(Received for publication, September 11, 1995; and in revised form, December 4, 1995)
From the
DREF, a transcription regulatory factor which specifically binds
to the promoter-activating element DRE (DNA replication-related
element) of DNA replication-related genes, was purified to homogeneity
from nuclear extracts of Drosophila Kc cells. cDNA for DREF
was isolated with the reverse-transcriptase polymerase chain reaction
method using primers synthesized on the basis of partial amino acid
sequences and following screening of cDNA libraries. Deduced from the
nucleotide sequences of cDNA, DREF is a polypeptide of 701 amino acid
residues with a molecular weight of 80,096, which contains three
characteristic regions, rich in basic amino acids, proline, and acidic
amino acids, respectively. Deletion analysis of bacterially expressed
DREF fused with glutathione S-transferase (GST-DREF) indicated
that a part of the N-terminal basic amino acid region (16-115
amino acids) is responsible for the specific binding to DRE. A
polyclonal and four monoclonal antibodies were raised against the
GST-DREF fusion protein. The antibodies inhibited specifically the
transcription of DNA polymerase promoter in vitro.
Cotransfection experiments using Kc cells demonstrated that
overproduction of DREF protein overcomes the repression of the
proliferating cell nuclear antigen gene promoter by the zerknüllt gene product. These results confirmed
that DREF is a trans-activating factor for DNA replication-related
genes. Immunocytochemical analysis demonstrated the presence of DREF
polypeptide in nuclei after the eighth nuclear division cycle,
suggesting that nuclear accumulation of DREF is important for the
coordinate zygotic expression of DNA replication-related genes carrying
DRE sequences.
A number of enzymes involved in DNA replication have been suggested to form an enzyme complex for this purpose(1) . Genes for these enzymes are expressed in proliferating cells and repressed in quiescent cells reaching confluency or in association with cellular differentiation(2) . Therefore, it is of interest to clarify the genetic mechanisms governing the coordinate induction or repression of DNA replication-related genes in relation to growth or differentiation signals.
In budding yeast, the MluI cell cycle box and the specific binding factor, DSC-1, are responsible for cell cycle-dependent transcription of a number of DNA replication-related genes(3, 4, 5, 6) .
The mRNAs for
human DNA polymerase , PCNA, (
)murine DNA polymerase
-primase complex, and thymidylate synthetase are present
throughout the cell cycle and increase slightly prior to the S
phase(7, 8, 9, 10) . The critical
promoter regions of DNA polymerase
, dihydrofolate reductase
(DHFR), and thymidine kinase genes contain binding sites for the E2F
family (11, 12, 13) . Mutagenesis of the
promoter of the DHFR gene has provided strong evidence that the E2F
element is responsible for the promoter activation in late
G
(14) . Furthermore, the E2F-binding site was shown
to be involved in activation of the DHFR and thymidine kinase genes
following serum stimulation(15, 16) . The fact that
the active form of E2F accumulates in late G
toward the S
phase (17, 18, 19) provides further evidence
that E2F family members are likely candidates for involvement in
transcriptional regulation of specific G
/S-phase-activated
genes that are required for DNA replication. The E2F binding sites of
the Drosophila genes for DNA polymerase
and PCNA are
also important for their proliferation-related
expression(20, 21) . However, little is known about
regulation mechanisms of DNA replication-related genes during the
transition from G
into the cell cycle or the transition
from proliferating to quiescent states.
We have analyzed upstream
regulatory regions of Drosophila genes for the DNA polymerase
180-kDa catalytic subunit (22) and the PCNA(23) ,
and found a novel transcription regulatory sequence consisting of an
8-bp palindromic sequence (5`-TATCGATA), called DRE (DNA
replication-related element) and a specific binding factor, DREF
(DRE-binding factor)(24) . Three DREs and one DRE are present
in the DNA polymerase
and PCNA genes, respectively. Transient CAT
expression and gel mobility shift assay using cultured Kc cells
indicated that DRE stimulates the promoter activities and that DREF can
bind specifically to DRE(24) .
Another aspect is that promoters of Drosophila DNA replication-related genes are repressed by the product of the zerknüllt (zen) gene, a homeobox gene which regulates the differentiation of the optic lobe and the amnioserosa in the dorsal region of the Drosophila embryo(25, 26, 27) . Repression of promoter activities by Zen protein has been observed not only in cultured Kc cells but also in transgenic flies carrying the PCNA gene promoter-directed lacZ gene(28, 29) . Recently we obtained evidence indicating that overexpression of Zen protein results in reduction of DREF activities in the cell(29) . Therefore, DREF may be one of the key transcription regulatory factors involved in proliferation- and differentiation-related control of DNA replication genes.
We earlier purified DREF as a homodimer of an approximately 86-kDa polypeptide to near homogeneity(24) . In the present study, we isolated cDNA for DREF and demonstrated the involvement of DREF in the high level of expression of DNA replication-related genes with DRE sequences.
To amplify a sequence containing a complete open reading frame, a set of PCR primers with a BamHI site was synthesized: 5`-specific oligonucleotide, 5`-ACAGGATCCAAGATGAGCGAAGGGGTACCA, and 3`-specific oligonucleotide, 5`-ATCCTAATTGTTGTGATGATGCT, where sequences with underlining indicate the BamHI site, translation initiation codon, and stop codon of the DREF cDNA, in that order.
Full-length cDNA clones for DREF
were isolated by screening two kinds of gt10 cDNA libraries
constructed from mRNAs obtained from 0-3-h-old and
3-12-h-old Drosophila embryos under high stringency
conditions using a
P-labeled 1.8-kb cDNA fragment excised
from pDCDREF1.8 as a probe. The obtained cDNA clones (2.7 kb) had a
single EcoRI site and, therefore, two EcoRI fragments
of 1.0 and 1.7 kb were excised from
gt10 DNA. Each EcoRI
fragment was subcloned into the EcoRI site of pBluescript II
SK(-) (pDCDREF1.0 and pDCDREF1.7) and sequenced.
A cDNA
containing a complete open reading frame without 5`- and
3`-untranslated sequences was obtained with 30 cycles of PCR (1 min at
94 °C, 1 min at 55 °C, and 2 min at 72 °C) using 10 ng of
DNA of gt10 clone carrying full-length cDNA as a template and 5`-
and 3`-specific oligonucleotides as primers. The resultant DNA fragment
was digested with BamHI and subcloned into BamHI-SmaI sites of pBluescript II SK(-)
(pDCDREF2.2).
We produced recombinant DREF fused to GST. A construct containing the full-length DREF (amino acids 1-701; pGST-DREF1-701) was created by inserting a 2.2-kb cDNA fragment from pDCDREF2.2 with BamHI and EcoRV into BamHI and SmaI sites of pGEX-2T. A construct containing amino acid residues 16-608 (pGST-DREF16-608) was obtained by excising a 1.8-kb cDNA fragment from pDCDREF1.8 with EcoRV and BamHI, filling the protruding-end with Klenow fragment and inserting it into the SmaI site of pGEX-3X. This expression plasmid was used for large scale preparation of the DREF polypeptide, which was then applied as an antigen to raise antibodies. Extraction and purification of recombinant protein were carried out as described previously(35) .
Constructs
containing the N-terminal basic region (amino acids 16-242;
pGST-DREF16-242) and C-terminal acidic region (amino acid
240-608; pGST-DREF240-608) were prepared by partially
digesting pGST-DREF16-608 with EcoRI and religating the
plasmid DNA with T4 DNA ligase. cDNAs of the other GST-DREF mutants
(see Fig. 2) were amplified by PCR with EcoRI-BamHI sites and inserted into pGEX-2T. All
constructs were sequenced. DREF-fusion proteins were produced in E.
coli essentially as described earlier(36) . Lysates of
cells were prepared by sonication in buffer D containing 0.6 M KCl, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml
each of pepstatin, leupeptin, and aprotinin. Lysates were clarified by
centrifugation at 12,000 g for 20 min at 4 °C, and
used for analysis by SDS-polyacrylamide gel electrophoresis, gel
mobility shift assay, or footprinting.
Figure 2:
DRE
binding of deletion mutants of GST-DREF. A, expression of
deletion mutants of the GST-DREF fusion protein. E. coli carrying plasmids expressing deleted DREF fused to GST were
inoculated in 2 ml of LB medium containing 50 µg/ml ampicillin, and
synthesis of GST-DREF was induced at 37 °C for 1 h by adding 1
mM -D-isopropyl-thiogalactopyranoside. E.
coli were pelleted by centrifugation from 100 µl of culture
medium, resuspended in SDS sample buffer, boiled for 3 min and
subjected to electrophoresis on an 8% SDS-polyacrylamide gel. B, DRE binding activities of deletion mutants of GST-DREF
fusion protein. E. coli lysates expressing GST-DREF were
prepared as described under ``Experimental Procedures.'' A
gel mobility shift assay with
P-labeled DRE-P
oligonucleotide (DRE-containing sequence from the PCNA gene) (24) was performed using Kc cell nuclear extract (Kc),
lysates of E. coli containing GST-DREF fusion proteins and E. coli lysate containing GST (GST).
Figure 1: cDNA and schematic structure of DREF. A, nucleotide sequence and the deduced amino acid sequence for DREF cDNA. The open reading frame with initiation and stop codons, at nucleotide numbers 571 and 2683, respectively, encodes a protein of 701 amino acid residues (shown in single letter code below the nucleotide sequence). Peptide sequences determined by microsequencing after lysyl-endopeptidase digestion of DREF are underlined. A putative polyadenylation signal is shown by bold letters. B, schematic structure of DREF. The region rich in basic amino acids (61-203 amino acids) and the region rich in acidic amino acids (218-390 amino acids) are shown by the hatched and shaded boxes, respectively. The proline-rich region (99-217 amino acids) is also indicated. NLS represents a putative nuclear localizing sequence (143-151 amino acids; RRRRTPPRK). The amino acids essential for DRE-binding (16-105 amino acids) are also indicated by a bracket.
Reverse-transcriptase-PCR
with all possible combinations of degenerated primers deduced from
amino acid sequences was performed to isolate cDNA fragments. PCR
products of 1.7 and 1.8 kb were obtained with primer III in combination
with primers VR and IIR, respectively. The 1.8-kb cDNA fragment was
used as a probe to screen cDNA libraries (gt10) prepared with
mRNAs extracted from 0-3-h or 3-6-h embryos. Eight clones
were isolated from 4
10
phages.
Southern blot analysis of Drosophila genomic DNA using the 1.8-kb cDNA fragment as a probe indicated that the DREF gene is present as a single copy (data not shown).
Figure 6: Effect of DREF overproduction on repression by zen. Two-µg aliquots of p5`-168DPCNACAT plasmid were cotransfected with the indicated amounts of pAct5C-zen and pUAS-DREF1-701 plasmids into Kc cells. After 48 h, cell extracts were prepared to determine the CAT expression. CAT activity was normalized to the protein amount. Quantification was with an imaging analyzer BAS 2000. Averaged values obtained from four independent transfections are given as CAT activity relative to that of transfection without effector plasmids.
The open reading frame encodes a polypeptide of 701 amino acid residues with a predicted molecular weight of 80,096 and pI of 8.5. A data base search using the FASTA program did not cover any significant homology with reported conserved motifs.
DREF contains three characteristic regions, 1) rich in basic amino acids (28.7% between amino acid residues 61 and 203), 2) rich in proline (17.9% between amino acid residues 99 and 217), and 3) rich in acidic amino acids (18.0% between amino acid residues 218 and 390), as shown in Fig. 1B. DREF also contains a putative consensus sequence for the nuclear localization signal (RRRRTPPRK) at 143-151 residues. However, typical structures reported to be responsible for DNA binding, transactivation, and dimerization were not found.
The DRE binding activities of these mutant polypeptides were examined by gel mobility shift assay using DRE-P as a probe (Fig. 2B). Truncated polypeptides encoding from the 16th amino acid residue up to the C termini at 242, 230, 205, 185, 165, 145, and 125 amino acid residues exhibited DNA binding activities as effective as GST-DREF16-608. C-terminal deletion up to amino acid residue 105 resulted in a reduction of DNA binding activity to approximately half that of the 16-125 construct, and deletion up to amino acid residue 85 resulted in only weak activity, less than 1% that of the 16-125 form. Further C-terminal deletion up to residue 49 caused complete loss of DNA binding activity. These results indicate that the region essential for strong DRE binding activity is located between amino acid residues 16 and 115, although amino acid residues 16-85 are sufficient for weak DRE binding.
Deletion of 32 amino acid residues from the N terminus resulted in an almost complete ablation of DNA binding activity, although we detected very weak signals of DRE-protein complexes with N-terminal deletion mutants, 32-230, 32-145, 32-125, and 32-105 after long-term exposure of gels to the imaging plate. Interestingly, their mobilities in the gel were clearly faster than those of DREF mutants with similar sizes carrying the 16-32 region. One possible explanation is that amino acid residues 16-32 are required for dimer formation and also for the strong DRE binding activity.
Figure 4:
Characterization of antibodies against
DREF. A, immuno-Western blotting analysis. Crude extracts from E. coli with plasmids expressing GST-DREF16-242 (N) (lanes 1, 3, and 5) and
GST-DREF240-608 (C) (lanes 2, 4, and 6) were fractionated by SDS-polyacrylamide gel
electrophoresis, transferred onto a poly(vinylidene fluoride) membrane
sheet, and immunostained with anti-DREF antiserum (lanes 1 and 2), culture supernatants of hybridoma lines 1 (lanes 3 and 4) and 2 (lanes 5 and 6) as primary
antibodies and alkaline phosphatase-conjugated second antibodies. B, effects of antibodies on DRE-DREF complex formation. A gel
mobility shift assay was performed using P-labeled DRE-P
oligonucleotide as the probe incubated without (lanes 1, 3, 5, 7, and 9) or with (lanes
2, 4, 6, 8, and 10) Kc cell
nuclear extract in the absence (lanes 1 and 2) or
presence (lanes 3, 4, 5, 6, 7, 8, 9, and 10) of various
antibodies. Used antibodies were: lanes 3 and 4,
culture supernatant of a mouse hybridoma cell line (4-2D) which
produces anti-chick primase antibody (6 µl); lanes 5 and 6, culture supernatant of mouse hybridoma cell line 1 (6
µl); lanes 7 and 8, culture supernatant of mouse
hybridoma cell line 4 (6 µl); lanes 9 and 10,
rabbit anti-DREF polyclonal antibody (50 µg/ml IgG, 6 µl).
Nuclear extract from Kc cells (2 µg) was mixed with each antibody,
incubated for 2 h on ice, added to mixtures containing
P-labeled DRE-P oligonucleotides (10
cpm) and
1 µg of poly(dI-dC), incubated for 15 min on ice, and analyzed on a
4% polyacrylamide gel.
Effects of antibodies on DRE-DREF complex formation were
examined by gel mobility shift assay, in which the Kc cell nuclear
extract was incubated with each antibody prior to adding the probe (Fig. 4B). Monoclonal antibody 1 inhibited complex
formation. Western blotting analysis with a set of truncated GST-DREF
proteins indicated that the epitope for monoclonal antibody 1 is
located within the DNA binding region between amino acid residues 72
and 125 (data not shown), confirming that this region plays a role in
DNA binding. Addition of monoclonal antibody 4 as well as the
polyclonal antiserum resulted in supershifts of the DREDREF
complexes. Monoclonal antibodies 2 and 3 had no effect on DRE
DREF
complex formation (data not shown). The results confirmed that the cDNA
isolated in this study encodes the DREF polypeptide.
Figure 5:
Requirement of DREF for in vitro transcription. Kc cell nuclear extract (40 µg of protein) was
mixed with 2 µg of BSA (lane 2), preimmune rabbit IgG (lane 2), anti-chick DNA polymerase monoclonal IgG,
anti-DREF polyclonal IgG (lane 4), or anti-DREF monoclonal 1
IgG (lane 5) and incubated for 30 min on ice, and then
supplemented with a mixture for in vitro transcription using
supercoiled plasmid containing DNA polymerase
promoter as a
template. Transcripts were detected by primer extension. Arrow indicates the position of precisely transcribed RNA. DNA size
markers (M) are shown in lane
1.
Figure 7:
Changes in DREF mRNA during Drosophila development. Twenty µg of RNAs prepared from Drosophila bodies at various developmental stages were fractionated on a 1%
agarose gel containing formaldehyde, transferred to a sheet of
GeneScreen Plus filter, and subjected to hybridization. A, RNA
blot hybridized with the P-labeled 1.8-kb cDNA fragment
derived from pDCDREF1.8. Used RNAs were derived from: lane 1,
unfertilized eggs; lane 2, 0-2-h embryos; lane
3, 2-4-h embryos; lane 4, 4-8-h embryos: lane 5, 8-12-h embryos; lane 6, 16-20-h
embryos, lane 7, 1st instar larva; lane 8, 2nd instar
larva; lane 9, 3rd instar larva; lane 10, pupa; lane 11, adult male flies; lane 12, adult female
flies. B, amounts of DREF mRNA as determined using an imaging
analyzer (closed circles). The amount of DNA polymerase
mRNA is also shown by the shaded bars, determined by
rehybridization of the filter with a 1.5-kb PstI fragment of
DNA polymerase
cDNA.
Figure 8:
Immunocytochemical localization of DREF in Drosophila early embryos. Polyclonal antibody against DREF and
alkaline phosphatase-conjugated goat anti-rabbit IgG were used as the
primary and secondary antibodies, respectively. Stages in nuclear
division cycles were determined by counting the numbers of DAPI-stained
nuclei. A, unfertilized egg; B, DAPI-staining of the
unfertilized egg shown in A; C, cycle 5; D,
DAPI-staining of the embryo shown in C; E, cycle 8: F, cycle 9; G, cycle 10; H, cycle 11; I, embryo stained without the primary antibody. Views at an
internal focal plane (A-D) or at a surface focal plane (E-I) are shown. In all cases, the anterior ends of the
embryos are on the left. Magnification
284.
The ooplasm of unfertilized eggs was weakly stained with the antibody (Fig. 8A), confirming maternal storage of DREF. Up to nuclear division cycle 7, the nuclei and the surrounding cytoplasmic region were stained weakly, but still more strongly than other regions of the syncytial cytoplasm (Fig. 8C). The signals differed from those observed with anti-PCNA antibody, which was uniformly strong in nuclei at these cycles(39) . After cycle 8, strong and uniform nuclear staining was observed (Fig. 8E). By cycle 11, staining in the cortical cytoplasm underneath surface nuclei had mostly faded, suggesting that most maternally stored DREF had been translocated into nuclei from the cytoplasm by this point.
In our previous studies, promoters of DNA replication-related
genes such as those encoding PCNA and DNA polymerase 180-kDa
catalytic subunit) were found to be positively regulated by DRE and a
specific binding factor, DREF(24, 41) . We have
searched for the TATCGATA sequence in a Drosophila data DNA
base and found 60 genes carrying this sequence within 600-bp upstream
regions from the transcription initiation sites. Interestingly, more
than 30 of these genes are related to cell proliferation, suggesting
that DRE is a common regulatory element responsible for the coordinated
expression of many proliferation-related genes(42) .
Furthermore, overexpression of the zerknullt gene product
results in repression of promoters of both PCNA and DNA polymerase
genes by impeding the DREF activity in cells(29) . These
findings suggest that DNA replication related genes are both positively
and negatively regulated via DRE and DREF. The present isolation of
cDNA for DREF, and preparation of specific antibodies should provide
important clues toward further understanding how DREF functions in this
regulation.
The DREF polypeptide contains three characteristic domains, respectively rich in basic amino acids, proline, and acidic amino acids. Although no significant similarity with any other proteins in data bases was found, these characteristic regions may be required for the function of DREF as a transcription regulatory factor. The DNA binding domain was mapped between 16-105 amino acid residues in the basic amino acid-rich region of 90 amino acid residues, but no characteristic feature similar to those previously reported could be identified. Structural analysis with nuclear magnetic resonance imaging is now under way using the DNA binding domain of the recombinant DREF polypeptide.
Transactivation of DRE-containing promoters by DREF
overproduction was not observed in the co-transfection experiment with
DREF expression and CAT plasmids. We suppose that DREF might be almost
saturated in Kc cells. However, anti-DREF antibodies inhibited in
vitro transcription activity of Kc cell nuclear extract,
indicating that DREF is required for the high level of transcription
from the DNA polymerase gene promoter. In addition,
over-expression of DREF protein in Kc cells overcame repression of the
PCNA gene promoter by Zen protein. We have obtained similar results
with the DNA polymerase
gene promoter (data not shown). The
evidence indicates that DREF is one of the positive factors required
for DNA replication-related genes and that it might be an important
transcription regulatory factor involved in proliferation- and
differentiation-related control. We have isolated the gene for DREF and
analysis of its promoter region is in progress. We preliminary obtained
results suggesting that zen protein represses the DREF
promoter.
In the present study, the fluctuation of DREF mRNA content
during development was similar to those of DNA polymerase and
PCNA, providing further evidence that DREF is an important
transcription regulatory factor for DNA replication-related genes. The
DREF polypeptide is distinctly localized in nuclei after the nuclear
division cycle 8. A pulse-labeling experiment of zygotic-transcribed
RNA demonstrated that nuclei become competent for transcriptional
activation at cycle 10 while synthesis of rRNA, tRNAs, 5sRNA, snRNA,
and poly(A) RNA is first detectable during cycles 11 or
12(43) . Transcripts for Krüppel, hunchback, and hairy genes, members of the gap gene
and pair-rule gene families, are first detected at cycle 11 and their
products begin to be expressed at cycles
13-14,(44, 45, 46) . The histone genes
become transcriptionally competent during cycle 10(43) .
Northern hybridization analysis has revealed that the amounts of mRNAs
for both PCNA and DNA polymerase
start to increase significantly
in embryos by about 2 h after fertilization (cycles
12-15)(22) . Therefore, the appearence of DREF in nuclei
at and after cycle 8 might contribute to such zygotic transcription of
DNA replication-related genes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D78373[GenBank].