From the
Rb represses E2F-mediated transcription in part by blocking the
trans-activation domain of E2F. In addition, Rb can convert an
E2F binding site from a positive to a negative element. To examine the
effect of a Rb-DNA-bound complex on transcription, full-length Rb was
fused to the DNA binding domain of GAL4. Here, we report that GAL4-Rb
can repress transcription mediated by either Sp1, AP-1, or p53,
dependent upon the presence of both the GAL4 DNA binding domain and
GAL4 binding sites. Moreover, GAL4-Rb inhibited the activity of the
herpes simplex virus tk promoter from GAL4 binding sites
located at a distance from the promoter. In contrast, GAL4-Rb was
unable to repress basal transcription. Cotransfection of specific
cyclins and cyclin-dependent kinases or SV40 T-antigen abolished the
repressive activity of GAL4-Rb. The domains of Rb involved in mediating
the repression of transcription were mapped to regions that are
overlapping, but not identical, to those required for the interaction
with E2F. We propose that Rb can function as a general repressor of
transcription when bound to the promoter region.
The protein product of the retinoblastoma susceptibility gene,
Rb, plays a key role in the regulation of cell growth and
differentiation
(1) . Rb appears to control cell growth, in
part, by regulating gene expression (for review see Ref. 2). Indeed, Rb
is able to regulate the transcription of a variety of genes encoding
growth-regulatory factors, such as c- fos (3) ,
c- myc (4) , TGF-
Rb is believed to repress transcription by interacting
directly with transcription factors, such as E2F, and blocking their
trans-activation domains
(18) . In addition, Rb has
been shown to convert an E2F site from a positive to a negative
regulatory sequence, suggesting an additional role for E2F-bound Rb
besides blocking the trans-activation domain of E2F
(19) . To examine the effect of a Rb-DNA-bound complex on
transcription, full-length Rb was fused to the DNA binding domain of
GAL4. In this manner, Rb can be brought to the promoter through the
interaction with GAL4 binding sites without having to interact with
specific transcription factors. Our results demonstrate that GAL4-Rb,
when bound to DNA through GAL4 binding sites, can repress transcription
mediated by several transcription factors. An interaction with SV40
T-antigen or phosphorylation by specific cyclins prevented GAL4-Rb from
repressing transcription. Moreover, GAL4-Rb proteins with deletions or
point mutations within domains A and B of the Rb pocket were deficient
in their ability to repress transcription. Our results suggest that Rb
can function as a general repressor of activated transcription when
bound to the promoter region.
To examine
the effect of GAL4-Rb on activated transcription, multiple copies of
the consensus binding sites for Sp1, AP-1, and p53 were inserted
between the GAL4 binding sites and the E1b TATA box in G5BCAT.
Cotransfection of GAL4-Rb and Sp1, AP-1, and p53 reporters into CCL-64
cells resulted in a significant repression of transcription in a
dose-dependent manner (Fig. 1). However, GAL4-Rb was not able to
repress transcription from the Sp1-CAT and AP1-CAT reporters lacking
GAL4 binding sites (Fig. 1, B and E). The GAL4
DNA binding domain alone, expressed from the pSG147 plasmid, and Rb
protein were unable to repress transcription from the GAL4-Sp1-CAT
reporter (Fig. 1 A). The levels of Rb and GAL4-Rb
proteins expressed in the transfected cells were shown to be similar by
Western blot analysis using an anti-RB antibody (data not shown). Thus,
the repression of transcription mediated by GAL4-Rb requires both Rb to
be fused to the GAL4 DNA binding domain and for GAL4 binding sites to
be present in the reporter plasmid. These results also demonstrate the
ability of GAL4-Rb to repress transcription mediated by three distinct
transcription factors, Sp1, AP-1, and p53.
Rb appears to regulate gene expression by interacting and
modulating the activity of specific transcription factors
(6, 7, 10, 13, 14, 16) .
It has been suggested that Rb suppresses E2F-mediated transcription
through direct physical association with E2F trans-activation
domain. However, it also has been shown that the Rb-E2F complex, when
bound to an E2F site, can inhibit the activity of adjacent promoter
elements and repress transcription
(19) . This result suggested
that Rb may regulate transcription through an additional mechanism(s)
besides simply blocking the trans-activation domain of E2F.
To determine the function of Rb when it is a part of a DNA-bound
complex, we have bypassed the requirement of specific transcription
factors for bringing Rb to the DNA by fusing Rb to the DNA binding
domain of the yeast transcription factor GAL4. The cotransfection
experiments have shown that GAL4-Rb, when bound to GAL4 sites, was able
to repress Sp1-, AP-1-, and p53-mediated transcription as well as
transcription mediated by the HSV tk promoter. The inhibition
of transcription by GAL4-Rb was not the consequence of steric hindrance
since GAL4-Rb repressed the activity of the HSV tk promoter
from GAL4 binding sites located either 760 bp upstream or 1000 bp
downstream from the site of transcriptional initiation. This effect was
specific to GAL4-Rb in that wild-type Rb protein was unable to repress
transcription, and GAL4 binding sites in the reporter plasmid were
required for GAL4-Rb-mediated transcription. Thus, GAL4-Rb can function
as a general repressor of transcription when bound to the DNA,
independent of an interaction with a specific transcription factor.
We demonstrated that the binding of SV40 T-antigen to GAL4-Rb was
able to at least partially abrogate GAL4-Rb-mediated repression. The
interaction of SV40 T-antigen with GAL4-Rb may prevent Rb from
interacting with a cellular factor(s) involved in conferring the
repressive effect of GAL4-Rb. The ability of SV40 T-antigen to abrogate
the repressive effect of GAL4-Rb suggested that the ``small
pocket'' of Rb was involved in repressing transcription. In
addition, we demonstrated that GAL4-Rb-mediated repression was
abolished when cyclin D1-cdk4 or cyclin E-cdk2 were coexpressed with
GAL4-Rb. Thus, the abrogation of GAL4-Rb-mediated repression by
cyclin-cdk complexes could be the result of the phosphorylation of
GAL4-Rb.
Deletion analyses have identified the domains of Rb
important for binding to certain viral oncoproteins such as adenovirus
E1A, SV40 T-antigen, and HPV E7
(45, 46) . These domains
extend from amino acid 379 to 571 (domain A) and from 649 to 792
(domain B). The E2F binding region in Rb coincides with the large
pocket extending from residues 379 to 928 excluding the spacer region
located between amino acids 571 and 649
(47, 48) .
Deletion of domain A of the pocket partially abolished GAL4-Rb-mediated
repression, whereas deletions or point mutations within domain B were
defective in repressing transcription. However, an intact spacer region
was not required. These results demonstrate the importance of domains A
and B for GAL4-Rb-mediated repression, consistent with the ability of
SV40 T-antigen to block repression. It is important to note that
GAL4-Rb proteins with deletions in the carboxyl-terminal domain of Rb
within the E2F binding domain also were able to inhibit the activity of
HSV tk promoter. Thus, the sequences of Rb involved in
mediating the repression of transcription map to ``small
pocket'' and are overlapping but not identical to those required
for the interaction with E2F.
Our results demonstrate that Rb, when
brought to the promoter by a GAL4 DNA binding domain, can significantly
repress transcription. Normally during the cell cycle, Rb is brought to
the promoter region through interactions with specific transcription
factors such as E2F. The interaction of Rb with E2F appears to block
its ability to activate transcription, in part, by blocking its
trans-activation domain. We propose that besides blocking the
trans-activation domain of E2F, Rb can negatively regulate
transcription through an alternate mechanism. Given that GAL4-Rb
represses activated but not basal transcription, we have examined the
ability of Rb to interact with a panel of different coactivator
proteins or TAFs (TBP-associated protein) including
TAF
Two models for how GAL4-Rb can regulate
transcription are shown in Fig. 6. Rb can be brought to the promoter by
the GAL4 DNA binding domain or through an interaction with a specific
transcription factor. Rb then can interact either with other
transcription factors to block their interaction with TFIID or interact
with TFIID directly through binding to a coactivator(s) such as
TAF
We thank Drs. Martin Schmidt and Steve Phillips for
critically reviewing the manuscript. We also thank Di Jiang, Dennis
Templeton, Ivan Sadowski, Charles Sherr, and Steve Reed for providing
important reagents.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1
(5) , TGF-
2
(6) , IGF-II
(7) , IL-6
(8) , and neu (9) . It appears that Rb regulates transcription by
interacting with specific transcription factors and modulating their
activity. Rb can interact in vivo with E2F
(10, 11, 12) , MyoD
(13) , and Elf-1
(14) and in vitro with ATF-2
(6) , N-Myc
(15) , c-Myc
(15) , PU.1
(16) , and UBF
(17) .
Plasmid Constructs
The plasmids G5BCAT,
pBLCAT2, and pSG424 have been previously described
(3, 7, 20) . E1b-CAT was provided by Dr. M.
Green
(21) . The GAL4-TK-CAT, GAL4-UAS, and
GAL4-UAS
reporter plasmids were provided by Dr. F.
Rauscher
(22) . GAL4-Sp1-CAT, Sp1-CAT, GAL4-AP1-CAT, AP1-CAT,
and GAL4-p53-CAT were constructed using Sp1 (CTAGGGGGCGGGGC), AP-1
(CTAGGTGACTCAGCGCG), and p53 (CTAGAGGCATGTCT) double-stranded
oligonucleotides. Briefly, single-stranded oligonucleotides were
annealed, and phosphorylated for 1 h at 37 °C in presence of 10
m
M ATP and 18 units of T4 bacteriophage kinase and then
partially ligated using T4 DNA ligase. The ligation products were
separated by an agarose gel (SeaKem 1% + NuSieve 2%), and
fragments of different sizes were isolated. The fragments with three
binding sites of a specific transcription factor were purified and
ligated to G5-E1b-CAT (for the construction of GAL4-Sp1-CAT,
GAL4-AP1-CAT, and GAL4-p53-CAT) and to E1b-CAT (for the construction of
Sp1-CAT and AP1-CAT) at the XbaI site. SVE, a SV40 early
promoter-based expression vector, and the human Rb expression plasmid
were provided by Dr. D. Templeton
(23) . The 1137 plasmid
encodes for a truncated T-antigen (amino acids 1-121) containing
the Rb/p107 binding domain
(24) . The 1137rb plasmid harbors a
single amino acid change Glu-107 to Lys that abolishes the Rb/p107
binding ability of 1137. GAL4-Rb was constructed by inserting the
4.5-kilobase EagI-filled in- SacI fragment from the
human Rb expression plasmid into SmaI and SacI sites
of pSG424. The GAL4-Rb plasmid contains almost the entire region of Rb
(10-928 amino acids) fused in-frame to the GAL4 (1-147) DNA
binding domain. All of the GAL4-Rb deletion mutants were constructed by
replacing the wild-type sequences in GAL4-Rb with the mutated
sequences, individually, from their expression plasmids, which were
provided by Dr. D. Templeton
(23) . GAL4-Rb
(706) and
GAL-Rb
(592) encodes for a GAL4-Rb fusion protein with a single
amino acid change at amino acids 706 of Rb and a deletion of exon 22
(amino acids 738-775), respectively. The 706 and 592 expression
plasmids were provided by Dr. J. M. Horowitz
(25, 26) .
The GAL-Rb (295-928) was provided by Dr. M. Green
(27) .
Cell Culture and Transfection
CCL-64 and
COS-7 cells were maintained in Dulbecco's modified Eagle's
medium plus 5 and 10% fetal calf serum, respectively. CCL-64 and COS-7
cells were transfected using a calcium phosphate protocol
(28) .
48 h post-transfection, the protein extracts were prepared as
previously described
(6) and used for chloramphenicol
acetyltransferase (CAT)(
)
assay. The acetylated
and nonacetylated forms of [
C]chloramphenicol
were separated by thin-layer chromatography and counted directly on a
Betagen counter. The level of CAT activity was calculated as the
percentage of the two acetylated forms of chloramphenicol relative to
the total amount of [
C]chloramphenicol. For
normalization of transfection efficiencies, a luciferase expression
plasmid pSV
-luc was included in each transfection as an
internal control. The transfection experiments were repeated at least
three times.
Western Blotting
2 10
COS-7 cells were transfected with 5 µg of each expression
plasmid as described above. 48 h post-transfection, the cells were
collected and resuspended in phosphate-buffered saline and then lysed
by mixing with equal volume of 2
Laemmli sample buffer. Equal
amounts of total proteins from each sample were separated on 8%
SDS-polyacrylamide gels and transferred to nitrocellulose membranes by
standard techniques
(29) . The membranes were then probed with a
rabbit polyclonal antibody to GAL4 (kindly provided by I. Sadowski)
followed by incubating with a horseradish peroxidase-conjugated
anti-rabbit antibody (Sigma). Cross-reactive proteins were detected by
the ECL system (Amersham Corp.).
GAL4-Rb Represses Sp1-, AP-1-, and p53-mediated
Transcription from Adjacent GAL4 Binding Sites
Rb protein
regulates the expression of a number of genes encoding
growth-regulatory factors by interacting with specific transcription
factors and modulating their activity
(3, 4, 5, 6, 7, 8, 9) .
To better understand the mechanism through which Rb protein regulates
transcription when it is a part of a DNA-bound complex, Rb was targeted
to the promoter by fusing Rb to the heterologous GAL4 DNA binding
domain. A GAL4-Rb expression vector was constructed by inserting the Rb
cDNA in-frame downstream of the yeast transcription factor GAL4 DNA
binding domain (GAL4 1-147) in the plasmid pSG424. G5BCAT,
containing 5 GAL4 binding sites upstream of E1b TATA box, was used as a
basal promoter reporter plasmid. Cotransfection of the GAL4-Rb
expression vector with the G5BCAT reporter plasmid into CCL-64 cells
did not affect transcription of G5BCAT (data not shown), suggesting
that GAL4-Rb is unable to regulate basal transcription.
Figure 1:
GAL4-Rb represses transcription
mediated by Sp1, AP-1, and p53. CCL-64 cells were transfected with SVE
(an SV40 expression plasmid), pSG147 (a control plasmid encoding for
GAL4 DNA binding domain), or GAL4-Rb expression plasmid and 1.5 µg
of the following reporter plasmids: GAL4-Sp1-CAT ( panel A), Sp1-CAT ( panel B), GAL4-p53-CAT
( panel C), GAL4-AP1-CAT ( panel D),
or AP1-CAT ( panel E). The results shown are
representative of at least three separate experiments. The results are
presented as relative CAT activity with SVE given a value of 1. The
structure of the reporters are shown below the corresponding
graphs.
GAL4-Rb Represses Transcription from a
Distance
To examine whether GAL4-Rb was able to repress
transcription from a distance, reporter plasmids carrying GAL4 binding
sites adjacent to the HSV tk promoter (GAL4-TK-CAT) and either
760 bp upstream (GAL4-UAS) or 1000 bp downstream
(GAL4-UAS
) of the transcription initiation site
were tested. The results of the cotransfection experiments with the
different reporters showed that the repression of the activity of HSV
tk promoter by GAL4-Rb was dependent upon the presence of the
GAL4 binding sites but was independent of their position (Fig. 2).
Again, repression of transcription was observed only with GAL4-Rb and
not with Rb (Fig. 2 A). These results demonstrate the
ability of GAL4-Rb to repress transcription from GAL4 binding sites
positioned at a distance from HSV tk promoter. These results
also rule out the possibility that GAL4-Rb was inhibiting transcription
by preventing the binding of Sp1, AP-1, or p53 to their
sequence-specific binding sites.
Figure 2:
GAL4-Rb represses the transcription from
GAL4 binding sites located at a distance from the transcription
initiation site. CCL-64 cells were transfected with SVE, pSG147,
GAL4-Rb, or Rb expression plasmid and 1.5 µg of the following
reporter plasmids: GAL4-TK-CAT ( panel A), pBLCAT2
( panel B), GAL4-UAS( panel C), and GAL4-UAS
( panel D). The results shown are representative of at least
three separate experiments. The results are presented as relative CAT
activity with SVE given a value of 1. The structure of the reporters
are shown below the corresponding
graphs.
SV40 T-antigen Prevents GAL4-Rb From Repressing the
Transcription
Rb protein interacts with several viral
oncoproteins such as SV40 T-antigen
(30) , human papillomavirus
(HPV), E7 protein
(31, 32) , and adenovirus E1A
(33) . The interaction of Rb with the viral oncoproteins alters
normal Rb function, presumably by dissociating Rb complexes. To
determine the effect of viral oncoproteins on the repression of
transcription mediated by GAL4-Rb, a pair of truncated SV40 T-antigen
mutants differing in their ability to bind to Rb were used in the
transfection assay (Fig. 3). The truncated SV40 T-antigen dl1137
prevented GAL4-Rb from repressing transcription in a dose-dependent
manner. In contrast, the mutant dl1137rb, deficient in its ability to
interact with Rb, did not block repression by GAL4-Rb at all input
doses. The reason for the marginal stimulation of GAL4-Tk-CAT by 1137
at the high input dose in the absence of GAL4-Rb is unclear but may
reflect an interaction of 1137 with endogenous p107, p130, and/or Rb.
Taken together, these results demonstrate the ability of SV40 T-antigen
to at least partially block GAL4-Rb-mediated repression and suggest
that the T-antigen binding pocket of Rb may be required for the
observed repression.
Expression of Cyclins and cdks Abrogates
GAL4-Rb-mediated Repression
Rb activity is regulated by
serine/threonine phosphorylation during the cell cycle
(34, 35, 36) . Several cdks and their regulatory
subunits (cyclins) have been shown to interact with and to
phosphorylate Rb
(37, 38, 39, 40, 41, 42, 43, 44) .
To determine the effect of Rb phosphorylation on GAL4-Rb-mediated
repression, cdk4, cdk2, and their respective regulatory subunits,
cyclin D1 and cyclin E, were cotransfected, either alone or in
combination, with GAL4-Rb (Fig. 4). Cotransfection of GAL4-Rb with
either cyclin D1 and cdk4 or cyclin E and cdk2 abrogated
GAL4-Rb-mediated repression, restoring HSV tk promoter
activity. In contrast, no significant effect on GAL4-Rb-mediated
repression was observed when cyclin D1, cyclin E, cdk4, and cdk2 were
cotransfected separately with GAL4-Rb. Moreover, in the absence of
GAL4-Rb, the overexpression of cyclin D1, cyclin E, cdk4, and cdk2,
either alone or in combination, did not affect the activity of the
GAL4-TK-CAT reporter. Thus, the abrogation of GAL4-Rb-mediated
repression by cyclin D1-cdk4 and cyclin E-cdk2 complexes may be the
result of the phosphorylation of GAL4-Rb.
Mapping the Domains Required for GAL4-Rb-mediated
Repression
To map the domain(s) of GAL4-Rb involved in the
repression of transcription, a panel of GAL4-Rb deletion mutants were
tested (Fig. 5). The levels of expression of the GAL4-Rb mutants were
determined by Western blot analysis using an anti-GAL4 antibody
(Fig. 5 C). Except for dl37-89 and dl309-343
(deleted for the amino acids indicated), the GAL4-Rb mutants were
expressed at a similar level in COS-7 cells and in CCL-64 cells. The
GAL4-Rb mutants were also tested for their ability to interact in
vivo with adenovirus E1A protein. E1A has been shown to activate
transcription when brought to the promoter through an interaction with
GAL4-Rb
(27) . Therefore, the ability of E1A to activate
transcription from G5BCAT when brought to the promoter through an
interaction with the GAL4-Rb mutants was determined. E1A was able to
activate transcription in the presence of the full-length GAL4-Rb and
from the majority of the GAL4-Rb mutants with an intact pocket domain
(Fig. 5 C). In contrast, deletions or point mutations within
domain A and domain B resulted in an inability of E1A to activate
transcription. However, four deletion mutants outside the pocket region
failed to support E1A activation, two of which were expressed at a
lower level (dl37-89 and dl309-343). The reason of the
decrease of the ability of E1A to activate transcription from the
mutants dl202-248 and dl248-309 is not clear.
Figure 5:
Rb ``small pocket'' is required
for GAL4-Rb-mediated repression. A, structure and the amino
acid positions of the mutant GAL4-Rb proteins. B, effect of
GAL4-Rb and GAL4-Rb mutants on HSV tk promoter activity.
CCL-64 cells were transfected with 1.5 µg of GAL4-TK-CAT reporter
and 2 µg of SVE, GAL4-Rb, or GAL4-Rb mutant expression plasmid. The
GAL4-Rb proteins designated by the annotation dl are deleted for the
amino acids indicated. The GAL4-Rb mutants 295-928 and 706*
encode for Rb ``large pocket'' and a naturally occurring
mutation at position 706. The results are presented as an average of
four separate experiments. C, top: level of
expression of GAL4-Rb and GAL4-Rb mutants. COS7 cells were transfected
with 5 µg of each of the GAL4-Rb plasmids. 2 days
post-transfection, the cells were lysed with RIPA buffer, and equal
amounts of total proteins from each sample were separated on 8%
SDS-polyacrylamide gels. The nitrocellulose membrane was probed with an
anti-GAL4 antibody. Bottom, activation of transcription by E1A
through its interaction with GAL4-Rb and GAL4-Rb mutants. NIH3T3 cells
were cotransfected with 4 µg of G5BCAT reporter plasmid, 2 µg
of SVE, GAL4-Rb, or GAL4-Rb mutant expression plasmid, and 4 µg of
E1A expression plasmid. The results are presented as relative -fold
activation with SVE given a value of 1.
The
ability of the GAL4-Rb deletion mutants to repress the transcription
from GAL4-TK-CAT reporter was examined in CCL-64 cells
(Fig. 5 B). GAL4-Rb proteins with deletions within the
amino-terminal domain of Rb did not alter GAL4-Rb-mediated repression.
The reduction in the repressive activity of GAL4-Rb dl37-89 and
dl309-343 may be the result of the low cellular level of the
proteins (Fig. 5 C). Deletion of domain A (mutant
dl389-580) resulted in a significant reduction in
GAL4-Rb-mediated repression. In addition, deletion or point mutation
within domain B (dl614-662, dl662-775, and the naturally
occurring mutants 592 (dl738-775) and 706) resulted in the
abrogation of GAL4-Rb-mediated repression. Deletion of 34 amino acids
in the spacer region (dl580-614) did not affect the repressive
activity of GAL4-Rb. Furthermore, the GAL4-Rb mutants truncated in the
carboxyl-terminal region of Rb (dl775-817, dl817-839, and
dl839-892) retained their ability to repress transcription. These
results demonstrate the requirement for domains A and B, but not for
the amino- and carboxyl-terminal regions of Rb, for full inhibition of
HSV tk promoter activity. Interestingly, the mutants
dl202-248 and dl248-309, which supported only weak E1A
activation, were able to repress tk promoter activity to the
same level as with the full-length GAL4-Rb.
250, TAF
150, TAF
110,
TAF
70, TAF
32, TAF
30-
, and
TAF
30-
. TAF
250 was able to bind with
high affinity to Rb and to GAL4-Rb in vivo and in vitro (49) and in the yeast two-hybrid system (data not shown).
Thus, Rb can bind to TFIID through an association with
TAF
250.
250. In the first model, Rb might interact with other
factors involved in transcription such as adjacently bound
transcription factors. In the second model, Rb may function as an
adapter-like molecule that can regulate transcription through
protein-protein interactions, bridging specific transcription factors
such as E2F with coactivators such as TAF
250. Since our
reporter plasmid contains only a binding site for a single
transcription factor, such as Sp1, AP-1, or p53, that has not been
shown to bind directly to Rb, we favor the latter model.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.