(Received for publication, May 22, 1995)
From the
To investigate the mechanisms governing the expression of DNA
topoisomerase II, the Chinese hamster topoisomerase II
gene
has been cloned and the promoter region analyzed. There are several
transcriptional start sites clustered in a region of 30 base pairs,
with the major one being 102 nucleotides upstream from the ATG
translation initiation site. Sequencing data reveal one GC box and a
total of five inverted CCAAT elements (ICEs) within a region of 530
base pairs upstream from the major transcription start site. Sequence
comparison between the human and Chinese hamster topoisomerase II
gene promoter regions shows a high degree of homology centered at the
ICEs and GC box. In vitro DNase I footprinting results
indicate protection by binding proteins at and around each ICE on both
DNA strands. However, no obvious protection was observed for the GC
box. Competition gel mobility shift assays with oligonucleotides
containing either the wild-type or mutated ICE sequences suggest that
identical or similar proteins specifically bind at each ICE, although
with different affinities for individual ICE sequences. Chloramphenicol
acetyltransferase assays employing nested 5`-deletions of the
5`-flanking sequence of the gene demonstrate that the sequence between
-186 and +102, which contains three proximal ICEs, is
sufficient for near wild-type level of promoter activity. When these
three ICEs were gradually replaced with sequences which do not interact
with the binding proteins, reducing promoter activity of the resulted
constructs was observed. In conjunction with results from footprinting
and gel mobility shift studies, the transient gene expression finding
suggests that the ICEs are functionally important for the
transcriptional regulation of the topoisomerase II
gene.
Mammalian DNA topoisomerase II (Top II) ()is an
essential nuclear enzyme which changes the topology of DNA by passing
an intact helix through a transient double-stranded break made in a
second helix followed by religation of the DNA break (reviewed in (1) and (2) ). The enzyme functions as a homodimer and
in an ATP-dependent manner(3) . A feature of Top II function is
the covalent attachment of the enzyme to the 5`-termini of DNA breaks
via a tyrosine-DNA phosphodiester linkage. Top II has been implicated
in a number of cellular processes such as synthesis and transcription
of DNA (4) and chromosomal segregation during
mitosis(5) . Top II enzyme also plays a structural role in
organizing both mitotic chromosomes and interphase
nuclei(6, 7) . Use of specific antibodies has
demonstrated that Top II is a major component of the mitotic
chromosomes and the interphase nuclear-matrix fractions(7) .
Moreover, specific DNA scaffold-attachment sites have been found to
contain the consensus cleavage sequence for Top II(8) .
Top
II is also the target of several classes of anti-cancer drugs such as
anthracyclines, amsacrine, and epipodophyllotoxins. These drugs
stabilize the cleavable complex formed between Top II protein and DNA,
resulting in increased DNA scission and concomitant inhibition of the
rejoining reaction(9) . The drug-induced DNA breaks are
reversible after drug removal. However, most of the cells are arrested
in the G phase and eventually die(10) .
Resistance to agents that target Top II is a major problem in cancer chemotherapy. In addition to the classical multidrug resistance, which is due to overexpression of the multidrug resistance transporter (mdr protein or P-glycoprotein)(11) , atypical multidrug resistance (at-MDR) has been described and is associated with altered Top II activity that is due to either mutated enzyme or a decrease in the amount of the enzyme(11, 12, 13) . It is likely that lower Top II levels result in fewer drug-induced DNA lesions and diminished cytotoxicity of Top II-targeting drugs(14, 15) . A correlation between cellular expression of Top II and the in vitro sensitivity to Top II active anti-tumor drugs has been found in a VM-26-resistant human cancer KB cell line(16) , the 9-hydroxyellipticine-resistant Chinese hamster lung fibroblast cell line DC3F/9-OHE(10, 17) , and in a panel of seven human lung cancer cell lines(18) .
In human and probably in
other mammals, Top II occurs in two isoforms, the 170-kDa form
and the 180-kDa
form, which are encoded by two discrete
genes(19, 20) . These isoforms have different in
vitro sensitivities to antineoplastic agents, different cleavage
sites, thermal stability, and inhibition by AT-rich oligonucleotides (21) . Recent work has demonstrated that the expression of the
170-kDa form is quantitatively cell cycle-regulated and cell
proliferation-related(21, 22) . The level of
expression peaks in the late G
to M phases and is greater
in rapidly proliferating cells. In proliferating granulocyte
precursors, the levels of 170-kDa in vivo were 2-3-fold
higher than mature cells and approached the levels in neoplastic cell
lines of the same lineage(22) . In ras-transformed
cells, the proportion of 170-kDa Top II is higher and depends less on
growth state than in untransformed cells(23) . The ras-transformed cells were also more sensitive to the
cytotoxic effects of teniposide and merbarone, drugs which selectively
inhibit the 170-kDa form of Top II, indicating a possible link between
drug sensitivity and expression of the 170-kDa form(23) . The
changes in amounts of the mRNA coding for the 170-kDa enzyme were
similar to the changes in the 170-kDa enzyme levels, suggesting that
the regulation might be mainly at the transcriptional
level(23) .
In order to investigate the cell cycle-regulated
expression of the top II gene and the mechanisms of
altered top II expression in drug-resistant cells, genomic
clones for the top II
gene of Chinese hamster were
isolated, and the 5`-flanking region of the gene was analyzed. These
studies have identified and characterized a group of inverted CCAAT
elements, which are present in the proximal promoters of both human and
Chinese hamster top II
genes, and are functionally
important for the transcriptional regulation of the top
II
gene.
To prepare the constructs with more adjacent 5`-flanking sequence, the 4.0-kb HindIII-SalI fragment in pBluescript plasmid was cut with PstI and the 1.0-kb PstI gemomic fragment (one PstI site was from the vector) was ligated to the PstI-digested pCAT-747 DNA in the sense orientation to give the construct pCAT-1700. Nested-deletions were performed on the KpnI-XhoI-digested pCAT-1700 DNA with Exo III exonuclease (Stratagene) for different time points, mung bean nuclease (Stratagene)-treated, ligated, and transformed into Escherichia coli strain BB4. Screening of transformed cells for clones with different sized 5`-flanking sequence yielded the pCAT-1000, pCAT-366, pCAT-223, pCAT-49, and pCAT-0 constructs.
Construct pCAT-186 was prepared by the polymerase chain reaction of the pCAT-747 template with the primer, 5`-CGCTCTCGAGAAGACTCTCCCGCCTCC-3`, and the above reverse primer. The amplified DNA was cloned into the pCAT-(HB) plasmid at XhoI-HindIII sites. The construct pCAT-152 was prepared by PstI-BstBI digestion of the pCAT-747 DNA, recovering the vector-containing DNA, blunting the ends with T4 DNA polymerase, and self-ligation.
Figure 1:
The 5`-genomic region of the CHO top II gene. A, the partial restriction maps of
the two genomic clones, Top II-21 and Top II-93, are shown. The
restriction map of the 4.0-kb HindIII-SalI fragment
of the 5`-genomic region is expanded in the middle (the SalI
site is from the vector). Open boxes represent noncoding
sequences, while the stippled boxes show the coding regions of
the first, second, and third exons in this region. The arrow on top of the NcoI site represents the translation start
and the orientation of the gene. Beneath the 4.0-kb HindIII-SalI fragment are the three fragments (open boxes) employed for the DNase I footprinting and gel
mobility shift assays, as well as the region of DNA sequence (solid
box) shown in B. Abbreviations: B, BamHI; Bs, BstBI; E, EcoRI; H, HindIII; N, NcoI; P, PstI; S, SalI. B, DNA sequence of the 5`-portion (solid box in A) of the CHO top II
gene. The coding sequence
of the first exon is shown in boldface. The arrow indicates the major transcriptional start site (+1). The five
inverted CCAAT sequences are boxed, and both the GC box and
the TATA-like sequence are underlined.
Figure 2:
Determination of the transcriptional start
site of the CHO top II gene. A, primer extension
experiment and B, RNase protection assay were performed as
described under ``Materials and Methods.'' Shown are
reactions with 2 µg of CHO poly(A)
RNA (lane
1) and 5 µg of yeast RNA (lane 2). The arrows indicate the minor extension and RNase protection products, while
the arrowheads represent the major reaction products. In A, the sequence ladder next to the reaction lanes was produced
by sequencing a 5`-genomic clone with the same 21-mer primer. There
were consistent GC compressions observed in the sequence ladder at
positions close to the major transcription site. Another sequence
ladder produced by dITP sequencing is shown on the left to resolve the
sequence around this region. In B, the sequence ladder
produced by sequencing M13 mp18 DNA with universal primer is marked as
size marker.
Figure 3:
Comparison of the CHO top II
5`- genomic sequence to other homologous sequences. A, the
highest scored comparisons of the CHO top II
5`-genomic
sequence in a GenBank
data base search using the
``Blast'' program are presented. Shown are the best parts of
alignments of the CHO sequence with the human top II
promoter sequence (alignments 1-3), with mouse top
II
cDNA sequence (alignment 4), and with the gene
for human IgM heavy chain (alignment 5). The subject sequences
are numbered as they appear in the GenBank
data base.
Identical nucleotides in the comparisons are indicated by colons.
B, the alignment of Chinese hamster and human promoter sequences
for the top II
gene with the ``Bestfit''
program from the GCG sequence analysis package. Identical nucleotides
are indicated by colons in the alignment. Both sequences are
numbered with respect to the major transcriptional start site (as
+1). The ATG translation initiation site, inverted CCAAT
sequences, GC box, and the TATA-like sequences are underlined.
Figure 4:
DNase I footprinting analysis of the
5`-flanking region of the CHO top II gene. Experiments
were performed as described under ``Materials and Methods''
with either the coding strand (A) or the noncoding strand (B) labeled. For all panels, Maxam and Gilbert (A +
G)-sequence ladders are electrophoresed on the side, with the positions
of the sequence elements indicated: numbers 1-5 for the
inverted CCAAT sequences, GC for the GC box, and TA for the TATA-like sequence. On the right side of the footprinting
lanes, the footprints at each ICE and the juxtaposed positions are
grouped together and marked with romanic numerals I-V.
For A, the footprinting reactions with the 535-bp EcoRI-NcoI fragment labeled at the NcoI site (a) and the 285-bp EcoRI-BstBI DNA labeled
at the BstBI site (b) are shown. The reactions were
performed without nuclear extracts (lane 1), with 0.3 µl (lane 2), 3 µl (lane 3), and 10 µl (lane
4) of the nuclear extracts, respectively. For B, the
footprinting reactions with the 535-bp EcoRI-NcoI
fragment labeled at the EcoRI site (a) and the 383-bp BstBI-BamHI DNA at the BstBI site (b) are shown. Electrophoresis of the 535-bp EcoRI-NcoI reactions for both short and longer time
are shown to present the footprints at the sequence elements. The
reactions were carried out without nuclear extracts (lane 1),
with 3 µl (lane 2), and 10 µl (lane 3) of the
nuclear extracts, respectively.
Figure 5:
Gel mobility shift assays of DNA fragments
from the 5`-flanking region of the top II gene. The
reactions with the labeled proximal fragment, the 383-bp BstBI-BamHI DNA (lanes 1-5), and with
the labeled distal fragment, the 285-bp EcoRI-BstBI
DNA (lanes 6-10) are presented. The labeled DNAs were
incubated without nuclear extracts (lanes 1 and 6) or
with 5 µg of nuclear extracts (lanes 2-5 and 7-10). The binding of proteins to the labeled DNAs was
competed with a 50-fold molar excess of proximal fragment DNA (lanes 3 and 8), distal fragment DNA (lanes 4 and 9), or the multicloning region of pBluescript DNA (lanes 5 and 10). The free labeled DNAs (F)
and the nucleoprotein complexes are
indicated.
Since footprints were mostly observed at the ICEs, the ICEs are probably the binding sites for the nuclear factors in the complex formation of both fragments. To demonstrate this, we synthesized five pairs of oligonucleotide duplexes which encompass the first through five ICEs and their complementary sequences, respectively (Fig. 6B). These oligonucleotide duplexes were used in the competition gel mobility shift assays (Fig. 6A, lanes 2-7 and lanes 11-18). In an experiment employing the proximal fragment, the first, second, and the third ICE-containing oligonucleotides were effective in competing for the formation of complexes A and B (Fig. 6A, lanes 2-7). Experiments with the fourth and the fifth ICE-containing oligonucleotides also demonstrated competition for formation of complexes A and B (data not shown). In the assay with the labeled distal fragment (Fig. 6A, lanes 10-18), excess amounts of ICE-containing oligonucleotides could deplete the formation of complexes A` and C` and partially the complex B`. The first ICE-containing oligonucleotide functioned as a competitor at least as well as the ICE-containing oligonucleotides derived from the distal fragment. The addition of 50-fold molar excess of ICE-containing oligonucleotide competitors resulted in low level complex formation of B` (Fig. 6A, lanes 11, 13, 15, and 17). However, the bands were not completely depleted even with a 500-fold excess of competitors (Fig. 6A, lanes 12, 14, 16, and 18), suggesting that a portion of B` was derived from complexes formed by non-CCAAT binding activity. These findings suggest that the complexes A, B, A`, C`, and partially B` were composed of proteins which recognized the common sequences of all the five ICE-containing oligonucleotide competitors. Since the sequence 5`-ATTGG-3` (its complementary sequence is 5`-CCAAT-3`) is the common sequence represented by all five ICEs, the binding proteins are likely to be CCAAT-binding proteins. The second (lanes 4 and 5) and fourth (lanes 13 and 14) ICE-containing oligonucleotide competitors were not as effective as other ICE-containing competitors in the competition. To confirm that the complexes are formed by CCAAT-binding proteins, an oligonucleotide duplex containing the same flanking sequences as the first ICE but with the core ATTGG sequence mutated to CTGGA was employed in the competition gel mobility shift assay (Fig. 6A, lanes 8, 9, 19, and 20). A 50-fold molar excess of the mutated ICE-containing competitor did not compete for complex formation, while large excess amounts (500-fold) of the mutated competitor could compete for the formation of complexes A and A`.
Figure 6: Competition gel mobility shift assays with various ICE-containing oligonucleotides. A, the reactions with the labeled proximal fragment (lanes 1-9) and the distal fragment (lanes 10-20) are shown. The incubations were carried out with 5 µg of nuclear extracts. The binding was competed with 50-fold (lanes 2, 4, 6, 8, 11, 13, 15, 17, and 19) or 500-fold (lanes 3, 5, 7, 9, 12, 14, 16, 18, and 20) molar excess of competitor DNAs. The competitor DNAs used were the first (lanes 2, 3, 17, and 18), second (lanes 4 and 5), third (lanes 6, 7, 11, and 12), fourth (lanes 13 and 14), fifth (lanes 15 and 16), and the mutated (lanes 8, 9, 19, and 20) ICE-containing oligonucleotides. B, the sequences of the five ICE-containing oligonucleotides and the mutant ICE-containing oligonucleotide are aligned together. Identical nucleotides which are present in the same position of four or more aligned sequences are shown in boldface.
To analyze the CCAAT-binding activities to the ICEs, the six pairs of oligonucleotide duplexes were radiolabeled and employed in the gel mobility shift assay (Fig. 7). A DNA-protein complex was formed with each ICE-containing oligonucleotide duplex but not with the mutated ICE-containing oligonucleotide duplex (lane 12). The mutated ICE-containing oligonucleotide duplex was also not an effective competitor for the complex formation (lane 14). These are consistent with the previous results (Fig. 6) that the bindings to the ICE-containing oligonucleotides were by CCAAT-binding proteins. All of the ICE-specific complexes comigrated in the gel, suggesting that the same CCAAT-binding factors or proteins of similar electrophoretic properties were bound to the oligonucleotides. There was less complex formation with the fourth ICE-containing oligonucleotide duplex (lane 8), despite the fact that equal amounts of radiolabeled oligonucleotides were used. There were also some faster migrating complexes observed in the gel mobility shift assays, which might be due to protein degradation or some unknown protein bindings not specific to the ICEs.
Figure 7:
Gel mobility shift assays of the
radiolabeled ICE-containing oligonucleotides. The reactions with the P-labeled first (lanes 1, 2, and 13-15), second (lanes 3 and 4), third (lanes 5 and 6), fourth (lanes 7 and 8), fifth (lanes 9 and 10), and mutated (lanes 11 and 12) ICE-containing oligonucleotides are
shown. The incubations were carried out without (lanes 1, 3, 5, 7, 9, and 11) or
with 5 µg (lanes 2, 4, 6, 8, 10, and 12-15) of nuclear extracts. The binding
of nuclear proteins to the labeled first ICE-containing oligonucleotide
was competed with a 50-fold molar excess of the first (lane
13) and mutated (lane 14) ICE-containing oligonucleotides
or the nonspecific multicloning region of pBluescript DNA (lane
15). The specific DNA-protein complexes are indicated by the arrow.
Figure 8:
Transient expression activity of the
upstream sequence of the CHO top II gene. The result of
one representative experiment is shown in A. Lane 1, pCAT-(HB)
as negative control; lane 2, pCAT-0; lane 3, pCAT-49; lane 4, pCAT-152; lane 5, pCAT-186; lane 6,
pCAT-223; lane 7, pCAT-366; lane 8, pCAT-747; lane 9, pCAT-1000; lane 10, pCAT-1700; lane
11, the positive control, pSV2CAT. B, the constructs
employed in the assay and the average percentage CAT activity for each
construct (relative to the CAT activity elicited by the pCAT-749
construct) are presented. The drawings are not to scale. The panel on
top of the constructs shows the sequence elements (solid
bars). The first to the fifth inverted CCAAT elements are
represented by numbers 1-5, TATA-like sequence by TA, and the GC box by GC. The numbers below indicate
the positions of the sequence elements in the top II
sequence. The upstream sequences are represented by open
boxes, the 5`-untranslated sequences are shown by shaded
boxes, and the CAT reporter sequences are shown by solid
boxes. The arrow represents the transcriptional start
site. The results represent the average of at least three independent
transfections with results differing by no more than
25%.
Since the pCAT-186 construct encompasses three ICEs and CCAAT-binding proteins were observed to interact with the ICEs in the previous experiments, the functional role of ICEs in promoter activation was analyzed by site-directed mutagenesis and transient gene expression (Fig. 9). When the first ICE was mutated, about 40% of pCAT-186 promoter activity was lost (Fig. 9, lane 4). Mutation of the third ICE alone also gave similar results (data not shown). When both the first and third ICEs were mutated (lane 5), 72% of the promoter activity was lost. With further mutation of the second ICE (lane 6), the promoter activity was diminished to about 23%.
Figure 9:
Site-directed mutagenesis and transient
gene expression assay. The result of one experiment is shown in A.
Lane 1, pCAT-(HB); lane 2, pCAT-152; lane 3,
pCAT-186; lane 4; pCAT-186 -1 (the first ICE mutated); lane 5, pCAT-186
-1/
-3 (the first and third ICEs
mutated); lane 6, pCAT-186
-1/
-2/
-3 (all of the
ICEs mutated). B, the average CAT activities of the constructs
relative to that of the pCAT-186 construct are shown. At least three
transfection experiments were performed for the presented
results.
We have isolated genomic clones that contain the promoter
elements of the Chinese hamster top II gene. Comparison
of the available genomic sequence of the human top II
gene (32) with the Chinese hamster gene suggests that the
genomic structures of these two genes may be similar. In both genes,
the first exon is comprised of about 90-102 nucleotides as an
untranslated region, and 21 nucleotides as the coding sequence. The
first and second introns are of similar sizes and are of the same
class. (
)The promoters of these two genes share a high
degree of homology (67% sequence identity). The highly homologous
regions are centered around and between the transcription and
translation initiation sites, and the ICE areas in the 5`-flanking
region (Fig. 3). The genomic sequence for the mouse top
II
gene is not available, but comparison of the Chinese
hamster sequence with the 5`-end of the mouse cDNA sequence
demonstrates 86% sequence identity (Fig. 3). The 5`-flanking
sequence, however, does not share any homology to the Drosophila and yeast sequences (data not shown). This suggests that mammalian top II
genes may share the same transcriptional
regulation machinery.
The Chinese hamster top II gene
promoter has a moderately high GC content, no canonical TATA box
sequence, and the transcriptional start sites are scattered in several
discrete positions. These are the characteristics of promoters of genes
that have housekeeping and growth-related functions(34) . Like
many housekeeping genes, the promoter of Chinese hamster top
II
gene contains a GC box with the potential for binding of
the transcription factor Sp1. However, footprinting analysis of the top II
promoter did not reveal any bona fide protection of the GC box. GC box elements that do not bind Sp1 are
also observed in other promoters such as the herpesvirus
immediate-early 3 (ICP-4) promoter(35) .
Five ICEs were
found scattered within the 400-bp proximal promoter region in the
Chinese hamster top II gene. DNase I footprinting and gel
mobility shift assays demonstrated the binding of sequence-specific
proteins at and around the ICEs. The CCAAT sequence is a moderately
conserved transcriptional regulatory element in many eukaryotic
promoters, such as histone(36) , albumin(37) ,
globin(38) , major histocompatibility complex class
II(39) , and viral gene promoters(40, 41) ,
and has been shown to function in either orientation(40) .
Inverted CCAAT elements are important for the cell-cycle regulation of
transcription in the human thymidine kinase gene (42, 43) and the serum induction of transcription from
the human DNA polymerase
gene (44) and transcription from
the long terminal repeat of Rous sarcoma virus(41) . Several
proteins that specifically recognize CCAAT elements have also been
characterized(37, 39, 45) . In the analysis
of the ICEs in the top II
promoter, competition with
ICE-containing oligonucleotides in the gel mobility shift assays
employing labeled proximal and distal fragments suggests that complexes
A, B, A`, C`, and part of B` are ICE-binding complexes (Fig. 6A). Although they may be formed by different
proteins, the combined results of Fig. 6and Fig. 7suggest that the depletable complexes are more likely
formed by the same or very similar ICE-binding proteins to the ICEs
with different affinities. For example, in the gel mobility shift assay
with the proximal fragment, the ICE-binding protein would bind to the
first ICE with greater affinity to form complex B. Additional binding
of the ICE-binding protein to the second ICE of the proximal fragment
with lower affinity produced the less intense complex A. In the
competition assays, addition of the ICE-containing oligonucleotides
would easily compete out the binding to sites with lower affinity and
disrupt the formation of higher ordered complexes. The fourth and
second ICEs have less affinity to the ICE-binding proteins since the
fourth ICE-containing oligonucleotide formed less complex (Fig. 7), and the fourth and second ICE-containing
oligonucleotide competitors were less effective in the competition gel
mobility shift assays (Fig. 6A). This is consistent
with the result of DNase I footprinting, in which the fourth ICE
exhibited lesser protection from DNase I digestion (Fig. 4). The
different affinities of the ICE-binding protein to the ICEs can be a
function of the interaction with the flanking sequences around the core
ATTGG sequence. This may also account for the partial competition of
500-fold molar excess of mutated ICE-containing oligonucleotide for the
complex formation (Fig. 6A). All five ICEs are similar
in that they have a pyrimidine-rich 3`-flanking sequence (Fig. 6B), and like many CCAAT elements, they are
asymmetrical. However, alignments of the ICE sequences did not show any
obvious flanking sequence residues which suggest distinction between
the high affinity ICEs and the low affinity ICEs.
Transient gene
expression assays have delineated the 5`-limit of the functional
promoter to the region between 186 and 152 bp upstream of the major
transcription site. 5`-Deletion beyond this limit significantly reduces
the promoter activity. The three ICEs in this core promoter region were
analyzed by site-directed mutagenesis and transient gene expression (Fig. 9). Mutations of the ICEs elicited reduction in basal
promoter activity, although a residual promoter activity remained when
all three ICEs were mutated. This suggests the activation role of ICEs
in the transcriptional regulation of the top II gene and
some other elements may be present in the core promoter for the
residual activity. The reduction in basal promoter activity was
additive for the first and third ICE mutations, whereas the mutation of
the second ICE had a minimal effect on the decrease of promoter
activity. Thus, the in vitro binding activity of the ICEs is
likely consistent with their in vivo activation activity.
In summary, these studies have characterized the promoter region of
the Chinese hamster topoisomerase II gene and the five
protein-binding ICEs which have promoter activation function. Further
study is required to characterize CHO topII
ICE-binding
protein(s) and compare them to other CCAAT-binding proteins, as well as
other embedded elements in the promoter and the upstream regions which
regulate both the basal and cell cycle-regulated expression of the top II
gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34196 [GenBank]