(Received for publication, January 29, 1996; and in revised form, March 13, 1996)
Cyclin-dependent kinase 2 is a serine/threonine protein kinase
essential for progression of the mammalian cell cycle from G to S phase. CDK2 mRNA has been shown to be induced by
serum in several cultured cell types. Therefore, we set out to identify
elements that regulate the transcription of the human CDK2 gene and to characterize its structure. This paper describes the
cloning of a
2.4-kilobase pair genomic DNA fragment from the
upstream region of the human CDK2 gene. This fragment contains
five transcription initiation sites within a 72-nucleotide stretch. A
200-base pair sub-fragment that confers 70% of maximal basal promoter
activity was shown to contain two synergistically acting Sp1 sites.
However, a much larger DNA fragment containing
1.7 kilobase pairs
of upstream sequence is required for induction of promoter activity
following serum stimulation. The intron exon boundaries of seven exons
in this gene were also identified, and this information will be useful
for analyzing genomic abnormalities associated with CDK2.
Cyclin-dependent kinases (CDKs) ()are the catalytic
subunits of a family of serine/threonine protein kinase complexes that
are also composed of a cyclin regulatory
subunit(1, 2, 3) . Most members of the CDK
family are involved in regulating the progression of the eukaryotic
cell cycle at various stages throughout G
, S,
G
, and M phases(4) . Other CDKs are involved in
regulation of other processes in the cell, including phosphate
metabolism (5) and transcription(6, 7) .
CDK2 is a member of the CDK family whose activity is restricted to
the G/S phase of the cell cycle. Several experiments
demonstrated that CDK2 is essential for the mammalian cell cycle
progression; micro-injection of antibodies directed against CDK2
blocked the progression of human diploid fibroblasts into S
phase(8, 9) , and overexpression of a CDK2 dominant negative mutant in human osteosarcoma cells had a similar
effect(10) .
CDK2 is subject to an elaborate series of post-translational modifications. Although it has no kinase activity itself, kinase activity is conferred by association of CDK2 with a regulatory subunit, cyclin A or cyclin E, and by phosphorylation of Thr-160. Conversely, CDK2 activity is repressed by phosphorylation of Thr-14 or Tyr-15. Another layer of complexity is added to the regulatory scheme by CDK inhibitory proteins that can bind to CDK2 and inhibit the activity of the cyclin-kinase complex(4) .
While
much attention has been given to the post-translational regulation of
CDK2, we and others have found that CDK2 is also regulated at
the transcriptional level. Horiguchi-Yamada et al.(11) reported a 3-fold increase in CDK2 mRNA in
HL60 cells following stimulation with the phorbol ester
12-o-tetradecanoyl 13-acetate. Other groups (12) had
similar findings with serum-stimulated human keratinocytes and human
lung fibroblasts. Tanguay et al.(13) found induction
of CDK2 expression in primary B lymphocytes following anti-IgM
stimulation. These data suggest that transcriptional regulation of CDK2 could be important in the transition of cells from
G to S phase.
Our interest in CDK2 transcriptional regulation originated from our observation that
CDK2 protein is undetectable by immunohistochemistry in sections of
normal rat carotid arteries but is rapidly induced in smooth muscle
cells of rat carotid arteries after balloon injury(14) . This
manuscript reports the cloning of the human genomic DNA upstream of the
coding region of CDK2. Most (70%) of the basal transcriptional
activity of this promoter was localized to a 210-base pair (bp)
fragment. Two Sp1 sites in this region were shown to contribute
cooperatively to this transcriptional activity. The serum-induced
activity of the promoter is located in a 1.7-kilobase pairs (kb)
region starting 680 bp upstream of the most proximal transcription
initiation site.
Figure 1: Restriction map and amplification strategy of the upstream region of human CDK2 gene. The lower part of the figure shows a restriction map of the 5`-end and upstream region of the human CDK2 gene. Restriction sites are indicated. Solid arrows represent primers used in inverse PCR reactions. The position of intron I is indicated with a triangle. Fragment A is an inverse PCR product; unfilled arrows at the ends of this fragment indicate that the ends are joined. Fragment B is a PstI-AvrII subclone derived from fragment A.
Figure 5: DNase I protection assay of the CDK2 promoter. The nucleotide sequences of the protected regions are indicated on the left. Purified Sp1 protein (Promega) was used according to manufacturer's instructions. Poly(dI:dC) concentrations were 0.3 µg/µl. DNase I concentrations were 0.5 ng/µl (lanes 1), 0.75 ng/µl (lanes 2), 1.0 ng/µl (lanes 3), and 1.25 ng/µl (lanes 4). Panel A, wild type CDK2 promoter region; panel B, CDK2 promoter with Sp1 site I mutated (DSC67); panel C, CDK2 promoter with Sp1 site II mutated (DSC68).
Figure 2: Nucleotide sequence of the 5`-end region of the human CDK2 gene. The 5`-end of deletion derivatives and restriction enzyme sites are indicated above the nucleotide sequence. DNase I protected regions are boxed. Putative transcription factor binding sites are indicated below the sequence. Transcription start sites are indicated with horizontal arrows. The translated portion of the gene is indicated by the amino acid sequence below the nucleotide sequence.
Figure 3: Mapping of the human CDK2 transcription initiation site. Autoradiogram of RNase protection analysis. Lane 1, 20 µg of RNA from human umbilical vein cells; lane 2, 20 µg of RNA from yeast. Protected fragments and their sizes are indicated.
Figure 4: Deletion analysis of CDK2 promoter activity. Luciferase constructs are depicted on the left side. The 5`-end of each of construct relative to the proximal transcription start site is indicated. Luciferase activity was divided by the activity of the cotransfected SEAP-expressing construct to correct for differences in transfection efficiency (see ``Materials and Methods'') and is expressed as a percentage of DSC37 activity. Bars represent standard errors of the mean.
DNase I protection analysis of the
region contained in DSC40 using HeLa nuclear extracts identified two
protected regions, each of which contained Sp1-like binding sequences
(data not shown). To test the importance of these Sp1 sites, a
conserved GG sequence in each of the Sp1 sites was independently
mutated to AA. A DNase I protection assay (Fig. 5A)
demonstrated that the wild type DNA fragment was protected by purified
Sp1 protein from DNase I digestion at two distinct regions (I and II);
these regions were the same as those detected with HeLa nuclear
extract. Mutating each of these Sp1 sites individually resulted in loss
of protection in the mutated Sp1 site but did not affect Sp1 binding to
the remaining wild type Sp1 site (Fig. 5, B and C). Transient transfection of NIH3T3 cells with constructs
analogous to DSC409-17, except containing mutations in
either one of the Sp1 sites (Fig. 6, DSC67 and DSC68), generated
luciferase activity that was less than 25% of the activity generated by
the full-length CDK2 promoter construct (DSC37), or
approximately 30% of that generated by DSC40
9-17. These
results indicate that each of these Sp1 sites contributes to the
observed transcription activity. Moreover, it also suggests that these
sites act synergistically to generate transcriptional activity that is
greater than the sum of activities each site can generate by itself.
Figure 6: Mutational analysis of CDK2 basal promoter activity. Luciferase activity was corrected by dividing by the activity of the cotransfected SEAP-expressing construct (see ``Materials and Methods'') and is expressed as a percentage of DSC37 activity. Constructs are depicted on the left side, and the 5`-end of each construct relative to the proximal transcription start site is indicated. Bars represent standard errors of the mean.
Figure 7: Time course of serum induction of CDK2 promoter. NIH3T3 cells stably expressing constructs DSC37 (open circles) and DSC40 (closed circles) were serum starved for 72 h. Luciferase activity was determined at the end of starvation (time = 0) and at the indicated times after serum stimulation. Each point represents the average of three independent experiments. Bars represent standard errors.
Figure 8: Exon map of the human CDK2 gene. Boxes correspond in length to the exon size. The end of exon VII was not determined. Nucleotides are numbered relative to the most proximal transcription initiation site. Below the map, the exon/intron boundaries are aligned with each other and with the consensus splice acceptor and splice donor sequences. 100% conserved nucleotides are underlined.
In this study, we have cloned and sequenced the upstream
region of the human CDK2 gene and determined the transcription
start sites for this gene by ribonuclease protection assay. Five
transcription start sites spread over a 72-bp region were identified (Fig. 3). No consensus TATA box was identified in the entire
upstream sequence. Thus, this promoter falls into the category of
TATA-less promoters similar to all other cell cycle genes analyzed to
date including: cdc2(27) , cyclin A(28) ,
cyclin D1(29, 30) , cyclin D2 and cyclin
D3(31) , as well as Xenopus laevis cdk2(32) .
A YY1 box, which in some TATA-less promoters is responsible for
determining the transcription start site(26) , is present just
upstream from the three start sites located at positions +1,
-5, and -9. An Sp1 site was identified upstream of each of
the remaining transcription start sites (-33 and -71),
suggesting that these Sp1 regions may be responsible for localizing the
start of transcription at these sites (26) . Other putative
transcription factor binding sites were also identified (Fig. 2). The presence of a c-Myb binding site is intriguing
since c-myb was shown to transactivate the closely related
human CDC2 gene(33) . This could indicate that a
transcription factor that positively regulates a G event,
like CDC2 induction, might also regulate a G
event
such as CDK2 induction. Two putative p53 binding sites were
identified within 200 bp of the 3` or most proximal transcription start
site. p53 is a known tumor suppressor gene that has been
postulated to be involved in induction of cell cycle arrest. It is
perplexing to assume that p53 would induce CDK2 since
this induction would most likely result in an accelerated cell cycle
rather than a cell cycle arrest. Interestingly, a p53 site was also
identified in the promoter region of the cyclin A gene, a regulatory
partner of CDK2(28) . Further investigation of the
possible involvement of p53 in CDK2 regulation is required.
Functional analysis of the promoter region revealed that a construct
(DSC409-17) that contains DNA extending from nucleotide
-100 to +108 is sufficient for strong basal promoter
activity (about 30% of the SV40 early promoter, data not shown). DNase
I footprint analysis of the CDK2 upstream region with HeLa
nuclear extract (data not shown) revealed only two protected regions,
both of which are Sp1 like sites, contained within the
DSC40
9-17 clone. Further analysis indicated that these sites
in fact bind purified Sp1 protein (Fig. 5). Furthermore,
individually mutating each site abolished the DNase I protection only
in the mutated site but not in the adjacent wild type site. This
information indicates that Sp1 can bind to each of these sites in an
independent fashion. The transcriptional activity of reporter gene
constructs equivalent to DSC40
9-17, but with individually
mutated Sp1 sites (Fig. 6, DSC67 and DSC68), was less than 25%
of the activity generated by the full-length CDK2 promoter
construct (DSC37) and approximately 30% of that generated by
DSC40
9-17. This suggests that each of these Sp1 sites
contributes to the basal activity of the CDK2 promoter. It
also suggests that their combined effect is synergistic, since both
sites generate transcriptional activity that is greater than the sum of
the activities generated by each site independently.
The level of CDK2 mRNA induction following stimulation of quiescent cells
was reported to be
2-3-fold(11, 12, 13) . Our attempts to
detect this low level of serum-induced promoter activity using a
transient transfection cell culture system produced ambiguous results,
presumably because there is plasmid loss over time, and this loss masks
the serum-induced promoter activity of the retained plasmids. To
overcome this problem, NIH3T3 cell lines stably expressing the
luciferase enzyme driven by various CDK2 promoter constructs
were established. The basal luciferase activity of the cell lines in
this study was comparable; however, only cells which contained about
2.4 kb of the upstream region of the CDK2 gene (DSC37) were
induced by serum. The level of induction following serum starvation and
maximal growth factor stimulation was about 3-fold, as was expected
from the published literature and our own unpublished observations. The
next longest deletion derivative, DSC40, which expressed full basal
promoter activity in a transient transfection assay, was not induced by
serum and growth factor stimulation. These data suggest that the
information needed for serum induction resides in a 1.7-kb
segment, which starts 682 nucleotides upstream of the most proximal
transcription start site.
We found that the human CDK2 gene is made up of at least seven exons. However, our characterization would not detect exons located 3` to position 1295 in the published cDNA sequence(15) . All the intervening sequences that were identified are contained within the coding region of the gene. Exon I is longer in CDK2 than in the characterized CDC2 genes (27, 34) and is conserved in X. laevis cdk2(32) . Other differences between the CDK2 and the CDC 2 gene structure in
clude two
additional introns located at amino acids 105 and 196 of the human CDK2 gene that are not present in the Sacchromyces pombe
CDC2 gene. The CDK2 gene structure and sequence
information published here may be useful for designing primers to
investigate possible CDK2 gene mutations and rearrangements.
Although CDK2 has not been implicated in oncogenic
transformation, one of its regulatory partners, cyclin A, has been
implicated in human hepatocellular carcinoma(35) , and its
other regulatory partner, cyclin E, has been shown to accelerate
G progression if overexpressed(36) . It is thus
plausible to assume that CDK2 mutations might play a role in
malignancy and may prove worthwhile targets for exploration of genetic
instability in tumors.
In summary, the elements required for basal
expression and serum induction of the human CDK2 promoter were
localized to a 2.4-kb fragment. Basal level expression of the CDK2 promoter is fully contained within 290 bp upstream of the
most proximal transcription start site (DSC40
6-3), and
approximately 70% of the activity can be generated by a 200-bp fragment
containing only 100 bp upstream of the most proximal transcription
start site. Two Sp1 DNA binding sites identified in this region
synergistically contribute most of the basal promoter activity of this
region. The elements required for serum inducibility lie about 700 bp
further upstream and are contained in a
1.7-kb fragment. Multiple
sites with homology to known transcription factor binding sites are
located in the promoter region of the human CDK2 gene. Further
analysis of these sites and their corresponding transcription factors
is necessary for a more complete understanding of the transcriptional
regulation of this gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U50730[GenBank].