(Received for publication, June 6, 1996, and in revised form, December 13, 1996)
From the Plk (polo-like kinase) is a serine-threonine
kinase that appears to function in mitotic control in mammalian cells.
We demonstrated previously that PLK mRNA expression is
low at the G1-S transition, increases during S phase, and
is maximally expressed during G2-M. In the present study,
we have cloned the human PLK gene and analyzed the
structure and function of 2 kilobases of its 5 The data accumulated during the last few years have clearly
demonstrated that the cyclin-dependent kinases regulate
many important functions during cell cycle progression, and new
cyclin-dependent kinases continue to be identified.
However, it has become evident that cell cycle progression also
involves the activities of a variety of cyclin-independent kinases,
some of which regulate cyclin-dependent kinase activity and
some with activities entirely unrelated to cyclin-dependent
kinase functions.
Recently, a family of cell cycle-regulated, cyclin-independent protein
kinases, known as polo-related kinases, has been identified. Polo, the
prototype enzyme, was originally identified in Drosophila as
a cell cycle gene with an essential function during G2-M
(1). Mutant alleles of the polo gene caused formation of
monopolar and multipolar mitotic spindles and abnormal segregation of
chromosomes. In Saccharomyces cerevisiae, mutant alleles of
the polo homologue CDC5 cause impaired spindle
formation, whereas mutations in the Shizosaccharomyces pombe
polo homologue Plo1 caused failure both in the
formation of the F-actin ring during cytokinesis and in deposition of
septal material (2, 3). We and others have cloned a putative mammalian
polo homologue, polo-like kinase
(PLK)1 (4-8). The kinetics of
Plk kinase activation (9), its localization at the spindle poles and
postmitotic midbody (10), and its association with CHO1/MKLP-1 (11)
suggest that Plk, like polo, CDC5, and Plo1, is involved in chromosome
segregation.
It has been shown previously that steady-state PLK message
and protein levels are coordinately regulated, steadily rising from a
low in G1 to a peak during G2-M (9, 11). Lake
and Jelinek (8) suggested that posttranscriptional regulation is responsible for the cell cycle-associated fluctuations in
PLK mRNA abundance based on the results of nuclear
run-off experiments. More recently, Zwicker (12) showed that
S-G2-specific transcription of cdc25C, cdc2, and
cyclin A is regulated by two repressor elements known as CDE
(cell cycle-dependent element) and CHR (cell cycle gene
homology region). Mutation of the CDE and CHR elements in these genes
allowed elevated transcription during G1 and consequent loss of cell cycle-regulated expression. Several cell cycle genes expressed at the G1S transition are regulated by members of
the E2F/DP family (13, 14). The E2F/DP transcription factors are repressed in G0-G1 due to their association
with RB family members. In late G1, transcription of
E2F-regulated genes such as DHFR, E2F-1, and DNA
polymerase is activated following dissociation of E2F/DP complexes from
RB (15-17). Similarly, transcription of cdc2 is repressed
during G1 by binding of E2F-4-p130 complexes at the CHR/CDE
region (18).
To investigate the mechanisms regulating fluctuations in steady-state
PLK message, we have cloned the 5 NIH3T3 and
HeLa cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and penicillin. For transient
transfections, 3 × 105 cells/dish (60-mm diameter)
were plated 24 h prior to transfection. Cells were then
transfected with 5 µg of various luciferase gene constructs using
liposome-mediated
N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammoniummethyl sulfate reagent as described by the manufacturer (Boehringer Mannheim). After 6 h, the transfection media was replaced with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for
48 h. Cells were then harvested using Reporter lysis buffer (Promega Corp.). For synchronization, cells were treated with mimosine
(300 µM), thymidine (2 mM) or nocodazole (100 ng/ml) for 24 h to arrest them at G1,
G1-S, or prometaphase, respectively.
HeLa cells were
transfected with PLK/luciferase constructs using
N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammoniummethyl
sulfate reagent, and then a mixture of reporter plasmid (10 µg) and
pRSV-neo (0.5 µg) was added. After 8 h, the medium was replaced
with fresh medium Following incubation for 24 h, the cells were
incubated in selection medium containing 0.8 mg/ml Geneticin (G418)
(Life Technologies, Inc.). Growing colonies (20-30 per 106
cells) were cloned, expanded, and tested for luciferase activity.
Untransfected and
stably transfected HeLa cells were grown on 100-mm dishes to 30%
confluence and synchronized at the G1-S boundary by a
double thymidine block protocol. The first block was for 19 h,
followed by a 9-h release and a 17-h second block (19). Cells were
harvested at various times after release from the second block and used
for cell cycle analysis, luciferase assays, and Northern blot analysis.
For cell cycle analysis, cells were harvested, washed in ice-cold
phosphate-buffered saline, and lysed in 138 mM NaCl, 5 mM KCl, 440 µM
KH2PO4, 335 µM
Na2HPO4, 1 mM CaCl2,
500 µM MgCl2, 400 µM
MgSO4, 24 mM Hepes, 0.2% w/v BSA, and 0.4%
w/v Nonidet P-40. Cells were then treated with RNase, and cellular DNA
was stained with propidium iodide. Cell cycle determination was
performed using a Becton-Dickinson fluorescence-activated cell
analyzer, and data were interpreted using the SFIT model program
provided by the manufacturer.
Northern blot analysis was performed
with 20 µg of total RNA prepared with RNeasy spin columns (Qiagen).
After transfer to Hybond-N membrane (Oncor) and UV cross-linking, the
blot was hybridized with human PLK cDNA probe generated
by random primer labeling (Amersham Corp.). After hybridization, the
blots were washed twice at 42 °C in washing buffer 1 (2 × SSC,
0.1% SDS) and twice in washing buffer 2 (0.2 × SSC, 0.1%SDS),
and exposed to Kodak x-ray film.
A genomic library of the
human fibroblast cell line was screened with a 5 Cells were lysed in 200 µl of 25 mM Tris-phosphate buffer, pH 7.5, containing 1% Triton
X-100. Soluble extracts were prepared by centrifugation (14,000 × g for 15 s). Protein concentrations were determined
with a protein assay kit (Bio-Rad), and concentrations were normalized.
Luciferase assays were performed with luciferase assay reagent (Promega
Corp.), and light intensity was measured for 10 s on a Berthold
LB9510 luminometer. The luciferase assay results shown in the figures
are representative of at least three independent experiments.
Ten pmol of antisense
oligonucleotide 5-21 bp relative to the translation start site was end
labeled with [32P]ATP. The labeled primer and HeLa
poly(A)+RNA (5 µg) were denatured for 10 min at 65 °C and then
incubated at 42 °C overnight. Primer extension was carried out in a
total volume of 20 µl containing 50 mM Tris, pH 8.3, 75 mM KCl, 10 mM dithiothreitol, 3 mM
MgCl2, 400 µM deoxynucleotide triphosphates,
2 units RNasin, and M-MuLV reverse transcriptase (Promega). After
incubation for 30 min at 42 °C, the reaction was stopped with EDTA
and purified with phenol-chloroform isoamyl alcohol. The cDNA
product was then separated by electrophoresis on a 6% polyacrylamide
sequencing gel containing 8 M urea.
PCR reactions contained 1 ng of template DNA, 100 pM oligonucleotide primers, 200 µM deoxynucleotide triphosphates, and 2.5 units of
Taq DNA polymerase in reaction buffer (50 mM
KCl, 10mMTris-HCl, pH 8.3, 1.5 mM MgCl2, and
0.01% (w/v) gelatin). The reaction was carried out with a DNA thermal
cycler (Perkin-Elmer Corp.) for 30 cycles of denaturation at 94 °C
for 30 s, annealing at 55 °C for 1 min, and polymerization at
72 °C for 2 min.
Inserts were cloned into the multiple cloning site
between the SacI and BglII sites of pGL2-Basic
(Promega Corp.). The 3 HeLa cells (4 × 107) treated with mimosine or nocodazole were washed twice
with phosphate-buffered saline, collected by trypsinization, and
resuspended in 200 µl of cold buffer A (10 mM HEPES, pH
7.9, 10 mM KCl, 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 0.2 mM
EGTA, 0.5 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride). Following incubation on ice for 15 min,
the cells were lysed with 5 strokes of a 1-ml syringe (25-gauge
needle). Each sample was centrifuged for 40 s in a microcentrifuge
to collect the nuclear pellet. The pellet was resuspended in 100 µl
of cold buffer C (20 mM HEPES, pH 7.9, 0.4 M
NaCl, 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 0.2 mM EGTA, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and
25% glycerol). Samples were incubated for 30 min on ice with frequent
gentle mixing. The nuclear extracts were centrifuged for 20 min at
4 °C to remove insoluble material, and the supernatant was stored at
We first examined the expression of
PLK mRNA by Northern blot analysis of synchronized cell
populations. HeLa cells were synchronized at the G1-S
boundary by double thymidine block and harvested at various times after
release. Flow cytometric analysis was performed on propidium
iodide-stained cells to determine DNA content as a control for
synchrony.
HeLa cells entered S phase immediately after release from thymidine,
were predominantly in G2 at 8 h, and had the highest mitotic index at 10-12 h (Fig. 1C). Northern
blots were hybridized with PLK probes (Fig. 1A).
PLK mRNA accumulates in S phase, peaks in M phase,
(10-12 h after release from the G1S block), and decreases as cells enter G1 phase, 14-18 h after release. Levels of
PLK mRNA varied 6-8-fold during the cell cycle (Fig.
1A). To confirm the drop in PLK message during
G1, we examined PLK expression after release
from a nocodazole-induced prometaphase block. HeLa cells synchronized
with nocodazole (16 h) were released from nocodazole and harvested at
indicated times. The level of PLK mRNA decreased after
release of block and reached its nadir at 8 h (Fig.
1B). Flow cytometry analysis showed that 90% of cells
arrested by nocodazole were in G2-M phase and 80% of the
cells entered G1 phase by 8 h after release (data not
shown). These results confirm that the steady-state level of
PLK mRNA is tightly cell cycle-regulated.
To determine if the differences in mRNA levels might
be regulated through promoter function, we isolated the 5
The transcription initiation site was located using a primer extension
assay. Using 32P-labeled oligonucleotide and 5 µg poly(A)
RNA from HeLa cells, one major band was found 78 bp upstream of the ATG
codon (Fig. 3). No primer-extended product was seen
using tRNA as control. Previously, Brauninger et al. (21)
reported that the human PLK gene contains three potential
transcription initiation sites. The site we have identified corresponds
to one of the three sites they identified, but we have found no
indication of any other sites. This difference may be cell
type-specific and was not further investigated.
To
determine the upstream sequences required for promoter activity, we
cloned the 5
To
examine the cell cycle-regulated activity of the PLK
promoter, PLK promoter-luciferase constructs (pGL-815,
pGL-389, pGL-195, and pGL-93) that have full promoter activity were
transfected into HeLa cells, and luciferase activity was assessed in
cultures synchronized by a thymidine block and release. After release
from the thymidine block, cells were harvested at indicated times (Fig. 5). Luciferase activity was measured and normalized for
protein content. The cells were predominantly in G1-S at
the start of the experiment. At this point, the level of luciferase
activity was low. As the cells entered S phase, luciferase activity
rose and peaked between 10 and 12 h after release, when the
majority of cells were in G2-M. After 15 h, when the
majority of cells entered G1, luciferase activity had
substantially decreased. At 24 h after release, luciferase
activity had fallen to slightly below the levels detected at the start
of the experiment. The lowest level of luciferase activity was detected
in G1, an increased level in S phase, and the maximal level
in G2-M, corresponding to a pattern paralleling the level
of PLK mRNA in cycling cells.
We next examined luciferase activity in extracts made from cells
transfected with various constructs (pGL-815, pGL-165, pGL-148, pGL-93,
pGL-65, and pGL-19) and blocked at different points in the cell cycle.
The transfected cells were treated with mimosine, thymidine, or
nocodazole to arrest them in G1, G1-S or
G2-M (prometaphase) phase, respectively, and flow cytometry
was used to confirm the arrest points (data not shown). The arrested
cells were harvested and assayed for luciferase activity (Fig.
6). The cells transfected with the four largest promoter
fragments had 5-6-fold greater luciferase activity when arrested in
G2-M (prometaphase) than cells arrested in G1
or at G1-S. The pGL-65 construct demonstrated about 50% of
the promoter activity compared with pGL-93 when arrested at
G1 and a 3-4-fold increase at G2-M
(prometaphase). pGL2-Basic (data not shown) and pGL-19 had essentially
no activity in transfected cells in any phase of the cell cycle. Thus,
experiments using either double thymidine block and release, or simple
overnight block with mimosine, thymidine, or nocodazole, produced
comparable results in the transiently transfected cells and indicated
that the PLK promoter is cell cycle-regulated.
To confirm the results obtained from transient transfections, we
established six cell lines that stably expressed four different promoter/luciferase constructs (pGL-815, pGL-241, pGL-195, and pGL-93)
(Fig. 7A). These cell lines were synchronized
at the G1-S boundary by double thymidine block and
harvested at time intervals after release (Fig. 7B).
Luciferase activity rose as the cells entered S phase and peaked
between 10 and 12 h after release when the majority of cells were
in G2-M. After 15 h, when the majority of cells
entered G1, luciferase activity dropped. By 24 h after release, luciferase activity had decreased to approximately the same
levels detected prior to release from the G1-S block.
Levels of PLK promoter activities varied 4-5-fold during
the cell cycle. A control SV40 promoter enhancer construct
(pGL-control) showed minimal differences between the mimosine- and
nocodazole-blocked cell extracts (data not shown). Both the timing of
PLK promoter activity and induction levels in the stable
lines were essentially identical with the results obtained in
transiently transfected cells. The results of the experiment in Fig. 7
confirm that the changes in luciferase activity during the time course
are not simply due to elevation and subsequent decrease after transient transfections.
The studies described above indicate that the basic
PLK promoter is present in the region downstream from
We identified at least three positive regulatory elements in the
PLK promoter. The first spans nucleotides The third element spans nucleotides Zwicker et al. (12, 22) reported recently
that transcription of SG2-specific genes such as
cdc25C, cdc2, and cyclin A are mediated by a cell
cycle-regulated repressor element (CDE) and a cell cycle gene homology
region (CHR). An alignment of the PLK promoter sequence with
the CDE and CHR of these S-G2-specific genes shows that
the PLK promoter sequence is perfectly matched with the CHR
consensus sequence (Fig. 9). Three base pairs at the
center of a putative PLK CDE region (GCG) are identical with the consensus sequence, but the perimeter is different. However, the
spacing between these two putative elements in PLK and their locations within the promoter region are very similar to that reported
for the other genes. These results suggest that the putative CDE and
CHR region of PLK function as a G1-specific
repressor as they do in other SG2-specific genes. To
confirm the repressor function of these elements, we performed
mutagenesis of CDE and CHR in the PLK promoter-luciferase
construct pGL-93 (Fig. 10A). Wild-type and
two mutant constructs were transiently transfected into HeLa cells, and
luciferase activity was determined in cells arrested with mimosine
(G1) or nocodazole (prometaphase) for 16 h (Fig.
10B). The wild-type construct showed low activity in
extracts from cells in G1, but this activity was elevated
approximately 5-fold in extracts from prometaphase cells. The CHR
mutant constructs had elevated activity in the G1 extracts
but demonstrated only 1.5-fold increases in the prometaphase extracts.
In contrast, the CDE mutant constructs caused insignificant elevation
in G1 extracts. The CHR mutations decreased the ratio of
the promoter activity measured in prometaphase versus
G1 cells by approximately 3-fold, whereas the CDE mutations
decreased the ratio by less than 2-fold (Fig. 10C). These
results indicate that the CHR element in PLK acts as a
G1-specific repressor, whereas the CDE-like element in
PLK makes little contribution to cell cycle-specific
transcription.
Several groups have reported that steady-state PLK
message levels vary dramatically during cell cycle progression, from
very low or undetectable amounts in G0-G1,
steadily increasing amounts detected during S phase onward, to a peak
at mitosis (5, 8, 11). Recently, Brauninger et al. (21) have
analyzed the structure of both the human and murine PLK
promoter regions and have shown that the 5 Our work, to the extent that it overlaps with the results of Brauninger
et al. (21), is in good agreement with their findings. However, in addition to addressing the question of basal PLK
promoter activity, we have examined cell cycle-specific regulation of
promoter function and have identified the responsible sequence elements within the core promoter region.
The results of our experiments demonstrate that cytoplasmic
PLK mRNA levels, like those of several other cell
cycle-regulated genes, correlate with the cell cycle. The hypothesis
that these fluctuations in mRNA levels are based, at least in part,
on promoter function is supported by results from both transient
transfections with various promoter/luciferase constructs and by
experiments using the stably transfected cell lines. Finally, both the
presence in the PLK promoter of CDE and CHR sequences
similar to those found in cdc2, cyclin A, and
cdc25C promoters (12, 22, 23), and the fact that mutations
of the CDE and CHR sequences of PLK caused elevated
G1 luciferase activity, also support a role for promoter
function in the control of mRNA levels.
Our results and conclusions conflict with the nuclear run-off results
of Lake and Jelinek (8), who detected no indication of cell
cycle-specific transcriptional regulation of PLK after serum
stimulation of starved NIH 3T3 cells. However, in their experimental
system, they only tested cells starved for 48 h and at 6 and
24 h following serum stimulation. The first two points comprise
cells in G0 and G1 when little PLK
mRNA is detected, whereas the 24-h point was expected to be mainly
G2-M but could possibly have contained substantial numbers
of G1 cells. Thus, the limited nature of the time course
they examined may account for the differences between their data and
the results presented here.
The results of our studies support the hypothesis that the increase in
PLK mRNA levels seen at G2-M is largely if
not entirely due to cell cycle-specific transcriptional regulation. In
our experiments using transient transfections and stable cell lines containing the full promoter region, we observed approximately a 5-fold
increase of luciferase activity at G2-M phase. We also observed that the PLK promoter sequences between nucleotides
Using deletion analysis within this region, we have identified elements
responsible for transcriptional regulation of the PLK gene.
We identified a critical region required for minimal expression of the
PLK gene between nucleotide position The first positive element spans nucleotides The second positive element contains a putative SP1 site (24).
Potential binding sites for SP1 were previously identified in the
promoters of cyclin A, cdc2, and cdc25C genes
(12, 22). However, we did not detect cell cycle regulated binding to
the GC-rich SP1 containing PLK oligonucleotide in
electrophoretic mobility shift assays, nor did mutation of the SP1 site
block cell cycle regulation of the promoter/luciferase construct (data not shown). Nevertheless, deletion of the entire GC stretch region reduced promoter activity by 20%.
The third positive element found between Transcription of the cdc2 gene is controlled by multiple
regulatory regions, including upstream activating sequences and one repressing (CDE/CHR) element. The isolated regulatory region of the
cdc25C promoter contains SP1 and NF-Y binding sites and CDE and CHR
repressor elements (12, 22, 23). The CDE/CHR element of the
cdc25C gene inhibits the transcription activity of the SV40
promoter (23), demonstrating that repressor elements that interfere
with the basal transcription machinery will affect all promoters.
Plk kinase activity is subject to very tight controls during cell cycle
progression. In parallel with increased steady-state message and
protein levels, kinase activity rises gradually from S phase to an
abrupt peak at M phase that is a consequence of activating
phosphorylation on one or more serine residues. At the end of mitosis,
Plk protein levels drop with only slightly slower kinetics than cyclin
B protein (11).2 The present manuscript
demonstrates that cell cycle-specific transcriptional regulation is at
least partly responsible for the tight regulation of Plk kinase
activity. One area of future interest will be to determine whether
specific programs exist to degrade Plk protein at the end of mitosis or
if postmitotic loss of the midbody is sufficient to account for the
decrease in protein levels.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U78073[GenBank].
Laboratory of Leukocyte Biology,
-flanking region. Using
synchronized cultures of HeLa cells transfected with PLK promoter/luciferase constructs, we show that the promoter of
PLK is activated at S phase and is maximal at
G2-M phase. Using various PLK
promoter/luciferase constructs, we show that three activating regions
are located between 35 and 93 base pairs upstream of the transcription
initiation site. We identified a repressor element (CDE/CHR) in the
region of the transcription start site, and mutations within this
element diminished cell cycle regulation of transcription.
region of the human PLK gene and analyzed its functions. Here we show that
PLK expression is transcriptionally regulated in a cell
cycle-dependent manner. This transcriptional regulation is
achieved through interactions between repressor regions and at least
three activating regions.
Cell Culture and Transient DNA Transfections
-terminal probe (100 bp) derived from the human PLK cDNA sequence.
Prehybridization and hybridization were performed as described
previously (6). A PstI fragment of 2 kb and an EcoRI overlapping fragment of 3 kb were subcloned into
pBluescript, and the nucleotide sequence was determined by the dideoxy
termination method (Sequenase; U. S. Biochemical Corp.) using
synthetic primers as described previously (6).
-end of all inserts corresponded to position
+63, 10 bp before the translation initiation site. PCR primers with
SacI and BglII sites artificially installed at 5
and 3
ends, respectively, were used to synthesize inserts that were
subcloned into the pGL2-Basic vector. Amplification of the promoter
sequences was performed using an oligonucleotide primer at the 3
end
of the promoter, +63 (5
-ACCTCTGCAGACCTCGATC),
and one of the following oligonucleotide primers for truncated 5
ends
of the promoter;
982 (5
end at nucleotide
982),
AGCGAGCTCTCTTTTAAA;
815,
5
-AGCAGCCGGGCATGGTG;
615,
5
-AGCTTTGGCTATGACCTG;
477,
5
-AGCTCTGGTACTGTGC;
389,
5
-AGCGGTAGGGCCTG;
295,
5
-AGCGTAAAAGCTTCCTG;
241,
5
-AGCGAGAAAGGGAGAA;
195,
5
-AGCTCTCCGCATCCAC;
165,
5
-AGCAGGCTATCCCACGT;
148,
5
-AGCGGGCGTCCGTGTAA;
115,
5
-AGCGTCCGGGTTTAAAGG;
93,
5
-AGCCGCAGGGCGCTCCA;
75,
5
-AGCTGCCGCGCGGCGG;
65,
5
-AGCCGGGTTTGGATTTTAAT;
45,
5
-AGCCCCCGCGGCCAAT;
35,
5
-AGCCAATCAGTGGCGCGC; and
19,
5
-AGCCGTTCCCAGCGCCGCG. The
nucleotides underlined represent the restriction site
(BglII and SacI). Site-directed mutagenesis of
the SP1, CCAAT, CDE, and CHR sites in the pGL-93PLK-LUC was
carried out by a PCR-based method. The primers to generate specific
mutations in these sites were as follows: mutation in the SP1 site,
5
-GGCGGTCAGTGGCGC (M2) and 5
-
CCGCGGCCAATTGGCGC (M3); CDE site,
5
GTTCCCTCCGCGTTTGAA; and CHR site, 5
-
CGCCGCGTTATTCGGGGGA. The underlined nucleotides represent those which were mutated. The first PCR reactions were carried out between the mutated primer and +63 primer by using pfu
polymerase (Stratagene). The second PCR reactions were carried out
using the
93 primer and the first PCR products using Taq polymerase. Then the PCR products were digested with SacI
and BglII and ligated into pGL2-Basic vector. Mutations were
confirmed by DNA sequencing.
70 °C. The protein concentrations were determined with a protein
assay kit (Bio-Rad).
Induction of PLK mRNA Expression during
G2-M
Fig. 1.
Steady-state PLK mRNA
expression during the cell cycle. A, Northern blot of
PLK mRNA from HeLa cell line after release from double
thymidine block. Total RNA (20 µg) was prepared at indicated times
and hybridized with a PLK probe. The lower panel shows ethidium bromide staining of the same gel to confirm that similar
amounts of RNA were loaded in each lane. B, Northern blot analysis. HeLa cells were treated with nocodazole for 18 h and collected by shake-off. Cells were washed four times and replated with
normal medium. Total RNA (20 µg) was prepared at indicated times and
hybridized with PLK probe. C, percentage of cells
in different stages during the cell synchrony experiment after release of double thymidine block. Flow cytometric analysis of the DNA content
of the cells at indicated times (in hours) after release from the
second thymidine block is shown. Quantitation of the percentage of
cells in different stages of the cell cycle was based on DNA
content.
[View Larger Version of this Image (29K GIF file)]
Flanking
Sequence
upstream
sequences of the human PLK gene. A human fibroblast genomic
library was screened with the human 5
PLK cDNA probe
(100 bp). Two positive clones were analyzed by Southern blotting using
the same probe. These two clones, containing inserts of about 20 kb,
were found to be overlapping (Fig. 2A). An
EcoRI fragment of 3 kb and a PstI fragment of 2 kb were subcloned into the pBluescript vector and sequenced. Fig.
2B shows the sequence of these two overlapping clones and
identifies several structural elements. As shown in Fig. 2B,
we found a 486-bp sequence corresponding to exon 1, containing 408 bp
encoding the N-terminal amino acids of Plk. The 2943-bp PLK sequence had 10 nucleotides different from those
published in a previous report (21). A region upstream of the
transcriptional initiation site contains several sequences similar to
previously identified transcription regulatory elements. There is a
TATA-less promoter and a CCAAT box at
40. Further upstream at
70 is
a GC stretch containing a putative SP1 consensus element. A c-myc binding site is located at
160.
Fig. 2.
Structure of the PLK regulatory
region. A, physical map. PG1 and PG17 indicate isolated
genomic fragments, which hybridized with 5-PLK cDNA.
The EcoRI restriction sites in the DNA fragment are denoted.
The bottom two lines show fragments (2 kb of PstI fragment and 3 kb of EcoRI fragment) that were subcloned
into pBluescript. The solid box indicates EXON 1. The major
transcription initiation site is indicated by an arrow. ATG
is the translation initiation site. E, EcoRI; P,
PstI; Sa, SacI; H, HindIII; A,
AccI; S, SmaI. B, sequence of the 5
upstream region of the human PLK gene. Underlined bases
indicate the potential transcription factor binding sites. The
nucleotides corresponding to transcription initiation sites are
indicated by asterisks as a position +1. The deduced amino
acid sequence is given above the nucleotide sequence. The
italic underlined sequence indicates the first intron.
[View Larger Version of this Image (51K GIF file)]
Fig. 3.
Transcription start site of the human
PLK gene. Primer extension assay was performed using
32P-labeled 17-mer oligonucleotide primer 5-21 bp upstream
from translation initiation sites. Labeling oligonucleotide was
annealed to 5 µg of mRNA from HeLa (RNA) and tRNA
(control). Sequence ladder (lanes G, A, T, and C) was
obtained with the same primer. The +1 on the right shows the
major transcription initiation site.
[View Larger Version of this Image (33K GIF file)]
Upstream Region of PLK
upstream sequences of the PLK gene into a
Luciferase reporter vector, pGL2-Basic (Fig.
4A). The plasmid containing the 5
upstream
sequences was then transfected into HeLa cells using liposome reagent.
To identify functional elements, a series of deletion mutants of the
fragment were examined for transcriptional activity. Fragments deleted
up to nucleotide position
93 exhibited full promoter activity (Fig.
4B). The
65 (pGL-65),
35 (pGL-35), and
19 (pGL-19) deletion
mutants exhibited approximately 40, 4, and 0.5% activity with respect
to
93 (pGL-93). These results indicate that the
93 to +63 fragment
of the PLK promoter has full promoter activity and that
there are at least two positive regulatory regions (
93 to
65 and
65 to
35) in the PLK promoter.
Fig. 4.
Activity of truncated fragments of the
PLK promoter. A, the full-length PLK
promoter (982 to +63) was truncated to generate various fragments and
subcloned into promoterless luciferase vector (pGL2-Basic). The SV40
promoter and enhancer sequences (pGL2-SV40) and promoterless sequence
(pGL2-Basic) were used as positive and negative controls. B,
HeLa cells were transfected with PLK promoter-luciferase
plasmids using Lipofectin. Transfected cells were incubated for 48 h and assayed for luciferase activity. Values were normalized to the
activity of pGL2-Basic.
[View Larger Version of this Image (30K GIF file)]
Fig. 5.
Cell cycle activity of the human
PLK promoter. HeLa cells were transfected with the
indicated plasmids containing the promoter of PLK upstream
of the luciferase reporter gene (pGL2-Basic). 18 h after
transfection, cells were synchronized with thymidine (2 mM)
for 16 h. Cells were released from the block (time, 0 h) and
harvested at the indicated times for luciferase assays. Luciferase values were normalized to protein amounts.
[View Larger Version of this Image (21K GIF file)]
Fig. 6.
Promoter activity of the upstream sequences
of PLK. HeLa cells were transfected with the indicated
PLK-promoter-luciferase plasmids. 18 h after
transfection, cells were treated for 18 h with mimosine to block
in G1, thymidine to block at G1-S, and nocodazole to block in G2-M (prometaphase). Cells were then
harvested and assayed for luciferase activity. Luciferase values were
normalized to protein amounts.
[View Larger Version of this Image (36K GIF file)]
Fig. 7.
Promoter activity of stably transfected cell
lines. In A, HeLa cells were transfected with deletion
plasmids. After selection by geneticin, colonies were examined for
luciferase activity. Six independent cell lines that expressed
luciferase activity were cloned. Open box, c-myc binding
site; solid box, GC stretch (SP1) and CCAAT;
oval, CDE/CHR element. B, Each stable transfectant, HeLa-815(1), HeLa-815(2), HeLa-241(3), HeLa-195(4), HeLa-93(5), and HeLa-93(6), was synchronized at G1-S phase
by double thymidine block. Cells were released (time, 0 h) and
harvested at the indicated times for luciferase and protein assays.
Luciferase values were normalized to the activity at time = 0.
[View Larger Version of this Image (31K GIF file)]
93.
This region includes a GC stretch region containing a consensus SP1
site and a CCAAT box. To identify the sequences within this region
responsible for conferring the cell cycle-regulated transcription of
PLK, we generated a series of truncated or mutated plasmids
spanning the PLK promoter (Fig.
8A). To assess the activity of the GC stretch (SP1) site and CCAAT box sequences, we introduced substitutional mutations in the sequences. GGCGGG was converted to GAATGG (pGL-93, M1), and CCAATCAG was converted to CAGCTCAG or CCAATTCA (pGL-93, M2 and
pGL-93, M3, respectively). These constructs were subcloned into
pGL-Basic and were transfected into HeLa cells. Transfected cells were
then blocked with either mimosine or nocodazole.
Fig. 8.
Deletion and mutation analysis of
PLK gene regulatory sequences. A, diagrammatic
representations of the luciferase reporter gene constructs. The
constructs are named according to the length of the PLK
sequence upstream of the transcription initiation site.
Arrow, the region containing the transcription start site; box, a GC stretch sequence containing the SP1 site;
oval, the CCAAT box; X, mutation sites. The M1
mutation is GGG from the original sequence GGGCGG. The
M2 and M3 mutations CTCTG and CCAAT,
respectively, were made from the original sequence CCAATCAG. B, HeLa cells were transfected with 11 PLK-promoter-Luciferase plasmids and control plasmid
(pGL-Basic). 18 h after transfection, cells were treated with
mimosine to block in G1 and nocodazole to block in
G2-M (prometaphase) for 18 h. Cells were then
harvested and assayed for luciferase activity. Luciferase values were
normalized to protein amount.
[View Larger Version of this Image (21K GIF file)]
93 to
75 and has about 40% of the G1 activity of the pGL-93 construct
(Fig. 8B). Truncation of the SP-1 site in construct pGL-65
resulted in a decrease to about 20% of the G1 activity of
pGL-93, whereas the larger deletion in pGL-45 caused no additional
effect. The second region spanning nucleotides
65 to
75 contains a
portion of the 17-bp GC stretch sequence
(GCCGCGCGGCG) containing a potential SP1 site
(underlined). Mutation of the SP1 site (pGL-93, M1) did not reduce
G1 or G2-M phase promoter activity. These
results suggest that the GC stretch region is involved in
transcriptional activation, but that the putative Sp1 site contained
within it is not.
45 to
33 and has about 30% of
the G1 activity of the pGL-93 construct (Fig.
8B). Taking into account the lack of basal promoter activity
with pGL-35 and pGL-19, or in mutants of pGL-65 (pGL-65, M2 or pGL-65,
M3) which contain the putative CCAAT box, the results suggest that the
CCAAT box binding factor NF-Y is likely important for PLK
promoter activity.
Fig. 9.
Sequence homologies of the CDE/CHR element of
PLK. Alignments of cdc25C, cyclin A, cdc2, cyclin B,
and PLK promoter sequences in the region of the CDE and CHR
elements (12, 29). Core sequences are underlined. The
lower panel shows the consensus sequences described by
Zwicker et al. (12). Numbers indicate the positions relative
to the transcription initiation sites.
[View Larger Version of this Image (22K GIF file)]
Fig. 10.
Effect of mutations within the CDE and CHR
elements on promoter activity. A, diagrammatic
representations of the luciferase reporter gene constructs.
Arrow, the region containing the transcription start site;
box, CDE/CHR elements; X, a mutation site.
B, transient transfection experiments with PLK
promoter-Luciferase construct. Wild-type (pGL-93) and mutant constructs
(pGL-93mCDE and pGL-93mCHR) were transfected into HeLa cells. 18 h
after transfection, cells were treated 18 h with mimosine to block
in G1 and nocodazole to block in G2-M
(prometaphase). Cells were then harvested and assayed for luciferase
activity. C, relative luciferase activity of
G2-M versus G1 cells is given for
the wild-type and mutant constructs. Bars, S.D.
[View Larger Version of this Image (26K GIF file)]
flanking sequences contain
two regions of homology. The largest region extends about 400 bp
upstream of the translation initiation site and includes both a CCAAT
box and an SP1 site. Deletion of the 3
112 bp, including the CCAAT box
and SP1 site from a human promoter/CAT construct, abolished promoter
activity. The second region included 90 bp located 1.8 kb 5
of the
core homology region. Deletion of the 5
-most 500 bp, including the conserved 90 bp, reduced the promoter activity of the full 2.3-kb clone
by 50%. Their studies thus identified two regions that significantly contributed to basal human PLK promoter activity and showed
that these elements are conserved in both the human and mouse
genes.
93 and +65 were sufficient to cause similar 5-6-fold induction
during G2-M, which is comparable to the increases in
steady-state levels of the endogenous human PLK
mRNA.
35 and
95. This
region contains at least three positive regulatory elements, the first
with no known consensus site, the second containing a GC-rich element
that includes an SP1 site, and the third containing the CAATT box.
93 to
75 and has about
40% of the activity of the largest PLK construct. As mentioned above, we did not identify any consensus sequences for known
transcription factors in this region. However, Tommasi and Pfeifer
(18), using genomic footprinting, identified 11 protein binding sites
in the cdc2 promoter. Six of these sites correspond to known regulatory
elements, including an inverted SP1, inverted CCAAT box, and est-2 and
E2F-4 sites. The identities of the other five factors are unknown. We
found that the PLK promoter region between
93 to
75
contains a sequence similar to one of the binding sites for unknown
factors identified in the cdc2 promoter.
45 and
33 contains a CCAAT
box (NF-Y binding site) (25-28). This region of the basal promoter has
approximately 40% of the activity of the largest PLK
construct. (Fig. 4). Mutation of the NF-Y binding site did not abolish
cell cycle-regulated transcription (Fig. 8). Also, we did not observe
cell cycle-regulated binding to the NF-Y oligonucleotide in
electrophoretic mobility shift assay, although we confirmed that NF-Y
does bind to the site by supershift analysis (data not shown).
*
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article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
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To whom correspondence should be addressed. Tel.:
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1
The abbreviations used are: PLK, polo-like
kinase; CDE, cell cycle-dependent element; CHR, cell cycle
gene homology region; bp, base pair(s); kb, kilobase(s); PCR,
polymerase chain reaction.
2
D. K. Ferris, unpublished observation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.