B
reporter constructs were plotted for the HT1080 and HepG2 cells.
Panel B, the relative luciferase activities for wild-type
(Wt) and mutated hRFC-A reporter constructs were plotted for
the HT1080 and HepG2 cells. The star indicates that the
differences between wild-type and mutant activities were statistically
significant (p < 0.05). The error bars
represent the S.E.
View larger version (18K):
[in a new window]
Fig. 7.
Effects of co-transfected Sp1 and Sp3
isoforms on hRFC-B promoter activity in Drosophila SL2
cells. SL2 cells were co-transfected with 1 µg of the hRFC
promoter constructs in pGL3-Basic and pPacSp1, pPacSp3, and pPacUSp3 or
empty pPacO vector. Luciferase activity was assayed after 48 h as
described under "Materials and Methods." Panel A,
luciferase activity was measured in cotransfections of pPacSp1 with
hRFC-pGL3-Basic constructs including hRFC-B/-2016, hRFC-B/-1088, and
hRFC-A/-501. Data are also shown for transfections in which empty
pGL3-Basic was substituted for the promoter-pGL3 constructs.
Panel B, dose-dependent transactivation of
hRFC-B/-1088 with pPacSp1, pPacSp3, and pPacUSp3. Transfections were
supplemented with pPacO vector so that the total of pPacSp plus pPacO
vectors was 110 ng. Panel C, cotransfections were performed
with hRFC-B/-1088 and a total of 110 ng of expression vector, as shown.
The error bars represent S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
501 to
455
and
1088 to
1043, respectively. Gel mobility shift assays including
competition and supershift analysis were used to identify the
critical transcriptional elements in these core promoter regions. Thus, specific binding by members of the Sp family of transcription factors (Sp1 and Sp3 isoforms) to the highly conserved GC-box in
promoter B and members of the superfamily of bZip DNA-binding proteins
(CREB-1, ATF-1, and c-Jun) to an CRE/AP-1-like element in promoter A
could be demonstrated. This promoter duality should provide a
potentially powerful means of ensuring adequate levels of hRFC
transcripts (and protein) in response to requirements for folate
cofactors or in response to cell- or tissue-specific signals.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Guntram Suske (University of Marburg, Germany) and Dr. Robert Tjian (University of California, Berkeley, CA) for providing the pPacSp3, pPacUSp3, pPacSp1, and pPacO plasmids for our studies.
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FOOTNOTES |
---|
* This study was supported by National Institutes of Health Grant CA53535 (NCI).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 correspondence should be addressed: Experimental and Clinical Therapeutics Program, Karmanos Cancer Institute, 110 E. Warren Ave., Detroit, MI 48201. Tel.: 313-833-0715 (ext. 2407); Fax: 313-832-7294; E-mail: matherly@kci.wayne.edu.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M008074200
2 J. R. Whetstine and L. H. Matherly, manuscript in preparation.
3 J. R. Whetstine and L. H. Matherly, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: RFC, reduced folate carrier; hRFC, human RFC; Mtx, methotrexate; 5'-UTRs, 5'-untranslated regions; bp, base pair(s); CRE, cAMP-response element; CREB, CRE-binding protein.
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