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(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
Cyclin-dependent kinases (CDKs) ( CDK2 is a member of the CDK family whose activity is restricted to
the G 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 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
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 DSC40
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 Functional analysis of the promoter region revealed that a construct
(DSC40 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 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 In summary, the elements required for basal
expression and serum induction of the human CDK2 promoter were
localized to a
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U50730[GenBank].
Volume 271,
Number 21,
Issue of May 24, 1996 pp. 12199-12204
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
PROMOTER ANALYSIS AND GENE STRUCTURE (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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) . /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) . to S phase.1.7-kilobase pairs (kb)
region starting 680 bp upstream of the most proximal transcription
initiation site.
Plasmids and Constructs
pGL2-Basic (Promega) was
used to generate luciferase reporter gene constructs. pCMV/SEAP
(Tropix), which contains the secreted alkalaine phosphatase (SEAP) gene
driven by the cytomegalovirus (CMV) promoter, was used in
cotransfection experiments. pBR-
-Puromycin, a plasmid expressing
the puromycin resistance gene driven by the -actin promoter, was a
kind gift of L. Lee of the S. N. Cohen Lab (Stanford University) and
was used to generate stably transfected cell lines by cotransfection.
DSC34 was generated by cloning a 2.4-kb AvrII-PstI fragment (Fig. 1, fragment
B) from inverse PCR-amplified fragment A into pUC19 (New England
Biolabs) digested with PstI and XbaI. DSC36 was
generated by cloning a blunt-ended 2.4-kb PstI-Asp718 fragment of DSC34 into HindIII-digested, blunt-ended pGL2-Basic, such that the CDK2 promoter directs transcr
iption away from the luciferase
gene. DSC37 was constructed the same way as DSC36, except that the CDK2 promoter directs transcription toward the luciferase
gene. DSC40 was generated from DSC37 by deleting from the BamHI site in the insert to a BglII site in the
poly-cloning region of pGL2-Basic. DSC40
4-1,
DSC406-3, DSC409-17, DSC4010-10, and
DSC4010-16 were generated by exonuclease III/mung bean
nuclease deletions (15) using NheI/SacI-digested DSC40. The end points of the
deletions were determined by sequencing. DSC42 was generated from DSC37
by deletion of a BglII-Eco47III fragment. DSC51 was
generated from DSC40 by deleting an Eco47III-Bsp120I
fragment. DSC67 and DSC68 were generated from DSC409-17 by
site-directed mutagenesis (see below).
PCR Amplifications
The positions of the 5`-end of
all primers are given in numbers corresponding to the published cDNA
sequence(16) . Inverse PCR (17) was carried out using
primers 5`-GCCAGAAACAAGTTGACG-3` (231, sense) and
5`-ACACAACTCCGTACGTGC-3` (225, antisense) as described(18) .
For intron exon mapping, the following pairs of primers were used:
introns I and II, 5`- GCCAGAAACAAGTTGACG-3` (231, sense) and
5`-GATGAGGGGAAGAGGAATG-3` (481, antisense); intron III,
5`-CTGCTGGATGTCATTCAC-3` (365, sense) and 5`-AGTACGAACAGGGACTCC-3`
(644, antisense); introns IV and V, 5-CTAGCTTTCTGCCATTCT-3` (513,
sense) and 5`-GGAAGAGCTGGTCAATCT-3` (810, antisense); intron VI,
5`-CCTATTCCCTGGAGATTC- 3` (772, sense), 5`-GAGAGGGTGAGATTAGGG-3` (1106,
antisense), and 5`-GTGGTGGAGGCTAACTTA-3` (1295, antisense). PCR and
reverse transcription-PCR were carried out using Perkin Elmer kits.RNase Protection Assays
The 250-bp Eco47III-PstI fragment from DSC34 was cloned into
pT7T318U (Pharmacia Biotech Inc.) digested with HincII and PstI. The resulting plasmid was linearized with EcoRI
and transcribed in vitro with T3 RNA polymerase in the
presence of [
-
P]ATP to generate an
antisense probe. Ribonuclease protection was performed as described (19) using RNA isolated from human umbilical vein cells (ATCC),
according to Chirgwin et al.(20) . Yeast RNA (Sigma) was used as a negative control. The size of
the protected products was determined from a sequencing ladder run
alongside the samples.DNase I Protection Assays
Protection assays were
carried out as described(21) , using purified Sp1 protein
(Promega). An XmaI fragment from DSC40 (for Fig. 5, panel A) and a Bsp120I fragment from DSC68 and DSC69
(for Fig. 5, panels B and C) were radiolabeled
using [
-P]ATP and T4 polynucleotide kinase.
These labeled fragments were digested with PvuII (for a
fragment derived from DSC40) or BglI (for fragments derived
from DSC68 or DSC69) to obtain fragments exclusively labeled at the
5`-end of the bottom strand. A primer (5`-CCGGGTCGGGATGGAACG-3`)
starting at the 5`-end of the XmaI fragment was used to
generate a parallel sequencing ladder.
Site-directed Mutagenesis
DSC409-17 was
mutagenized using the U. S. E. Mutagenesis Kit from Pharmacia with the
following oligonucleotides: 5`-TTTCCCTGGCTCCGAACCAGGC-3` and
5`-CACCAGAGGCCCCGAACTGCTTCCCGCGTTT-3`, which are the Sp1 mutagenic
oligonucleotides, and 5`-CATCGGTCGATGGATCCAGAC-3`, which was used to
mutate the SalI site in the vector (mutated nucleotides are
underlined). The SalI site change was used to enrich and
screen for mutated plasmids. Mutagenesis was verified by sequence
analysis.Cell Culture Methods
All tissue culture reagents
were purchased from Life Technologies, Inc., except where indicated.
NIH3T3 cells were grown in Dulbecco's modified essential medium
containing 10% calf serum, 100 units/ml penicillin, G and 100 µg/ml
streptomycin. For serum stimulation experiments, cells were serum
starved by growing them for 72 h in Dulbecco's modified essential
medium containing 0.5% calf serum. Cells were stimulated with growth
medium containing 10 ng/ml basic fibroblast growth factor and 1 ng/ml
epidermal growth factor. Cells were transfected using LipofectAMINE
(Life Technologies, Inc.) according to manufacturer's
instructions. For transient assays 1.5 µg of the
luciferase-expressing plasmids were cotransfected with 0.5 µg of
pCMV/SEAP. Conditioned media were collected 24 h after transfection and
assayed for SEAP using the Phospha-Light kit (Tropix). Cells were
harvested and assayed for luciferase activity using the Luciferase
Assay System (Promega). Transient transfections were repeated at least
two independent times in duplicates. Stable cell lines were generated
by cotransfecting 1.8 µg of a luciferase-expressing construct and
0.2 µg of pBR--Puromycin, a plasmid expressing the puromycin
resistance gene. Resistant cells were selected with puromycin (2
µg/ml, Sigma) 24 h after transfection. After
5-10 days of selection, single resistant colonies were isolated
and expanded.
Cloning Genomic Upstream Sequences of the Human CDK2
Gene
Inverse PCR (17) was employed to clone a 4.2-kb
genomic DNA fragment upstream of the known cDNA sequence corresponding
to a BglII-BclI fragment (Fig. 1, fragment
A). Sequence analysis revealed that this fragment contains an
intron, located just upstream of the first BglII site in the
coding region. A 2.4-kb AvrII-PstI subclone (fragment B), containing only exon sequences upstream of the
translational start site, was used for subsequent transcription
analyses. The cloned inverse PCR product was shown to be part of the CDK2 gene by identifying the sequence junction of the
published CDK2 cDNA and the upstream sequence. In addition, in situ hybridization (data not shown) mapped the cloned CDK2 upstream sequence to the chromosomal locus 12q13,
corresponding to a previously published report(22) .Sequencing the Upstream Region of the Human CDK2 Gene and
Mapping the 5`-End of the mRNA
The nucleotide sequence 1.1 kb
upstream of the ATG translation initiation codon was determined from
both strands as shown in Fig. 2. To determine the transcription
start site, a ribonuclease protection assay was performed using RNA
isolated from human umbilical vein cells and an in vitro transcribed RNA probe extending from the PstI site just
upstream of the translation initiation codon to the Eco47III
site 250 bp upstream (Fig. 3). Five transcription start sites
were identified. The most downstream site was designated as nucleotide
+1 in Fig. 2. Three transcription start sites are clustered
at positions +1, -5, and -9. Two additional sites are
located at positions -33 and -71. The -33 site maps
close to the 5`-end of the longest published human CDK2 cDNA(23) , which was isolated from HeLa cells, whereas the
-9 start site maps close to the 5`-end of a different cDNA clone (15) that was also isolated from HeLa cells. No consensus TATA
box was identified upstream to any of the transcription start sites nor
was one identified anywhere else in the sequenced upstream region.
Putative transcription factor binding sites were identified using
manual scanning and the TFD data base (24) in conjunction with
the MacPattern program (25) (see Fig. 2). Two consensus
Sp1 elements were found to lie in proximity to the two upstream
transcription start sites. Sp1 is known to guide initiation in some
TATA-less promoters(26) , and so we hypothesized that these
elements might be functionally important in the human CDK2 gene. A binding site for YY1, another factor also known to
determine the sites of initiation in some TATA-less
promoters(26) , was also identified upstream of the three
transcription start sites clustered at positions +1, -5, and
-9. Other putative transcription factor binding sites identified
in the upstream region of the human CDK2 gene include multiple AP-2, E2F, and p53 binding sites as well as
single sites for AP-1, c-myb, oct,
HiNF-A, and NFY/CTF, a CCAAT box binding factor.
Functional Analysis of the Basal Activity of the CDK2
Promoter
The CDK2 promoter region was analyzed by
transient transfection of luciferase reporter gene constructs into
NIH3T3 cells. Luciferase activity was corrected for differences in
transfection efficiency by cotransfection with a plasmid expressing the SEAP gene driven by the CMV promoter (see ``Materials and
Methods''). Deletion analysis of the CDK2 promoter (Fig. 4) revealed that a 210-bp fragment containing 100 bp
upstream of the most proximal transcription start site
(DSC409-17) contains the required elements for approximately
70% of the promoter activity that is generated by a full-length
construct (DSC37). A further deletion to nucleotide -15
(DSC4010-10) reduced the activity to less than 3% of that
generated by the full-length construct (DSC37). This activity was
similar to the background activity generated by the vector alone
(pGL2-Basic). An internal deletion that removes all the transcriptional
start sites (DSC51) also had no promoter activity above background as
did a reporter construct containing the full-length sequence in the
reverse orientation (DSC36).
9-17, 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 DSC409-17. 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.
Analysis of Serum-induced Activity of CDK2
Promoter
To analyze the serum inducibility of the cloned CDK2 promoter region, stably transfected NIH3T3 cell lines
expressing luciferase from CDK2 promoter deletion derivatives
were established. Cells were serum starved for 72 h prior to being
exposed to serum and growth factors (Fig. 7). Luciferase
activity increased 3-fold 12 h after serum stimulation of cells stably
expressing the full-length construct (DSC37). In contrast, no induction
by serum was observed with cells stably expressing DSC40, which
exhibits full basal activity, but is about 1.7 kb shorter than DSC37.
The same results were obtained with two independently isolated cell
lines stably expressing the same constructs (data not shown).
CDK2 Gene Structure
PCR amplifications with pairs
of primers that overlap most of the published human CDK2 cDNA
sequence were used to determine the intron/exon junctions of this gene.
Human genomic DNA and total human RNA were used as amplification
substrates. Fragments from DNA amplification that were larger than the
respective fragments amplified from RNA were cloned. Each cloned
fragment was sequenced from both ends until an exon/intron boundary was
reached. Seven exons were identified, and their positions are indicated
in Fig. 8.
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. 9-17) 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
DSC409-17 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 DSC409-17, 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
DSC409-17. 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.1.7-kb
segment, which starts 682 nucleotides upstream of the most proximal
transcription start site. 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.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 (DSC406-3), 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.
)
We thank H. Donis-Keller and R. Veile for in situ hybridization data and R. M. Lawn for helpful discussions and
suggestions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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E. J. Cram, B. D. Liu, L. F. Bjeldanes, and G. L. Firestone Indole-3-carbinol Inhibits CDK6 Expression in Human MCF-7 Breast Cancer Cells by Disrupting Sp1 Transcription Factor Interactions with a Composite Element in the CDK6 Gene Promoter J. Biol. Chem., June 15, 2001; 276(25): 22332 - 22340. [Abstract] [Full Text] [PDF]
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