(Received for publication, October 29, 1996, and in revised form, December 16, 1996)
From the Department of Pediatrics, § Ohio
State Biochemistry Program, and ¶ Department of Medical
Biochemistry, Children's Hospital, The Ohio State University,
Columbus, Ohio 43205
The promoter of the human POLD1 gene
encoding the catalytic subunit of DNA polymerase is G/C-rich and
does not contain a TATA box. Transient transfection analysis in HeLa
cells employing POLD1-luciferase chimeric plasmids revealed
a core promoter region extending 328 base pairs (bp) from the major
transcription initiation site. Multiple elements in this region
including two 11-bp direct repeats located between nucleotide positions
92 and
22, play an important role in POLD1 promoter
activity. Deletion or linker-replacement mutations of the repeats
drastically reduced the promoter activity. A 70-bp DNA fragment
containing the two repeats could stimulate the expression of the
POLD1 or a heterologous promoter in an
orientation-independent manner. DNase I footprinting and band-shift
assays showed that HeLa nuclear extracts contained proteins
specifically binding to the repeat sequences. Southwestern blot and UV
cross-linking analyses identified Sp1 and two 85-kDa proteins that
bound to the repeats. Additionally, screening of HeLa cDNA
expression libraries for the sequence-specific DNA-binding protein
using the 11-bp repeat sequences as the probe, identified a cDNA
that corresponds to Sp3, a member of the Sp1 family. Cotransfection
studies in Drosophila SL2 cells showed that both Sp1 and
Sp3, but not Sp2, could activate the POLD1 promoter through
the repeat sequences. The POLD1 promoter activity was
induced about 4-fold at the late G1/S boundary in
serum-stimulated cells. The 11-bp repeats together with an E2F-like
sequence, located adjacent to the major transcription initiation site,
were important for the stimulation. Taken together, this study provides
a direct evidence for transcriptional regulation of the human
POLD1 gene.
The DNA replication of eukaryotic chromosomes is a complex but highly regulated process. Through the cooperation of multiple protein factors and enzymes including DNA polymerases, each chromosome replicates once during the S phase of the cell cycle (reviewed in Ref. 1). Presently, the mechanisms underlying this cell cycle regulation of DNA replication are not completely understood.
DNA polymerase (pol
)1 is one of the
major enzymes involved in the synthesis of mammalian nuclear DNA (2,
3). It was reported as a new type of DNA polymerase with an intrinsic
3
to 5
exonuclease activity (4), suggesting that it possesses exonucleolytic proofreading ability (5, 6). Purified pol
is
composed of a 125-kDa catalytic subunit and an associated 48-kDa small
subunit whose function has not been defined (7). In addition, a 36-kDa
factor was shown to convert the pol
activity from low to high
processivity (8). This factor was subsequently shown to be identical to
the proliferating cell nuclear antigen (9, 10).
Previously, we, in collaboration with Lee's group (11), and others
(12) isolated the full-length cDNA for the catalytic subunit of
human pol . In addition, the coding sequences of the pol
catalytic subunit have been isolated from bakers' yeast (13), fission
yeast (14), malaria parasite (15, 16), calf thymus (17), and mouse
cells (18). Sequence analysis showed that the catalytic subunit of
human pol
contains 1,107 amino acids and is related to other
eukaryotic DNA polymerases (11). Recently, we also cloned the gene for
the catalytic subunit of human pol
(POLD1) and its
5
-flanking sequences (19). The human POLD1 gene contains 27 exons and 26 introns. Transcription of the gene appears to be initiated
at multiple sites with the major initiation site 53 nucleotides
upstream of the ATG start codon. As found in the promoters of many
mammalian housekeeping genes (for reviews, see Refs. 20-22), the
POLD1 promoter is G/C-rich and does not contain a TATA box.
Several potential binding sites for transcription factors AP2, CTF,
Ets1, GCF, MBF-1, NF-E1, and Sp1 are present in the 5
-flanking region
of the POLD1 gene (19). However, the significance of these
elements in regulating POLD1 promoter activity is presently
not known.
Studies on the expression of the genes involved in nucleotide metabolism and DNA synthesis reveal the presence of multiple regulatory elements including the GC boxes, which are required for maximal promoter activity (23-30). The transcription factor Sp1 (31, 32) has been shown to regulate the transcription through the GC boxes. Recently, several Sp1-related proteins, Sp2, Sp3, and Sp4, that bind to the GC box or GT motif were identified (33-35); however, their role in promoter regulation is less defined. Sp3 and Sp4 share a high degree of amino acid sequence homology with Sp1 (33). The high degree of structural conservation among Sp1, Sp3, and Sp4 suggests that these transcriptional regulators act through binding to similar DNA elements. Like Sp1, both Sp3 and Sp4 can bind to the GC box or GT motif, and Sp4 can activate several Sp1-responsive promoters (34, 36, 37). However, the Sp4 mRNA is detected only in brain (33). On the other hand, Sp3 is ubiquitously expressed and can either activate or repress transcription (34, 36, 38-40). In addition, Sp3 can repress Sp1-mediated activation (34, 36, 39). Another Sp1-family member, Sp2, appears to share less homology with other members and has not been shown to act as an activator or a repressor (35). In addition, the binding affinity of Sp2 to the GT motif is much weaker than that of Sp1 and Sp3. Nonetheless, it has been hypothesized that these Sp1-family transcription factors may play a concerted role in regulating different GC or GT sequence in the promoter (36). Since the POLD1 promoter contains potential Sp1-binding sites, it would be important to examine if the Sp1-family proteins can regulate POLD1 transcription.
POLD1 expression is induced in serum-stimulated cells,
consistent with its involvement in DNA replication (11). Northern blot
analysis of RNA from populations of human Molt 4 cells separated by
counterflow centrifugal elutriation showed that POLD1
mRNA content is regulated during the cell cycle (41). Similarly, expression of the gene for the human pol catalytic subunit is stimulated upon serum addition to starved cells, and multiple elements
in its promoter appear to mediate the full serum response (30).
Presently, the response of the POLD1 promoter to serum induction has not been studied. No serum-responsive element (42) or DNA
replication-related element, identified in the Drosophila genes for pol
and proliferating cell nuclear antigen (43), is
present in the POLD1 upstream region. Thus, it is not known which elements in the POLD1 gene are responsible for serum
induction and cell cycle regulation.
To understand the regulation of the human POLD1 gene, we
examined the cis-acting sequences controlling its expression
in cycling and serum-stimulated cells. We identified two 11-bp direct
repeats located in the 5 promoter proximal region, which could
function as an activator sequence. We also found that Sp1, Sp3, and two 85-kDa proteins could bind to the repeats, and showed that both Sp1 and
Sp3 could activate the POLD1 promoter. In addition, we reported that expression of the POLD1 promoter was induced
at the G1/S boundary and the induction was mediated through
the 11-bp repeat sequences and an E2F-like sequence located near the
major transcription initiation site.
The pGL2-(
1758) plasmid
(previously named pGL2-
P1), containing the 1.8-kb 5
-flanking DNA of
the POLD1 gene linked to the luciferase expression cassette
from pGL2-basic (Promega), has been described (19). A series of
5
-unidirectional deletion mutations within the POLD1
promoter DNA of pGL2-
(
1758) were created using the Erase-a-Base
system (Promega) or the Bal31 nuclease (44). Some 5
and 3
deletion
mutants of the POLD1 promoter were constructed by deleting
appropriate restriction fragments or by polymerase chain reaction (PCR;
Ref. 45). The linker replacement mutants within the 11-bp repeat region
were constructed by ligating an XhoI linker to the proper
deletion clones created by Bal31 deletion or by PCR. To construct the
pGL2-HSVtkbasal plasmid, the
EcoRI-PstI fragment of the minimal herpes simplex
virus thymidine kinase (HSVtk) promoter containing the TATA sequence
and 60-bp upstream DNA from pMC1 (Stratagene) was isolated and ligated
to the luciferase expression cassette. The chimeric promoter plasmids, pGL2-
R2R1-HSVtkbasal and
pGL2-
R1R2-HSVtkbasal, were created by inserting the
70-bp PstI-SacII fragment of the POLD1
promoter DNA 5
to the HSVtkbasal promoter in the sense and
antisense orientation, respectively. The exact locations of the
deletion end points or appropriate fusions in all of these plasmids
were determined by sequencing with POLD1-specific primers or
primers derived from the pGL2 vector (46).
The Sp1 expression plasmid, pPacSp1 (47), was generously provided by
Dr. Robert Tjian of University of California at Berkeley. It contains a
2.6-kb Drosophila actin 5C promoter, a 700-bp Ubx cDNA
fragment consisting of the 5 untranslated region (UTR) plus the first
eight codons of the Ubx open reading frame (ORF), a 2.1-kb Sp1 cDNA
fused with the Ubx ORF, and a 1.1-kb 3
UTR of the actin 5C gene
providing the polyadenylation (poly(A)) signal sequence. Expression
plasmids for Sp2 and Sp3 were generated based on pPacSp1 (47). Briefly,
to construct the Sp2 expression plasmid, a BamHI linker was
inserted immediately 5
to the Sp2 translation initiation codon in the
Sp2 cDNA plasmid, Sp2-pKS(
) (American Type Culture Collection;
Ref. 35), and an XhoI linker was inserted into the
SacI site at nucleotide position 2062. The
BamHI-XhoI fragment containing the entire Sp2
coding region was excised and used to replace the
BamHI-XhoI Sp1 fragment in pPacSp1 to generate the pPacSp2 plasmid. To construct the Sp3 expression plasmid, an Sp3
cDNA clone was isolated from the screening of a cDNA expression library (see below). A 2.1-kb EcoRI-XhoI Sp3
cDNA fragment was excised and used to replace the
EcoRI-XhoI Sp1 cDNA in pPacSp1 to generate
the pPacSp3 plasmid.
Human HeLa cells and mouse C3H10T1/2 cells
(American Type Culture Collection) were routinely grown in Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal bovine serum
(FBS). Drosophila Schneider line (SL2) cells (48) were grown
in Schneider medium (Life Technologies, Inc.) supplemented with 10%
FBS at 25 °C. Transfection assays were performed by the calcium
phosphate technique (49) with slight modifications (50). Each
POLD1-luciferase plasmid (20 µg) was transfected into a
100-mm dish of actively growing HeLa cells. To standardize the
transfection efficiency, 2 µg of the pCH110 plasmid (Pharmacia
Biotech Inc.), containing the SV40 early promoter driving
-galactosidase gene, were also included as an internal control in
each transfection. Calcium phosphate-DNA coprecipitate mixtures were
prepared and added to cells in the presence of medium. Following
overnight incubation, the medium was replaced with fresh growth medium.
At 48 h after transfection, cells were harvested in reporter lysis
buffer and the luciferase and
-galactosidase activities were
measured using the reporter assay system (Promega). For all
transfection assays, at least three independent experiments were
performed. The luciferase activity expressed from each promoter
construct was normalized to the
-galactosidase activity. For
transfection of Drosophila SL2 cells, 5 µg of each
POLD1-luciferase plasmid and various amounts of the Sp1,
Sp2, or Sp3 expression plasmid were used. The total amounts of DNA were
compensated to 20 µg with the control pPacU vector (47). An equal
amount of cell extracts from each transfection was used for measuring
the luciferase activity. To detect the expression of the Sp1-family
protein, the same amount (80 µg) of protein extracts from each
transfection of SL2 cells was electrophoresed in an 8%
SDS-polyacrylamide gel and electroblotted onto a PVDF-Plus filter
(Micron Separations) using a Hoefer Transphor electrophoresis unit
(44). The filter slice containing extracts from cells transfected with
each Sp1-family expression plasmid was stained with the corresponding antibody against each expressed protein (anti-Sp1 (IC6), anti-Sp2 (K-20), or anti-Sp3 (D-20) antibody from Santa Cruz), followed by a
secondary antibody conjugated with alkaline phosphatase using the
ProtoBlot AP system (Promega).
Nuclear
extracts from HeLa spinner cells (American Type Culture Collection)
were prepared as described (51). Briefly, HeLa cell nuclei were
extracted with 0.35 M KCl and nuclear extracts were
dialyzed against the buffer containing 10 mM HEPES, pH 7.9, 100 mM KCl, 20% glycerol, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol.
The protein concentration of the extract was determined by using the
Bio-Rad protein assay dye reagent and was approximately 5 mg/ml. For
preparing nuclear extracts from transfected SL2 cells, a rapid
micropreparation method was used (52). SL2 cells were transfected with
5 µg of the Sp1, Sp2, or Sp3 expression plasmid, and nuclear extracts were prepared 48 h after transfection. EMSA (53) was performed according to the Gel Shift Assay System (Promega) with minor
modifications. Briefly, various amounts of nuclear extracts were
incubated with 1 µg of poly(dI-dC)·poly(dI-dC) (Pharmacia) in 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2,
0.5 mM EDTA, 0.5 mM DTT, 50 mM
NaCl, and 4% glycerol in a total volume of 18 µl. After incubation
at room temperature for 5 min, 2 µl of 32P-labeled
double-stranded DNA probe (0.8 ng) was added and the mixture incubated
for an additional 20 min. The reaction products were electrophoresed in
a 5% polyacrylamide gel (acrylamide:bisacrylamide ratio = 60:1).
The probes used were the 70-bp PstI-SacII
fragment (position 92 to
23 relative to the major transcription
initiation site) containing the two 11-bp repeats of the
POLD1 promoter or the linker replacement mutants with one or
both copies of the repeats replaced by the XhoI linker. For
antibody supershift experiments, 1 µg of the anti-Sp1 or anti-Sp3
antibody (Santa Cruz) was added to the binding reaction described
above, and the reaction mixture was incubated overnight at 4 °C
prior to electrophoresis.
The POLD1 promoter
DNA containing the sequence between 261 and +49 relative to the major
transcription start site was isolated from pGL2-
(
261) by digestion
with KpnI and HindIII. This substrate DNA was
treated with calf intestinal alkaline phosphatase (Boehringer Mannheim), gel-purified, and 5
end-labeled with T4 polynucleotide kinase or 3
end-labeled with Klenow fragment of E. coli DNA
polymerase I (44). The end-labeled DNA was digested with
XhoI to remove 21-bp DNA from the distal end of the
POLD1 promoter. The resulting DNA fragment, labeled only at
the proximal end, was purified using a quick-spin G50 column
(Boehringer Mannheim). The purified probe (0.3 pmol) was incubated with
100 µg of HeLa nuclear extracts in a volume of 50 µl containing 25 mM Tris-HCl, pH 8.0, 50 mM KCl, 6.25 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 10% glycerol, at room temperature for 10 min.
50 µl of a solution containing 5 mM CaCl2 and
10 mM MgCl2 were added and incubated at room
temperature for another 1 min. Subsequently, the reaction mixture was
treated with DNase I at the concentration between 1.5 and 6 units (or 0.15 and 0.6 unit for the control DNA without nuclear extracts) at room
temperature for 1 min. The reaction was terminated by adding 90 µl of
the stop solution containing 200 mM NaCl, 30 mM EDTA, 1% SDS, and 100 µg/ml yeast tRNA. The DNase I-digested DNA was
purified by phenol extraction and ethanol precipitation, and then
electrophoresed in an 8% polyacrylamide gel containing 8 M
urea. Sequencing G ladders were generated by methylating the probe with
dimethyl sulfate (Aldrich), followed by cleavage with piperidine
(Sigma).
Nuclear extracts from HeLa (75 µg) or transfected SL2 cells (80 µg) were electrophoresed in an 8% SDS-polyacrylamide gel. Fractionated proteins in the gel were then electro-transferred to a PVDF-Plus filter (Micron Separations). After transferring, the filter was prehybridized with a 100-ml hybridization buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, and 5% nonfat dry milk at room temperature for 30 min, and then rinsed twice with the hybridization buffer containing 0.25% nonfat dry milk. Subsequently, the filter was hybridized with an end-labeled probe in 25 ml of hybridization buffer containing 0.25% nonfat dry milk and 250 µg of poly(dI-dC)·poly(dI-dC) at room temperature for 2 h. The hybridized filter was washed four times with the hybridization buffer containing 0.25% nonfat dry milk, blot-dried, and exposed overnight to an x-ray film.
UV Cross-linking AnalysisThe 70-bp POLD1
promoter DNA fragment containing the two 11-bp repeats was end-labeled
using Klenow fragment in the presence of bromodeoxyuridine triphosphate
(Sigma) and [-32P]dCTP (44). The labeled probe was
incubated with 12.5 µg of HeLa nuclear extracts in a volume of 20 µl to allow binding to reach equilibrium as described above for EMSA.
Each reaction mixture was then pipetted onto a parafilm and irradiated
for various times at a distance of 5 cm using an Ultraviolet
Transilluminator with a 310 nm filter (Ultra-Lum). Samples were not
treated with nucleases since the substrate DNA was labeled at its 3
ends (32). Cross-linked complexes were analyzed by electrophoresis in
an 8% SDS-polyacrylamide gel and visualized by autoradiography.
Two HeLa
cell cDNA expression libraries constructed in either the Uni-ZAPTM
(Stratagene) or EXloxR (Clontech) vector were
screened for clones encoding proteins that can bind to the
POLD1 DNA containing the 11-bp repeats as described by Singh
et al. (54) and Vinson et al. (55). The probe
used was a trimer of the 70-bp POLD1 fragment (
92 to
23 relative to the major transcription initiation site), which was 32P-labeled using the nick translation kit (Boehringer
Mannheim). About 106 phages from each library were plated
out. After filter lift, without the treatment to denature and renature
the proteins, the filters were prehybridized, hybridized, and washed
using the same conditions as those for the Southwestern blot analysis.
One positive clone was obtained from the HeLa cDNA library in
Uni-ZAPTM. The cDNA insert from the positive clone was rescued
according to the In Vivo Excision Protocol (Stratagene) and
sequenced.
For synchronization experiments, C3H10T1/2 cells were plated out at a density of 5 × 105 cells/100-mm dish and transfected the next day with 20 µg of each POLD1-luciferase plasmid plus 2 µg of the control pCH110 plasmid as described previously (50). Calcium phosphate-DNA coprecipitates were added to cells in the presence of medium overnight. Transfected cells were then washed twice with Ca2+- and Mg2+-free phosphate-buffered saline (PBS) and changed to DMEM supplemented with 0.1% FBS. After arresting at low serum for 3 days, transfected cells were restimulated with growth medium containing 10% FBS. At various times after serum stimulation, cells were harvested and the same amount of protein extracts was used for reporter enzyme assays.
For flow cytometry, synchronized cells were harvested by trypsinization at various time points, washed in PBS containing 1 mM EDTA, pH 8.0, and 1% bovine serum albumin, and then fixed and permeabilized with ice-cold 70% ethanol. After washing with PBS containing 1% bovine serum albumin, fixed cells were incubated in propidium iodide (PI) staining solution (10 µg/ml of PI and 0.5 mg/ml of RNase A) at 37 °C for 30 min. and then 4 °C in the dark for at least 1.5 h. Flow cytometry analysis was performed on a Coulter EPICS Elite flow cytometer. For detection of PI, stained cells were excited at 488 nm with a 15-milliwatt air-cooled argon laser and fluorochrome emission was absorbed with a 610-nm long pass filter. Extended analysis of DNA content was performed on Multicycle software (Phoenix Flow System).
Previously, we showed that the 1.8-kb
5-flanking DNA of the human POLD1 gene could direct the
expression of the luciferase reporter gene when transfected into
actively growing HeLa cells (19). In order to define the
cis-acting elements required for the transcription of the
POLD1 promoter, a series of unidirectional deletion mutants
were created within the POLD1 promoter segment of the
pGL2-
(
1758) plasmid (Fig. 1). The promoter activity
of each deletion mutant was then evaluated by luciferase assay of extracts from transfected HeLa cells. The experiment was repeated more
than three times, and the results are shown in Fig. 1. By reference to
the major transcription start site as +1, deletions of the 5
upstream
sequence between nucleotide positions
1758 and
328 did not affect
or slightly reduced the promoter activity (compare pGL2-
(
328) to
pGL2-
(
1758) in Fig. 1). On the other hand, a 3
deletion to
position
93 (pGL2-
(
1758 ~
93)) reduced the promoter activity
to about 20% of the 1.8-kb promoter activity. Another 3
deletion to
position
247 (pGL2-
(
1758 ~
247)) further reduced the
promoter activity to about 14%. Further 3
deletion to position
322
(pGL2-
(
1758 ~
322)) almost completely abolished the promoter
activity. These results indicate that the core promoter region of the
human POLD1 gene is located within the
328 region of its
5
-flanking DNA.
Further 5 deletion analysis showed that deletions between positions
328 and
92 gave rise to slightly reduced promoter activities (Fig.
1). Deletion to position
92 (pGL2-
(
92)) decreased the promoter
activity to about 34% of that of the 1.8-kb promoter. Further deletion
to position
22 (pGL2-
(
22)) drastically reduced the promoter
activity to less than 2% of that produced by the 1.8-kb promoter.
Deletion to position
10 (pGL2-
(
10)) reduced the promoter
activity to 1% of the 1.8-kb promoter, while deletion to position
2
(pGL2-
(
2)) reduced the promoter activity to 0.3%, similar to that
of pGL2-Basic (Fig. 1). These results suggest that while multiple
regions are requires for full promoter activity, the sequence between
nucleotides
92 and
22 plays an essential role in the expression of
the human POLD1 gene.
To examine the regulatory element(s) within the
92 to
22 region, a series of fine deletions within the
POLD1 promoter segment of the pGL2-
(
92) plasmid were
constructed and used to test their promoter activities as described
above (Fig. 2A). By comparison to the
promoter activity expressed from pGL2-
(
92), deletion to position
81 did not affect the promoter activity. In contrast, deletion to
position
75 or
68 decreased the promoter activity to about 46% and
28% of that expressed by pGL2-
(
92), respectively. However,
further deletion to position
58 resulted in partial recovery of the
promoter activity from 28% to 50%. These result suggest that a
sequence located between positions
81 and
68 positively regulates
the POLD1 promoter expression, while there is a negative
regulatory sequence located between positions
68 and
58. Further
analysis showed that an additional deletion to position
48 resulted
in a similar level of promoter activity as that of the
58 deletion
(Fig. 2A). In contrast, deletion to position
40 led to a
large reduction (7%) of the promoter activity and deletion to position
22 decreased the promoter activity to about 5% of that produced by
pGL2-
(
92). These results suggest the existence of another positive
regulatory sequence located around position
48 to
40.
Interestingly, examination of the sequences within the two positive
regulatory regions revealed the presence of two identical 11-bp
sequences (5
-GGGGCGTGGCC-3
, located at position
81 to
71 and
position
49 to
39), arranged in the same orientation in each region
(Fig. 2A).
To test if the two 11-bp repeats are essential for POLD1
promoter activity, several linker replacement mutations, with an XhoI linker replacing part of the 11-bp repeat sequence in
pGL2-(
92), were constructed and tested (Fig. 2B). As
compared to the promoter activity expressed from pGL2-
(
92),
mutants with a linker replacing either the left (
(
92)M2R1-1) or
right half (
(
92)M2R1-2) of the 5
copy of the 11-bp repeat
sequence, reduced the promoter activity by about 2-fold. Similarly, a
mutant with a linker replacing the middle portion of the 3
copy of the
11-bp repeat (
(
92)R2M1) also reduced the promoter activity by
about 2-fold. Interestingly, mutations in both copies of the 11-bp
repeats resulted in drastic reduction of the promoter activity by
10-12-fold (
(
92)M2M1-1 and
(
92)M2M1-2 in Fig.
2B). Additionally, the promoter activities of these double
replacement mutants were similar to that of the deletion mutant without
both repeats (compared to pGL2-
(
40) in Fig. 2A). All of
these results indicate that the two 11-bp repeats are important
transcriptional regulatory elements for the POLD1
promoter.
The role of the two 11-bp direct
repeats as a positive regulatory sequence was further investigated by
placing them upstream of a heterologous promoter. A 70-bp
POLD1 DNA containing the two repeats was inserted in both
orientations into the plasmid containing the luciferase reporter gene
controlled by the HSVtk basal promoter (Fig. 3). The
resulting plasmids were transfected into HeLa cells and tested for
their promoter activities. As expected, the HSVtk basal promoter
expressed relatively low activity since it contained only the TATA
sequence and 60-bp upstream DNA (compare pGL2-HSVtkbasal to
pGL2-(
1758) in Fig. 3). Insertion of the two 11-bp repeats in the
sense orientation upstream of the HSVtk basal promoter resulted in
stimulation of the expression by about 16-fold (compare pGL2-
R2R1-HSVtkbasal to pGL2-HSVtkbasal).
Interestingly, insertion of the two repeats in the antisense
orientation upstream of the HSVtk basal promoter also stimulated the
expression by about 7-fold (compared
pGL2-
R1R2-HSVtkbasal to pGL2-HSVtkbasal).
These results indicate that the two 11-bp direct repeats can function
as an activator on a heterologous promoter in an
orientation-independent manner. Consistently, the 70-bp
POLD1 DNA containing the two direct repeats also stimulated
the POLD1 promoter activity when inserted in the antisense
orientation (compare pGL2-
(
92)R1R2 to pGL2-
(
22)). However, it
should be noted that the two 11-bp direct repeats, when placed in the
original orientation, appeared to activate the POLD1 or
HSVtk basal promoter to a higher extent than when inserted in the
opposite orientation (Fig. 3).
Specific Nuclear Proteins Bind to the POLD1 Promoter DNA
To
localize the protein binding sites on the POLD1 promoter
DNA, DNase I footprint analysis was performed using crude HeLa nuclear
extracts and a 310-bp POLD1 DNA (nucleotide position 261 to +49) as the substrate (Fig. 4). When the sense strand
of the POLD1 DNA was examined, the nuclear extracts
protected three regions (Fig. 4A). Protected region I
extended from position
117 to
97. Analysis of the sequence in this
region revealed the presence of a putative Sp1-binding site. Protected
region II extended from position
85 to
66 and protected region III
extended from position
52 to
34. Remarkably, the 11-bp activator
sequence is located at the center of the protected region II and III
(designated R2 and R1; see Fig. 4A). Analysis of the
antisense strand of the POLD1 DNA demonstrated protection of
the same three regions (Fig. 4B). These results indicate
that the specific nuclear proteins bind to the POLD1
promoter DNA, including the two 11-bp repeats.
To corroborate the results of the DNase I footprint analysis and to
examine the specificity of the binding proteins, electrophoretic mobility shift assays were carried out using HeLa nuclear extracts and
the 70-bp POLD1 DNA (position 92 to
23, designated
R2R1), containing the two 11-bp repeat sequences (but not the upstream Sp1 site detected by the DNase I footprint analysis) from
pGL2-
(
92), as the probe (Fig. 5). With low amounts
of nuclear extracts, one major (designated CI) and one
minor (designated CIV) protein-DNA complexes were observed
(Fig. 5A, lanes 2 and 3). With
increasing amounts of nuclear extracts, four complexes, designated
CI-CIV, were identified. With excess amount
(12 µg) of nuclear extracts, the CIV complex became the
predominant species, while the CI complex became the minor
species (lane 4). The specificity of the protein-DNA interaction was indicated by the disappearance of the shifted bands in
the presence of a 50-fold molar excess of unlabeled R2R1 DNA
(lane 6). In contrast, incubation with a 50-fold excess of a
DNA fragment containing the POLD1 initiator region (position
22 to +49, designated DI) did not result in any decrease of the intensity of the shifted species (lane 7).
To further examine the characteristics of the four complexes, the 70-bp
DNA fragments containing one or two mutated copies of the 11-bp repeat
from the linker replacement mutants ((
92)M2R1-2,
(
92)R2M1,
and
(
92)M2M1-2 in Fig. 2B) were used in the EMSA. When
the M2R1 DNA with the linker-replacement mutation in the 5
copy of the
11-bp repeat sequence was used as the probe, the CI complex
was detected as the major species even when excess amounts of HeLa
nuclear extracts were used (Fig. 5A, lanes
8-11). Similarly, when the R2M1 DNA with a mutation in the 3
copy of the 11-bp repeat sequence was used as the probe, the
CI complex remained as the major species, while some
slow-migrating complexes could also be detected when excess amounts of
nuclear extracts were used (lanes 12-15). These results
suggest that the CI complex was formed only when one copy
of either repeat was occupied by the protein. In contrast, no
protein-DNA complex was detected when the M2M1 DNA containing mutations
in both copies of the repeat sequences was used (lanes
16-19). All of these results indicate that the 11-bp repeats in
the POLD1 promoter are bound by specific nuclear
proteins.
Since the left half of the 11-bp repeat sequence (5-GGGGCGTGGCC-3
)
resembles the consensus Sp1-binding site (5
-GGGCGG-3
; Refs. 32 and
56), we also conducted an antibody supershift experiment to determine
if the specific protein-DNA complexes detected in EMSA contained the
Sp1 protein. By adjusting the amount of HeLa nuclear extracts and the
R2R1 DNA as the probe, four protein-DNA complexes similar to those
observed above (Fig. 5A) were detected (Fig. 5B).
Addition of an anti-Sp1 antibody resulted in supershifting of most, but
not all, of these complexes. When either M2R1 or R2M1 DNA was used as
the probe, addition of the anti-Sp1 antibody also led to supershifting
of most of the CI complex (Fig. 5B). The
addition of an anti-E2F antibody as a control (50) had no effect on the
complexes (data not shown). Taken together, the results indicate that
specific nuclear proteins, including Sp1, can bind to the 11-bp repeat
sequence in the POLD1 promoter DNA.
To further characterize the 11-bp repeat-binding
proteins, Southwestern blot analysis of HeLa nuclear extracts was
carried out using the same 70-bp R2R1 DNA containing the two repeats as the probe (Fig. 6). In addition, a double-stranded 22-bp
oligonucleotide with an Sp1 site
(5-ATTCGATCGGGGCGGGGCGAGC-3
; Ref. 57) was also used as a
probe in a comparative analysis. When the R2R1 DNA was used, a doublet
of major DNA-binding proteins with molecular masses of around 85 kDa
and a minor DNA-binding protein of 105 kDa were detected (Fig.
6A, lane 1). However, when the Sp1
site-containing oligonucleotide was used, a major DNA-binding protein
of 105 kDa, which corresponds to the size of the reported Sp1 protein
(57), was identified (Fig. 6A, lane 2). Note that
this Sp1 protein co-migrated with the 105-kDa band detected by the R2R1
probe (compare lane 2 to lane 1 in Fig.
6A), suggesting that the 105-kDa band is the Sp1 protein. In
addition, the Sp1 oligonucleotide also detected a minor band of around
53 kDa in the analysis.
We also conducted a Southwestern blot experiment using the mutant
POLD1 DNA probes containing one or two mutated copies of the
11-bp repeat from the linker replacement mutants, like those used in
the previous EMSA (Fig. 5). In contrast to R2R1, the M2R1 DNA with the
linker-replacement mutation in the 5 copy of the 11-bp repeat sequence
or the R2M1 DNA with the mutation in the 3
copy only detected the
105-kDa protein (Fig. 6B). In addition, the intensity of
this 105-kDa band detected by these single-replacement mutant DNAs was
much stronger than that detected by the wild-type R2R1 DNA. When the
M2M1 DNA containing mutations in both copies of the repeats was used,
only the 105-kDa band with reduced intensity, was observed (Fig.
6B). These results suggest that the binding of the 85-kDa
proteins requires both copies of the 11-bp repeat sequences.
Because Southwestern blot employs a denaturation step, some DNA-binding
proteins sensitive to this treatment might not be detected in this
analysis. To exclude this possibility and to confirm the results
obtained from the Southwestern blot analysis, UV cross-linking
experiments were conducted. The same 32P-labeled R2R1 DNA
containing the two 11-bp repeats was mixed with HeLa nuclear extracts
and binding was allowed to reach equilibrium. The mixture was then
subjected to UV irradiation for various time and the cross-linked
material analyzed by electrophoresis. A doublet of major bands
migrating at about 110 kDa and a minor band with slightly higher
molecular mass were detected (Fig. 7). The pattern of
these bands is similar to that observed in the Southwestern blot
analysis (Fig. 6), albeit with slower mobilities due to the presence of
the probe DNA in the cross-linked complexes. A band with molecular mass
of around 64 kDa was also seen. The intensity of all of these bands
increased linearly with increasing time of irradiation, consistent with
a simple interaction between the activated DNA and closely associated
proteins. In addition, production of all of these bands could be
inhibited by competition with the unlabeled homologous DNA (data not
shown). Taken together, these results indicate that in addition to Sp1,
the 85-kDa proteins in HeLa nuclear extracts can bind
specifically to the 11-bp repeat sequences of the
POLD1 promoter DNA.
Sp1 and Sp3, but Not Sp2, Can Activate the POLD1 Promoter
To identify the gene encoding the protein that binds to the 11-bp repeat sequences, we screened HeLa cDNA expression libraries with a concatenated 70-bp R2R1 DNA, which contains the two 11-bp repeats as the probe. One positive clone containing a 2.1-kb cDNA insert was obtained, and sequence analysis showed that the cDNA sequence is identical to that of Sp3, a member of the Sp1 family (33, 35). The sequence of the 2.1-kb cDNA covers almost the entire Sp3 ORF (33, 35), starting at position +46, relative to the first nucleotide of the published Sp3 cDNA sequence designated as +1 (33), and ending at +2109, 15 bp downstream of the translation termination codon.
To test if the Sp1-family proteins can activate the POLD1
promoter, expression plasmids for Sp1, Sp2 (35), and Sp3 were constructed (Fig. 8A) and used in
cotransfection of Drosophila SL2 cells, which lack
endogenous Sp factors and have been useful in studying the function of
the Sp1-family proteins (34, 47, 58). When the
POLD1-luciferase construct pGL2-(
92) containing the two
11-bp repeats was cotransfected with increasing amounts of each
Sp1-family expression vector, both Sp1 and Sp3, but not Sp2, stimulated
the POLD1 promoter activity (Fig. 8B). Maximal stimulation was detected when 5 µg of the Sp1- or Sp3-expression plasmid were used. To determine if the stimulation of the
POLD1 promoter activity by Sp1 and Sp3 was mediated through
the two 11-bp repeats, the POLD1-luciferase plasmid with
(pGL2-
(
92)) or without (pGL2-
(
22)) the two repeats was
cotransfected with 5 µg of each Sp1-family expression plasmid into
SL2 cells. In addition, the pGL2-Basic plasmid was used as the vector
control. As expected, only the Sp1- and Sp3-expression plasmid
stimulated the promoter activity of pGL2-
(
92) containing the two
repeats (Fig. 8C). Deletion of the two 11-bp repeats
(pGL2-
(
22)) drastically reduced the ability of Sp1 or Sp3 to
stimulate the POLD1 promoter activity. Intriguingly, both
Sp1 and Sp3 also stimulated the luciferase activity expressed by the
control pGL2-Basic plasmid (Fig. 8C), suggesting that this
reporter plasmid contains cryptic promoter elements responsive to Sp1
and Sp3 stimulation in Drosophila SL2 cells. Nonetheless, by
comparing to the level of stimulation for each reporter construct, the
luciferase activity of the pGL2-
(
22) plasmid without the two 11-bp
repeats was stimulated to the extent similar to that of the pGL2-Basic
plasmid by Sp1 or Sp3, while in the presence of the two repeats
(pGL2-
(
92)) a much larger stimulation of the luciferase activity
was observed (Fig. 8C). It should be pointed out that all
three Sp1-family proteins were expressed at the similar level in
transfected SL2 cells (Fig. 8D). Taken together, these
results indicate that Sp1 and Sp3, but not Sp2, can activate the
POLD1 promoter through the two 11-bp repeats.
To examine if the stimulation of the POLD1 promoter activity
by Sp1 and Sp3 correlates with their ability to bind to the 11-bp repeat sequence, nuclear extracts from transfected SL2 cells expressing each Sp1-family protein were tested for the binding ability to the
POLD1 R2R1 DNA. As compared with the protein-DNA complexes detected in HeLa extracts (Fig. 9A,
lanes 2 and 3), extracts from SL2 cells
transfected with the control pPacU vector gave rise to two
fast-migrating complexes with relatively low intensity (Fig.
9A, lane 4), representing some endogenous binding
activities from the Drosophila cells. On the other hand,
extracts from Sp1- or Sp3-expressing SL2 cells produced multiple
slow-migrating complexes, which migrated to similar positions as those
seen in HeLa extracts (Fig. 9A, lanes 5 and
6). In contrast, extracts from Sp2-expressing SL2 cells did
not give rise to any slow-migrating complexes and only showed
endogenous binding activities (Fig. 9A, lane
7).
Two additional experiments were carried out to confirm that the slow-migrating complexes are resulted from the binding of the POLD1 R2R1 DNA by Sp1 or Sp3 expressed in transfected SL2 cells. First, an antibody supershift experiment using the anti-Sp1 or anti-Sp3 antibody was conducted. Similar to those observed in HeLa extracts (Fig. 5A), multiple protein-DNA complexes were detected with increasing amounts of extracts prepared from Sp1- or Sp3-expressing SL2 cells (Fig. 9B). Addition of the anti-Sp1 antibody to the binding reaction containing Sp1-expressing SL2 extracts led to supershift all of the slow-migrating complexes to form higher-ordered complexes (compare lane 4 with lane 5 in Fig. 9B). Similarly, addition of the anti-Sp3 antibody to the binding reaction containing Sp3-expressing SL2 extracts also led to supershift all of the slow-migrating complexes to form higher-ordered complexes (compare lane 8 with lane 10 in Fig. 9B). Second, a Southwestern blot using the R2R1 DNA as the probe was also conducted. When extracts from Sp1-expressing SL2 cells were used, a 75-kDa DNA-binding protein was detected (Fig. 9C), consistent with the calculated molecular mass from the Sp1 ORF in the expression plasmid. Note that the molecular mass of this SL2 cell-expressed Sp1 is smaller than that of the reported Sp1 protein, presumably due to N-terminal 82-amino acid truncation of the Sp1 ORF in the expression plasmid (47). Similarly, when extracts from Sp3-expressing SL2 cells were used, a 90-kDa DNA-binding protein was detected (Fig. 9C), consistent with the calculated molecular mass from the Sp3 ORF (33, 35) in the expression plasmid. In addition, the molecular mass of the Sp1 or Sp3 protein detected here is identical to that observed in the Western blot analysis (Fig. 8D). Intriguingly, although both Sp1 and Sp3 bind well to the R2R1 DNA in EMSA (Fig. 9, A and B), the intensity of the Sp3 band detected in this Southwestern blot is much weaker than that of Sp1 (Fig. 9C). In contrast, when extracts from SL2 cells transfected with the pPacSp2 or the control pPacU plasmid were used, only an endogenous 50-kDa protein with weak intensity was detected (Fig. 9C). From all of these analyses, we conclude that Sp1 and Sp3, but not Sp2, can bind to the 11-bp repeat-containing R2R1 DNA.
The POLD1 Promoter Is Regulated during the Cell CycleYang
et al. (11) previously showed that the human
POLD1 mRNA level is increased 10-fold in
serum-stimulated IMR90 cells. To test if the POLD1 promoter
can respond to serum stimulation, the POLD1-luciferase
plasmid pGL2-(
1758) containing the 1.8-kb POLD1
promoter was transfected into C3H10T1/2 cells. After
transfection, cells were growth-arrested and then restimulated into the
cell cycle with 10% serum. The stimulated cells were harvested for reporter enzyme assays at various time points after serum addition. As
shown in Fig. 10A, the 1.8-kb
POLD1 promoter was induced during the cell cycle and the
increase in the POLD1 promoter activity began at 8-12 h
after serum stimulation and reached to the highest level (about 4-fold)
at 24-28 h, corresponding to the late G1/S phase as
indicated by flow cytometry analysis (Fig. 10B).
To define the cis-acting elements responsible for serum
stimulation of the POLD1 promoter during the cell cycle,
several promoter deletion mutant derivatives of pGL2-(
1758) were
studied. Deletion of the 5
upstream sequence between nucleotide
positions
1758 and
92 appeared to have little or no effect on serum
stimulation of the POLD1 promoter activity. Further deletion
to position
22 (pGL2-
(
22)), which removes the two 11-bp repeats,
reduced but still showed some response to serum stimulation (about
2-fold; Fig. 10B). These results indicate that the sequence
between position
92 and
22 containing the two repeats is important
for serum stimulation of the POLD1 promoter. To confirm the
role of the two 11-bp repeats in serum response, linker-replacement
mutations in both copies of the repeats were introduced into
pGL2-
(
1758) (Fig. 11A). The resulting
pGL2-
(
1758)M2M1 mutant was studied for the serum response as
described before. Similar to the deletion mutant pGL2-
(
22) without
the two repeats, pGL2-
(
1758)M2M1 also showed reduced response to
serum stimulation (Fig. 11B).
Since the POLD1 promoter without the two repeats
(pGL2-(
22)) still gave rise to some response to serum stimulation
(Fig. 10B), we examined if there was any additional element
involved in the regulation. Examination of the POLD1
sequence between position
22 and +49 in pGL2-
(
22) revealed two
sequences that resemble the binding site for the transcription factor
E2F, known to be involved in cell-cycle regulation of many S-phase
genes (21, 59, 60). One such sequence is located adjacent to the major transcription initiation site at position
13 to
6 with the sequence of 5
-TT
CGCGC-3
(one nucleotide (underlined) differs
from the consensus sequence of the E2F-binding site 5
-TTTSSCGC-3
,
where S = G/C). The other is located in the 5
UTR at position +37
to +30 with the sequence of 5
-TTTCCCGC-3
, which is arranged in the
antisense orientation and completely matches with the consensus sequence of the E2F-binding site (Fig. 11B). To examine if
these two sequences are important for serum stimulation of the
POLD1 promoter, mutation in each sequence was introduced
into pGL2-
(
1758) (Fig. 11A). Mutation of the putative
E2F-binding sequence in the leader region (pGL2-
(
1758)Lm) did not
affect the response of the POLD1 promoter to serum
stimulation, while mutation of the E2F-like sequence at the initiator
region (pGL2-
(
1758)Im) reduced the response to about 2-fold,
suggesting that the E2F-like sequence at the initiator region is also
important for serum stimulation.
To examine if both the 11-bp repeats and the E2F-like sequence at
the initiator region can confer full serum response of the POLD1 promoter, the double mutant pGL2-(
1758)M2M1/Im
containing mutations in both of these sequences was constructed (Fig.
11A) and analyzed. As shown in Fig. 11B, this
mutant plasmid did not show any response to serum stimulation. Taken
together, these results indicate that the human POLD1
promoter is induced during serum stimulation, and both the 11-bp
repeats and an E2F-like sequence adjacent the major transcription
initiation site are important for the regulation.
In this paper, we have analyzed the promoter of the gene for the
catalytic subunit of human DNA polymerase (POLD1). We
have demonstrated that the core promoter of the POLD1 gene
is located within 328-bp DNA upstream from the major transcription
initiation site. Multiple elements including two 11-bp direct repeats
located between nucleotide position
92 and
22, play an important
role in POLD1 promoter activity.
Several lines of evidence indicate that the two 11-bp repeats function as an activator sequence for the POLD1 promoter. First, deletion of one copy of the two repeats reduced the POLD1 promoter activity by about 2-4-fold, while deletion of both copies nearly abolished the promoter activity. Second, linker-replacement mutations in either one or both copies of the two repeats gave rise to a similar effect on the promoter activity as the deletion mutants. Third, a 70-bp POLD1 DNA containing the two 11-bp repeats can enhance the expression of both the POLD1 and the heterologous HSVtk basal promoters in an orientation-independent manner.
The sequence of the 11-bp repeat (5-GGGGCGTGGCC-3
) does not show
complete identity with any known transcription factor-binding sites
(61-63). However, the left half of the repeat sequence
(5
-GGGCGT-3
) resembles the consensus sequence of the
Sp1-binding site (5
-GGGCGG-3
; 31, 32). Indeed, several
experiments indicated that Sp1 can bind to the 11-bp repeat sequence of
the POLD1 promoter DNA. Southwestern blot analysis showed
that a 105-kDa protein, comigrating with the Sp1 protein, could bind to
the repeat sequence. An anti-Sp1 antibody could supershift most of the
protein-DNA complexes containing the 11-bp repeats in EMSA. In
addition, the Sp1 protein expressed in Drosophila SL2 cells
could bind to the repeat sequence. Similar to Sp1, another Sp1-family
protein, Sp3, could also bind to the 11-bp repeats. A cDNA clone
encoding Sp3 was obtained by screening the HeLa cDNA expression
library for the sequence-specific DNA-binding protein using the 11-bp
repeats as the probe. Also, the Sp3 protein expressed from this
cDNA could bind to the repeat sequence. The role of these
Sp1-family proteins in regulating the POLD1 promoter activity was demonstrated by the fact that both Sp3 and Sp1 can activate the POLD1 promoter through the two 11-bp repeats.
In contrast, Sp2 can not bind to the repeat sequence and as a
consequence, has no effect on the POLD1 promoter
activity.
In addition to the Sp1-family proteins, a doublet of 85-kDa protein was
found to bind to the 11-bp repeats in both the Southwestern blot and UV
cross-linking analyses. Consistently, although a linker-replacement mutation in the left half of the 5 copy of the repeat sequence that
resembles the Sp1-binding site reduced the POLD1 promoter activity, mutation in the right half produced a similar effect. In
addition, deletion of the entire 5
copy of the 11-bp repeat resulted
in a greater reduction of the promoter activity, suggesting the
requirement of the entire 11-bp repeat sequence and the possible involvement of the 85-kDa proteins for POLD1 promoter
regulation. It should be pointed out that these 85-kDa proteins are not
degradation products of Sp1, since they were not detected by the Sp1
site-containing oligonucleotide DNA. Additionally, the molecular
weights of these proteins are not the same as those of the Sp1-family
members (33-35) or the small GT/GC box-binding proteins BTEB1, BTEB2,
and EKLF (64-66).
Intriguingly, the 85-kDa binding proteins were the predominant species that bind to the 11-bp repeats in Southwestern blot analysis, while most, but not all, of the protein-DNA complexes detected by EMSA could be supershifted by the anti-Sp1 antibody. Similarly, Sp3 appears to bind well to the 11-bp repeat sequence in EMSA but only binds weakly in Southwestern blot analysis as compared to Sp1, which shows strong binding in both assays. It is possible that Sp1 may have stronger binding affinity to the 11-bp repeat sequence in EMSA, while the conditions in Southwestern blot analysis allow the detection of other subtle DNA-binding proteins such as the 85-kDa proteins. Importantly, mutation analysis showed that binding of the 85-kDa proteins to the POLD1 promoter DNA required the presence of both copies of the 11-bp repeats. The molecular masses and the specific binding property of these 85-kDa binding proteins suggest that they are potentially novel transcription factors for the expression of the POLD1 gene. Further characterization of these DNA-binding proteins and their encoding genes should allow us to better understand the transcriptional regulation of the gene encoding this major DNA replication enzyme.
The result from DNase I footprinting analysis showed that, in addition
to the 11-bp repeat region, another protein-binding site was detected
in the upstream region of the POLD1 promoter. Sequence
analysis identified two Sp1-binding sites in the POLD1 core
promoter region (19), and one of these sites coincides with the
protein-binding site detected in the footprinting analysis. It appears
that these Sp1 sites play a positive role in regulating the human
POLD1 gene, since deletion of these Sp1 site-containing regions gave rise to some reduction of the promoter activity. Besides
the positive elements identified in the human POLD1
promoter, a negative element located within the region from nucleotide
position 68 to
58 appears to exist since deletion of this region
resulted in an increase of promoter activity. Interestingly, a sequence within this region (5
-CACTTG-3
at position
63 to
58) shares similarity to the binding sites of the basic region helix-loop-helix and basic region helix-loop-helix-zipper proteins, defined as the E-box
sequence motif (5
-CAXXTG-3
in which the central 2 bp are
specified by each protein; Refs. 67-73). At present we have not
identified the protein that binds to this element using crude HeLa
nuclear extracts. Nonetheless, the presence of both positive and
negative elements and their interactions may play an important role in
regulating the expression of the POLD1 gene in the
proliferating state (11).
Our previous analysis showed that the human POLD1 mRNA
level is increased 10-fold in serum-stimulated IMR90 cells (11). In
this study, the POLD1 promoter is induced about 4-fold
during serum stimulation, suggesting that regulation of the
POLD1 gene during the cell cycle is not limited to the
transcriptional level. Posttranscriptional regulation has been found to
be important for growth stimulation of several genes involved in
nucleotide metabolism and DNA synthesis (20, 22). It should be noted that the POLD1 gene contains 26 introns, and seven of them
are smaller than 100 bp. It is possible that correct processing of the
pre-mRNA and, perhaps, the sequence within the intron may contribute to the regulation of the POLD1 gene. In addition,
Zeng et al. (41) showed that the catalytic subunit of human
pol is a phosphoprotein that is most actively phosphorylated during the S phase. Thus, regulation of POLD1 expression appears to
be mediated at multiple levels.
Interestingly, both the 11-bp repeats and an E2F-like sequence located immediately upstream to the major transcription initiation site are responsible for serum stimulation of POLD1 promoter activity. The E2F transcription factor is involved in the transcription of several cellular genes necessary for proliferation and is one of the targets of the retinoblastoma family of growth suppressors (reviewed in Ref. 74). The retinoblastoma protein, which negatively regulates cell cycle progression from G1 into S phase, binds to E2F, and the functional consequence of this interaction is to prevent E2F from activating transcription. The presence of the E2F site at or adjacent to the transcription initiation site has been found in a few growth-regulated genes (for reviews, see Refs. 20, 21, 59, and 60). Although the sequence of this E2F-like element in the POLD1 promoter has one nucleotide difference from the consensus sequence of the E2F-binding site, it is possible that E2F may regulate the POLD1 gene through this element for its timely expression during the cell cycle. Experiments are in progress to investigate if indeed, E2F can bind and activate this POLD1 element. Recently, Lin et al. (75) and Karlseder et al. (76) found that E2F1 and Sp1 both functionally and physically interact with each other, and suggested that through this interaction, Sp1 and E2F1 may regulate transcription of genes containing binding sites for either or both factors. Given the fact that Sp1 can bind and activate the POLD1 promoter through the 11-bp repeat sequence, and both the 11-bp repeats and the E2F-like sequence near the initiator region are involved in serum stimulation of the POLD1 promoter activity, we are presently conducting experiments to examine if Sp1 and E2F can cooperatively regulate POLD1 expression. Nevertheless, the present study provides direct evidence for transcriptional regulation of the human POLD1 gene.
We sincerely thank Dr. Robert Tjian of University of California at Berkeley for the Sp1 expression plasmid, and O. Joseph Trask, Jr. and Dr. Fredika Robertson of the Flow Cytometry Laboratory of the Ohio State University Comprehensive Cancer Center for their expert FACS analysis. We greatly appreciate Drs. Ing-Ming Chiu, Tsonwin Hai, Vincent V. Hamparian, Lee Johnson, and Lingyun Zhu for critical reading of the manuscript, and members of the Chang laboratory for stimulating discussions.