(Received for publication, August 14, 1995)
From the
Cyclin B1 mRNA expression varies through the cell cycle with its
peak in G/M. In cycling mammalian cells, its lowest level
is in G
with a steady increase in S until a level 50-fold
greater than that in G
is reached. In order to characterize
the transcriptional component to this variation in expression, we
cloned the upstream region 872 base pairs upstream from the start site
of the cyclin B1 gene and have demonstrated that it confers cell
cycle-dependent regulation onto two reporter genes, both
chloramphenicol acetyltransferase and luciferase. Its activity was
25-fold greater in G
/M than in G
in HeLa cells
with intermediate activity in S. This cyclical activity could be seen
with sequences encompassing only 90 base pairs upstream from the start
site. Protein binding to this region was demonstrated using
electrophoretic mobility shift assays, and the binding profiles
appeared to vary depending upon the phase of the cycle in which the
extracts are made. Thus, transcriptional control plays an important
role in determining cyclin B1 mRNA levels, and cell cycle-dependent
activity is regulated through interactions with the region 90 bases
upstream from the start site.
Cyclin B1 expression is required for the activation of mitosis
or maturation promoting factor. This activation, which triggers the
progression into mitosis is dependent both upon the binding of Cdk-1
(synonymous with p34) to cyclin B1 as well as a
series of phosphorylation and dephosphorylation
events(1, 2, 3, 4, 5) .
Cyclin B1 was first identified as a protein whose level oscillated
during the sea urchin oocyte cell cycle(6) . Subsequently, it
was shown that cyclin B1 was expressed ubiquitously in all eukaryotes
examined and that the level of cyclin B1 was minimal in G
,
began to rise in S phase, and peaked at the G
/M
transition(5, 7) . The increase in cyclin B1 protein
in somatic cycling mammalian cells is mirrored by oscillations in the
expression of the cyclin B1 mRNA which increases by over 50-fold as
cells progress through G
and S into G
/M (7, 8, 9) . Thus, the increase in cyclin B1
protein that is required to activate maturation promoting factor is
likely to depend upon its mRNA level.
The importance of the
fluctuations of cyclin B1 mRNA in controlling the cell cycle raises the
question of how cyclin B1 mRNA level is regulated. Understanding cyclin
B1 mRNA regulation could be important in understanding the mechanisms
leading to cell cycle control in G/M. Both transcriptional
and post-transcriptional controls are likely to be involved. Pines and
Hunter(7) , using nuclear run-on assays, suggested that the
transcription of cyclin B1 was increased in cells synchronized by
thymidine-aphidicolin treatment in G
/M as compared to cells
in G
. Maity et al.(9) showed that the
stability of cyclin B1 mRNA also fluctuated through the cell cycle with
a half-life of 1-2 h in G
, 8 h in S, and 13 h in
G
/M. While changes in the cyclin B1 mRNA stability are
clearly significant, they alone are not of sufficient magnitude to
account for the variation in level of 50-fold that is seen in many
cycling cells. To further investigate the role of transcription in the
cell cycle regulation of the human cyclin B1 gene, we have cloned its
5` upstream regulatory region and in this report describe its cell
cycle-regulated promoter activity. We have also defined a short region
of the promoter that can confer cell cycle-regulated transcription and
that shows variation in protein binding through the cell cycle as
evaluated by electrophoretic mobility shift assays.
Figure 1: Sequence of the 5` upstream region of the human cyclin B1 gene. Underlined bases indicate the CAAT box and TATA-like motif. Underlined bases also show the start site identified by Pines and Hunter (7) and the first methionine of the open reading frame.
Figure 2:
Cell cycle position following arrest with
mimosine, thymidine, or nocodazole. Representative flow cytometric
profiles of cell cycle position as determined by DNA content after
treatment with the following. A, 24-h treatment with 400
µM mimosine arrests 65-75% of the cells in late
G; B, 16-h treatment with 2 mM thymidine
arrests 55-65% of the cells in S phase; C, 24-h
treatment with 0.04 µg/ml nocodazole arrests 85-90% of the
cells in G
/M.
Figure 3:
Promoter activity of the upstream
sequences of cyclin B1. HeLa cells were transfected with one of the
following CAT plasmids: the SV40 promoter and enhancer sequences (pCAT-CONTROL), no promoter or enhancer sequences (pCAT-BASIC), or the 949-bp upstream region of cyclin B1
(pCAT-upcB) upstream from CAT. A -galactosidase plasmid
(pSV
-gal) was included in the transfection as an internal control
for transfection efficiency. 18 h after transfection, the cells were
treated with either mimosine to block in G
, thymidine to
block in S, or nocodazole to block in G
/M. Cells were then
harvested and assayed for CAT and
-galactosidase activity. After
normalization to
-galactosidase values, the ratios of CAT activity
in different phases of the cell cycle were the same as the ratios when
comparing absolute CAT activity levels. CAT activity shown here has not
been normalized.
To be sure that the cell cycle differences
we observed were specific for the cyclin B1 upstream sequences and not
an artifact of the experimental assay, we tested several other
promoters in the same system. We transfected plasmids containing the
human DNA polymerase , the human cyclin D1, the human thymidine
kinase, or the hamster histone H3.2 promoters (14, 15) upstream of the CAT reporter gene into HeLa
cells and then treated the cells with either nocodazole, mimosine, or
thymidine. Each of these promoters has been characterized as being
specific for S phase(14, 15) . The human DNA
polymerase
and the human cyclin D1 promoters have both been
characterized as being dependent upon E2F
activation(14, 21, 22, 23, 24) .
Since HeLa cells are effectively deficient for the retinoblastoma
protein (pRb) due to the binding of pRb to the E7 protein of human
papilloma virus(25) , and the regulation of E2F transactivation
is affected by its interaction with pRb, these cells may not regulate
E2F-activated genes in the same way as cells having normal pRb
activity(26) . Cells deficient in pRb, for example, bypass the
requirement for cyclin D1 in G
(26) . In evaluating
these experiments, it should also be noted that the CAT protein has a
half-life of about 50 h (27) . The long half-life can affect
the interpretation of experiments because once a cell has synthesized
the CAT protein, CAT activity would be detectable for the duration of
the experiment. Since the majority of the cells in an exponentially
growing population of HeLa cells are in either G
or S
phase, the cells in S would lead to considerable synthesis of the CAT
protein by an S phase-specific promoter. The CAT protein because of its
long half-life would remain detectable as cells progressed into
G
. In contrast, few cells would be in G
/M and
spend little time at that point since it is the shortest component of
the HeLa cell cycle. Thus, this method should clearly distinguish
transcription in G
/M from that in G
or S, but
would not be expected to clearly separate transcription in G
from S phase.
After transient transfection and cell cycle
blockade, the S phase-specific promoters showed 1.3-3.4 times
more CAT activity in S phase than in G and slightly more
CAT activity in S than in G
/M, as shown in Fig. 4.
In contrast, however, the cyclin B1 upstream region vector had markedly
higher CAT activity in G
/M compared to other phases of the
cell cycle. Thus, it is unlikely that the cell cycle-regulated activity
of the cyclin B1 promoter is an artifact of our experimental assay or
of the drugs used to induce blockage in the cell cycle. These
experiments indicate that the upstream region of the human cyclin B1
gene has promoter activity that is low in G
, higher in S,
and maximal at G
/M.
Figure 4:
Promoter activity of S phase-specific
promoters. HeLa cells were transfected with S phase-specific
promoter-CAT plasmids (human DNA polymerase , human cyclin D1,
human thymidine kinase, or hamster histone H3.2) and treated with
either mimosine to block in G
, thymidine to block in S, or
nocodazole to block in G
/M. Cells were then harvested and
assayed for CAT activity. Cotransfection with pSV
-gal indicated
equivalent transfection efficiency in all
samples.
To further confirm the cell
cycle-regulated activity of the cyclin B1 promoter, we subcloned the
cyclin B1 promoter upstream of the luciferase reporter gene in the
pGL2-BASIC vector (pGL2-upcB). While the CAT mRNA has a short
half-life, its protein has a long half-life of 50 h. In contrast, both
the luciferase mRNA and protein have short half-lives. The half-life of
the luciferase protein is approximately 3 h(27) . Thus, we
could transfect a luciferase expression vector driven by the cyclin B1
promoter, follow the cells as they cycled, and assess the activity of
the cyclin B1 promoter throughout the cell cycle by monitoring
luciferase activity. These experiments did not depend upon
pharmacological blockade of the cell cycle. HeLa cells were
cotransfected with pGL2-upcB and pSV-gal (as an internal control
for transfection efficiency) and treated with aphidicolin for 16 h.
After the cells were released from the aphidicolin block, cells were
harvested every 3 h for a 24-h period. Cell lysates were assayed for
luciferase and
-galactosidase activity and by flow cytometry for
DNA content. As shown in Fig. 5A, the cells were
predominantly in G
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. Luciferase activity peaked (at 9 times
greater than the level at the time of release) 10 h after release when
the majority of cells were in G
/M. Once the cells exited
mitosis and re-entered G
, luciferase activity began to
drop. At 21 h after release, only 11% of the cells remained in
G
/M while 62% were in G
. At this point, the
luciferase activity had fallen to a level only twice that of the level
at the time of release from the aphidicolin block. This decline in
luciferase activity was not a result simply of activity declining with
time after transfection, but was a result of cell cycle progression.
This can be seen from the experiment shown in Fig. 5B.
Some of the transfected synchronized cells from the experiment shown in Fig. 5A were also treated with nocodazole. By blocking
the cells in G
/M and following them with time, we could
assess the effect that increasing time after transfection had on the
luciferase activity. As shown in Fig. 5B, nocodazole
blocked the cells in G
/M, yet luciferase activity remained
high throughout the remainder of the experiment. Thus, the cyclin B1
promoter conferred cell cycle variation to the expression of the
luciferase reporter gene. It resulted in the lowest level of luciferase
activity in G
, an increasing level in S phase, and the
maximal level in G
/M corresponding to a pattern paralleling
the level of cyclin B1 mRNA in cycling cells.
Figure 5:
Cyclical activity of the cyclin B1
promoter. A, HeLa cells were co-transfected with a plasmid
containing the promoter of cyclin B1 upstream of the luciferase
reporter gene (pGL2-upcB) and a -galactosidase plasmid
(pSV
-gal) as an internal control for transfection efficiency.
Cells were then synchronized with aphidicolin for 16 h, released, and
harvested for luciferase and
-galactosidase activity and flow
cytometry every 3 h for a 24-h period. To control for transfection
efficiency, luciferase values were normalized to
-galactosidase
values. B, some of the transfected synchronized cells were
treated with nocodazole to keep them arrested in G
/M. Cells
were then harvested for luciferase and
-galactosidase activity and
flow cytometry.
Figure 6:
Promoter activity of truncations of the
cyclin B1 promoter. The full-length cyclin B1 promoter(-872) was
truncated to generate fragments of 207, 140, and 90 bp upstream of the
transcriptional start site and subcloned into a promoterless CAT vector
(pCAT-BASIC). HeLa cells were transfected with these truncated cyclin
B1 promoter-CAT plasmids and treated with nocodazole to block in
G/M or mimosine to block in G
. Cells were then
harvested and assayed for CAT activity indicated as counts/min.
Figure 7:
Electrophoretic mobility shift assays
using the minimal cyclin B1 promoter. Electrophoretic mobility shift
assays were performed using double-stranded oligonucleotides
corresponding to the cyclin B1 sequence from -90 to +1 as
the radiolabeled probe. Nuclear extracts were prepared from HeLa cells
that were blocked in late G with mimosine or in
G
/M with nocodazole. HeLa cells were synchronized in early
G
by a double block of thymidine and
aphidicolin(35, 36) . After release from the final
block, the cells were cultured until they had progressed through the
cell cycle into early G
(2-3 h after completion of
mitosis)(9) . These cells were used in the preparation of the
early G
extracts. Extracts prepared from cells in these
different phases of the cell cycle were incubated with the probe as
described under ``Materials and Methods.'' Arrows indicate a faint, but reproducible band seen in assays from late
but not early G
cells.
In this study, we have described the isolation of the 5`
upstream sequences of the human cyclin B1 gene, and, using two
different experimental assay systems, we have shown that these
sequences show promoter activity that is strongly cell cycle-regulated.
Its activity was lowest in G, rose to an intermediate level
in S phase, and reached a peak in G
/M. This activity
parallels the pattern of expression of the cyclin B1 mRNA. We found
that the cyclin B1 promoter was 11-26 times more active in
G
/M than in G
. Previous studies have shown that
the level of cyclin B1 mRNA present during G
/M is up to
50-fold greater than the amount of mRNA present in G
. Data
from our laboratory have shown that variations in cyclin B1 mRNA
stability during the cell cycle contribute to the vast differences in
the cyclin B1 mRNA level(9) . The half-life of the cyclin B1
message is 13 h in G
and 2 h in G
. The cyclical
activity of the cyclin B1 promoter, along with the differences in the
cyclin B1 half-life, suggest that both changes in the rate of
transcription and changes in mRNA stability are responsible for the
cell cycle-regulated expression of this gene.
Piaggio et al.(11) have recently cloned the upstream region of the human
cyclin B1 gene. They found that the upstream region had promoter
activity that was minimal in quiescent or G cells, but
increased in activity gradually after serum stimulation. However, they
were unable to show any cell cycle-specific regulation of activity. In
their experiments, the activity of the cyclin B1 promoter did not fall
following mitosis and entry into G
. This may have been due
to their use of chloramphenicol acetyltransferase (CAT) as their
reporter gene, since CAT has a half-life that is considerably longer
than the cell cycle time of about 24 h in the cells they used. High
levels of CAT protein present during mitosis would still be present
once cells cycled back into G
.
Cogswell et al.(12) have also reported the isolation of a cyclin B1
promoter element using the upstream sequences from the cyclin B1 gene.
They found that the activity of the cyclin B1 promoter was enhanced in vivo during G/M as compared to S. Their
experiments also use CAT as the reporter gene and hence do not show
cyclical activity of the cyclin B1 promoter. In this study, Cogswell et al.(12) identified a region as being important for
cell cycle regulation that is distal to the -90 region that we
studied. This region contained a binding site for the upstream
stimulatory factor and is required for in vitro transcription
of the cyclin B1 gene. Using an electrophoretic mobility shift assay,
they were able to identify upstream stimulatory factor binding to this
region. This binding was greater in extracts from G
/M phase
cells than in S phase cells, suggesting that upstream stimulatory
factor stimulates transcription of cyclin B1 during G
/M.
This is in contrast to our finding that suggests the presence of
inhibitory protein(s) that repress cyclin B1 transcription during
G
. Together these experiments suggest that regulation of
the cyclical activity of the cyclin B1 promoter may be controlled by
several regions and that their action may be redundant.
The
isolation of the cyclin B1 promoter and its further characterization
will provide us with a better understanding of how the cyclin B1 gene
is regulated and thus how transcription may be triggered in a cell
cycle-specific fashion. The cdc25C promoter has been
identified as being specific for G/M
transcription(28) . Cdc25C dephosphorylates the inhibitory
phosphorylations on p34
and is required for
activation of maturation promoting factor and for transition into M and
G
(3, 5) . Its mRNA cycles with the highest
levels in G
/M and the lowest in G
, yet in spite
of the cyclical expression of the mRNA, the protein itself appears to
be present in constant amounts through the cell cycle making the
physiological significance of the cell cycle regulation of cdc25C mRNA difficult to understand(29, 30) . The cdc25C promoter lacks a canonical TATA element(28) .
An element termed the cycle-dependent element was identified to act as
a cis-acting repressor element that is responsible for the low
level of expression of cdc25C during G
and
G
. This cycle-dependent element contains the sequence GGCGG
that when mutated resulted in increased expression of the promoter in
G
. The cyclin B1 gene contains in the 5`-untranslated
region of its mRNA a GGCGG sequence. However, the adjacent bases are
not homologous to the cycle-dependent element of cdc25C.
Furthermore, the GGCGG sequence that was identified as important in the
cycle-dependent element is located downstream of the transcriptional
start site of cyclin B1 making it less likely to play a role in the
regulation of cyclin B1, but not excluding that possibility.
The
finding of promoters that are actively transcribed during
G/M raises another interesting question with respect to
cell cycle-dependent transcription. Generally, transcription is turned
off during mitosis at which point the chromosomes are
condensed(31, 32) . Since the cyclin B1 gene is
maximally active during this phase of the cell cycle, there must be
novel mechanisms that allow transcription of this gene to occur under
conditions in which transcription generally is repressed. Further study
of this promoter should reveal important insights into transcriptional
control during chromosome condensation and perhaps will reveal regions
protected from chromosome condensation.
Currently, the B-type
cyclins are a well-characterized class of proteins, but the mechanisms
underlying their transcriptional regulation are poorly understood.
Although genes have been identified in yeast that regulate the
transcription of cyclin B1, their mammalian homologs are
unknown(33, 34) . A trans-acting factor that
acts positively during the G/M transition or negatively
during G
has yet to be identified in mammalian cells. The
existence of such proteins is likely given that the cyclin B1 promoter
is maximally active during G
/M and given the differential
patterns of gel retardation found through the cell cycle using portions
of the cyclin B1 promoter.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U36838[GenBank].