ABSTRACT
The
Drosophila
gene for cyclin A is expressed in dividing cells throughout development. This expression pattern is similar to those of genes related to DNA replication, suggesting
involvement of some common control mechanism(s). In the upstream region (-71 to -64 with respect to the transcription initiation site) of the
CycA
gene, we found a sequence identical to the DNA replication-related element (DRE; 5
'
-TATCGATA), which is important for high level expression of replication-related genes such as those encoding DNA polymerase
[alpha]
and proliferating cell nuclear antigen. Transient expression assays with
chloramphenicol acetyltransferase (CAT) were carried out to examine the
function of the DRE sequence of the
CycA
gene. Deletion or base substitution mutations resulted in an extensive
reduction in CAT expression. Furthermore, monoclonal antibodies against DRE
binding factor (DREF) diminished or supershifted the complex of the DREF and DRE-containing fragment. The results indicate that the
Drosophila
CycA
gene is under the control of a DRE/DREF system, as are DNA replication-related genes.
It is now well established in eukaryotes that a number of CDK/cyclin complexes
play major roles in cell cycle progression. Cyclin A is first expressed at the
G
1
-S transition and is required for entry into the S and M phases. Therefore, it
may be involved in the regulation of DNA replication (
1
,
2
) and also transcriptional control during S phase (
3
,
4
). Cyclin A is found in dividing cells throughout development of
Drosophila melanogaster
(
5
) and its constitutive expression has been associated with tumorigenesis (
6
,
7
), while, inversely, abolition of its expression was found to cause growth
arrest of cells (
8
). Thus, the expression profile of the gene encoding cyclin A is similar to
those of other proliferation-related genes, such as genes involved in DNA replication.
Eukaryotic genes encoding proteins involved in DNA replication appear to be coordinately expressed in response to signals for cell growth
and/or cell cycle progression. Furthermore, common transcription regulatory mechanisms have been found to function in expression of
various DNA replication-related genes. For example, replication-related genes of budding yeast are expressed depending on cell cycle progression and a common sequence (
Mlu
I cell cycle box) present in promoter regions of these genes and the specific
binding factor DSC1, the complex consisting of products encoded by the
SWI
6 and
MBP
1 genes, are known to be required for their transient expression at the G
1
-S boundary (
9
,
10
).
In mammalian cells, the transcription factor E2F binds to the E2F recognition site (5'-TTTCGCGC) and regulates transcription of a group of genes whose products are necessary for cell
proliferation (
11
,
12
). This includes the genes encoding DNA polymerase [alpha], dihydroforate reductase, thymidine kinase, c-Myc, c-Myb, Cdc2, proliferating cell nuclear antigen (PCNA) and also
cyclin A (
13
-
17
).
We have isolated
Drosophila
genes for the 180 kDa catalytic polypeptide (
18
) and 73 kDa subunit polypeptide (
19
) of DNA polymerase [alpha] as well as PCNA (
20
). The promoters of these genes contain regions featuring a common 8 bp
palindromic sequence (5'-TATCGATA), named the DNA replication-related element (DRE) (
21
). The DRE requirements for promoter activation have been confirmed in both
cultured cells (
21
) and transgenic flies (
22
). Furthermore, we found a specific DRE binding factor (DREF) consisting of an
80 kDa polypeptide homodimer (
21
), whose cDNA has recently been cloned (
23
).
Involvement of DRE/DREF in regulation of a considerable variety of genes has
been suggested by the results of DNA database searches (
24
). It is, therefore, of interest to determine whether the DRE/DREF system is also utilized in the transcription of cell proliferation-related genes other than those directly relevant to DNA replication. To
answer this question, we decided to study genes with a role in the cell cycle,
because these, like their DNA replication-related counterparts, are expressed dependent on proliferation status.
Since the mammalian genes for cyclin A and DNA replication enzymes are commonly
controlled by E2F, as mentioned above, we have focused on this gene. A cDNA and
the gene for
Drosophila
cyclin A have been cloned and sequenced (
5
,
25
). We found a sequence identical to DRE in the region of nucleotide positions -71 to -64 with respect to the transcription initiation site and have
examined its role in promoter activity.The obtained results indicate that the
Drosophila
CycA
gene is indeed under the control of the DRE/DREF system, like DNA replication- related genes.
Kc cells derived from
D.melanogaster
embryos were grown at 25oC in M(3)BF medium supplemented with 2% fetal calf serum in the presence of
5% CO
2
(
26
).
To obtain a fragment containing the promoter of the
CycA
gene (nucleotide positions -260 to +12 with respect to the transcription initiation site) by the polymerase chain reaction (PCR), the following
primers were chemically synthesized: 5'-ACACTCGAGAAGCTTAGAACTAAATAAATATGCAC-3' (containing the region between -260 and -234 and the
Xho
I site); 5'-TTCCCGCGGTAAAAGCAATTGCTGGCTCTTTTTGA-3' (containing the region between -14 and +12 and the
Sac
II site).
The sequences of double-stranded 30 bp oligonucleotides containing the DRE sequence or its base-substituted derivatives in the
CycA
gene promoter were defined as follows:
DRE-CA,
5'-gatccACGACCTATCGATAGCTGGAa-3'
3'-gTGCTGGATAGCTATCGACCTtctag-5';
DRE-CAmut
5'-gatccACGACCTAT
where mutated bases are underlined and lower case letters indicate the linker sequence.
The double-stranded 30 bp oligonucleotide DRE-P contains the 24 bp DRE-containing sequence of the PCNA gene promoter and the 6 bp
linker sequence, while DRE-PM contains a 2 bp substitution in the DRE sequence of DRE-P (
21
).
To construct the plasmid used for the CAT transient expression assay, a DNA
fragment containing the upstream region from position -260 to position +12 of the
CycA
gene was obtained by PCR using
Drosophila
Canton S genomic DNA as a template and the above-defined primer set, digested with
Sal
I and
Sac
II and then placed between the
Sal
I and
Sac
II sites of plasmid pSKCAT (
27
). The resultant plasmid was named p-260DCYCACAT. A set of 5'-end deletion derivatives of plasmid p-260DCYCACAT were constructed by digestion with
Escherichia coli
exonuclease III and S1 nuclease, as described earlier (
28
). Deletion break points of these derivatives were determined by nucleotide
sequencing.
To construct the plasmids p-260DCYCACATmutI and p-260DCYCACATmutII containing mutations in the DRE sequence, p-260DCYCACAT was digested at the center of the DRE sequence
with
Cla
I and then blunt-ended using T4 DNA polymerase, followed by self-ligation using T4 DNA ligase. After this treatment, p-260DCYCACATmutI had an unexpected additional 1 bp at the
center of the DRE sequence (TATC
Kc cells (2 * 10
6
cells/dish) were grown in 60 mm plastic dishes for 24 h and co-transfected with 2 [mu]g
CycA
promoter-CAT plasmid as the reporter and 100 ng luciferase plasmid as an internal
control by the calcium phosphate co-precipitation method, as described earlier (
29
). Cells were harvested 48 h after DNA transfection and cell extracts for
determination of CAT activities were prepared as previously reported (
30
). Radioactivities of spots corresponding to acetylated [
14
C]chloramphenicols were quantified with an imaging analizer BAS2000 (Fuji Film).
The luciferase assay was carried out using a PicaGene assay Kit (Toyo Inc Co.)
following a documented protocol (
31
). All assays were performed within the range of concentrations showing a linear
relation of activity to incubation time and protein amount. CAT activitiy was
normalized to the luciferase activitiy.
The gel mobility shift analysis was performed as reported previously (
21
), with minor modifications. Kc cell nuclear extract and
E.coli
lysate containing GST-DREF(16-608) fusion protein were prepared as described elsewhere (
23
). These were then added to a reaction mixture containing 15 mM HEPES, pH 7.6,
60 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 12% glycerol, 0.5 [mu]g poly(dI-dC), 0.5 [mu]g sonicated calf thymus DNA (average size 0.2 kb) and double-stranded
32
P-labeled synthetic oligonucleotides (10 000 c.p.m.) and incubated for 15 min on ice. When necessary,
unlabeled DNA fragments were added as competitors at this step. DNA-protein complexes were electrophoretically resolved on 4% polyacrylamide gels in 50 mM Tris-borate, pH 8.3, 1 mM EDTA and 2.5% glycerol at 25oC. Gels were dried and autoradiographed.
The gel shift assay was also performed with anti-DREF monoclonal antibody no.1, anti-DREF monoclonal antibody no.4 (
23
) and anti-chick DNA polymerase [alpha] monoclonal antibody 2-4D (
32
), as a control. Kc cell nuclear extract was mixed with each antibody, incubated
for 2 h on ice, added to mixtures containing
32
P-labeled synthetic oligonucleotides (10 000 c.p.m.) and 0.5 [mu]g poly(dI-dC) and then incubated for 15 min on ice as decribed above.
The presence of a single transcription start site in the
CycA
gene has been determined previously using mRNA from fly bodies at various
developmental stages (
25
). In the upstream region (nucleotide positions -71 to -64 with respect to the transcription initiation site) of the
CycA
gene, we found a sequence identical to the DRE (5'-TATCGATA) (Fig.
1
), which is important for the regulation of DNA replication-related genes, such as those encoding DNA polymerase [alpha] and PCNA (
21
).
The genomic sequence corresponding to the -260 to +12 nucleotide positions was amplified by PCR and placed adjacent
to and upstream of the CAT gene to construct plasmid p-260DCYCACAT. Transient CAT assays with this plasmid and its 5'-end deletion derivatives were carried out to determine which
sequences are important for promoter activity (Fig.
1
). An extensive reduction in CAT activity was observed when deletions were
extended from position -120 to -92. The deleted region contains a TATA-like sequence (
25
), although we have not determined that this sequence itself is essential for
promoter activation. A further reduction in CAT activity was observed when a
deletion extended from position -82 to -63. This region contains the 8 bp palindromic sequence which is
identical to DRE (5'-TATCGATA). Since this sequence is important for promoter activation
and DREF binding, as described later, it was given the name
CycA
DRE.
To investigate the requirement for the
CycA
DRE for activation of the promoter of the
CycA
gene, we introduced 1 and 2 bp insertional mutations at the center of this
sequence and carried out CAT assays. The mutations resulted in extensive
reductions in CAT activity (Fig.
2
), indicating the necessity for an intact sequence for promoter activation.
We are grateful to Drs M. Yamaguchi for discussion and encouragements and M. Moor for critical reading of the manuscript. This work was supported in part by grant-in-aid from the Ministry of Education, Science and Culture, Japan.
REFERENCES
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