From the
An overlapping inverted repeat sequence that binds the
eukaryotic transcription factor E2F is 100% conserved near the major
transcription start sites in the promoters of three mammalian genes
encoding dihydrofolate reductase, and is also found in the promoters of
several other important cellular and viral genes. This element,
5`-TTTCGCGCCAAA-3`, is comprised of two overlapping, oppositely
oriented sites which match the consensus E2F site
(5`-TTT(C/G)(C/G)CGC-3`). Recent work has shown that E2F binding
activity is composed of at least six related cellular polypeptides
which are capable of forming DNA-binding homo- and heterodimers. We
have investigated the binding of cellular E2F activity and of homo- and
heterodimers of cloned E2F proteins to the inverted repeat E2F element.
We have demonstrated that mutations in this element that abolish its
inverted repeat nature, while preserving a single consensus E2F site,
significantly decrease the binding stability of all of the forms of E2F
tested. The rate of association of E2F-1/DP-1 heterodimers with the
inverted repeat wild type site was not significantly different from
those with the two single site mutated probes. Furthermore, the
mutations decrease in vitro transcription and transient
reporter gene expression 2-5-fold, an effect equivalent to that
of abolishing E2F binding altogether. These data suggest a functional
role that may explain the conservation of inverted repeat E2F elements
among the DHFR promoters and several other cellular and viral
promoters.
E2F is a eukaryotic transcription factor that binds to the
consensus sequence element 5`-TTT(C/G)(C/G)CGC-3`. It was first
discovered as a protein in adenovirus-infected HeLa cells that binds to
and regulates transcription from the adenovirus E2a early promoter
(1, 2, 3, 4, 5) .
DHFR
Recent data show that the E2F
binding activity in mammalian cells consists of a family of proteins
which bind DNA as both homo- and heterodimers. Three groups
independently cloned a protein termed E2F-1
(16, 17, 18) , and closely related proteins,
E2F-2 and E2F-3, have also been characterized
(19, 20) .
E2F-4 has also been recently cloned and characterized
(59) .
Additionally, the proteins DP-1, DP-2, and DP-3 have also been shown to
be a part of cellular E2F binding activities
(14, 21, 22, 23) . Each of these cloned
E2F polypeptides contain amino acid sequences that potentially mediate
dimerization, and E2F-1/DP-1 heterodimerization has been directly
demonstrated
(14, 17, 18, 19, 20, 22, 24) .
Interactions between E2F family members appears to contribute to
regulation of gene expression since E2F-1 and DP-1 cooperatively
activate an E2F-responsive promoter
(23, 24, 25) , and E2F proteins may differ in
their ability to bind to the Rb and Rb-related p107 tumor suppressor
proteins
(19, 24, 26, 59) .
Binding
affinity could represent an important way in which expression of
E2F-regulated genes is modulated. This notion is supported by the
unique example of the adenovirus E2 promoter, which contains two
oppositely oriented E2F sites spaced 16 base pairs apart
(1, 2, 3, 4, 5) . Both the
spacing and orientation of these sites were found to be critical for
proper control of this promoter by the product of the adenovirus E1A
immediate early gene
(28) . E1A dissociates E2F from other
cellular proteins and allows it to bind the 19-kDa E4 gene product,
which appears to stabilize cooperative binding of E2F molecules to the
two sites in the E2 promoter
(29, 30, 31, 32, 33, 34, 35, 36, 37, 38) .
E2F binding to the E2 promoter was shown to be very unstable in the
absence of E4 protein, and E4 did not stabilize E2F binding to a single
E2F site
(28, 39) .
We have investigated the role of
a conserved overlapping inverted repeat E2F sequence motif found in the
promoters of the mammalian DHFR genes
(40, 41, 42, 43, 44) . Studies
have shown that E2F is required for efficient basal transcription of
the DHFR gene
(8) , for cell cycle-regulated activation
(44) , and/or repression
In Vitro Transcription and
Translation-Run-off transcription of DHFR-CAT constructs and
analysis of start sites by primer extension assay was performed as
described previously
(51) . Transcription products were
quantitated by laser densitometry of autoradiographs. For production of
mRNA encoding E2F-1 protein fragments, pBSK-E2F1-121 (described
above) was digested to completion with either BamHI (for amino
acids 88-437) or PvuI (amino acids 88-292),
phenol-chloroform extracted twice, ethanol precipitated, resuspended in
diethylpyrocarbonate-treated water and incubated with T7 RNA polymerase
(Promega) in standard transcription reactions essentially as
recommended by the manufacturer. The RNA was phenol-chloroform
extracted twice, precipitated, resuspended in
diethylpyrocarbonate-treated water, and added directly to in vitro translation reaction mixtures. RNA was added to 40 µl of
rabbit reticulocyte lysate (Promega) in the presence of RNase inhibitor
(40 units of RNasin, Promega) and amino acids (1 mM each), in
a total reaction volume of 50 µl. Where indicated, the
pBSK-E2F-121, pGC or a control luciferase-encoding plasmid were added
directly to a coupled transcription/translation system (TNT, Promega)
as recommended by the manufacturer. To prepare
For the E2F-1 and
DP-1 gel shift experiments, programmed rabbit reticulocyte lysates were
added to reactions instead of nuclear extract, and the reactions were
done in the presence of 1.5% Nonidet P-40 and 3 mg/ml of bovine serum
albumin. A labeled E2F site oligonucleotide probe (see above) was used
as the probe in the cotranslation experiments depicted in Fig. 3.
For the off-rate experiments, the amount of labeled wild type or single
site mutant promoter fragment probe DNA was increased to 80,000 cpm
(0.2-0.4 ng), and the amount of competitor oligonucleotide to 80
ng. For the E2F-1 experiments, five µl of programmed lysate (TNT,
Promega) was added to the reaction mixture; for the E2F-1/DP-1
experiments, 1 µl of lysate was added and the amount of cold
competitor oligonucleotide increased to 400 ng. Native polyacrylamide
gels (6% for E2F-1 and 4% for E2F-1/DP-1) were run at 250-400 V
for 4 h for E2F-1 and 2 h for E2F-1/DP-1. Otherwise, the off-rate
experiments were performed exactly as described above.
Computer densitometry
analysis of video-captured autoradiographs was performed using the
Image program (NIMH Research Services Branch). The half-life of
E2F-1-DP-1 complex and the rates of association were determined by
direct quantitation of radioactivity in bands on dried gels using a
Molecular Dynamics PhosphorImager system.
The 12-nucleotide inverted repeat E2F sequence near the major
transcription start site is 100% conserved among the DHFR promoters in
mouse, hamster ( shaded boxes, Fig. 1A), and human
(40, 41, 42, 43, 44) .
Additionally, inspection of E2F sequences in a number of other gene
promoters, including the E1A promoters of at least eight serotypes of
adenovirus and seven different cellular promoters, including both the
human and mouse E2F-1 promoters (12, for review; 52, 53), reveals
inverted repeat-like sequences (Fig. 1 C). Finally, in
two studies several E2F sites selected from among random
oligonucleotide populations by proteins associated with Rb have
inverted repeat character (Fig. 1 C; 46, 47). Therefore,
we set out to determine whether the DHFR inverted repeat sequence
conferred any changes in E2F binding relative to binding to sequences
that contained only a single site. Using site-directed mutagenesis, we
created mutations in the DHFR E2F site that allowed us to abolish
either the 5` site or the 3` site separately; each of the these
mutations preserved only a single site. The sites that remain
(5`-GCGCCAAA-3` in the Site 1 mutant and 5`-TTTCGCGC-3` in the Site 2
mutant) each represent single consensus E2F sites. An additional
mutation at the center of the inverted repeat (the double site mutant)
abolished both E2F sequences (Fig. 1 B); this double site
mutation completely eliminates E2F binding activity
(8) . E2F
binding to the single site mutant probes produced gel mobility shift
complexes indistinguishable in mobility from those produced by binding
to the wild type inverted repeat element (Fig. 2, lanes
1, 6, and 10). Extensive investigation of the
adenovirus E2 promoter, which consists of two oppositely oriented E2F
sites, had established that extracts from adenovirus-infected cells
gave a slower mobility gel shift complex characteristic of simultaneous
binding of E2F molecules to two separate sites
(29, 30, 31, 32, 33, 34, 35, 36, 37, 38) .
However, titration experiments showed that E2F complexes with identical
gel mobility formed with the wild type and single site mutant DHFR E2F
sites even at high protein concentration (data not shown).
Initial results confirmed earlier studies of the E2
promoter
(28, 34) indicating that E2F binding to DNA
in vitro is unusually unstable: binding of HeLa cell E2F to
all three of the probes used in our study was virtually abolished
within 5 min of addition of the competitor. However, our more detailed
examination of binding revealed that the DHFR inverted repeat site
conferred a significant increase in binding stability. In three
separate experiments, the half-life of HeLa cell E2F binding to the
inverted repeat sequence was found to be an average of 35.3 s. The
half-life of binding to the Site 1 mutant sequence was 18 s, while the
half-life of binding to the Site 2 sequence was 19 s (Fig. 2).
Furthermore, the single site mutant sequences compete for binding to
the wild type probe (data not shown), indicating that the the same
factor(s) binds all three sites. The amount of binding to the three
probes in this experiment does not appear to be significantly
different. In contrast, in the experiment presented in Fig. 5,
the amount of E2F bound at equilibrium is different (see
Fig. 5
legend) between the wild type and mutant probes under the
conditions of that experiment. The difference in these results is due
to differences in the concentrations of probe and E2F in the binding
reactions; probe is clearly in at least 5-fold excess in the binding
reactions presented in Fig. 2, whereas, probe is limiting in the
binding conditions of the experiment shown in Fig. 5. With probe
in excess, the dissociation rate difference does not result in a
different amount of binding at equilibrium because the reaction is
driven by the rate of association.
The results of these experiments are shown in
Fig. 3B. The protein fragments encoded by these RNA
transcripts produced distinct gel shift complexes that could be
distinguished from the endogenous E2F binding activity characteristic
of reticulocyte lysates (45, 54; Fig. 3 B, lane 4 versus lanes 1 and 3). When the
different-sized E2F-1 transcripts were translated simultaneously, a new
gel shift complex of intermediate mobility was observed, in addition to
those seen with 88-437 or 88-292 alone
(Fig. 3 B, lane 2, indicated by bold
arrow). This complex most likely results from a dimer consisting
of one molecule of each of the two different-sized fragments of E2F-1.
There is an extra band in lane 2 which is the result of
persistence of a portion of the larger E2F after homodimer formation
between the large and small forms. It should also be noted that the E2F
binding activity native to reticulocyte lysates is more active than
that of the E2F-1 homodimers.
E2F-1 DNA binding was analyzed further
in off-rate experiments similar to those described above for HeLa cell
E2F activity. Off-rate analysis of the gel mobility shift complex
consisting of the E2F-1 (88-437) homodimer
(Fig. 3 B) demonstrated that E2F-1 homodimers also bind
significantly more stably to the DHFR inverted repeat site than to the
single sites. The half-life of binding to the wild type site in three
experiments averaged 222 s, compared to 67 s for the Site 1 mutant and
83 s for the Site 2 mutant (Fig. 3 C), suggesting that
the inverted repeat sequence confers additional stability on the DNA
binding of a defined homodimeric form of E2F-1. It should be noted that
while the binding stability difference is significant, the much longer
absolute binding half-lives of the complexes bound to each of the three
probes (relative to those observed with the HeLa cell E2F activity,
Fig. 2
) probably reflects different binding conditions related to
the relatively large amount of reticulocyte lysate required to
visualize the E2F-1 homodimers, which bind to DNA much lessavidly than the ``native'' and probably heterodimeric
(see below) E2F activity in the lysate or in cellular extracts. As
shown in Fig. 3 B, the complex that is due to E2F in the
lysate is the slowest mobility band in these gel shifts, and its
binding parallels that of the E2F-1 homodimer on the three probes,
supporting the notion that binding conditions affect the absolute
measurement of the t
With the
availability of cloned DP-1, we addressed whether heterodimers of
E2F-1/DP-1, produced by cotranslation, also displayed enhanced binding
to the DHFR inverted repeat E2F sequence. A coupled in vitro transcription-translation system was used to produce E2F-1 and
DP-1 proteins in rabbit reticulocyte lysates. The production of
proteins was monitored by incorporation of
[
Finally, we tested the
effects of our Site 1 and Site 2 mutations that reduced E2F binding
stability in in vitro transcription and reporter gene
expression assays. Fig. 6, A and B show in
vitro transcription analysis in a HeLa cell extract of wild type
and mutant DHFR promoter constructs driving expression of the bacterial
CAT gene. This experiment showed that the effect on in vitro transcription of the Site 1 and Site 2 mutations is roughly the
same as the effect of a double site mutation that completely abolishes
E2F binding; in all three mutants, transcription was reduced
approximately 5-fold. Therefore, two separate mutations which leave
intact single E2F sites, and which clearly allow E2F binding, have
virtually the same effect on basal transcription of the DHFR gene as a
double site mutation, which completely abolished detectable E2F
binding.
Because the
inverted repeat E2F sequence is located just 3` of the major
transcription start site of the hamster DHFR promoter, we also tested
whether the mutations we created in the sequence affected the start
site. The transcripts produced in the in vitro transcription
assay were subjected to primer extension analysis. The results showed
that none of the mutations introduced in the inverted repeat
E2F-binding site affected the nucleotides at which transcription was
initiated. As with the wild type construct
(51) , approximately
80% of the transcripts originated from a major start site cluster at
positions
In the promoters of three mammalian DHFR genes, the inverted
repeat sequence 5`-TTTCGCGCCAAA-3` is absolutely conserved. As shown in
Fig. 1C, similar or identical sequences are found in the
E1A promoters of at least eight serotypes of adenovirus and in several
cellular gene promoters, including those of the human and mouse E2F-1
genes
(52, 53) . This sequence conservation indicates
the functional significance of the inverted repeat E2F binding
sequence. We demonstrate in this report that this element confers
significantly enhanced stability on the binding of the cellular
transcription factor E2F, relative to binding to slightly shorter
sequences which match the consensus E2F-binding site
(5`-TTT(C/G)C(/G)CGC-3`). This finding applies to HeLa cell E2F binding
activity, as well as to homo- and heterodimers of cloned E2F proteins
expressed in vitro. Interestingly, inverted repeat-like E2F
sequences also are selected from among random populations of
oligonucleotides by Rb-associated proteins
(46, 47) .
Since Rb binds E2F, this indicates that these sequences confer the most
stable DNA binding for E2F.
Previous studies of the adenovirus E2
promoter have strongly suggested that enhancing the stability of E2F
binding can provide a means of activating gene expression. During
adenovirus infection, the adenovirus E4 ORF 6/7 protein acts as a
molecular clamp to stabilize the binding of E2F heterodimers to
separate sites, an effect that is correlated with enhanced E2 gene
expression
(28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) .
Interaction with the E4 protein can increase the half-life of E2F
binding to sites in this particular configuration from a period of less
than 5 min to at least 30 min
(28, 34) . In this study,
we have shown that the overlapping inverted repeat E2F sites, which are
found in the DHFR and many other cellular and viral promoters, provides
a more stable binding site for E2F homo- and heterodimers than the
single E2F sites found in many other promoters. The difference in
affinity is due to the difference in dissociation rate since the
association rate is the same. While the stability difference conferred
in vitro on E2F binding by the inverted repeat sequence
investigated in this report is much more subtle than that in the E2
gene in adenovirus infected cells, the functional effects of mutations
altering the repeat structure are significant. At the level of basal
transcription, the inverted repeat sequence element is essential for
optimal DHFR gene expression. Mutations which merely abolish the
inverted repeat but leave intact either of the two overlapping
consensus E2F sites in this element have virtually the same functional
effect as a mutation that completely eliminates E2F binding.
Neither
the findings presented here nor the previous studies of E2 promoter
activation necessarily imply that E2F binds to promoters with a
half-life of less than 5 min in vivo. Interactions among E2F
and other factors on gene promoters very likely would serve to enhance
E2F's binding stability. Not unexpectedly, we found that by
varying the experimental conditions, such as the total protein
concentration in the binding reactions, we could influence the measured
half-life of binding, which probably explains the longer overall
half-life of binding by the E2F-1 homodimers (and lysate under these
conditions) to all three sites as shown in Fig. 3 C.
However, we suggest that it is the difference in binding
stability between the inverted repeat sequence and the shorter
consensus sequences preserved by our Site 1 and Site 2 mutant promoters
that is significant; it is reasonable to argue that a conserved
sequence element which can effectively double the stability of binding
of a transcription factor and apparently increase its ability to
activate transcription in vitro could confer the same relative
effects in vivo. This is the first study to implicate
sequence-related differences in E2F binding stability as a mode of
differential gene regulation. Given the considerable variability in the
sequences of reported and proposed E2F sites
(9, 10, 12, 27) , it will be interesting
to determine whether sequence variations contribute to the range of
E2F's regulatory capabilities. Expression clones of additional
forms of E2F will facilitate such studies.
The DHFR inverted repeat
E2F sequence is located immediately 3` of the major transcription
initiation site in the hamster and human DHFR promoter (51;
Fig. 6B). In most E2F-regulated promoters, the E2F
sequences act from a position much farther 5` of the transcription
start site(s)
(12) . In studies of the mouse DHFR promoter in
which the entire initiation region has been deleted or subjected to
multiple point mutations, the initiation site can be changed
(27, 55) ; however, our study indicates that point
mutations that either abolish E2F binding or diminish its stability
affect only the efficiency of transcription, not the sites of
initiation (Fig. 6 C). This finding, as well as a recent
study in which wild type and mutant DHFR initiation region sequences
were placed in a heterologous promoter background, suggests that E2F
itself does not act as a DNA-binding ``initiator'' protein
(56, 57) .
Our footprinting studies of the DHFR
inverted repeat E2F sequence element show that the entire region is
somewhat resistant to chemical or enzymatic cleavage even in the
absence of cellular proteins and that E2F binding creates a distinct
footprint that is virtually identical on the wild type, Site 1 mutant,
and Site 2 mutant promoters (8 and data not shown). Resistance to
DNA-cleaving agents suggests the possibility that the inverted repeat
element can form unusual structures. Interestingly, recent work has
shown that binding of E2F-1 and HeLa cell E2F produces pronounced DNA
bending with a flexure angle of more than 125°
(58) . The
inverted repeat sequence may favorably influence such bending.
Alternatively, the inverted repeat motif, by providing a consensus
sequence element on each side of the DNA helix, could provide a double
binding site for E2F dimers that rapidly dissociate from and then
reassociate with the DNA in this region; however, the finding that the
rate of association to the three different sites was the same argue
against this interpretation. It would appear that both sites can be
occupied by a single E2F dimer.
Finally, oligomeric interactions
between E2F family members and a number of other cellular proteins
(including pRb, p107, p130, cyclin A-cdk2, and cyclin E-cdk2) point to
another area in which the stability of E2F binding could influence gene
regulation. Binding of Rb to E2F, which represses E2F-dependent
transcription, increases the half-life of E2F-DNA complexes
10-15-fold
(58) . The binding characteristics of other
multimeric E2F-DNA interactions have not been reported. Future studies
will address whether the inverted repeat overlapping sites on the DHFR
promoter influence binding of multimeric complexes. Sequences which
enhance the binding stability of such complexes would also be expected
to influence their regulatory functions.
We acknowledge Dr. Joseph Nevins and members of his
laboratory for helpful discussions, Drs. R. Girling and N. B. La
Thangue for the DP-1 plasmid, and Drs. Al Baldwin, Bill Marzluff, and
Adrian Black and our laboratory colleagues for comments on the
manuscript. We also thank Dr. Adrian Black for helpful suggestions on
the kinetic studies, Carol Hoover for technical assistance with the
association rate studies, and Dr. Bill Kaelin for sharing data prior to
publication.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
and c- myc were the first cellular
genes shown to be subject to regulation through E2F
(6, 7, 8) . More than a dozen cellular and viral
genes have known or potential E2F-binding sequence elements in their
promoter regions
(9, 10, 11, 12, 13, 14) .
A link between E2F and growth control has been suggested by a large
number of studies demonstrating the direct binding of E2F to proteins
implicated in the regulation of cellular growth control, including
cyclins and cyclin-dependent kinases, as well as tumor suppressor-like
proteins which can repress transcription through an E2F-binding site
(11, 14, 15) .
(
)
of DHFR, and
for adenovirus- and human cytomegalovirus-mediated activation of the
gene
(10, 45) . In one of these previous reports, we
suggested that the E2F sequence found near the major transcription
start site of the three characterized mammalian DHFR genes,
5`-TTTCGCGCCAAA-3`, was a ``full site'' for optimal E2F
binding and function, and that the sites found in the E2 promoter,
5`-TTTCGCGC-3`, represented a less stable site for binding of a dimeric
transcription factor
(10) . A functional role for inverted
repeat E2F elements is further supported by the presence of identical
or similar sequence elements in a number of other genes
(Fig. 1 C) and by two studies which show that inverted
repeat E2F elements are selected from among populations of random
oligonucleotides by Rb-binding proteins
(46, 47) .
Figure 1:
A, control elements in
the hamster DHFR promoter. The open boxes are binding sites
for the transcription factor Sp1. The hatched boxes are
structural control elements (12). The thin arrow represents
the minor transcription start site at position 107 relative to
the ATG translation initiation codon at +1. The bold arrow represents the major transcription start site cluster at positions
63,
64, and
66. The overlapping inverted repeat
E2F site at position
62 to
51 is depicted by the
shaded boxes. The promoter construct used for functional
studies contained all of the sequence represented here, except for
mutations in the E2F element as indicated. Promoter fragments used in
binding studies contained only the wild type or mutated E2F element.
B, sequences of wild type and mutant DHFR E2F sites used in
binding and functional studies. C, examples of inverted repeat
E2F sites in mammalian and viral genes and sequences selected from
degenerate oligonucleotides by Rb-associated proteins for subsequent
polymerase chain reaction amplification (see text for
references).
This paper reports the effect of mutations in the DHFR inverted
repeat E2F sequence element on the binding stability of cellular
form(s) of E2F and defined homo- and heterodimers of cloned E2F
proteins. Because this element consists of two overlapping, oppositely
oriented high affinity E2F binding sequences, we were able to use
site-specific mutagenesis to destroy the inverted repeat while leaving
intact one or the other of the two E2F sites within the element. We
demonstrate that all the forms of E2F dimers tested bind significantly
more stably in vitro to the inverted repeat sequence than to
``single'' E2F sites that do not possess inverted symmetry.
We also show that the rates of association are the same among the three
sites. Finally, altering the inverted repeat results in a
2-5-fold decrease in transcription from the DHFR promoter,
equivalent to the functional consequence of mutationally abolishing E2F
binding
(8) . These data support the hypothesis that the
inverted repeat overlapping E2F-binding sites in the DHFR and other
promoters represents a novel means by which E2F binding and gene
transcription are modulated.
Plasmids and Oligonucleotides
The wild-type and
E2F double site mutant DHFR-CAT reporter plasmids have been described
previously
(44) . Construction of the E2F Site 1 and Site 2
mutants also has been described
(10, 45) . Plasmids
containing wild type and single site mutant DHFR E2F site promoter
fragments (restricted to remove the upstream Sp1 sites) and the
synthetic oligonucleotide corresponding to the DHFR wild type E2F site
also have been described previously
(45) . cDNA encoding E2F-1
amino acids 88-437 was cloned into the pBSK vector (Stratagene)
by polymerase chain reaction amplification using the p121 primer
previously described to produce the pBSK-E2F1-121 construct
(16) . A plasmid encoding DP-1, pGC, was a kind gift of R.
Girling and N. B. LaThangue
(22) .
Cell Culture and Nuclear Extract Preparation
HeLa
S-3 cells were grown in Joklik's modified minimal essential media
(Life Technologies, Inc.) supplemented with 5% fetal calf serum in
suspension at a concentration of 4-8 10
cells/ml with daily 1:2 expansion of cells. Transcriptionally
competent nuclear extracts were prepared as described previously
(8, 48) .
Transient Transfection Assay
HeLa, Chinese hamster
ovary and Balb/c3T3 cells were transfected with the appropriate
DHFR-CAT reporter plasmids by the calcium phosphate method
(49) and assayed for CAT activity as described previously
(8, 50) . CAT activity was quantitated by liquid
scintillation counting of spots cut from thin layer chromatography
plates.
S-labeled
proteins for denaturing (sodium dodecyl sulfate) polyacrylamide gel
analysis, translation or transcription-translation reactions were
performed in the presence of methionine-minus amino acid mixtures,
and[
S]methionine was added.
Gel Mobility Shift Assays
For the analysis of
E2FDNA complexes formed in the presence of HeLa nuclear extract,
gel mobility shift reaction were performed essentially as described
previously
(8) . For off-rate experiments, 40 µl of reaction
mixtures were equilibrated on ice in the presence of 12 µg of
nuclear extract. DHFR wild type or mutant promoter fragment probes were
fill-in labeled by incubation with the Klenow fragment of DNA
polymerase I and [
P]dATP; 40,000 cpm
(0.2-0.4 ng) was added to each reaction mixture, and binding
reactions were incubated for 20 min at room temperature. (Longer
incubation times of up to 1 h had no effect on the intensity or
mobility of E2F complexes.) The reaction mixtures were then mixed with
3 µl of loading dye (50% glycerol, 0.5
Tris borate (TBE)
buffer, 0.0001% xylene cyanol, 0.0001% bromphenol blue) and transferred
to a 4 °C cold room. A 6-µl aliquot was immediately removed and
applied to a 4% native polyacrylamide gel that was already running at
100 V in a 4 °C cold room (zero time point). Unlabeled competitor
oligonucleotide (40 ng) was added to the remaining reaction mixture
with thorough mixing by repeated pipetting, and aliquots were removed
and loaded onto the running gel at the indicated time points. Addition
of probe to each reaction mixture was timed so that each probe was
incubated with nuclear extract for 20 min before competitor
oligonucleotide was added. Gels were run at 250 V for 1.5-2 h
after the loading dyes had migrated into the gel.
Figure 3:
Analysis of E2F-1 binding activity.
A, reducing 8% SDS-polyacrylamide gel electrophoresis analysis
of in vitro translated E2F-1 protein fragments. Lane
1, E2F-1 amino acids 88-437. Lane 2, E2F-1 amino
acids 88-292. The migration of molecular weight markers (in kDa)
run in parallel is shown at the left. B, gel mobility
shift assay of showing dimerization of in vitro translated unlabeled proteins using a labeled oligonucleotide
consisting of the wild type DHFR E2F site as a probe. Aliquots of
in vitro translation reaction mixtures were added directly to
gel shift reaction mixtures. mRNAs encoding E2F-1 (88-292)
( lane 1), E2F-1 (88-437) ( lane 3), or both
mRNAs ( lane 2) were added to the in vitro translation
reactions, which were then analyzed by gel shift. Non-denaturing 6%
gels were run at 300 V for 4 h to adequately separate the three
different E2F-1 binding activities from one another and from the
lysate. Lane 4 shows gel shift analysis of a control in
vitro translation reaction to which brome mosaic virus RNA (1
µg) was added, i.e. the endogenous E2F binding activity in
the rabbit reticulocyte lysate. The arrows show the migration
of proteinDNA complexes. As indicated, the lysate binding activity
is seen in all the samples. The bold arrow in the position
intermediate between the two protein fragments depicts the migration of
a complex consisting of an E2F-1 dimer composed of protein fragments of
different sizes in lane 2. The lysates in which only a single
mRNA was translated (those analyzed in lanes 1 and 3)
were programmed with 2 µl each out of 10 µl mRNA recovered from
the in vitro transcription reaction mixtures. The lysate
analyzed in lane 2 was programmed with 1 µl of E2F-1
(88-437) and 1 µl of E2F-1 (88-292) mRNA. C,
the half-life of binding of E2F-1 to wild type, Site 1 mutant, and Site
2 mutant DHFR E2F site promoter fragment probes. Gel shift reactions
using lysates programmed with E2F-1 were subjected to off-rate analysis
by addition of unlabeled E2F DNA-binding site as described for Fig. 2.
Densitometry analysis of the E2F-1 complex was used to derive a
half-life of binding to the three probes as described under
``Materials and Methods'' and in the legend to Fig. 2; the
average half-life of the E2F-1 complex bound to each of the probes in
the presence of unlabeled competitor oligonucleotides is shown. The
error bars depict standard error, n =
3.
The
association rate for E2F-1/DP-1 heterodimers was measured in three
separate experiments. Probes consisted of end-labeled DNA fragments
(1.5 nmol DNA, 10cpm/µg) containing the inverted
repeat E2F site or each of the single E2F sites as used in the off-rate
experiments; all probes had the same specific activity. Reactions (80
µl) were initiated with each of the three probes by the addition of
2 µl of reticulocyte lysate programmed with E2F-1 and DP-1 in
vitro transcribed mRNA, and aliquots were removed and applied to
running gels at 30-s intervals for 3 min.
Figure 2:
Off-rate assay of HeLa cell E2F binding
activity. A, a representative autoradiograph showing the
effect of addition of unlabeled competitor oligonucleotides on E2F
binding to labeled wild type E2F site, Site 1 mutant, or Site 2 mutant
promoter fragment probes. Competitor oligonucleotide (100-fold molar
excess) was added ( t = 0), and aliquots of gel mobility
shift reaction mixtures were removed at the indicated times after the
addition ( min.) and immediately applied to a continuously
running gel. The major E2F band is indicated by the arrow. The
dark band just above the E2F band represents nonspecific DNA binding by
extract protein. The free probe is the wide band at the bottom of the
gel. B, the half-life of E2F binding activity in the presence
of cold competitor oligonucleotides. The half-life of binding for
off-rate experiments was computed by plotting the disappearance of the
major E2F band as measured by densitometry analysis of autoradiographs
of gels. The graph depicts the average half-life value for the major
E2F band. The error bars represent standard error, n = 3.
Having
demonstrated that the inverted repeat sequence apparently did not serve
as two independent sites, we undertook a series of experiments to
determine whether it enhanced the stability of E2F binding relative to
binding to a single E2F site. E2F in HeLa cell extracts was allowed to
bind to radiolabeled promoter fragment probes containing the wild type,
Site 1 mutant or Site 2 mutant E2F sites (Fig. 1 B). The
binding reaction was allowed to reach equilibrium (15-30 min at
room temperature), and an excess of a competitor oligonucleotide
consisting of the wild type DHFR E2F site was added to the reaction
mixture. Aliquots of the mixture were then applied to a gel that was
already running at time points after addition of the competitor
oligonucleotide.
Figure 5:
E2F-1 and DP1 were cotranslated as
described for Fig. 5. At time 0, a binding reaction was started by the
addition of DNA probe (wild type or single site mutant). At the times
indicated, an aliquot was removed from the reaction and loaded onto a
running polyacrylamide gel. The gel was subjected to analysis by a
Molecular Dynamics Phosphorimager to obtain quantitative output. The
ratio of the amount of protein bound at each time point ( B) to
the amount bound at equilibrium ( B) (3 min) is
plotted. The experiment was repeated three times, and a representative
experiment is shown. The amount of binding at equilibrium to the three
probes is different. In these experiments, binding at equilibrium,
expressed as the ratio of the counts bound over the total counts and
averaged for three separate experiments is: 0.0285 for the wild type
probe, 0.0145 for the site 1 mutant probe, and 0.014 for the site 2
mutant probe.
The cloning of the E2F-1 protein
(16, 17, 18) allowed a direct test of E2F
dimerization. Using in vitro transcription of E2F-1 cDNA, we
synthesized run-off RNA transcripts encoding either E2F-1 amino acids
88-437 or 88-292. These transcripts were then translated
separately or together in rabbit reticulocyte lysates, and the
resulting protein products, which migrated in a denaturing gel at
approximately 45 and 16 kDa, respectively (Fig. 3 A),
were analyzed by gel mobility shift analysis using the wild type DHFR
E2F site as a probe.
but do not affect the
relative differences in stability between binding sites.
S]methionine into aliquots of the reaction
mixtures and visualized by SDS-polyacrylamide gel electrophoresis
followed by autoradiography as shown in Fig. 4 A. A
plasmid encoding full-length DP-1 produced a protein that migrated at
approximately 60 kDa, while a plasmid encoding the C-terminal 348 amino
acids of E2F-1 produced a protein that migrated as a doublet of
approximately 45 kDa (Fig. 4 A). The wild type DHFR
inverted repeat E2F sequence was used as a probe to analyze the DNA
binding characteristics of these proteins by gel mobility shift
(Fig. 4 B). The endogenous E2F binding activity contained
within the lysate produces three bands in a gel mobility shift
( lane 2). When E2F-1 was translated, an additional band of
slightly faster mobility was observed ( lane 3); when DP-1 was
translated, no additional bands over those seen with the control lysate
were seen ( lane 4), indicating that DP-1 alone binds very
weakly to the E2F sites. When E2F-1 and DP-1 were cotranslated, a novel
complex of faster mobility than that of the lysate E2F was observed
( lane 5, heterodimer indicated by the arrow). This
complex was the strongest signal seen in the three lanes, indicating
that heterodimer binding to the E2F site is stronger than that of
homodimer. The distinct gel mobility shift complex produced by
E2F-1/DP-1 heterodimers was subjected to off-rate analysis. A
representative experiment is shown in Fig. 4 C; due to
the shorter exposure of the gel and less material/lane as compared to
the gel in panel B, complexes resulting from endogenous E2F
activity in the lysate are only weakly visible and the only prominent
complex is that of the E2F-1/DP-1 heterodimer. The average of five
experiments is shown in Fig. 4 D; an E2F-1/DP-1
heterodimer dissociates from the inverted repeat site with an average
half-life of 43.9 s in the presence of excess competitor
oligonucleotide, compared to an average half-life of 21 s for binding
to the Site 1 mutant and 22.1 s for binding to the Site 2 mutant. The
data demonstrate that E2F-1 and DP-1 heterodimers, like the predominant
E2F binding activity in extracts from HeLa cells (Fig. 2) and
E2F-1 homodimers (Fig. 3), bind with optimal stability to an
inverted repeat sequence, relative to binding to a single E2F site.
Figure 4:
Analysis
of specific E2F-1/DP-1 DNA binding activity. A. radiolabeled
proteins produced in coupled in vitro transcription-translation reactions were resolved on an 8%
SDS-polyacrylamide gel electrophoresis gel under reducing conditions.
Lane 1, luciferase control protein. Lane 2, E2F-1
amino acids 88-437. Lane 3, DP-1. Lane 4, E2F-1
and DP-1. The migration of molecular mass markers in kDa is shown at
the left. B, gel shifts. The
transcription-translation reactions were performed as for panel A in the presence of unlabeled amino acids. The reticulocyte lysates
were programmed with RNA encoding E2F-1 ( lane 3), DP-1
( lane 4), or E2F-1 and DP-1 ( lane 5) and were
incubated with labeled wild type DHFR E2F site probes gel in mobility
shift reactions. In lanes 6 and 7, 400 ng of an
oligonucleotide corresponding to the wild type DHFR E2F site was added
to the reaction mixtures analyzed in lanes 3 and 5,
respectively; in lanes 8 and 9, 400 ng of an
oligonucleotide corresponding to the double site mutant DHFR E2F site
was added to the reaction mixtures analyzed in lanes 3 and
5, respectively. C, off-rate analysis of E2F-1/DP-1
DNA binding activity. One µl of the E2F-1/DP-1 lysate was incubated
with the wild type, Site 1 mutant, or Site 2 mutant DHFR E2F site
probes in a 40-µl reaction mixture, and aliquots of the mixture
were applied to a running gel at the indicated time points as in Fig. 2
after addition of competitor oligonucleotide (400 ng). The predominant
DNA binding activity is a single band from the E2F-1/DP-1 heterodimer
which is different from that observed in lane 5 of panel C because one-fifth of the reaction was loaded on the gel and the
exposure is shorter. D, bands corresponding to the
E2FDP-1 complex as shown in the autoradiography in panel C were directly counted using a Molecular Dynamics Phosphorimager
system, and the radioactivity of each complex measured to derive a
half-life of binding to each of the three probes, as in Figs. 2 and 3.
The average half-life of E2F-1/DP-1 binding to each of the three probes
is shown. Error bars depict standard error, n = 5.
To address whether the different E2F sites have different affinities
for E2F, the rates of association of E2F-1/DP-1 to the same sites were
measured. Binding reactions were initiated by addition of probe, and
aliquots were removed at 30-s intervals and immediately applied to a
gel that was already running. Equilibrium binding was achieved by 2.5
min. Data at each time point were expressed as the amount bound over
the amount bound at equilibrium (B/B) (Fig. 5). This
experiment was repeated three times and no significant difference was
observed in B/B
among the probes at any time point. The
amount of binding at equilibrium was different; the wild type sequence
bound approximately twice as much protein as did the two single site
mutants. These data support the hypothesis that there is little
difference between the rate of association and that the difference in
affinity between the inverted repeat site and the single E2F site is
due to the different rates of dissociation.
Figure 6:
The functional effects of mutations
abolishing either one or both of the E2F sites in the DHFR inverted
repeat motif. A, denaturing gel analysis of the products of
in vitro transcription reactions using HeLa nuclear extract
and CAT reporter gene templates under the control of the wild type DHFR
promoter or the same templates bearing Site 1, Site 2, or the E2F
double site mutations. A 494-base Sp6 transcript was included as an
internal control for sample recovery and is indicated by an
asterisk in the figure. The amount of DHFR major and minor
transcripts were quantitated in three separate experiments by laser
densitometry of the autoradiograms; the difference in transcript
produced by the three mutants as compared with the wild type construct
was 4.9 ± 0.83-fold. B, in vitro transcription
products assayed by primer extension to map initiation sites. The
transcription products analyzed are in the same order as shown in
panel A, and were electrophoresed alongside a sequencing
ladder run in parallel, which was used to map the start site positions.
For primer extension, in vitro transcription reactions were
performed in the presence of unlabeled ribonucleotides, and the
resulting transcripts were subjected to reverse transcription in the
presence of [P]
-ATP-labeled primer.
One-third of the radiolabeled DNA resulting from the wild type template
was analyzed on the gel, while all of the radiolabeled DNA resulting
from the mutant templates was analyzed. C, the promoter-CAT
constructs that were run-off transcribed in the experiments shown in
panels A and B were transfected into HeLa cells by
calcium phosphate coprecipitation, and CAT activity in cell lysates was
analyzed by measuring acetylation of
[
C]chloramphenicol after 48 h. An autoradiograph
of a representative thin layer chromatography plate is shown. The
experiment was repeated four times, with quantitation by direct liquid
scintillation counting of plates, and the range of activity of the
double-stranded and single-stranded mutants was 2-5-fold less
than the wild type. The three mutants were not significantly different
from one another ( p < 0.05), but were significantly from
the wild type ( p < 0.01).
The reduced efficiency of the single E2F site was confirmed
by the CAT assay shown in Fig. 6 C. The same promoter-CAT
constructs used for the in vitro transcription analysis in
Fig. 6A were transfected into HeLa cells, and CAT gene
expression was monitored by thin layer chromatography of
[C]chloramphenicol converted to the acetylated
form by the expressed CAT gene product. The assay demonstrates that CAT
gene expression was reduced by approximately equivalent amounts by the
Site 1, Site 2, and double site mutations in the inverted repeat E2F
site, relative to expression driven by the wild type promoter. Within
each of four separate transfection experiments, the CAT activities of
the mutated promoters (Site 1, Site 2, and double site) were not
significantly different from one another ( p < 0.05) but
were significantly less than the wild type ( p < 0.01), The
difference observed was 2-5-fold reduction in CAT activity from
the mutants as compared to the wild type construct. Very similar
results were obtained in Chinese hamster ovary and Balb/c 3T3 cells
(data not shown). The relatively wide range of difference is likely a
reflection of variability in the growth state or confluence of the
transfected cells in spite of efforts to plate exactly the same number
of cells in each experiment; the plating efficiency and degree of
dispersion of the cells will certainly affect growth state. The effect
of E2F on transcription varies according to the growth state of the
cell. If a cell is in late log phase, E2F acts as a repressor, whereas
if in mid-log cells, E2F stimulates transcription.
(
)
In a 48-h experiment, some of the cells may be reaching
late log in some areas of the tissue culture dish, which would abrogate
the stimulatory effect of E2F on DHFR transcription and reduce the
difference between the mutant and wild type constructs.
63,
64, and
66, while approximately 20%
originated from the minor initiation site at
107 for each of the
mutated promoters (Fig. 6 B). We conclude, therefore,
that neither completely abolishing binding, reducing the stability of
E2F binding to this site, nor eliminating its palindromic nature
affects the specificity of transcription initiation, but instead
affects the efficiency of transcription from the DHFR promoter. E2F
binding at this sequence thus appears to function to enhance the
efficiency of transcription, not to influence the selection of the
start sites.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.