Howard Hughes Medical Institute, Department of Genetics, Harvard Medical
School, Boston, Massachusetts 02115, USA
* Present address: Department of Exploratory Science, Biogen Inc., 12 Cambridge
Center, Cambridge, Massachusetts 02142, USA
Author for correspondence (e-mail:
leder{at}rascal.med.harvard.edu)
Accepted 23 October 2002
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Summary |
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Key words: DAXX, RNAi, Apoptosis, Transcriptional repression, NF-B, E2F1
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Introduction |
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Many reports have implicated DAXX in apoptosis, but whether it functions as
a pro- or anti-apoptotic molecule has been a matter of dispute. Targeted
deletion of Daxx in the mouse results in severe developmental
abnormalities in knockout embryos, highlighted by extensive apoptosis at
embryonic day (e) 7.5 and e8.5 (Michaelson
et al., 1999). Daxx-knockout embryonic stem cell (ES)
lines are similarly characterized by elevated levels of apoptosis
(Michaelson et al., 1999
).
Together, these studies imply that DAXX plays a protective role in preventing
apoptosis. Additional evidence implicating DAXX as anti-apoptotic include a
report showing the ability of DAXX to inhibit CD43-mediated apoptosis in
hematopoietic cells (Cermak et al.,
2001
), and a recent suggestion that histone deacetylase
inhibitor-induced apoptosis may be associated with downregulation of DAXX in
acute promyelocytic leukemia cells (Amin et
al., 2001
).
Overexpression studies, however, suggested that DAXX is capable of inducing
apoptosis. DAXX, initially identified as interacting with the Fas receptor
death domain, was shown to enhance Fas-mediated apoptosis when overexpressed
(Yang et al., 1997). DAXX was
proposed to function in a pathway independent of Fas-associated death domain
and to mediate apoptosis through activation of the Jun N-terminal kinase (JNK)
pathway via apoptosis signal-regulating kinase 1
(Chang et al., 1998
;
Chang et al., 1999
;
Ko et al., 2001
;
Yang et al., 1997
). Similarly,
a role for DAXX in transforming growth factor ß (TGF-ß)-induced
apoptosis and associated JNK activation was shown in B-cell lymphomas and
hepatocytes (Perlman et al.,
2001
). Interestingly, Fas-mediated apoptosis and JNK signaling was
shown to be independent of DAXX in lymphoid cells
(Villunger et al., 2000
).
Other studies have proposed that the ability of DAXX to induce apoptosis
relies not on the ability of DAXX to interact with Fas, but rather on a
nuclear apoptotic pathway, consistent with DAXX localization studies.
Furthermore, it was suggested that DAXX facilitates the induction of apoptosis
in primary splenocytes and keratinocytes from within PODs and only in the
presence of PML (Zhong et al.,
2000). Similarly, Torii et al. report that the ability of DAXX to
facilitate Fas-induced apoptosis requires DAXX localization to PODs
(Torii et al., 1999
).
The ability of DAXX to function as a transcriptional repressor has also
been shown. DAXX was shown to bind to Pax3, a member of the paired box
homeodomain family of transcription factors, and on overexpression DAXX
repressed Pax3-mediated transcriptional activity
(Hollenbach et al., 1999).
Interaction of DAXX with the ETS1 transcription factor similarly caused
repression of transcriptional activity (Li
et al., 2000b
). When tethered to a galactose 4 (Gal4)-DNA binding
domain, DAXX could inhibit basal transcription, possibly through recruitment
of histone deacetylases (Li et al.,
2000a
). In B cells, DAXX was shown to function as a co-repressor,
and under certain circumstances as a co-activator, of Pax5 (BSAP)
(Emelyanov et al., 2002
). A
recent report suggests that the ability of DAXX to interact with histone
deacetylases, core histones and the chromatin-associated protein DEK, provides
the mechanism by which DAXX represses transcription
(Hollenbach et al., 2002
).
We have used RNAi as a method to deplete cell lines of endogenous DAXX
protein in an effort to determine conclusively the function of DAXX in
apoptosis and transcriptional regulation. RNA interference (RNAi) is the
process of gene-specific post-transcriptional silencing following the
introduction of double-stranded RNA homologous to the gene of interest
(reviewed by Hunter, 2000).
The phenomenon, initially described in Caenorhabditis elegans, has
recently been successfully used as a tool to inhibit gene expression in
mammalian cells (Caplen et al.,
2001
; Elbashir et al.,
2001
). Specifically, transfection of 21-23-nucleotide
double-stranded RNAs into human and mouse cell lines was shown to efficiently
and specifically suppress the expression of target genes, either endogenous or
overexpressed.
Our analysis of cell lines depleted of DAXX by RNAi has revealed increased levels of apoptosis, confirming the role of DAXX as an anti-apoptotic protein. Furthermore, transcriptional studies in DAXX-depleted cells have shown that endogenous DAXX represses transcriptional activity, and has allowed for the identification of probable physiological targets of DAXX repression.
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Materials and Methods |
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HeLa (5x104), U2OS (6x104) and 293 (7.5x104) cells were grown in six-well dishes for 24 hours in medium without antibiotics before RNA transfection. Cells were transfected with 10 µl of 20 µM double-stranded RNA in Opti-Mem media (Gibco) using 3 µl Oligofectamine (Invitrogen) precisely according to the manufacturer's instructions. For FACS analysis, HeLa cells were plated at 3.5x105 per 10 cm plate and transfected with 40 µl of 20 µM RNA and 12 µl Oligofectamine.
For DNA transfections, HeLa cells were transfected with 3 µg DNA per well of a six-well dish (or 8 µg DNA for a 10 cm plate) using Fugene6 reagent (Boehringer Mannheim) in medium containing serum.
RNA oligonucleotides
21-nucleotide RNA oligonucleotides were obtained from Dharmacon
Research.
hDx1: CCCUCCCACACACCUCUCCdTdT, GGAGAGGUGUGUGGGAGGGdTdT
GenBank AF015956 bp 531-551.
hDx2: GGAGUUGGAUCUCUCAGAAdTdT, UCUGAGAGAUCCAACUCCdTdT
GenBank AF015956 bp 678-798.
mDx2: GGAGUUGGACCUGUCAGAGCdTdT, GCUCUGACAGGUCCAACUCCdT(dT)
GenBank NM_007829 bp 666-686.
RNA oligonucleotides were annealed as described previously
(Elbashir et al., 2001).
DNA constructs
The Daxx expression vector contains full-length mouse
Daxx with a C-terminal myc epitope tag expressed under the control of
the chicken ß-actin promoter in the pCAGGS vector
(Niwa et al., 1991). pEGFP-C1
(Clontech) was used for expression of green fluorescent protein (GFP). The
Bcl-2 expression vector contained full-length human Bcl-2 cloned into
pCDNA3.1. cMet-luciferase (Met-luc) contains luciferase under control of the
cMet promoter (Epstein et al.,
1996
). E2F1-luc and SP1-luc were kind gifts from P. Farnham
(University of Wisconsin Medical School, Madison, WI)
(Slansky et al., 1993
). Other
luciferase reporter constructs were obtained from Stratagene.
Western blot analysis
Protein extracts were prepared in non-denaturing buffer [1% NP40, 0.15 M
NaCl, 0.01 M NaPO4, 2 mM EDTA and 50 mM NaF with Complete protease
inhibitors cocktail (Boehringer Mannheim)]. Samples were electrophoresed on 8%
polyacrylamide gels and then transferred to a polyvinylidene fluoride (PVDF)
Immobilon-P membrane (Millipore). Affinity purified anti-DAXX polyclonal
antibody was used as described (Michaelson
et al., 1999). Monoclonal anti-ß-actin diluted 1:5000 (Sigma)
and rabbit anti-poly-ADP ribose polymerase diluted 1:500 (Santa-Cruz) were
also used as primary antibodies. Horseradish peroxidase linked sheep
anti-rabbit and anti-mouse immunoglobulin G (Amersham) were used as secondary
antibodies, followed by detection by electrochemiluminescence (ECL).
FACS analysis
Cells were collected 48, 72 or 96 hours following RNAi treatment, washed in
PBS and fixed in 70% ethanol. Samples were incubated with RNase A (0.5 mg/ml)
and propidium iodide (5 µg/ml), followed by analysis on a FACS Calibur flow
cytometer (Becton-Dickinson). A minimum of 1.5x105 cells were
analyzed for each FACS experiment sample. For GFP experiments, a minimum of
5x104 cells were analyzed.
Reporter assays
Lysates were prepared from six-well dishes 24 hours following DNA
transfection in 250 µl Passive Lysis Buffer (Promega; Madison, WI), and a
50 µl sample was analyzed on an Automat LB953 luminometer (Berthold) with
automatic injection of luciferase reagent (Fischer). All samples were
co-transfected with 50-100 ng ß-galactosidase reporter construct,
pCMVß (MacGregor and Caskey,
1989). Luciferase values were normalized for transfection
efficiency by measuring ß-galactosidase activity using the Galacto-star
system (Tropix). All transfections were performed in triplicate.
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Results |
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The specificity of the hDx2 effect was confirmed using a control RNA oligonucleotide (mDx2), the mouse homolog of hDx2 which differs in sequence by three nucleotides plus an additional nucleotide. Indeed, mDx2 had no effect on DAXX protein levels (Fig. 1C). Transient transfection of mouse Daxx cDNA following RNAi treatment revealed that hDx2 had no effect on accumulation of overexpressed mouse DAXX, whereas mDx2 treatment prevented overexpression (Fig. 1D). These studies confirmed the species specificity of hDx2 and mDx2. As shown in Fig. 1E,F, hDx2 was also effective in U2OS and 293 cells, with the resulting depletion of DAXX protein being slightly more pronounced following a second round of transfection. Taken together, these studies show that treatment of human cell lines with hDx2 results in a specific and significant depletion of endogenous DAXX protein.
Increased levels of apoptosis upon depletion of DAXX by RNAi
Whether DAXX functions as a pro- and/or anti-apoptotic molecule has been a
matter of dispute in the literature (reviewed by
Michaelson, 2000). Although
results from knockout studies show that DAXX probably plays a role in
preventing apoptosis in the early embryo and in ES cells, overexpression
studies suggested that DAXX may function as a pro-apoptotic molecule in other
cell types. Our ability to deplete DAXX using RNAi provided the opportunity to
directly assess the function of DAXX in cell lines previously analyzed in
overexpression studies (Chang et al.,
1998
; Chang et al.,
1999
; Ko et al.,
2001
; Torii et al.,
1999
; Yang et al.,
1997
).
The cell-cycle profile of HeLa cells transfected with hDx2 was analyzed by
FACS. The sub-G1 peak, indicative of the apoptotic fraction due to fragmented
DNA content, was modestly yet significantly increased in hDx2-compared with
mock-transfected cells (Fig.
2A). The increased levels of apoptosis were evident as early as 48
hours post-transfection and were more pronounced 72 and 96 hours following
RNAi transfection. The levels observed in the DAXX-depleted cells were similar
to those observed in Daxx-knockout ES cell lines
(Michaelson et al., 1999).
Elevated levels of apoptosis were similarly observed following transfection of
hDx2 into 293 cells (data not shown). To verify that the increased apoptosis
observed in HeLa cells was not a nonspecific RNAi effect, mDx2 was used in a
similar experiment and showed no apoptotic effect
(Fig. 2B).
|
As an additional method of assessing apoptosis, cleavage of the caspase target PARP was analyzed by western blot following RNAi treatment. In contrast to extracts prepared from mDx2-transfected HeLa cells, in which only full-length PARP (112 kDa) was evident, depletion of DAXX by hDx2 treatment resulted in the reproducible appearance of the cleaved version of PARP (85 kDa) (Fig. 2C), indicating activation of the caspase cascade.
It is well documented that Bcl-2, the founding member of a conserved family
of proteins that regulate cell death, functions to inhibit apoptosis (reviewed
by Adams and Cory, 2001). We
tested whether overexpression of Bcl-2 could rescue the apoptosis induced by
DAXX depletion. HeLa cells were transfected with Bcl-2 or vector control 4
hours post-RNAi treatment. Measurement by FACS analysis revealed a significant
rescue of the apoptotic fraction following transfection with Bcl-2
(Fig. 3A). The rescue was not
complete, as expected, given the limitation of transfection efficiency. To
monitor transfected cells only, HeLa cells were co-transfected with GFP in
addition to Bcl-2 or vector control following RNAi treatment. FACS analysis on
GFP-positive staining cells revealed a complete rescue of apoptotic cells
(Fig. 3B). Note that in
addition to rescue of hDx2-induced apoptosis, Bcl-2 also rescued the apoptotic
fraction generated presumably as a result of GFP toxicity
(Liu et al., 1999
), which was
also evident in mock-transfected cells.
|
RNAi depletion of DAXX results in transcriptional de-repression
Previous studies have shown that DAXX, when overexpressed, can mediate
transcriptional repression (Emelyanov et
al., 2002; Hollenbach et al.,
2002
; Hollenbach et al.,
1999
; Li et al.,
2000a
; Li et al.,
2000b
). We have assessed the ability of endogenous DAXX to mediate
repression by measuring transcriptional activity of putative targets in cells
depleted of DAXX by RNAi. Following RNAi treatment, HeLa cells were
transfected with Met-luc (Epstein et al.,
1996
), a luciferase reporter gene driven by the Pax-3-regulated
c-Met (hepatocyte growth factor) promoter. Cells treated with hDx2 RNAi
revealed significantly increased levels of luciferase activity relative to
mock- or mDx2-treated cells, indicating that depletion of endogenous DAXX
causes de-repression of the c-Met promoter
(Fig. 4A).
|
To show that the de-repression was solely a function of the loss of DAXX,
an attempt was made to revert the de-repression by reconstituting cells with
increasing levels of DAXX. Reconstitution was accomplished by transfection
with mouse Daxx, which is unaffected by hDx2 treatment (see
Fig. 1D). In mock-treated
cells, transfection of mouse Daxx cDNA resulted in decreased activity
of Met-luc (Fig. 4B),
indicative of repression of the c-Met promoter and consistent with previous
studies showing the repressive effects of DAXX overexpression
(Hollenbach et al., 1999;
Li et al., 2000b
). In
hDx2-treated cells, where increased levels of c-Met activity were observed
(Fig. 4B), transfection of
increasing levels of Daxx resulted in an incremental decreases in
luciferase activity (Fig. 4B).
In the presence of a high concentration of transfected Daxx (2.0
µg), significant repression of Met-luc was observed. These results confirm
that loss of DAXX is responsible for the de-repression observed in the RNAi
experiments.
The ability of endogenous DAXX to mediate repression of several other
luciferase reporter constructs was tested. Following depletion of DAXX by
hDx2, significantly increased luciferase activity from reporters driven by
NF-B and E2F1 elements (NF
B-luc and E2F1-luc) was observed
relative to mDx2-treated cells (Fig.
5A), indicating that NF-
B and E2F1 targets are probably
repressed by endogenous DAXX. In contrast, a basal promoter element (TATA-luc)
showed no difference in luciferase activity when treated with hDx2 as compared
with mDx2 (Fig. 5A). Similarly,
RNAi treatment had no significant effect on the transcriptional activity of
several reporter constructs, including AP1-luc and others
(Fig. 5A). Thus, it is likely
that endogenous DAXX does not regulate this subset of promoters in the given
cellular context.
|
The ability of DAXX to repress the panel of reporter constructs was also
tested using DAXX overexpression studies. When DAXX was overexpressed, the
Met-, NF-B- and E2F1-driven luciferase activities were accordingly
repressed, but there was no significant effect on TATA-luc activity
(Fig. 5B). These results are
consistent with the effect of DAXX observed in the RNAi studies
(Fig. 5A). However, in
overexpression studies, DAXX was capable of moderately repressing AP1-luc,
SP1-luc and cAMP-responsive element (CRE)-luc, reporters that were not
accordingly de-repressed following depletion of endogenous DAXX
(Fig. 5A). Taken together,
these studies probably identify a set of physiological targets of DAXX, which
include the Pax-3 regulated c-Met, as well as NF-
B and E2F1
targets.
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Discussion |
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We have taken advantage of the power of RNAi to evaluate the function of
DAXX. Only recently has RNAi been shown to be effective in inhibiting gene
expression in mammalian systems (Caplen et
al., 2001; Elbashir et al.,
2001
). As a technical application, our studies exploit RNAi as a
tool to evaluate mammalian gene function, namely apoptosis and transcriptional
regulation. Recently, a few examples of successful use of RNAi in assessing
cell growth and division were reported (Du
et al., 2001
; Harborth et al.,
2001
; Ohta et al.,
2002
). Our studies have further taken advantage of the specificity
of RNAi to enable human cells depleted of DAXX to be reconstituted with mouse
DAXX. By reintroducing DAXX into RNAi-depleted cells, we could show the
specificity of the RNAi effect in mediating transcriptional de-repression.
This type of reconstitution experiment is a powerful and effective method that
will undoubtedly prove useful in validating phenotypes observed following RNAi
treatment.
The ability to effect nearly complete depletion of endogenous protein was
crucial for these studies. The observations that Daxx heterozygous
mice develop normally and that Daxx heterozygous embryos as well as
ES cells show no increase in apoptosis, despite the reduced levels of DAXX
protein (Michaelson et al.,
1999), suggest that modest levels of DAXX within the cell are
probably sufficient for normal gene function. Therefore, it is likely that a
relatively complete reduction in protein levels is required to achieve
functional deletion of DAXX. Previous studies have attempted to use anti-sense
technology to assess DAXX function. For example, in a study by Gongora and
colleagues (Gongora et al.,
2001
), the significant levels of residual DAXX evident following
anti-sense treatment make it difficult to interpret the effect observed on
interferon-induced apoptosis in pro-B cells. Antisense studies were also
employed to assess TGF-ß-induced apoptosis in murine hepatocytes;
although protection from apoptosis was observed, the extent to which depletion
of DAXX was achieved was not reported
(Perlman et al., 2001
). The
RNAi studies reported here show nearly complete depletion of DAXX and thus
enable a more meaningful interpretation of resulting phenotypes.
RNAi studies have allowed us to evaluate the function of endogenous DAXX, a
matter that until now has been somewhat controversial. Previously, results
from the Daxx-knockout studies revealed that embryos and ES cell
lines lacking DAXX have increased levels of apoptosis
(Michaelson et al., 1999). In
contrast to the knockout findings, several studies suggested that when
overexpressed, DAXX could enhance Fas-mediated apoptosis
(Chang et al., 1998
;
Chang et al., 1999
;
Ko et al., 2001
;
Yang et al., 1997
), although
not in lymphoid cells (Villunger et al.,
2000
). Other studies argued for DAXX mediating apoptosis from
within the nucleus (Torii et al.,
1999
; Zhong et al.,
2000
). Taken together, the accumulating data were consistent with
the notion that DAXX might have a dual function with respect to apoptosis,
depending on the cellular context. Although DAXX may function in an
anti-apoptotic capacity in development and possibly in the lymphoid system, it
might be pro-apoptotic in fibroblasts and other cell types. The RNAi data
presented here, however, suggest that DAXX also has an anti-apoptotic effect
in cell lines such as HeLa and 293, in which overexpression studies previously
showed the opposite effect. We deduce that DAXX may be anti-apoptotic in a
variety of contexts, given that many of the studies arguing for a
pro-apoptotic role used overexpression methodology. Nevertheless, the
possibility that DAXX may be pro-apoptotic under certain circumstances cannot
be ruled out.
Indeed, the levels of apoptosis that we observed here are consistent with
the percentage of apoptotic cells measured in the Daxx-knockout ES
cell lines (Michaelson et al.,
1999). Although a truncated DAXX transcript and polypeptide
observed in the knockout was of potential concern, we have recently generated
a true DAXX null and the phenotype mimics that of the original knockout
(J.S.M. and P.L., unpublished observations). Nevertheless, although our
results suggest that the absence of DAXX results in a consistent level of
apoptosis in several cellular contexts, the levels of apoptosis observed both
in the RNAi studies and in the knockout ES lines are relatively modest. It is
possible that cells lacking DAXX are only susceptible to apoptosis at a
particular phase of the cell cycle. Alternatively, it is conceivable that
cells with an exceedingly low level of DAXX can escape apoptosis. Finally, as
discussed below, apoptosis may be secondary to the transcriptional effects of
DAXX, in which case the apoptotic outcome may be dictated by fine differences
in the transcriptional status of a given cell.
Significantly, we have found that Bcl-2 can rescue cells from the apoptosis
induced by the absence of DAXX. This observation confirms that the death
induced by DAXX depletion is indeed apoptotic. Furthermore, this finding
indicates that cells lacking DAXX are subject to an apoptotic pathway that is
Bcl-2 dependent. This would imply that the absence of DAXX cannot directly
induce effector caspase activation. The caspase cascade is, nevertheless,
probably involved in the apoptotic effect, given our observation of PARP
cleavage in RNAi-treated cells. The ability of Bcl-2 to rescue the apoptotic
effect of DAXX depletion is not surprising, given the ability of Bcl-2 to
rescue many apoptotic responses, including that presumably induced by GFP
overexpression (Liu et al.,
1999) as observed in this study
(Fig. 3B).
The RNAi studies also show the role of endogenous DAXX in mediating
transcriptional repression. It was previously observed that overexpression of
DAXX resulted in repression of targets of various transcription factors, such
as ETS and Pax family members (Hollenbach
et al., 1999; Li et al.,
2000a
; Li et al.,
2000b
). Our RNAi data show that endogenous DAXX can indeed mediate
transcriptional repression of a variety of targets, including the c-Met
promoter regulated by Pax 3. It is likely, however, that in our experimental
system, regulation of the c-Met promoter is through other factors, given that
Pax-3 is not expressed in HeLa cells. In cases where we observed
transcriptional de-repression following RNAi treatment, such as Met-luc,
NF-
B-luc and E2F1-luc, we detected corresponding levels of repression
when DAXX was overexpressed. These probably represent true targets of DAXX. In
contrast, several targets showed no change with RNAi treatment, indicating the
specificity of DAXX repression and arguing against DAXX being a general
repressor. Interestingly, modest repression of AP1-luc, SP1-luc and CRE-luc
was observed with DAXX overexpression. We consider it likely that these are
not physiological targets of DAXX, but rather that the repression observed is
an artifact of overexpression. Alternatively, they may be potential targets of
DAXX repression under certain circumstances. We believe that RNAi thus
provides an advantageous method to identify physiological targets of
transcriptional regulators.
Our results suggest that AP1 transcriptional regulation is probably not a
physiological target of DAXX. Previous studies had suggested that DAXX
mediated JNK activation (Chang et al.,
1998; Perlman et al.,
2001
; Yang et al.,
1997
), a process which results in increased transcriptional
activity of AP-1 (reviewed by Davis,
2000
). However, in our RNAi studies we observed no effect on AP-1
and, upon overexpression of DAXX, we actually detected modest repression of
AP-1. Our findings thus do not support the claim that DAXX induces JNK
activation. JNK activation by DAXX has similarly been challenged by several
other studies in both lymphoid (Villunger
et al., 2000
) and fibroblast cell lines
(Charette et al., 2000
;
Hofmann et al., 2001
;
Torii et al., 1999
).
The question remains regarding the link between the role of DAXX in
protecting from apoptosis and its function in transcriptional repression. It
is possible that these represent independent activities of DAXX. However, our
data provide a potential explanation for how the transcriptional effects of
DAXX may dictate an apoptotic outcome. For example, the strong repression of
E2F1 targets by DAXX and its derepression in the absence of DAXX correlate
with the positive role for E2F1 in inducing apoptosis (reviewed by
Black and Azizkhan-Clifford,
1999). Furthermore, the repression of NF-
B target genes by
DAXX may reduce the expression of proapoptotic genes, which, in the absence of
DAXX, are then upregulated to induce apoptosis. Alternatively, it is
conceivable that the activation of NF-
B in the absence of DAXX, as
observed in our transcriptional studies, may represent a response to apoptotic
signals. Such a mechanism would suggest that the transcriptional activities of
DAXX are secondary to its apoptotic function. Future experimentation will be
required to dissect the interplay between the role of DAXX in apoptosis and
its function in transcriptional repression.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, J. M. and Cory, S. (2001). Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci. 26,61 -66.[CrossRef][Medline]
Amin, H. M., Saeed, S. and Alkan, S. (2001). Histone deacetylase inhibitors induce caspase-dependent apoptosis and downregulation of daxx in acute promyelocytic leukaemia with t(15;17). Br. J. Haematol. 115,287 -297.[CrossRef][Medline]
Black, A. R. and Azizkhan-Clifford, J. (1999). Regulation of E2F: a family of transcription factors involved in proliferation control. Gene 237,281 -302.[CrossRef][Medline]
Caplen, N. J., Parrish, S., Imani, F., Fire, A. and Morgan, R.
A. (2001). Specific inhibition of gene expression by small
double-stranded RNAs in invertebrate and vertebrate systems. Proc.
Natl Acad. Sci. USA 98,9742
-9747.
Cermak, L., Simova, A., Pintzas, A., Horeji, V. and Andera, L. (2001). Molecular mechanisms involved in CD43-mediated apoptosis of TF-1 cells: roles of transcription, Daxx expression and adhesion molecules. J. Biol. Chem. 31,7955 -7961.
Chang, H. Y., Nishitoh, H., Yang, X., Ichijo, H. and Baltimore,
D. (1998). Activation of apoptosis signal-regulating kinase 1
(ASK1) by the adapter protein Daxx. Science
281,1860
-1863.
Chang, H. Y., Yang, X. and Baltimore, D.
(1999). Dissecting Fas signaling with an altered-specificity
death-domain mutant: Requirement of FADD binding for apoptosis but not Jun
N-terminal kinase activation. Proc. Natl Acad. Sci.
96,1252
-1256.
Charette, S. J., Lavoie, J. N., Lambert, H. and Landry, J.
(2000). Inhibition of Daxx-mediated apoptosis by heat shock
protein 27. Mol. Cell. Biol.
20,7602
-7612.
Davis, R. J. (2000). Signal transduction by the JNK group of MAP kinases. Cell 103,239 -252.[Medline]
Du, Q., Stukenberg, P. T. and Macara, I. G. (2001). A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization. Nat. Cell Biol. 3,1069 -1075.[CrossRef][Medline]
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411,494 -498.[CrossRef][Medline]
Emelyanov, A. V., Kovac, C. R., Sepulveda, M. A. and Birshtein, B. K. (2002). The interaction of Pax5 (BSAP) with Daxx can result in transcriptional activation in B cells. J. Biol. Chem. 17,11156 -11164.[CrossRef]
Epstein, J. A., Shapiro, D. N., Cheng, J., Lam, P. Y. and Maas,
R. L. (1996). Pax3 modulates expression of the c-Met receptor
during limb muscle development. Proc. Natl Acad. Sci.
USA 93,4213
-4218.
Gongora, R., Stephan, R. P., Zhang, Z. and Cooper, M. D. (2001). An essential role for Daxx in the inhibition of B lymphopoiesis by type I interferons. Immunity 14,727 -737.[CrossRef][Medline]
Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T. and
Weber, K. (2001). Identification of essential genes in
cultured mammalian cells using small interfering RNAs. J. Cell
Sci. 114,4557
-4565.
Hofmann, T. G., Moller, A., Hehner, S. P., Welsch, D., Droge, W. and Schmitz, M. L. (2001). CD95-induced JNK activation signals are transmitted by the death-inducing signaling complex (DISC), but not by Daxx. Int. J. Cancer 93,185 -191.[CrossRef][Medline]
Hollenbach, A. D., Sublett, J. E., McPherson, C. J. and
Grosveld, G. (1999). The Pax3-FKHR oncoprotein is
unresponsive to the Pax3-associated repressor hDaxx. EMBO
J. 18,3702
-3711.
Hollenbach, A. D., McPherson, C. J., Mientjes, E. J., Iyengar,
R. and Grosveld, G. (2002). Daxx and histone deacetylase II
associate with chromatin through an interaction with core histones and the
chromatin-associated protein Dek. J. Cell Sci.
115,3319
-3330.
Hunter, C. P. (2000). Gene silencing: shrinking the black box of RNAi. Curr. Biol. 10,R137 -140.[CrossRef][Medline]
Ishov, A. M., Sotnikov, A. G., Negorev, D., Vladimirova, O. V.,
Neff, N., Kamitani, T., Yeh, E. T., Strauss, J. F., III and Maul, G. G.
(1999). PML is critical for ND10 formation and recruits the
PML-interacting protein daxx to this nuclear structure when modified by
SUMO-1. J. Cell Biol.
147,221
-234.
Ko, Y. G., Kang, Y. S., Park, H., Seol, W., Kim, J., Kim, T.,
Park, H. S., Choi, E. J. and Kim, S. (2001). Apoptosis
signal-regulating kinase 1 controls the proapoptotic function of
death-associated protein (Daxx) in the cytoplasm. J. Biol.
Chem. 276,39103
-39106.
Li, H., Leo, C., Zhu, J., Wu, X., O'Neil, J., Park, E. J. and
Chen, J. D. (2000a). Sequestration and inhibition of
Daxx-mediated transcriptional repression by PML. Mol. Cell.
Biol. 20,1784
-1796.
Li, R., Pei, H., Watson, D. K. and Papas, T. S. (2000b). EAP1/Daxx interacts with ETS1 and represses transcriptional activation of ETS1 target genes. Oncogene 19,745 -753.[CrossRef][Medline]
Liu, H. S., Jan, M. S., Chou, C. K., Chen, P. H. and Ke, N. J. (1999). Is green fluorescent protein toxic to the living cells? Biochem. Biophys. Res. Commun. 260,712 -717.[CrossRef][Medline]
MacGregor, G. R. and Caskey, C. T. (1989). Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res. 17, 2365.[Medline]
Michaelson, J. S. (2000). The Daxx enigma. Apoptosis 5,217 -220.[CrossRef][Medline]
Michaelson, J. S., Bader, D., Kuo, F., Kozak, C. and Leder,
P. (1999). Loss of Daxx, a promiscuously interacting protein,
results in extensive apoptosis in early mouse development. Genes
Dev. 13,1918
-1923.
Niwa, H., Yamamura, K. and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108,193 -199.[CrossRef][Medline]
Ohta, T., Essner, R., Ryu, J. H., Palazzo, R. E., Uetake, Y. and
Kuriyama, R. (2002). Characterization of Cep135, a novel
coiled-coil centrosomal protein involved in microtubule organization in
mammalian cells. J. Cell Biol.
156, 87-99.
Perlman, R., Schiemann, W. P., Brooks, M. W., Lodish, H. F. and Weinberg, R. A. (2001). TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation. Nat. Cell Biol. 3,708 -714.[CrossRef][Medline]
Slansky, J. E., Li, Y., Kaelin, W. G. and Farnham, P. J. (1993). A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter. Mol. Cell Biol. 13,1610 -1618.[Abstract]
Torii, S., Egan, D. A., Evans, R. A. and Reed, J. C.
(1999). Human daxx regulates fas-induced apoptosis from nuclear
PML oncogenic domains (PODs) [In Process Citation]. EMBO
J. 18,6037
-6049.
Villunger, A., Huang, D. C., Holler, N., Tschopp, J. and
Strasser, A. (2000). Fas ligand-induced c-Jun kinase
activation in lymphoid cells requires extensive receptor aggregation but is
independent of DAXX, and Fas-mediated cell death does not involve DAXX, RIP,
or RAIDD. J. Immunol.
165,1337
-1343.
Yang, X., Khosravi-Far, R., Chang, H. Y. and Baltimore, D. (1997). Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell 89,1067 -1076.[Medline]
Zhong, S., Salomoni, P., Ronchetti, S., Guo, A., Ruggero, D. and
Pandolfi, P. P. (2000). Promyelocytic leukemia protein (PML)
and Daxx participate in a novel nuclear pathway for apoptosis. J.
Exp. Med. 191,631
-640.
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