From the Departments of Viral Oncology and Gene
Research, Cancer Institute, Japanese Foundation for Cancer Research,
1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan
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ABSTRACT |
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ELL was originally identified as a
gene that undergoes translocation with the trithorax-like
MLL gene in acute myeloid leukemia. Recent studies have
shown that the gene product, ELL, functions as an RNA polymerase II
elongation factor that increases the rate of transcription by RNA
polymerase II by suppressing transient pausing. Using yeast two-hybrid
screening with ELL as bait, we isolated the p53 tumor suppressor
protein as a specific interactor of ELL. The interaction involves
respectively the transcription elongation activation domain of ELL and
the C-terminal tail of p53. Through this interaction, ELL inhibits both
sequence-specific transactivation and sequence-independent
transrepression by p53. Thus, ELL acts as a negative regulator of p53
in transcription. Conversely, p53 inhibits the transcription elongation
activity of ELL, suggesting that p53 is capable of regulating general
transcription by RNA polymerase II through controlling the ELL
activity. Elevated levels of ELL in cells resulted in the inhibition of
p53-dependent induction of endogenous p21 and substantially
protected cells from p53-mediated apoptosis that is induced by
genotoxic stress. Our observations indicate the existence of a mutually
inhibitory interaction between p53 and a general transcription
elongation factor ELL and raise the possibility that an aberrant
interaction between p53 and ELL may play a role in the genesis of
leukemias carrying MLL-ELL gene translocations.
The ELL gene (also known as MEN) was
molecularly identified as a gene that was fused to the Drosophila
trithorax-like MLL gene (also called
ALL-1 or Hrx) in acute myeloid leukemia carrying the t(11;19)(q23;p13.1) chromosomal translocation (1, 2). The gene
product, ELL, was recently demonstrated to function as an RNA
polymerase II elongation factor that increases the overall rate of
transcription elongation by RNA polymerase II via suppression of
transient pausing by the polymerase at many sites along DNA (3).
The connection between transcription elongation factors and oncogenesis
was first provided by the finding that the product of VHL
tumor suppressor gene, VHL, associates with the complex of the B and C
regulatory subunits of another transcription elongation factor, elongin
(4, 5). Binding of VHL to the elongin BC complex inhibits its ability
to activate elongin A subunit in transcription elongation. The
observation that many of the naturally occurring VHL mutants exhibit
substantially reduced binding to the elongin BC complexes led to the
suggestion that tumor suppression by VHL may involve negative
regulation of elongin as a transcription elongation factor.
ELL is the second transcription elongation factor found associated with
human malignancy. The chimeric protein generated by MLL-ELL
gene translocation contains an N-terminal AT-hook DNA binding domain
and the methyltransferase-like domain of MLL that is fused to almost
the entire ELL sequences (1, 2). Expression of the MLL portion of the
fusion protein appears to be insufficient for inducing the leukemic
phenotype in mice (6), indicating a role for the fusion partner of MLL
in cellular transformation.
p53 is a tumor suppressor protein that is mutated in more than 50% of
human cancers (7). Wild-type p53 inhibits cell growth and suppresses
cellular transformation when ectopically expressed (8-11). p53 is also
known to play a crucial role in apoptosis (12-15) and DNA repair
(16-18). Evidence accumulates that the transcriptional activity of p53
is a major component of its biological effects. p53 has
sequence-specific DNA binding properties and transcriptionally activates p53 response genes that contain p53-binding/responsive elements (19-22). Such genes include those for p21
cyclin-dependent kinase inhibitor (also known as WAF1 or
Cip1) (23-25), MDM2 (26-28), GADD45 (29, 30), cyclin G
(31), Bax (32, 33), and IGF-BP3 (34). Transcriptional regulation of
these genes by p53 explains at least in part the pleiotropic functions
of p53 in cell growth control. This transcriptional activity of p53 is
negatively regulated by the MDM2 protein (35, 36) and by the adenovirus
E1B protein (37). These proteins bind to the N-terminal domain of p53
and inhibit its transcriptional activity. Recent studies further
demonstrated that MDM2-p53 interaction accelerates the
ubiquitin-mediated degradation of p53 (38, 39). Common p53 mutants in
tumors lack the sequence-specific DNA binding activity because of the
point mutations in the DNA-binding core domain and, hence, cannot
transactivate the p53-target genes (40).
p53 is also known to repress transcription from a wide variety of
promoters that do not possess p53 binding/responsive elements (41-46).
The potency of p53 as a transcriptional repressor is also thought to
play a role in its function as a tumor suppressor. Although detailed
mechanisms involved in this transrepression are not well understood, it
appears to be mediated at least in part by the physical interaction
between p53 and TATA-binding protein
(TBP)1 (47-50). However, the
observation that additional molecules other than TBP are also required
for the transrepression by p53 (51) indicates that p53 may sequester
factors that are necessary for efficient initiation and/or elongation
of RNA polymerase II-dependent transcription in addition to
TBP.
In order to understand the mechanisms through which the ELL
transcription elongation factor affects human malignancy, we have investigated molecules that physically interact with ELL. In this report, we show that p53 specifically binds ELL. Through the
interaction, ELL reduces the transcriptional capacity of p53, as well
as its action as a general transcriptional repressor. Conversely, p53 binding blocks the transcription elongation activity of ELL.
Furthermore, elevated levels of ELL in cells inhibit
p53-dependent induction of p21 and protect cells from
p53-mediated apoptosis. Our results indicate the existence of mutually
inhibitory interaction between p53 and ELL and raise the possibility
that aberrant interaction between p53 and ELL may be involved in
leukemogenesis associated with MLL-ELL gene translocation.
Yeast Two-hybrid Screening--
ELL cDNA was
amplified from human cDNA library by standard polymerase chain
reaction, and the sequence of entire coding region was confirmed. Bait
plasmid was constructed in pAS2C, a CEN-ARS version of pAS2,
using full-length ELL cDNA. Yeast two-hybrid screening
of a human B cell cDNA library was performed in the yeast strain Y190.
Plasmid Construction--
A cDNA encoding the N-terminal Myc
or hemagglutinin (HA) epitope-tagged human ELL (Myc-ELL or HA-ELL) was
constructed by using polymerase chain reaction technique and was cloned
into pcDNA3 or pSP65SR
pOPTET vector is an inducible cDNA expression vector containing
TcIP promoter, a modified tetracycline-regulatable promoter consisting
of the tetracycline-repressible enhancer element and the lac operator
(52, 53). cDNA expression from the TcIP promoter is repressed by
tetracycline (Tc) and is strongly induced by the lactose analog,
isopropyl- Cell Culture--
COS-7, L929, and SAOS-2 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. SAOS-2-derived clone 3 (SAOS-2/clone-3) cell was generated by stably introducing expression plasmids for tTA and LacI by
calcium-phosphate method. The 6-1 cell is a BaF3-derived mouse pro-B
cell line that stably co-express tTA and LacI (53, 54). Cells were
cultured in RPMI 1640 medium containing 10% fetal calf serum and 20%
WEHI-3B-conditioned medium (20% WEHI) as a source of interleukin 3. Stable transfectants that conditionally express Myc-ELL was created by
transfecting pOPTET-Myc-ELL by electroporation as described (53).
Expression of Myc-ELL protein in these transfectants was repressed in
medium containing 1 µg/ml Tc and was induced in medium containing 5 mM IPTG in the absence of Tc.
Immunoprecipitation and Immunoblotting--
Cells were
transfected with various combinations of expression vectors (2 µg
each) using the DEAE-dextran method for COS-7 or the calcium-phosphate
method for L929 or SAOS-2 and were lysed after 72 h in ELB buffer
(250 mM NaCl, 5 mM EDTA, 50 mM
HEPES, pH 7.0, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin). Lysates were then immunoprecipitated with anti-Myc (9E10)
or anti-p53 (PAb1801, NEO MARKERS) mouse monoclonal antibody.
Immunoprecipitates were recovered on protein A-agarose, washed five
times with ELB buffer, eluted by boiling in SDS-containing sample
buffer, resolved by electrophoresis in a 7.5% SDS-polyacrylamide gel,
transferred to PVDF membrane filters, and probed with goat anti-p53
polyclonal IgG (Santa Cruz Biotechnology, no. sc-1314) or anti-Myc
(9E10). Immunoblots were visualized using donkey anti-goat IgG or
anti-mouse IgG secondary antibodies conjugated to horseradish
peroxidase followed by chemiluminescence detection.
Immunofluorescence Staining--
SAOS-2 cells were grown on
slide flasks and transfected with pcDNA3-HA-ELL and/or
pcDNA3-p53 using calcium-phosphate method. Two days after the
transfection, cells were processed for immunofluorescence. The primary
antibodies were rabbit anti-HA IgG and anti-p53 monoclonal antibody
(Pab1801). The secondary antibodies were Texas red-conjugated goat
anti-rabbit IgG and FITC-conjugated anti-mouse IgG.
Protein Expression and Purification--
cDNAs encoding
entire human ELL and p53 were respectively subcloned into the
Escherichia coli expression plasmid pGEX-5X-1 and pGEX-3X.
Recombinant GST-ELL and GST-p53 fusion proteins were affinity purified
from bacterial cell extracts by incubating with 25 µl of
glutathione-Sepharose beads in suspension for 30 min on ice. Beads were
washed six times with NETN (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40).
Similarly, portions of ELL or p53 were expressed as GST fusion proteins
in E. coli. Protein concentration was determined using the
Bradford protein assay (Bio-Rad). 10× histidine-tagged human ELL
(His-ELL) was overexpressed in E. coli with pET16b vector,
and the recombinant ELL was purified according to the previously
described method (3). The purity of the recombinant protein was
assessed by Coomassie Blue staining of SDS-polyacrylamide gel
electrophoresis gels, and each of the recombinant proteins was detected
as a single major band with the expected molecular weight.
In Vitro Binding Assay--
Full-length or portions of
ELL or p53 cDNAs subcloned into pcDNA3
vector were used for in vitro transcription and translation (TNT coupled transcription/translation system, Promega). Immobilized GST fusion protein (5 µg) was mixed with 5 µl of in
vitro translated [35S]methionine-labeled lysate and
incubated for 30 min at 4 °C. The beads were then washed six times
with NETN. Bound protein was separated on a 7.5% SDS-polyacrylamide
gel and detected by fluorography.
Luciferase Assays--
Plasmids were transiently transfected
into the SAOS-2 or SAOS-2/clone-3 cells (2 × 106) by
calcium-phosphate method. For inducible cDNA expression with the
use of pOPTET vector, transfected cells were split into two 60-mm
dishes (5 ml each). To each plate, either Tc (final concentration, 1 µg/ml) or IPTG (final concentration, 5 mM) was added.
Luciferase assays were performed 48 h after the transfection. The
luciferase activities of equal amounts of the extracted proteins were
measured by Lumat 9507 luminometer (Berthold).
The Oligo(dC)-tailed Template Assay--
The oligo(dC)-tailed
template assay was performed with the use of pCpGR220A/P/X plasmid (100 ng) and RNA polymerase (0.01unit) according to the method described
(3). After 25 min of incubation, 100 µM cold CTP, 2 µM UTP and GST-ELL (100 ng), or His-ELL (600 ng) was
added with or without 800 ng of GST-p53, and the reactions were further
incubated. RNA transcripts were analyzed by electrophoresis through 6%
polyacrylamide/7 M urea gels.
Physical Interaction between ELL and p53 in Cells--
To isolate
molecules that interact with ELL, we employed a yeast two-hybrid screen
using ELL as a bait. By screening 6.5 × 106
independent cDNA clones prepared from an Epstein-Barr virus
transformed human B lymphocyte library, we isolated cDNA encoding
nearly full-length p53 that lacked the N-terminal 3 and the C-terminal
2 amino acids. This two-hybrid interaction was abolished by deleting 47 amino acids from the C terminus of
p53.2
The association of ELL and p53 in mammalian cells was then addressed by
assessing the interaction of transiently expressed Myc-ELL with
endogenous p53. In COS-7 cells expressing Myc-ELL, co-immunoprecipitation of endogenous p53 with ELL was readily detectable. Reciprocally, co-immunoprecipitation of ELL with endogenous p53 was detectable when the same lysates were immunoprecipitated with
anti-p53 (Fig. 1A). Because
COS-7 cells express SV40 large T antigen, which forms a complex with
p53, we also examined the p53-ELL interaction in SAOS-2 human
osteosarcoma cell or L929 mouse fibroblast cell, both of which do not
possess SV40 large T antigen. Upon transient expression of p53 and
Myc-ELL, reciprocal co-immunoprecipitation of ELL and p53 was again
demonstrated in these cells (Fig. 1, B and C). In
the absence of antibodies that specifically recognize ELL, it was
impossible to examine the interaction between endogenous ELL and
p53.
Nuclear Localization of ELL and p53--
The intracellular
localization of p53 and ELL was next examined by co-expressing p53 and
HA-ELL in SAOS-2 cells, which lack endogenous p53 (Fig.
2). Transfected SAOS-2 cells were doubly stained with anti-p53 (green) and anti-HA (red).
As described previously, ELL was expressed diffusely in the nucleus but
excluded from the nucleoli (55). Anti-p53 staining showed nuclear
staining that was almost indistinguishable from that of ELL. Merging
the two images produced yellow, suggesting co-localization of the two
proteins in the nucleus.
Delineation of Interaction Sites in p53 and ELL--
In order to
further characterize the ELL-p53 interaction, in vitro
binding assays were performed using p53 fused with glutathione S-transferase (GST-p53) and in vitro-translated,
35S-labeled ELL protein. As demonstrated in Fig.
3A, ELL was specifically bound
to immobilized GST-p53. 35S-Labeled p53 protein that forms
oligomers with GST-p53 was used as a positive control. Reciprocally,
in vitro-translated p53 was specifically bound to
immobilized GST-ELL (Fig. 3B). Addition of RNase A to the
binding reaction did not affect the formation of ELL-p53 complexes,
excluding the possible involvement of RNA in this interaction. A direct
interaction between ELL and p53 was also demonstrated by far Western
blotting analysis using histidine-tagged ELL (His-ELL) as a protein
probe (Fig. 3C).
Various deletion derivatives of p53 were used to map the part of p53
that is required for ELL binding. In accordance with the observed yeast
two-hybrid interaction, GST-ELL did not bind p53 that lacks the
C-terminal 47 amino acids (Fig. 4A,
lane 2). Likewise, GST-p53 lacking the C-terminal 47 amino acids
of p53 failed to bind ELL (Fig. 4B, lane 4). The results
indicate that the C-terminal 47 amino acids of p53 are required for ELL
binding.
Analysis using deletion derivatives of ELL revealed that a C-terminally
truncated mutant, ELL(1-363), retained the ability to bind p53 (Fig.
4C, lane 3). However, further C-terminal deletion to residue
250 decreased binding significantly. Internal deletion of ELL between
residues 35 and 249 strongly impaired p53 binding and further deletion
completely abolished the interaction (Fig. 4C, lanes 9 and
11). The results indicate that the N-terminal half of ELL, a
region where the transcription elongation factor activity resides (56),
is required for its binding with p53.
Effect of ELL on p53-dependent Transcriptional
Activation--
Because the C terminus of p53, to which ELL binds, is
known to be important for its transcriptional activity (19-22), we
next examined whether ELL is capable of inhibiting p53-mediated
transactivation of promoters containing p53-responsive elements. To
this end, a transient luciferase reporter assay was conducted in SAOS-2 cells using RGC-, cyclin G-, or MDM2-promoter
luciferase construct (28, 31, 57). As demonstrated in Fig.
5A, the
p53-dependent transactivation of the RGC
promoter was strongly repressed by co-expressing ELL. The inhibition
was comparable to that induced by MDM2 or HDM2 (the human homologue of
MDM2) under the experimental conditions employed. The effects of ELL on
p53-responsive promoters were also examined by using an inducible
cDNA expression system (53) in SAOS-2 cells. Again, induced
expression of ELL inhibited the p53-dependent
transactivation, whereas an ELL mutant that cannot interact with p53
failed to do so (Fig. 5, B and C). However, in
contrast to MDM2 (38, 39), expression of ELL did not reduce the
expression levels of p53 as examined by anti-p53
immunoblotting.2 Hence, the results indicate that ELL
inhibits the sequence-specific transactivation by p53 through the
complex formation with p53.
Effect of ELL on p53-dependent Transcriptional
Repression--
In addition to acting as a transactivator, p53
represses the activity of a variety of promoters lacking p53-binding
sites (41-50). Accordingly, the potential involvement of ELL in
p53-dependent transrepression was next addressed by
transient luciferase reporter assays in SAOS-2 cells.
Induced expression of ELL in the SAOS-2 cells stimulated the activity
of the p53-repressible CMV promoter, which lacks a p53-binding motif,
in a dose-dependent manner (Fig.
6A). The result was consistent with the fact that ELL potentiates general transcription by functioning as the transcription elongation factor. In contrast, p53 strongly suppressed CMV promoter activity (Fig. 6B). To address the
effect of ELL on p53-mediated promoter suppression, subsequent
experiments were performed under conditions in which induced expression
of ELL alone did not significantly affect the CMV promoter activity (Fig. 6A, 3 µg of pOPTET-ELL). As shown in Fig.
6C, the suppression of the CMV promoter by p53 was
efficiently reversed by the induced expression of wild-type ELL but not
by a mutant ELL that fails to bind p53. The same observation was
reproduced when SV40 promoter, which also lacks the p53-binding motif,
was analyzed (Fig. 6D). Furthermore, elevated levels of ELL
in SAOS-2 cells abolished p53-mediated transcriptional repression of
the CMV promoter (Fig. 6E). Hence, these data indicate that
ELL counteracts p53-dependent transcriptional
repression.
Effect of p53 on Transcription Elongation Activity of
ELL--
Given that the N-terminal half of ELL contains the
elongation activity (56) and also binds p53, it is possible that the association of p53 with ELL may modify the elongation activity of ELL.
To address this, the effect of p53 on the activation of transcription
elongation by ELL was examined in an in vitro RNA polymerase
II transcription assay using an oligo(dC)-tailed template (3, 58). This
promoter-independent assay has the advantage over the runoff assay
because it permits a direct measurement of the activity of the
elongation factor in the absence of basal transcription factors that
might otherwise interact functionally with p53. Addition of purified
GST-p53 itself to the assay did not have any effect on the RNA
polymerase II-dependent RNA elongation (Fig.
7, A and B),
indicating that p53 does not directly enhance or block transcription
elongation. In contrast, addition of GST-ELL (Fig. 7, A and
B) or His-ELL (Fig. 7C) in the assay
substantially stimulated the rate of elongation of RNA transcript as
reported previously (3). This ELL-dependent stimulation of
RNA transcription was strongly suppressed by full-length p53 but not by
a p53 mutant that cannot bind ELL (Fig. 7, B and
C). The result indicates that p53 is capable of regulating
general transcription by RNA polymerase II through its ability to bind
ELL.
Effect of ELL upon p53-dependent Induction of p21 and
Apoptosis--
To further pursue functional relationship
between ELL and p53 in vivo, we addressed whether ectopic
expression of ELL can compromise the transcriptional activation of a
p53-responsive p21 gene within its native chromosomal
context. To this end, we generated stable transfectant clones that
inducibly express Myc-ELL from 6-1 cell. (Fig.
8A). This 6-1 cell is a
subline of interleukin 3-dependent mouse pro-B lymphoid
cell line, BaF3, and stably co-expresses tTA and lacI (53, 54). The
BaF3 cell is reported to possess functional p53 and undergo
p53-dependent apoptosis by DNA-damaging agents, such as
X-irradiation and cisplatin (59, 60). Upon transient transfection of
p53-responsive luciferase reporter plasmids into the 6-1 cells or the
Myc-ELL-transfectants in an uninduced condition (i.e.
expression of Myc-ELL was repressed in culture medium containing 1 µg/ml Tc), the reporter gene was inducibly expressed in
response to cisplatin or X-irradiation.3
This indicated that, like BaF3 cells, the 6-1 cell and its
Myc-ELL-transfectants possess functional p53.
Myc-ELL was induced to express in one of representative transfectants,
13-1, by treating the cells with IPTG in the absence of tetracycline
for 27 h. The parental 6-1 and the Myc-ELL transfectant cells were
then treated with cisplatin. After 6 h of incubation, cell were
lysed, and expression of endogenous p53 or the p53-responsive p21
proteins was examined by anti-p53 or anti-p21 immunoblotting. As
demonstrated in Fig. 8B, both cells inducibly expressed
comparable amounts of endogenous p53 in response to cisplatin
treatment. In addition, induction of endogenous p21 was observed in the
parental 6-1 cells following cisplatin treatment. In contrast, the 13-1 cells expressing exogenous ELL failed to induce p21 despite the continued presence of functional p53 (Fig. 8C).
Finally, the effect of ELL on p53-mediated apoptosis was addressed. As
shown in Fig. 8D, elevated expression of ELL resulted in
greater protection from DNA damage-induced apoptosis. Hence, the
results indicate that the transcriptional activity of p53 is blocked
and that, as a result, p53-dependent apoptosis is inhibited by ELL.
We demonstrate in this work that the RNA polymerase II
transcription elongation factor, ELL, physically interacts with the p53
tumor suppressor protein. The interaction involves the C-terminal 47 amino acids of p53 and the transcriptional elongation activation domain
of ELL. The complex formation does not require DNA or RNA because it is
directly demonstrated by far Western blotting with the use of
recombinant proteins and is totally insensitive to RNase treatment.
The transcriptional activity of p53 is thought to play a crucial role
in its function as a tumor suppressor. We show here that by forming a
complex with p53, ELL inhibits p53-dependent transactivation of promoters containing p53-responsive elements. Furthermore, elevated expression of ELL in cells inhibits the function
of endogenous p53 to transcriptionally induce p21 in response to DNA
damage. The C-terminal p53 region, to which ELL binds, is known to be
required for the p53 oligomerization (61-63) and is capable of binding
DNA or RNA nonspecifically (64-71). These C-terminal activities are
suggested to be indispensable for transactivation of certain
p53-dependent promoters (21, 72, 73). Thus, through complex
formation, ELL might directly inhibit the transcriptional activity
associated with the C-terminal region of p53. Alternatively, ELL
binding might sterically alter the central core region of p53 and
impair the sequence-specific DNA binding activity of p53. This
possibility is supported by the observation that double-strand DNA
nonspecifically binds the C-terminal p53 and inhibits the sequence-specific DNA binding of p53 that is mediated by the central domain (74).
We also demonstrate here that ELL reverses p53-dependent
transcriptional repression of promoters lacking the p53-binding motifs. The p53-dependent transrepression also requires the
C-terminal p53 (41-46), to which ELL binds. Although precise
mechanisms underlying the transrepression by p53 are yet to be
understood, interaction of p53 with TBP is suspected to be involved
(47-50). Because the TBP-p53 interaction also requires the C-terminal
region of p53, it is possible that ELL competitively blocks the p53-TBP
complex formation and, as a result, inhibits p53-dependent
transrepression through TBP.
MDM2, a well characterized p53 interactor, specifically inhibits
p53-dependent transactivation by binding to the N-terminal region of p53 (35, 36, 75). In contrast, ELL suppresses both
p53-dependent transactivation and transrepression through its binding with the C-terminal tail of p53. This indicates that ELL is
a novel negative regulator of p53. Furthermore, in contrast to MDM2,
which promotes the rapid degradation of p53 (38, 39), ELL does not
appear to reduce the levels of p53. In normal cells, in which p53
levels are low, ELL may be involved in the down-regulation of the
residual transcriptional activity of p53. Induction of p53 in response
to genotoxic stress results in the accumulation of p53 in relative
excess to ELL and provokes p53-dependent transactivation as
well as transrepression. Alternatively, modifications such as
phosphorylation of ELL and/or p53 in response to DNA damage may induce
dissociation of the complex and convert p53 from a latent form to an
active form, as has been shown in retinoblastoma protein-E2F
interaction (76). Because the p53-mediated transcriptional activities
have been implicated in playing crucial roles in the tumor suppressor
function of p53, ELL is expected to act as an oncoprotein when it is
inappropriately overexpressed, as is the case of MDM2 (35, 36, 77).
Consistently, ectopic overexpression of ELL is capable of transforming
fibroblast cells (78). The MLL-ELL fusion protein in leukemic cells
contains almost the entire ELL protein sequence. Such a fusion protein
might aberrantly inhibit p53 via quantitative and/or qualitative
differences from the wild-type ELL. Consistent with this idea, leukemic
cells with the MLL-ELL gene translocations examined to date
contain wild-type p53 genes (79).
We demonstrate in this work that p53 interacts with the N-terminal half
of ELL and inhibits the elongation activity of ELL in vitro.
Our results thus indicate that p53 may have a role in controlling
transcription by regulating ELL activity as a transcription elongation
factor. In addition to the elongation activation domain, the N-terminal
ELL is reported to possess a novel RNA polymerase II interaction domain
that can inhibit RNA polymerase II activity in promoter-specific
transcription initiation (56). More recently, a large ELL-containing
complex that contains multiple proteins in addition to ELL was
identified (80). This ELL complex possesses the transcription
elongation activity but cannot inhibit RNA polymerase II activity in
promoter-specific transcription, suggesting the presence of the
ELL-associated proteins that suppress the transcriptional inhibitory
activity of ELL. Our work presented here raises a possibility that p53
is also involved in this ELL-mediated polymerase regulation. As
discussed, p53 has been suggested to inhibit initiation of transcription by directly binding TBP. Taken this together, our finding
provides a model in which p53 is capable of inhibiting general
transcription in both initiation and elongation processes by targeting
TBP and ELL, respectively. If this is the case, then elevated ELL
activity as a result of p53 loss, ELL overexpression, or
gain-of-function mutations of ELL may also contribute to
cellular transformation.
The p53-ELL interaction presented here parallels recent findings on the
VHL tumor suppressor gene, mutations of which are involved
in the development of sporadic as well as hereditary forms of renal
carcinoma. Wild-type VHL associates with the two regulatory subunits, B
and C, of another general transcription elongation factor, elongin, and
inhibits the elongation activity of the elongin A subunit, whereas
mutant VHL molecules fail to do so (4, 5). In the case of p53-ELL
interaction, common p53 mutants in tumors lose their transcriptional
activities and hence do not receive regulation by ELL anymore.
Furthermore, ELL mutants, such as the MLL-ELL fusion protein, may
aberrantly inhibit p53 activity. Together with the VHL-elongin
interaction, our results indicate the existence of functional
connections between transcription elongation factors and tumor
suppressors in the control of normal cell growth and in the prevention
of cellular transformation.
Finally, functional antagonism between p53 and ELL indicates that the
p53-mediated transcriptional control and the ELL-mediated transcriptional control should be executed in a mutually exclusive manner so as to exert coordinated expression of genes involved in cell
growth and differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 vector. Mammalian expression vectors
pcDNA3-p53, pcDNA3-ELL, pcDNA3-mdm2, pcDNA3-hdm, and
pcDNA3-
-galactosidase were constructed by inserting cDNAs
encoding human p53, human ELL, mouse MDM2, human homologue of MDM2
(HDM2), and
-galactosidase, respectively, into pcDNA3.
-thiogalactopyranoside (IPTG) in cells that ectopically
express the Tc-repressible transactivator (tTA) and the lac
repressor (LacI). A cDNA encoding p53, ELL, or mutant ELL was
cloned into pOPTET vector.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
In vivo association of ELL with
p53. A, whole cell lysates from COS-7 cells transfected
with pSP65SR 2 (lanes 1, 3, 5, and 7) or
pSP65SR
2-Myc-ELL (lanes 2, 4, 6, and 8) were
subjected to immunoprecipitation with anti-Myc (lanes 1 and
2) or anti-p53 (lanes 5 and 6). The
immunoprecipitated proteins were then analyzed by immunoblotting with
anti-p53 (lanes 1 and 2) or anti-Myc (lanes
5 and 6). Endogenous p53 (lanes 3 and
4) and ectopic Myc-ELL (lanes 7 and 8)
in the transfected COS-7 cells were detected by immunoblotting of the
whole cell lysates. B, whole cell lysates from SAOS-2 cells
transfected with pcDNA3-HA-ELL (lanes 3, 4, 7, 8, 11, 12, 15, and 16) and/or pcDNA3-p53 (lanes 2, 4, 6, 8, 10, 12, 14, and 16) were subjected to
immunoprecipitation with anti-HA (lanes 1-4) or anti-p53
(lanes 9-12). The immunoprecipitates were then analyzed by
immunoblotting with anti-p53 (lanes 1-4) or anti-HA
(lanes 9-12). Expression levels of p53 and HA-ELL were
analyzed by anti-p53 (lanes 5-8) or anti-HA (lanes
13-16) immunoblotting of the whole cell lysates. C,
whole cell lysates from L929 cells transfected with pSP65SR
2-Myc-ELL
(lanes 3, 4, 7, 8, 11, and 12) and/or
pcDNA3-p53 (lanes 2, 4, 6, 8, 10, and 12)
were subjected to immunoprecipitation with anti-p53 and the
immunoprecipitates were analyzed by immunoblotting with anti-Myc
(lanes 1-4). Expression levels of p53 and Myc-ELL were
analyzed by anti-p53 (lanes 5-8) or anti-Myc (lanes
9-12) immunoblotting of the whole cell lysates.
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Fig. 2.
Localization of ELL and p53 in the
nucleus. SAOS-2 cells transiently co-transfected with
pcDNA3-HA-ELL and pcDNA3-p53 were stained with mouse monoclonal
anti-p53 and rabbit anti-HA anti-sera. The secondary antibodies were
FITC-conjugated anti-mouse IgG (green) and Texas
red-conjugated goat anti-rabbit IgG (red).The merged,
two-color image is shown on the right; yellow
shows upon co-localization of green (p53) and red
(ELL) signals.
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Fig. 3.
In vitro binding assay between p53
and ELL. A, in vitro translated ELL,
-galactosidase (
-gal) or p53 was incubated with GST
beads (lanes 4-6) or GST-p53 beads (lanes 1-3).
Total inputs of in vitro translated proteins are shown in
lanes 7-9. B, in vitro translated p53
was incubated with GST beads (lanes 3 and 4) or
GST-ELL beads (lanes 1 and 2) in the presence
(lanes 2 and 4) or absence (lanes 1 and 3) of 100 µg/ml RNase A. Total inputs of in
vitro translated proteins are shown in lanes 5 and
6. C, far Western blotting. His-ELL or bovine
serum albumin (BSA) spotted onto a nitrocellulose filter was
incubated with GST-p53 (0.1 µg/ml). After washing, the filter was
probed with anti-p53, and the bound p53 was detected by the
chemiluminescence technique.
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Fig. 4.
Mapping of protein interaction domains.
A, in vitro translated wild-type p53
(p53wt) (lanes 1 and 3) or a mutant
p53 lacking the C-terminal 47 amino acids (p53 C)
(lanes 2 and 4) was incubated with GST beads
(lanes 3 and 4) or GST-ELL beads (lane
1 and 2). Total inputs of in vitro
translated proteins are shown in lanes 5 and 6.
B, in vitro translated full-length ELL was
incubated with GST-p53 beads with various deletions in the p53 portion
(lanes 1-7). Total input of in vitro translated
ELL is shown in lane 8. A schematic representation of each
deletion derivative of p53 is shown (bottom). C,
in vitro translated deletion derivatives of ELL were
incubated with GST beads or GST-p53 beads (lanes 1-12).
Total inputs of translated proteins are shown in lanes
13-18. A schematic representation of each deletion derivatives of
ELL is shown (bottom).
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Fig. 5.
Effect of ELL on p53-dependent
transactivation. A, p53-negative SAOS-2 cells were
transiently co-transfected with 4 µg of the p53-responsive RGC
promoter-luciferase reporter construct (pRGC-luciferase) and 1 µg of
pcDNA3-p53, along with 1 µg of pcDNA3-ELL, pcDNA3-mdm2,
pcDNA3-hdm or pcDNA3- -galactosidase (
-gal).
B, SAOS-2 cells were transfected with 4 µg of one of the
p53-responsive promoter-luciferase reporter constructs
(pRGC-luciferase, pCyclin-G-luciferase, or pMDM2-luciferase) along with
1 µg of pcDNA3-p53 and/or 1 µg of pcDNA3-ELL. C,
SAOS-2/clone-3 cells were transfected with 4 µg of pRGC-luciferase
along with 1 µg of pcDNA3-p53 and 3 µg of pOPTET-ELL or
pOPTET-ELL
, which expresses a mutant ELL, ELL
, that lacks
residues between 35-363 and hence cannot bind p53. Expression of ELL
or ELL
from the pOPTET vector was suppressed by 1 µg/ml Tc and was
induced by 5 mM IPTG. Values shown are the mean ± S.E. of three independent transfection experiments.
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Fig. 6.
Effect of ELL on p53-dependent
transrepression. A and B, SAOS-2/clone-3
cells were transiently transfected with pCMV-luciferase reporter
construct (4 µg) along with increased amounts of pOPTET-ELL
(A) or pOPTET-p53 (B). Expression of ELL or p53
from the pOPTET vector was suppressed by 1 µg/ml Tc and was induced
by 5 mM IPTG. C and D, SAOS-2 cells
were transfected with 4 µg of the reporter construct, pCMV-luciferase
(C) or pSV40-luciferase (D), along with 1 µg of
pcDNA3-p53 and 3 µg of pOPTET-ELL or pOPTET-ELL . Expression of
ELL or ELL
from the pOPTET vector was suppressed by 1 µg/ml Tc and
was induced by 5 mM IPTG. Values shown are the mean ± S.E. of three independent transfection experiments. E,
SAOS-2/clone-3 cells were triply transfected with 4 µg of
pCMV-luciferase, 3 µg of pOPTET-p53, and 12 µg of pcDNA3 or
pcDNA3-ELL. Expression of p53 from the pOPTET vector was suppressed
by 1 µg/ml Tc and was induced by 5 mM IPTG. Values shown
are the mean ± S.E. of three independent transfection
experiments.
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Fig. 7.
Effect of p53 on ELL-dependent
transcription elongation in vitro. A,
the oligo(dC)-tailed template assays were performed in the absence
(lanes 1-4) or presence of purified GST-p53 (lanes
5-7) or GST-ELL (lanes 8-10). Transcripts were chased
for 5, 15, and 30 min. B, the oligo(dC)-tailed template
assays were performed in the absence (lanes 1, 2, 5, and
6) or presence (lanes 3, 4, 7-12) of GST-ELL.
Purified GST-p53 (lanes 5, 6, 9, and 10) or GST-p53(1-346),
which cannot interact with ELL (lanes 11 and 12),
was added to the reaction. Transcripts were chased for 30 min.
C, the oligo(dC)-tailed template assays were performed in
the absence (lanes 1-4) or presence (lanes
5-13) of His-ELL. Purified GST-p53 (lanes 8-10) or
GST (lanes 11-13) was added to the reaction. Transcripts
were chased for 5, 15, and 30 min.
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Fig. 8.
Inhibition of p53-mediated p21 induction by
ELL. A, expression of ELL in 6-1 cells. Myc-epitope
tagged ELL was inducibly expressed in 6-1-derived stable transfectant
clones, 13-1 and 14-1, by culturing cells with 5 mM IPTG in
the absence or presence of tetracycline for 24 h. Total cell
lysates were prepared from cells and were subjected to anti-Myc
immunoblotting. Parental 6-1 cells were used as a negative control. A
position of Myc-ELL is indicated. B, following 27 h of
culture in the presence of IPTG, cells were treated with 8 µg/ml
cisplatin for additional 6 h. Lysates were prepared from the
cisplatin-treated and untreated cells and were subjected to
immunoblotting with anti-p53 antibody. A position of p53 is indicated.
C, the same samples used in B were immunoblotted
with anti-p21 antibody. A position of p21 is indicated. D,
cell viability of the parental 6-1 and ELL transfectants, 13-1 and
14-1, following cisplatin treatment. Cells (2 × 105/ml) were cultured in RPMI 1640 medium supplemented with
10% fetal calf serum and 20% WEHI-conditioned medium in the presence
of 5 mM IPTG and 5 µg/ml cisplatin. At 24 and 36 h
after the onset of culture, cell numbers were determined by trypan blue
dye exclusion assay. Cell viability (% viable cells/total cells) was
calculated and is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank L. Yamazaki, D. Cobrinik, and R. A. Weinberg for valuable comments and discussions; B. Vogelstein for p53 cDNAs; M. Oren for p53-responsible promoters; and B. Elenbaas for cDNAs encoding MDM2 and HDM2.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, Japan and by grants from Vehicle Racing Commemorative Foundation and Nippon Boehringer Ingelheim Co., Ltd.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81-3-5394-3880; Fax: 81-3-5394-3816; E-mail: mhatakeyama{at}jfcr.or.jp.
2 N. Shinobu, T. Maeda, and M. Hatakeyama, unpublished observations.
3 T. Kondo, T. Ito, and M. Hatakeyama, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
TBP, TATA-binding
protein;
HA, hemagglutinin;
Tc, tetracycline;
IPTG, isopropyl--thiogalactopyranoside;
GST, glutathione
S-transferase;
CMV, cytomegalovirus.
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REFERENCES |
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