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(Received for publication, May 22, 1995) From the
To investigate the mechanisms governing the expression of DNA
topoisomerase II
Mammalian DNA topoisomerase II (Top II) ( 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 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 In order to investigate the cell cycle-regulated
expression of the top II
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
Figure 2:
Determination of the transcriptional start
site of the CHO top II
Figure 3:
Comparison of the CHO top II
Figure 4:
DNase I footprinting analysis of the
5`-flanking region of the CHO top II
Figure 5:
Gel mobility shift assays of DNA fragments
from the 5`-flanking region of the top II
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
Figure 8:
Transient expression activity of the
upstream sequence of the CHO top II
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
We have isolated genomic clones that contain the promoter
elements of the Chinese hamster top II The Chinese hamster top II Five ICEs were
found scattered within the 400-bp proximal promoter region in the
Chinese hamster top II 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 In summary, these studies have characterized the promoter region of
the Chinese hamster topoisomerase II
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) U34196 [GenBank]
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 25850-25858
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gene (*)
, 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.
)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) .
phase and eventually die(10) . 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) . 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.
Cell Culture
Wild-type Chinese hamster ovary cells (CHO) were maintained
in -minimal essential media, supplemented with 10% fetal bovine
serum at 37 °C in the presence of 5% CO.Isolation of CHO Genomic Clones
The Chinese hamster ovary genomic library was purchased from
Stratagene, which was prepared by cloning CHO-K1 genomic DNA in
Lambda-Fix II vector. The phages were propagated in host
bacteria P2-392. The transformation and plating procedures were
according to the recommendation of the manufacturer. 10
plaques were screened under stringent conditions as described
elsewhere (24) with the CHO topII cDNA probes,
pC431 (5`-end probe) or pC42 (middle and 3`-end probe) (25) .
The cDNA probes were multiprimed labeled with the Klenow fragment of
DNA polymerase I in the presence of
[-P]dCTP(24) . Filter
hybridization, recovery of recombinant phage DNA, restriction mapping,
as well as the subcloning of genomic fragments into pBluescript and M13
vectors (Stratagene) were as previously described(24) .
Sequencing was performed on both strands of DNA by using a Sequenase
kit (U. S. Biochemical Corp.). Sequencing data were analyzed with
MacVector and GCG (Genetics Computer Group) sequence analysis programs.
Determination of Transcriptional Start Site
Primer Extension
A 21-mer oligonucleotide with
sequence 5`-CTCGTGAGTCCCGAAAGCGAC-3`, which is complementary to the
cDNA sequence 20-40 base pairs upstream of the ATG codon, was
labeled by T4 polynucleotide kinase and
[-
P]ATP. 5
10
cpm of
labeled primer were hybridized to 2 µg of CHO poly(A) RNA or 5 µg of control yeast RNA. The annealed primers were
extended by Superscript reverse transcriptase (Life Technologies,
Inc.), and the extended products were analyzed on a denaturing
polyacrylamide gel.
RNase Protection Assay
The 4.0-kb HindIII-SalI genomic fragment (the SalI site
was from the vector) was cloned into the pBluescript plasmid. This
plasmid was used in the polymerase chain reaction with a pair of
primers containing HindIII cloning sites,
5`-CGCTAAGCTTGCTGCAGAAGGCAGGCGGA-3` (the forward primer), and
5`-CGCTAAGCTTGGTGACGGTCCTGTAGGG-3` (the reverse primer), to amplify the
849-bp proximal fragment upstream from the ATG site. This fragment was
cloned in antisense orientation into the HindIII cut
pBluescript KS plasmid. Radiolabeled RNA was synthesized from this
plasmid by T7 RNA polymerase in the presence of
[P]UTP. Assays were performed with Ambion RPA
II
kit. 1.3
10 cpm (about 260 pg) of
labeled RNA was hybridized to 2 µg of CHO poly(A) RNA or 5 µg of control yeast RNA. The hybridized products
were digested at 37 °C for 30 min with a mixture of 20 units of
RNase T1 and 1 µg of RNase A. Protected fragments were
electrophoresed on a denaturing polyacrylamide gel and visualized by
autoradiography.
Chloramphenicol Acetyltransferase (CAT) Transient
Expression Assay
Preparation of Constructs
The positive control
for the experiment is the pSV2CAT plasmid, which has a simian virus 40
early promoter fused to the CAT coding sequence(26) . The
parent plasmid for the syntheses of other constructs is the pCAT-(HB)
which was prepared in this laboratory. The CAT coding sequence was
fused into the pBluescript SK plasmid at the HindIII and BamHI sites. The pCAT-(HB) plasmid has minimal CAT activity
and served as a negative control in the CAT assay. To synthesize other
constructs, the 849-bp HindIII fragment described in the RNase
protection assay was cloned in the sense orientation at the HindIII site upstream of the CAT coding sequence of the
pCAT-(HB) plasmid. The resulting plasmid is designated as pCAT-747. The
number designated to this and the following plasmids represents the
length of 5`-flanking genomic sequence (in base pairs) upstream from
the major transcription site in the constructs. Exceptions to this are
the pCAT-1000 and pCAT-1700 (see below), which represent only the
approximate sizes of the genomic fragments.Site-directed Mutagenesis of the pCAT-186
Plasmid
Transformer site-directed mutagenesis kit
(CLONTECH) was employed to mutate the three ICEs in the pCAT-186
plasmid according to the protocol of the manufacturer. The sequence of
the selection primer is 5`-GCCACCGCGGTGCATATGCAGCTTTTGTTCCC-3`. The
underlined sequence represents the NdeI recognition sequence,
which replaced the SacI sequence of the plasmid during the
mutagenesis reactions. The three mutagenic primers used to replace the
first, second, and the third ICE sequences were: 5`-CGACTCGGTGCTGGATT
CCTCTGAT-3`, 5`-GACCGTCCACGCTGGATTACTCTAAAC-3`, and 5`-CCTCC
TTTACCTACTGGATTCATTCGAACAG-3`, respectively.
DNA Transfection and CAT Assay
5 µg of test
constructs were cotransfected with 5 µg of -galactosidase
expression plasmid, pCH110 (Pharmacia Biotech Inc.), into 1
10 growing CHO cells by the calcium phosphate precipitation
method(26) . After 48 h, cells were harvested, and lysates were
prepared. Amounts of lysates employed for the CAT activity assays were
normalized to the -galactosidase activities. CAT activity assays
and -galactosidase activity assays were performed as described
elsewhere(26, 27) . Nonacetylated and acetylated
chloramphenicol spots on the TLC plates were quantitated with
PhosphorImager and analyzed by softwares from Molecular Dynamics Inc.Study of Binding Activities to the 5`-Flanking
Sequences
Preparation of Nuclear Extracts
Nuclear extracts
were prepared from growing CHO cells according to Dignam et
al. (28) . On average, 500 µg of proteins can be
obtained from 10
cells. Protein determination was performed
using the micro BCA protein assay kit (Pierce).DNase I Footprinting
DNase I footprinting
experiments were performed according to Goding et
al.(29) , except that no ammonium sulfate was added in the
incubation buffer. DNA was labeled at one end by a fill-in reaction
with the Klenow fragment of DNA polymerase I and the respective
radioactive nucleotides. For 3`-coding strand labeling, both the 535-bp EcoRI-NcoI DNA and the 285-bp EcoRI-BstBI DNA were labeled with
[-P]dCTP. For 3`-noncoding strand labeling,
the 535-bp EcoRI-NcoI DNA was labeled with
[
-P]dATP and the 383-bp BstBI-BamHI DNA was labeled with
[
-P]dCTP. After the incubation with nuclear
extract and the DNase I digestion steps, the DNA was phenol and
chloroform-extracted, ethanol-precipitated, and analyzed on a 6%
denaturing polyacrylamide gel. The (A + G)-sequencing ladders were
prepared with the same labeled DNA by the Maxam and Gilbert method (30) .
Gel Mobility Shift Assay
Gel mobility shift assays
were performed according to Goding et al. (29) with
minor modifications in the incubation buffer. The concentrations of the
KCl and EDTA were 50 mM and 0.1 mM, respectively.
After binding, the DNA-protein complexes were resolved by
electrophoresis on a native 4% polyacrylamide gel and exposed to
autoradiographic film. For the competition gel mobility shift assay,
the competitor oligonucleotide duplexes were as follows. The first
ICE-containing oligonucleotides were 5`-AGCGACTCGGTGATTGGTTCCTCTGAT-3`
and its complementary strand. The second ICE-containing
oligonucleotides were 5`-AGACCGTCCACGATTGGTTACTCTAAA-3` and its
complementary strand. The third ICE-containing oligonucleotides were
5`-CCTCCTTTACCTAATTGGTTCATTCGAACAGG-3` and reverse strand
5`-TTCGAATGAACCAATTAGGTAAAGGAGGCGGG-3`. The fourth ICE-containing
oligonucleotides were 5`-ACAGGAATAGACTATTGGTCTATCCTGAAGAC-3` and
reverse strand 5`-TCAGGATAGACCAATAGTCTATTCCTGTAGCA-3`. The fifth
ICE-containing oligonucleotides were
5`-TGGGCCTTTCTCATTGGCCAGATTTCCTGTA-3` and reverse strand
5`-GGAAATCTGGCCAATGAGAAAGGCCCATGTG-3`. The mutated ICE-containing
oligonucleotides are the first ICE-containing oligonucleotide with the
core 5`-ATTGG-3` sequence mutated to 5`-CTGGA-3` and its complementary
sequence.
Cloning of the 5`-Flanking Region of the Chinese
Hamster topII
The Chinese hamster genomic library was
screened under stringent conditions with either the pC431 (5`-end of
the cDNA) probe, or the pC42 (middle and 3`-end) probe, of the CHO top II gene cDNA(25) . A total of six overlapping
genomic clones were isolated. (The structure of the top II gene
will be described in detail elsewhere.) The clone Top II-93 is the only
clone which hybridized to the 5`-end cDNA probe and not to the pC42
probe. This and another overlapping clone, Top II-21, were further
characterized. The 5`-end of the cDNA was mapped to a 4-kb HindIII-SalI fragment of Top II-93. This fragment was
subsequently subcloned into pBluescript vector and analyzed. Sequencing
data revealed the location of the first, second, and third exons and
the 5`-flanking region of the top II gene (Fig. 1). The ATG translation initiation codon of the cDNA is
located at the NcoI site of the genomic fragment. The coding
sequence ends abruptly after 21 nucleotides at the PstI site
and is followed by the 1.01-kb first intron. The second and the third
exons are 153 and 91 bp, respectively. Sequencing of the 0.87-kb EcoRI and the 4.5-kb EcoRI fragments from the clone
Top II-21 confirmed the Top II-93 sequence.
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.
Determination of the Transcriptional Start Site of the
topII
To locate the transcriptional start site and
confirm the translation initiation site, primer extension was
performed. A GeneP-labeled 21-mer oligonucleotide
complementary to the cDNA sequence 20-40 base pairs upstream of
ATG was employed in the experiment. Several extension products were
observed (Fig. 2A). The major transcript, as deduced
from the predominant extension product, is being initiated from the
cytosine 102 nucleotides upstream from the ATG codon. Other minor
transcripts are initiated at adenine 99, cytosine 110, and thymines 119
and 129, respectively. To confirm the primer extension result, an RNase
protection assay was carried out. A chimeric clone containing the
genomic sequence upstream of the ATG site of the cDNA sequence was
subcloned into pBluescript vector (see ``Materials and
Methods''), and
P-labeled RNA in antisense
orientation was synthesized. Fig. 2B shows that several
protected RNA fragments with sizes ranging from 100 to 130 bases were
detected. The pattern of the RNase protected probes was similar to the
primer extension pattern, and the sizes of the protected bands agree
with the primer extension data, considering that there is a slight
difference in the electrophoretic mobility of the RNA probes and the
DNA marker. Downstream of the transcriptional start sites, the ATG
initiation codon deduced from the cDNA sequence (25) is the
first ATG codon in the genomic sequence, and the sequence, ACCATGG, is
a perfect match to the optimal sequence for initiation by eukaryotic
ribosomes as suggested by Kozak(31) . The major transcriptional
start site is designated hereafter as +1 unless otherwise stated.
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.
Sequence Analysis of the 5`-Flanking Region of the CHO
top II
The region between the transcriptional and
translation start sites and the 200-bp region immediately upstream of
the transcriptional start site have a moderately high GC content of 64
and 49%, respectively. There is no canonical TATA box sequence,
although an imperfect sequence of AATGAA was located 26 bp upstream of
the predominant transcriptional start site. Further upstream at the
-122 position, there is a TATA-like sequence (AATAAA). The 541-bp
5`-flanking sequence from the first upstream EcoRI site to the
translation initiation site was searched for potential binding sites
for transcription factors. The most prominent sequence motifs in this
immediate upstream region are one GC box with potential for Sp1 binding
on the coding strand and five CCAAT sequences on the opposite strand
(refer to Fig. 1B). These five inverted CCAAT elements
(ICEs) are designated one to five according to their proximities to the
ATG start codon. There are also two sequences at (60 to 66) and at
(-339 to -333), which are a one-nucleotide mismatch to a
canonical Ap1 sequence T(T/G)AGTCA. A pair of perfect direct repeat
sequences of AGAGCTGAG are located at positions -327 to
-319 and -317 to -309. Downstream from them there is
a pair of single mismatched inverted repeats at positions -300 to
-294 and -293 to -287. The 560-bp 5`-flanking and
first exon sequence was submitted to a search for homologous sequences
in the GenBank Gene data base. The Chinese hamster sequence
shares extensive homology with the human top II
promoter
sequence (32) and the 5`-end of the mouse top II
cDNA sequence (33) (Fig. 3A). The most
homologous parts between human and Chinese hamster promoters are the
region around the transcriptional start site (78% identity from
-18 to +47 of the Chinese hamster sequence), the region
immediately upstream of the ATG codon (85% from 59 to 106), and the
region around the fifth ICE (80% from -260 to -215). The
Chinese hamster and the human promoter sequences were aligned by the
Bestfit program and are presented in Fig. 3B. The GC
box, TATA-like element, and the first three ICEs of the hamster
sequence can be aligned to the corresponding elements of the human
sequence. However, the fourth hamster ICE element is positioned at a
sequence having one mismatch with the human gene. The fifth hamster ICE
is aligned with the fourth ICE of the human sequence, whereas the fifth
human ICE does not have a homologous counterpart in the hamster
sequence. Except for the fourth hamster ICE, regions around the
aforementioned sequence elements share a relatively high homology
between the hamster and human genes. The overall similarity between the
human and Chinese hamster promoter sequences is 67%. The sequence
similarity diminishes upstream of the Chinese hamster fifth ICE.
Interestingly, the translation start site sequence including part of
the first exon of the Chinese hamster top II gene shows a
21/23 identity with the translation start site of the gene for human
IgM heavy chain (Fig. 3A). It is not known though if
the translational regulation of both genes have any similarity.
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.
Analysis of the Proximal cis Elements in the 5`-Flanking
Region by in Vitro DNase I Footprinting
The 535-bp EcoRI-NcoI fragment encompassing the GC box and all
of the five ICEs was labeled at either the EcoRI or NcoI ends and subjected to in vitro DNase I
footprinting. To resolve the footprints away from the labeled ends, two
smaller fragments, the 285-bp EcoRI-BstBI fragment
and the 383-bp BstBI-BamHI fragment, were also
labeled, respectively, at the BstBI site for the footprinting
analysis of coding and noncoding strands. Analysis on both coding and
noncoding strands of the 535-bp region is presented in Fig. 4.
Footprints are observed at all the inverted CCAAT sequences and the
regions juxtaposed to them on both coding and noncoding strands. The
footprints of some flanking sequences, for example, around the fifth
ICE on the coding strand, are more pronounced than the inverted CCAAT
sequences. The fourth ICE has a lesser extent of protection than the
others. Whereas there were no footprints observed at the TATA-like
element and the GC box of the coding strand, analysis of the noncoding
strand demonstrated a marked footprint at the TATA-like element and a
small footprint at the 3`-end of the GC box (Fig. 4B).
There are some enhanced cleavages with nuclear extracts observed at
positions upstream of the fifth ICE and the TATA-like sequence,
respectively, as well as downstream of the third ICE region on the
noncoding strand footprinting. This may suggest protein-induced DNA
bending at these regions.
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.
Analysis of the Binding Activities to the Inverted CCAAT
Elements by Gel Mobility Shift Assay
The binding of protein
factors to the proximal DNA elements including the ICEs was analyzed
further by gel mobility shift assay. To facilitate an initial assay of
the entire 535-bp EcoRI-NcoI region, this region was
split into two fragments, the 383-bp BstBI-BamHI
(proximal) fragment, and the 285-bp EcoRI-BstBI
(distal) fragment, and were used separately for gel mobility shift
assays (Fig. 5). These two fragments bound to protein factors
from the nuclear extracts and migrated as distinct complexes on the
native polyacrylamide gel. There are in total three distinct
DNA-protein complexes observed in the assay with the 383-bp BstBI-BamHI proximal fragment (Fig. 5, complexes A, B, and C of lane 2).
There are also three bands of DNA-protein complexes observed in the
assay with the 285-bp EcoRI-BstBI distal fragment (complexes A`, B`, and C` of lane
7), although the complexes B` and C` are not well separated. All
the complexes were competed out when a 50-fold molar excess of the same
unlabeled fragments were included in the binding reaction (Fig. 5, lanes 3 and 9). The complexes formed
are sequence-specific because they were not depleted by the addition of
a 50-fold molar excess of nonspecific pBluescript competitor DNA (Fig. 5, lanes 5 and 10). Interestingly, when
the distal fragment was used as competitor DNA in the assay with
labeled proximal fragment (Fig. 5, lane 4), complex A
and most of complex B were depleted. If the proximal fragment was used
as competitor DNA in the assay with labeled distal fragment, the A` and
C` and some of the B` complexes were depleted (Fig. 5, lane
8). These results indicate that some proteins bound to the
proximally located elements may be identical or similar to those
binding to the more distally located elements.
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.
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.
Delineation of the 5`-End of the CHO top II
To search for a DNA
sequence important for in vivo promoter activity, constructs
with nested 5`-deletions of the 5`-flanking sequence through the
residue immediately upstream of the ATG codon were fused to the CAT
coding sequence and transfected into CHO cells. CAT activities were
assayed from the cell lysates (Fig. 8). pCAT-0 (construct with
deletion of sequence upstream of the transcriptional start site) and
pCAT-49 (construct with an intact GC box) did not have any measurable
promoter activity (compare Fig. 8, lane 1, the negative
control, with lanes 2 and 3), suggesting that the GC
box alone is not sufficient for promoter activity and additional
upstream elements are required. pCAT-152 (Fig. 8, lane
4) had promoter activity of about 25% that of the other constructs
with more upstream sequence. With only 34 bp of additional upstream
sequence, the construct pCAT-186 elicited near maximum promoter
activity which is similar to constructs with more upstream sequence
(compare Fig. 8, lane 5 with lanes 6, 7, 8, and 10). All constructs yielded CAT
activity of about one-third that directed by the simian virus 40 early
promoter (Fig. 8A, lane 11). Hence, the
5`-limit of the top II Promoter
and the Functional Analysis of the ICEs gene promoter can be localized
between the -186 and -152 region. Interestingly, the
pCAT-1000 plasmid (lane 9) consistently resulted in about 50%
decrease in promoter activity.
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%.
-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.
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. 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) . 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. 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. 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.
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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