From the Division of Hematology/Oncology, Cedars-Sinai Research Institute/UCLA School of Medicine, Los Angeles, California 90048
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ABSTRACT |
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Cyclin A1 is a recently cloned cyclin with high
level expression in meiotic cells in the testis. However, it is also
frequently expressed at high levels in acute myeloid leukemia. To
elucidate the regulation of cyclin A1 gene expression, we
cloned and analyzed the genomic structure of cyclin A1. It
consists of 9 exons within 13 kilobase pairs. The TATA-less promoter
initiates transcription from several start sites with the majority of
transcripts beginning within a 4-base pair stretch. A construct
containing a fragment from A growing family of cyclin-dependent kinases
(Cdk)1 regulates a wide
variety of cellular pathways (for review, see Ref. 1). Cdc 2 (Cdk 1)
and Cdk 2 play a central role in the cell cycle of mammalian cells.
Substrate specificity and activity of Cdks are controlled by their
interaction with different cyclins that trigger the initiation of cell
cycle events (2). Cdk 2 specifically interacts with cyclin E for
G1/S progression and with cyclin A during S and
G2/M phases. Cyclin B3 might be another partner for Cdc 2 and Cdk 2 during the G2/M phase (3). In accordance with their central role in mammalian cell cycle regulation, the levels of
cyclins A and E (among other cyclins) oscillate in most if not all
proliferating mammalian cells. Disruption of the murine cyclin
A2 (the homolog of human cyclin A) leads to early
embryonic death suggesting an essential role for this gene in embryonic cell cycle in mammals (4). The human cyclin A2 (also known as cyclin A) was initially cloned because its gene locus was
the site of integration by the hepatitis B virus in a case of
hepatocellular carcinoma (5). It has also been implicated to be
important in the recurrence of hepatocellular carcinoma (6).
Recently, we cloned a second human cyclin A-like partner for Cdk 2, termed cyclin A1, that exhibits a highly restricted pattern of
expression (7). The high level tissue-specific expression of the human
and murine cyclin A1 in testis suggests a specific role in
meiosis (7, 8). Very low levels are detected in other tissues by
reverse transcriptase-PCR; however, high levels of human cyclin A1 were
also found in acute myeloid leukemia cell lines (7) and myeloid
leukemia samples from patients (9). This intriguing observation might
suggest a possible role for cyclin A1 in proliferation and
differentiation of hematopoietic progenitors and/or in promotion of
growth of leukemic cells.
Cyclin A1 shows homology to cyclin A2 and forms in vivo
complexes with Rb as well as with E2F (10). Cyclin A1-Cdk 2 complexes phosphorylate these substrates in vitro (10). Our data
showing that cyclin A1 is expressed in hematopoietic progenitors (9) and interacts with Rb family members and E2F, suggest that it may
affect cell cycle progression in expressing cells.
The pattern of cyclin A1 expression indicates that the
regulation of its expression is different from that of cyclin A2.
Furthermore, overexpression of cyclin A1 in myeloid leukemia originates
at the transcriptional level. To elucidate the transcriptional
mechanisms that underlie the tissue-specific pattern of expression, we
cloned and analyzed the genomic organization of the cyclin
A1 gene and its promoter region. The highest transcriptional
activity was assigned to a 335-bp fragment that required intact GC
boxes located between Cloning of the Genomic Fragment of the Human Cyclin A1--
The
cyclin A1 gene was cloned by screening a genomic Fix II
lambda library made from placenta (Stratagene) using the cyclin A1
cDNA as a probe (7). Of the several phage clones obtained, one
contained all the exons and included a 1.3-kb region upstream of the 5'
end of the cDNA. A 2.2-kb NotI-BamHI fragment
from the 5' end of the gene was subcloned into the pRS316 cloning
vector. The construct was further digested using SmaI; and
three fragments were subcloned into PUC19. The fragments were sequenced
in both directions using cycle sequencing and an automated sequencer
(ABI373) or Sequenase 2.0 (Amersham Pharmacia Biotech). The positions
and lengths of the introns were determined by PCR amplification of the
entire cyclin A1 coding region with different primers
(detailed primer information will be provided on request).
Subsequently, PCR products were either subcloned using pGEM-T-Easy
(Promega) or directly sequenced using cycle sequencing. Boundaries of
the ~4.5-kb intron 2 were determined by direct sequencing of the
lambda phage clone.
Generation of Luciferase Reporter Constructs--
The initial
luciferase constructs were generated by PCR amplification of the pRS316
plasmid containing the 2.2-kb cyclin A1 fragment. A BglII
site at the 5' end and a BamHI site at the 3' end were
introduced and the Pfu-amplified fragment was cloned into
the BglII site of PGL3-Basic. The +145 fragment was
generated to include the potential E2F site at +138. The ATG in the
primer (the initiating codon for cyclin A1) was mutated to ATT to avoid the initiation of translation. All constructs were confirmed to have
the correct sequence by DNA sequencing. The 5' deletions were generated
by exonuclease III treatment using
KpnI/SacI-digested PGL3-Basic containing the
Cell Culture and Transfection--
HeLa cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum containing 100 units/ml penicillin and 100 µg/ml streptomycin.
For transfection, 5 × 105 cells were seeded into
60-mm plates 16 h before transfection. Transfection was carried
out using LipofectAMINE (Life Technologies, Inc.) according to the
manufacturer's protocol. Two µg of luciferase reporter plasmid was
transfected together with 300 ng of a CMV- Cell Cycle-dependent Promoter Activity--
HeLa
cells were transfected using LipofectAMINE as described above. After
transfection, cells were cultured in 0.1% fetal calf serum containing
medium. After 16 h, medium was exchanged, and cells were
synchronized essentially as described (11). Cells were arrested in
G1 by serum starvation (0.1% fetal calf serum), in early S
phase by aphidicolin (2 µg/ml), and in S phase by aphidicolin treatment and release into fresh medium 6 h before harvest. Cells were arrested in G2/M phase by nocodazole (0.1 µg/ml).
Appropriate synchronization was confirmed by DNA quantitation using
flow cytometry, and the experiments were performed at least three
times. For the cell cycle release experiments, HeLa cells were arrested
using aphidicolin as described above, and cells were harvested at the different time points after their release in fresh medium. The time
course experiments were independently performed two times. To analyze
cell cycle-regulated activity of different constructs, HeLa cells were
arrested by serum starvation for 36 h followed by aphidicolin
arrest and subsequently released for 18 h. At this time point,
most cells were in late S or in G2/M phase. The longer serum starvation of the cells led to better synchronization and a
higher induction after release. The release experiments were independently performed at least three times. All luciferase values were normalized using RACE and Primer Extension--
The rapid amplification of 5'
cDNA ends (RACE) was performed using a 5'-RACE system (Life
Technologies, Inc.). The procedure was performed as suggested in the
manufacturer's protocol using RNA of the myeloid leukemia cell lines
ML1 and U937. RNA was reversed-transcribed using the primer
5'-CCCTCTCAGAACAGACATACA (positions +981 to +961 of the cDNA) and
Superscript II reverse transcriptase (Life Technologies, Inc.).
Gene-specific cDNA was PCR-amplified using the gene-specific primer
5'-CTGATCCAGAATAACACCTGA (positions +460 to +440 of the cDNA) and
the universal 5'-RACE Abridged Anchor Primer
5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG. PCR amplifications from both
RNA samples yielded a single band of ~450 bp. The entire PCR product
was phenol/chloroform-extracted, precipitated using
NH4+ acetate, and finally cloned into
pGEM-T-Easy and sequenced.
The primer extension assay was carried out by reverse transcription of
10 µg of RNA (U937) using a 32P-labeled primer
5'-CTCCTCCCACCAGACAGGA corresponding to +97 to +79 on the cDNA.
Hybridization was carried out overnight at 58 °C. Superscript II was
used for reverse transcription at 42 °C for 50 min. Extension
products were resolved on a 8% sequencing gel with a sequencing
reaction being run in parallel. As negative controls, we used tRNA and
a sample without RNA.
Electrophoretic Mobility Shift Assays--
Nuclear extracts from
HeLa cells were prepared as described (12). For gel retardation
experiments, 1 ng of 32P-labeled double-stranded
oligonucleotides containing either GC boxes 1 + 2 (5'-CCTGCCCCGCCCTGCCCCGCCCAGCC) or GC boxes 3 + 4 (5'-CCTTCCCCGCCCTGCCCCGCCCGGCCC) were incubated for 20 min at room
temperature with 5 µg of HeLa nuclear extract. The final reaction
contained 10 mM Tris-HCl, pH 7.5, 5% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 100 mM NaCl, and 1 µg of
poly(dI-dC)·poly(dI-dC). For competition experiments, 100 ng of
double-stranded oligonucleotide containing either a Sp1 consensus site
(5'-ATTCGATCGGGGCGGGGCGAGC), the oligonucleotide used for gel
retardation (see above), or a nonspecific oligonucleotide
(5'-GAGACCGGCTCGAACGCAATCATGT) was preincubated for 15 min at room
temperature with the nuclear extracts before the addition of the
labeled oligonucleotide. For supershift experiments, 2-3 µg of
polyclonal antibody against Sp1 (Pep2, Santa Cruz Biotechnology) or Sp3
(D20, Santa Cruz Biotechnology) was preincubated with the nuclear
extracts. Reactions were loaded on a 0.5× TBE, 4% nondenaturing
polyacrylamide gel and run for 2-3 h at 10 V/cm. Gels were dried and autoradiographed.
Site-directed mutagenesis was performed according to the method from
Deng and Nickoloff (13) using the Transformer site-directed mutagenesis
kit (CLONTECH). In brief, phosphorylated
oligonucleotides containing the desired mutation were annealed on the
single-stranded PGL3-Basic plasmid (containing the fragment Genomic Cloning and Gene Structure of the Human Cyclin A1
Gene--
A human genomic lambda phage library was screened using the
cDNA of cyclin A1 as a probe. Several clones containing pieces of
the gene were obtained, and one clone with a 14.5-kb insert contained
the entire gene. A 2.2-kb fragment at the 5' end of the gene was
subcloned and sequenced (see "Materials and Methods"). The 2.2-kb
fragment contained the first intron and parts of exon 2. The other
exon-intron boundaries were analyzed by PCR amplification and
sequencing using sets of primers that span the entire coding region.
The human cyclin A1 gene consists of 9 exons and 8 introns that extend over ~13 kb (Fig. 1). All
the exon-intron boundaries adhere to the consensus sequence. The
translation initiation codon is located in the first exon. A
second in-frame ATG that resembles the starting codon of the murine
cyclin A1 is found in exon 2, 50 nucleotides downstream of
intron 1 of the human cyclin A1 gene.
Analysis of Transcription Start Sites--
Transcription start
sites were determined using primer extension analysis and 5'-RACE (Fig.
2). Both methods demonstrated the
existence of several transcription start sites. The PCR product from
the RACE reaction consisted of a single band of ~450 bp. Sequencing
of the inserts after cloning revealed that 80% of the RACE products
(20/25) started from a 4-base pair stretch, and thus the predominant
start site was assigned +1 (Fig. 2A). This site is 130 bp
upstream of the translation initiating ATG codon. Primer extension
analysis identified the same start sites, but minor products were also
seen further upstream (Fig. 2B). The major start site
coincides with the RACE results of the 5' end of the initially
described cDNA clone (7). We also looked for transcription start
sites upstream of the second ATG in intron 1. However, neither RACE
clones nor primer extension assays showed evidence for a second
transcript in myeloid leukemia cells (data not shown).
Potential Transcription Factor Binding Sites in the 5'-Upstream
Region--
Genomic sequences 1299 bp upstream of the transcription
start site were cloned and sequenced. No TATA box was found in
proximity to the putative transcription start site. The main
transcriptional start site is likely to function as an initiator region
(Inr) since the sequence "CCAGTT" is very similar to the consensus
Inr sequence "TCA (G/T) T (T/C)" (14). No DPE element was found downstream of the main transcriptional start site (14). Several potential binding sites for transcription factors occur within the
sequence (Fig. 3). An E2F site is located
at +140 and another possible site at +68. A site that resembles the
cycle-dependent element (CDE) of the cyclin A2
promoter was found at Functional Analysis of the Basal Activity of the Cyclin A1
Promoter--
Portions of the cyclin A1 promoter were
Pfu PCR-amplified and cloned into the promoterless
PGL3-Basic Luciferase vector. Promoter activity was analyzed after
transient transfection into HeLa cells. The construct containing
nucleotides from
Deletions from the 5' end were made for the Role of Sp1 and GC Boxes for Transcriptional Activity of the Cyclin
A1 Promoter--
TATA-less promoters frequently depend on GC boxes to
activate transcription (16, 17). One of the main factors binding to
these sites are Sp1 family proteins (18-20). The cyclin A1
promoter contains at least six potential GC boxes between 190 and 37 bp upstream of the transcription start site. To analyze the importance of
Sp1 for the activity of the cyclin A1 promoter, various
promoter constructs were transfected into the Drosophila
cell line S2 which lacks endogenous Sp1 and Sp3 (Fig.
5). When transfected alone, the activity
of all cyclin A1 promoter fragments was not significantly different from the control (Fig. 6,
dotted bars). The addition of an Sp1 expression plasmid
strongly activated transcription by 15-25-fold from the cyclin
A1 promoter (Fig. 5, solid bars). Interestingly,
increased transcriptional activity was observed only for constructs
containing sequences starting between
The relevance of these GC boxes for promoter activity was further
studied by mutational analysis. Point mutations were made in each GC
box. Each mutant was tested either alone with the remaining sites
unaltered or in combination with the other mutant sites. Luciferase
analyses demonstrated that a mutation in either GC box 1 or 2 reduced
promoter activity by about 40 and 75%, respectively, whereas a single
mutation of either GC box 3 or 4 did not have a major effect on
promoter activity (Fig. 7). Mutation of
GC box 1 and 2 together decreased promoter activity by 85%. The
presence of at least one of the two upstream GC boxes (boxes 3 and 4)
being intact was essential for cyclin A1 promoter activity as mutations in both reduced promoter activity by about 80%. Mutations of all four
GC boxes reduced activity of the cyclin A1 promoter by
95%.
Cell Cycle Regulation of Promoter Activity--
The concentration
of cyclins varies during the cell cycle, and one mechanism of their
regulation occurs at the transcriptional level (21). To analyze cell
cycle regulation of promoter activity, transiently transfected cells
were arrested in different phases of the cell cycle and were
subsequently analyzed for luciferase activity. Cell cycle-regulated
activity was found for the full-length promoter as well as for the
construct containing the
To define the regions that are relevant for cell cycle regulation of
the cyclin A1 promoter, we generated mutations in the presumed E2F sites and the suspected CDE. We also generated by PCR a 3'
deletion construct (
Hence, these E2F sites and the inverted CDE are unlikely to play a role
in cell cycle regulation of the promoter. Analysis of 5' deletions and
the constructs containing the mutated GC boxes revealed that the four
GC boxes are essential for cell cycle regulation (Fig. 9).
Interestingly, the activity of the construct containing the mutated GC
boxes showed 60% of the activity of the wild type reporter construct
in G1 phase. However, the activity of the construct failed
to increase when cells entered S phase and showed only 4% of the wild
type cyclin A1 promoter activity. Similar data were obtained
for the 5' deletion lacking the four GC boxes (Fig. 9).
Cyclin A2 (formerly cyclin A) is ubiquitously expressed in
proliferating cells and is required for cell cycle progression (11, 22,
23). A second cyclin A-like protein, cyclin A1, shows a highly
restricted pattern of expression suggestive not only of specific
functional activities but also of distinct mechanisms of regulation (7,
8). The human cyclin A1 gene and its promoter were cloned
from a genomic library to elucidate further the regions that regulate
expression. Similar to other cell cycle regulatory genes (24-27), the
cyclin A1 promoter does not possess a TATA box motif. The
nucleotides surrounding the transcriptional start site are likely to
function as an initiator. In addition, the upstream region contains a
GC-rich region with multiple Sp1-binding sites that are essential for
transcription from the cyclin A1 promoter. In contrast,
predicted GC boxes in the cyclin A2 gene are located more
than 120 bp upstream of the most 3'-transcriptional start site, and
these have not been shown to be essential for gene expression.
Three potential binding sites for Myb proteins are present within 100 bp of the transcription start sites of the cyclin A1 gene,
and one of them is located at +2. No consensus Myb sites have been
found for either the murine or human cyclin A2 promoter (24,
28). The Myb-binding sites may play an important role in expression of
cyclin A1 during spermatogenesis as well as hematopoiesis.
One major finding of this study demonstrates the importance of the GC
boxes and the Sp1 family transcription factors in the regulation of
cyclin A1 expression. Six GC boxes were found in the first 200 bp
upstream of the transcription start site. The 5' deletions revealed
that omitting the four GC boxes between The finding that these GC boxes are essential for expression of the
cyclin A1 gene raises some interesting questions. Is Sp1, as
the main activating factor of the Sp1 family, involved only in the
basic transcriptional activation of the cyclin A1 gene, or
do Sp1 family proteins also play a role in the tissue-specific expression of this gene? Sp1 has been shown to serve distinct roles in
transcriptional activation as follows: it can directly interact with
the basal transcription complex (29), and it can determine the
transcription start site in TATA-less promoters (16). However, Sp1 can
also function as a more general transcriptional activator. Whereas
these functions are not necessarily exclusive, recent studies
demonstrated that Sp1 and its other family members can play an
important role in directing tissue-specific expression (30-32). Levels
of the ubiquitously expressed Sp1 vary up to 10-fold in different
tissues (33). This could provide a basis for directing tissue-specific
expression, especially if the affinity of the cis-acting
Sp1-binding sites differ. For example, induction of Sp1 was found to be
associated with differentiation of embryonal carcinoma cells, and Sp1
was causally linked to expression of the fibronectin gene, providing
evidence for a role of Sp1 in differentiation (34). In adult tissue,
high levels of Sp1 have been reported in hematopoietic progenitors and
in the later stages of spermatogenesis (33). This pattern does not
entirely coincide with the much more restricted expression pattern of
cyclin A1, but Sp1 is obviously expressed at high levels in tissues
where cyclin A1 expression is found (8, 9). In addition, Sp1 was found to bind in vivo to two myeloid-specific promoters only
in myeloid cells and was thus implicated in directing
myeloid-specific promoter activity (31, 35).
Another mechanism of tissue-directed expression depends on the ratio of
Sp1 family members to each other resulting in either activation or
repression of transcription (19, 36). We showed that Sp3 can bind to
the GC boxes in the cyclin A1 promoter. The Sp3 protein is
known to function either as transcriptional activator or repressor
depending on the context of the binding site in the promoter (37, 38).
When Sp3 binds to a single site, it can activate transcription, but
binding to multiple sites can lead to strong transcriptional repression
(36).
Gene expression from the cyclin A1 promoter is cell
cycle-regulated with the lowest promoter activity found in
G0/G1 and highest activity during the S and
G2/M phases. Fragments containing nucleotides Further analyses of the mechanism of cell cycle regulation revealed
that the four GC boxes are critical for cell cycle-regulated transcription from the cyclin A1 promoter. No increase in
promoter activity in S phase was found when the four sites were
mutated. Interestingly, the effect of the GC box mutations on promoter activity in G1 phase was not prominent with only a 40%
reduction compared with the wild type construct. These findings are
consistent with repression of Sp1-mediated activity in the
G1 phase of the cell cycle. Selective repression of
Sp1-mediated activity by Sp3 has been demonstrated to be relevant in
cell cycle-regulated promoters containing several Sp1 sites. The
dihydrofolate reductase promoter contains four Sp1 sites and is
specifically repressed by Sp3 (36). Besides repression by Sp3, other
mechanisms probably contribute to repression of the cyclin
A1 promoter in G1. Studies have shown that repression
of glutamine-rich activators such as Sp1 and NF-Y is the predominant
mechanism of cell cycle regulation for several promoters (39, 40).
However, none of the known repressor elements (CDE, CHR, E2F) appears
to be relevant for the cyclin A1 promoter. Similar to the
cyclin A2 gene, two potential E2F sites are downstream of
the transcriptional start site of cyclin A1. These E2F sites are not
required for repression of cyclin A2 transcription in the
G1 phase (15, 28). The introduction of mutations in these sites in the cyclin A1 promoter did not alter cell cycle regulation. Further evidence that these E2F sites are not relevant for cell cycle
regulation was shown using a 3' deletion ( Besides the Sp1 family members, other mechanisms are likely to be
important for expression of cyclin A1 in vivo. So far, the promoter of cyclin A1 showed activity in all the cell lines
that we have analyzed including MCF-7, PC3, Cos-7, U937, KCL22, Jurkat, and others. Most of these cells expressed relatively low levels of
cyclin A1 mRNA. Three possibilities could explain the discordance between cyclin A1 expression in vivo and promoter activity
in vitro. First, regulatory sequences further upstream of
the cloned fragment could repress expression in vivo in
selected types of tissue. However, negatively regulating sequences are
often located within a few hundred bases of the transcription start
site as is the case for cyclin A2 (15, 41, 43) including those for many
myeloid-specific genes (31, 44). In addition, preliminary data from a
transgenic murine model suggest that the cyclin A1 promoter
fragment Taken together, we have cloned and analyzed the genomic organization of
the human cyclin A1 gene and its promoter region. The
analyses of the promoter region revealed that the cyclin A1 promoter critically depends on four GC boxes upstream of the
transcriptional start site. The promoter is cell cycle-regulated with
maximum activity in the S and G2/M phases. Our data suggest
that cell cycle regulation depends on periodic repression of promoter
activity in the G1 phase. The binding of both Sp1 and Sp3
implicates Sp1 family members in the regulation of tissue-specific and
periodic expression of the cyclin A1 gene.
190 to +145 showed the highest
transcriptional activity. Transfection of cyclin A1
promoter constructs into S2 Drosophila cells demonstrated
that Sp1 is essential for the activity of the promoter. Sp1, as well as
Sp3, bound to four GC boxes between nucleotides
130 and
80 as
observed by gel shift analysis. Mutations in two or more of the four GC
boxes decreased promoter activity by >80%. The promoter was found to
be cell cycle-regulated with highest activities found in late S and
G2/M phase. Further analyses suggested that cell cycle
regulation was accomplished by periodic repression of the GC boxes in
G1 phase. Taken together, our data show that cyclin
A1 promoter activity critically depends on four GC boxes, and
members of the Sp1 family appear to be involved in directing expression
of cyclin A1 in both a tissue- and cell cycle-specific manner.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
60 and
120 bp upstream of the main
transcriptional start sites. These sites are also essential for cell
cycle regulation of the promoter.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1299 to +145 fragment and the Erase-a-base kit (Promega). The end
points of the deletions were determined by sequencing. The
37
fragment was constructed by digesting the
190 to +145 containing
PGL3-Basic with NaeI and HindIII and subsequent cloning of the 200-bp fragment into PGL3-Basic digested with
SmaI and HindIII.
-gal expression vector
used for standardization. Cells were harvested and assayed for
luciferase and
-galactosidase activity after 48 h. All
experiments were carried out in duplicate and were independently performed at least three times. Data of luciferase assays are shown as
mean ± S.E. of three independent experiments unless stated otherwise. The Drosophila cell line SL2 was obtained from
ATCC and grown at room temperature in Schneider's insect cell medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Insect cells were transfected using Superfect (Qiagen). Briefly, 5 × 105 cells were seeded into 6-well plates and the
Superfect/DNA mixture was added dropwise. One µg of the luciferase
reporter was transfected with or without 100 ng of the Sp1 expression
vector pAC-Sp1, which was a kind gift from Dr. E. Stanbridge
(University of California, Irvine). Luciferase activity was analyzed
after 48 h. Luciferase values could not be standardized using
-galactosidase activity because the viral promoters in the available
plasmids also depended strongly on Sp1 for adequate expression. All
experiments were carried out in duplicate and independently performed
at least three times.
-galactosidase activity as described above.
190 to
+145) together with the oligonucleotide 5'-AATCGATAAGAATTCGTCGACCGA
that changes the unique BamHI site to an EcoRI
site. The complementary strand was extended and completed from the
annealed oligonucleotides using T4 polymerase and T4 ligase. Selection
for the mutant plasmid was performed by two rounds of digestion with
BamHI and subsequent transformations first into the
repair-deficient strain BMH 71-18 mutS and finally into
DH5
. The entire promoter fragment was sequenced to verify desired
mutations and to exclude second site mutations. Because of the short
distances between GC boxes 1 + 2 and 3 + 4, oligonucleotides were
designed to mutate both GC boxes simultaneously. Mutations in all four
GC boxes were introduced by simultaneously adding oligonucleotides 1 + 2 and 3 + 4. All oligonucleotides used in these experiments were
5'-phosphorylated. The following oligonucleotides were used (mutated
bases underlined): GC box 1, CCCCGCCCTGCCCCTTACAGCCGGCCACC; GC box 2, CCAACCCTGCCCTTACCTGCCCCG; GC box 3, CCCTGCCCCTTCCGGCCCGGCC; GC box 4, CTGCCCTTCCCTTCCCTGCCCC; GC boxes 1 + 2, GCCCAACCCTGCCCTTACCTGCCCCTTACAGCCGGCCACCTC; GC boxes 3 + 4, CTTCCCTGCCCTTCCCTTACCT
GCCCCTTACGGCCCGGCCCGGCC. The suspected CDE in the cyclin A1
promoter was mutated using the following oligonucleotide,
CCACCTCTTAACAAGCTTCCTCCAGTGCA.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Genomic organization and exon-intron
boundaries of the human cyclin A1 gene locus.
Exon-intron boundaries are indicated by capital letters for
coding sequences and lowercase letters for intronic
sequences. Preserved consensus sequences are underlined. The
entire sequence was obtained for the introns shown with an exact base
number; in all other cases, the intron length was estimated by the size
of the PCR product.
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Fig. 2.
Transcriptional start sites.
A, primer extension was carried out as outlined under
"Materials and Methods." Both a sample without RNA and a tRNA
sample (10 µg) were used as negative controls. B, the
primer extension products shown in A are indicated by an
asterisk above the appropriate nucleotide of the indicated
sequence. Starting points of the RACE products are indicated by an
arrow below the sequence. The number of RACE
clones (total 25) starting at a particular base is indicated by the
number shown below the arrows. The
site where 44% (11/25) RACE clones started was assigned +1.
28 (15). However, this element was located on
the antisense strand. No cell cycle genes homology region (CHR) was
found. Potential Myb sites are predicted at positions +2,
30, and
90. The nucleotide sequence contains two CpG islands of up to 90% GC
content reaching from
1000 to
700 and from
550 to
50. Multiple
GC boxes are found in this region, and six GC boxes grouped as three
double sites are located between nucleotides
150 and
45.
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Fig. 3.
5' upstream region of the human cyclin
A1 gene. Indicated are the first bases of the different
fragments as well as potential transcription factor binding sites
between 190 to +145. The transcriptional start site is marked with an
arrow and the translational initiation codon is
boldface.
1299 to +145 from the 5' cyclin A1
upstream region showed significant promoter activity when cloned in the
sense direction (Fig. 4). The same
fragment cloned in the opposite direction or a construct containing
solely exon 1 and intron 1 did not show promoter activity (data not
shown).
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Fig. 4.
Transactivation activity of promoter
fragments in HeLa cells. Activity of 5' deletion constructs was
analyzed in luciferase assays. Values are expressed as fold activation
(PGL3-Basic = 1), and means and S.E. of three independent
experiments are shown.
1299 to +145 fragment
using exonuclease III treatment (Fig. 4). Transient transfection and
subsequent luciferase assays revealed the strongest activity occurred
in the construct containing the fragment from
190 to +145 bp (Fig.
4). Both the
1299 and the
190 constructs exhibited promoter
activity in a variety of cell lines including Cos-7, MCF-7, U937,
KCL22, and Jurkat (data not shown). In all of these mammalian cell
lines, luciferase activities generated by the
190 construct were
higher than those by the
1299 construct. Constructs with a 5' end
containing less than 190 bp upstream of the transcription start site
showed a progressive loss of promoter activity. A construct containing
bp
37 to +145 showed only 2-fold higher activity than the
promoterless vector PGL3-Basic.
1299 and
112 bp upstream of
the transcription start site. The construct containing the nucleotide
sequences between
37 and +145 did not show any increase in activity,
suggesting that Sp1-binding sites between
112 and
37 are essential
for Sp1-mediated transcriptional activity of the cyclin A1
promoter in Drosophila cells. This region contains four GC
boxes that are grouped in two pairs (Fig. 3). To test whether Sp1 and
other Sp1 family members could bind to these sites, gel-shift
experiments were performed (Fig. 6). Several specific complexes in HeLa
cell nuclear extract bound to these sites (lanes 1 and
7). These complexes were competed away by an excess of cold
oligonucleotides containing either the original site (lanes
2 and 8) or an Sp1 consensus site (lanes 3 and 9). A 100-fold excess of a nonspecific oligonucleotide
did not alter complex binding (lanes 4 and 10).
Supershift experiments with antibody against either Sp1 or Sp3
demonstrated the presence of Sp1 in one complex (lanes 5 and
11) and the presence of Sp3 (lanes 6 and
12) in two other complexes. The composition of the fastest migrating complex is unknown.
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Fig. 5.
Activity of the cyclin A1
promoter fragments in the Drosophila cell line
S2. Activity is indicated as fold activation of PGL3-Basic as
compared with reporter gene activity without addition of Sp1 expression
plasmid. The punctated and solid bars represent
activities without and with Sp1 co-expression, respectively.
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Fig. 6.
Gel shift analysis of GC box binding proteins
in HeLa nuclear extract. Lanes 1-6 show complexes
binding to a 32P-end-labeled oligonucleotide containing GC
boxes 1 and 2, and the labeled oligonucleotide in lanes
7-12 contains GC boxes 3 and 4. Lanes 1 and
7 show binding of 5 µg of HeLa nuclear extract to the
respective oligonucleotide. Binding is competed away in lanes
2 and 8 with a 100-fold excess of cold Sp1 consensus
oligonucleotide and in lanes 3 and 9 by a
100-fold excess of cold oligonucleotides using either GC boxes 1 + 2 (lane 3) or GC boxes 3 + 4 (lane 9). A 100-fold
excess of a nonspecific oligonucleotide did not compete the specific
complexes away (lanes 4 and 10). Antibodies
against Sp1 were added to samples in lanes 5 and
11 and antibodies against Sp3 were present in reactions for
lanes 6 and 12. Identified complexes are marked
with an arrow, and the respective protein is named on the
left-hand side of the figure.
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Fig. 7.
Effects of GC box mutations on promoter
activity. Individual GC boxes or their combinations were mutated
and transiently transfected into HeLa cells. Activity of the wild type
construct containing nucleotides 190 to +145 was set as 100%. Wild
type GC boxes are indicated in white, and mutated GC boxes
are shown in black.
190 to +145 fragment. The cyclin
A1 promoter activity was relatively low during the
G0/G1 phase. It increased after the cell cycle
progressed beyond the G1/S boundary (Fig.
8A). The highest levels of
activity were observed in the S and G2/M phases. Recently,
we showed that RNA levels of cyclin A1 accumulated during S phase with
the highest levels present at the S and G2/M phases (10).
When transiently transfected HeLa cells were released from an
aphidicolin block, luciferase values started to increase after 6 h
and reached a maximum after 12-16 h (Fig. 8B). The maximum
promoter activity corresponded to the percentage of cells present in
the S and G2/M phases (Fig. 8C).
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Fig. 8.
Cell cycle-regulated activity of the
cyclin A1 promoter. A, HeLa cells were
cell cycle-arrested after transfection with a luciferase construct
containing nucleotides 190 to +145 of the cyclin A1 promoter. Cells
were subsequently analyzed for luciferase activity. Cell cycle
synchronization was confirmed by flow cytometry (data not shown). The
bars represent means and S.E. of at least three independent
experiments. Promoter activity at 0 h was set as 1. B,
following transient transfection and serum starvation, HeLa cells were
synchronized at the G1/S boundary using aphidicolin. Cells
were released from the block and harvested at the indicated time points
for luciferase and cell cycle analyses. The graph depicts
data from a representative experiment. C, cell cycle
distribution at the different time points of the time-release
experiment. The shaded, open, and solid
bars represent G1, S, and G2/M phases,
respectively.
190 + 13) that deleted the two presumed E2F sites
downstream of the transcriptional start site. Mutations in these two
presumed E2F sites, the mutation in the inverted presumed CDE and the
3' deletion, showed an indistinguishable pattern of cell cycle
regulation when compared with the wild type (Fig.
9 and data not shown).
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Fig. 9.
Effects of point mutations and deletions of
the cyclin A1 promoter on cell cycle-regulated
activity. Upper figure, activity of the wild type
construct in aphidicolin-arrested cells was set as 1.0 and compared
with the other constructs. Only a 40% decrease was detected for the
construct containing the four mutated GC boxes. Nucleotides in the
suspected CDE in antisense direction were mutated in the construct
called mutation 19 to
24 (for details see "Materials and
Methods"). The lower panel depicts the increase in
promoter activity after release from a G1/S block by
aphidicolin. Constructs lacking the four GC boxes either due to
mutation or 5' deletion are not induced upon entering the S phase. No
significant difference was observed between wild type and the mutation
19 to
24 construct.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
112 and
37 almost
completely abrogated promoter activity. Analysis of these fragments in
insect cells demonstrated that Sp1 can reconstitute cyclin
A1 promoter activity in all fragments that involve the GC boxes
1-4. These experiments show that Sp1 (or at least a member of the Sp1
family) is required for cyclin A1 promoter activity through
interaction with elements located between
112 and
37. A more
detailed analysis of the GC boxes 1-4 showed that the two closest to
the transcriptional start sites are most critical. Of the GC boxes 3 and 4, only one of these was necessary for a basal level of
transcriptional activity of the promoter. Gel-shift experiments
demonstrated that Sp1 and Sp3 can bind to GC boxes 1 + 2 and 3 + 4.
1299 to
+145,
190 to +145, or
190 to +13 performed similarly in these
experiments (data not shown). The levels of cyclin A1 mRNA and
protein paralleled the promoter activity (10). Both increased during S
phase and peaked at G2/M phase (10).
190 to +13) that showed
cell cycle regulation similar to the constructs containing both E2F
sites (data not shown). Likewise, an 8-bp sequence that resembles the
CDE of the human cyclin A2 gene was found in an antisense
direction at position
19 to
24 (TCGCGG) of the cyclin A1 promoter.
No significant differences in cell cycle regulation were found when
these nucleotides were mutated (Fig. 9). This is consistent with the
finding that these elements need to be in a 5'
3' orientation to be
functional (15, 41, 42). Taken together, our data regarding the
mechanisms of cell cycle regulation of the cyclin A1
promoter suggest that cell cycle regulation is accomplished by periodic
repression of the Sp1 site mediated activity. Repression is likely to
be accomplished by Sp3 and an as yet unidentified repressor mechanism
that does not depend on E2F, CDE, or CHR elements.
1299 to +145 is sufficient to direct tissue-specific expression.2 Second, some
genes (e.g. c-myb) that show limited or low
levels of expression in vivo are frequently expressed at
higher levels in cell lines (45-47). The broad range of expression of
such a transcription factor in cell lines could provide an explanation for the prominent promoter activity in cell lines, despite the limited
expression pattern in the tissues from which the cell lines were
derived. Third, methylation within the CpG island upstream of the start
site or variations in the chromatin structure might contribute to the
tissue-specific expression of cyclin A1 in vivo.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Eric Stanbridge for the pAC-Sp1 plasmid. We thank Dr. Adrian F. Gombart for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institutes of Health, the U. S. Army, and the C. and H. Koeffler and the Parker Hughes Funds.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number AF124143.
Recipient of a fellowship of the German Research Foundation (DFG).
To whom correspondence should be addressed: Davis Bldg., RM5066,
Division of Hematology/Oncology, Cedars-Sinai Research Institute/UCLA
School of Medicine, 8700 Beverly Blvd., Los Angeles, CA 90048. Tel.:
310-855-7736; Fax: 310-652-8411; E-mail: muellerc{at}CSMC.edu.
§ Holds the Mark Goodson Chair in Oncology Research and is a member of the Jonsson Cancer Center.
2 C. Müller, C. Readhead, and H. P. Koeffler, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: Cdk, cyclin-dependent kinases; RACE, rapid amplification of 5' cDNA ends; kb, kilobase pair; bp, base pair; PCR, polymerase chain reaction; CDE, cycle-dependent element; CHR, cell cycle homology region.
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REFERENCES |
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