1Department of Genetics, St. Jude Children's Research
Hospital, 332 North Lauderdale, Memphis, TN 38105, USA
* Present address: Department of Infectious Diseases, Parke Davis
Pharmaceuticals, 2800 Plymouth Road, Ann Arbor, MI 28105, USA
Author for correspondence (e-mail:
gerard.grosveld{at}stjude.org
)
Accepted 20 May 2002
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Summary |
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Key words: Daxx, HDAC II, Histone, Dek
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Introduction |
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In addition to the originally proposed role for hDaxx in promoting
apoptosis, it was also suggested that it acted as a link between FasR and ASK1
(Chang et al., 1998), a
downstream signaling kinase involved in Fas-dependent apoptosis. This
observation predicted a cytoplasmic cellular localization for hDaxx. However,
it has since been demonstrated that hDaxx is a strictly nuclear protein
(Hollenbach et al., 1999
;
Kiriakidou et al., 1997
;
Pluta et al., 1998
), with at
least two identified functions in the nucleus. First, hDaxx has been
identified as a component of nuclear promyelocytic leukemia protein (PML)
oncogenic domains (PODs) (Everett et al.,
1999
; Zhong et al.,
2000
). It was demonstrated that the observed sensitivity of cells
overexpressing hDaxx to apoptosis is mediated through the nuclear interaction
of hDaxx with PODs and not through a direct interaction with the Fas receptor
in the cytoplasm (Torii et al.,
1999
; Villunger et al.,
2000
; Zhong et al.,
2000
). Daxx associates with PODs through a direct interaction with
PML, a critical component of PODs (Everett
et al., 1999
; Ishov et al.,
1999
; Li et al.,
2000a
). The interaction is a dynamic, cell cycle regulated event
and is dependent on the post-translational modification of PML by the small
ubiquitin-related modifier SUMO-1 (Ishov
et al., 1999
; Lehembre et al.,
2001
; Li et al.,
2000a
).
In addition to its presence in PODs, we showed that hDaxx could act as a
transcriptional co-repressor. Consistent with this role hDaxx contains four
structural domains commonly found in transcriptional regulatory proteins: two
predicted paired amphipathic helices, an acid-rich domain and a Ser/Pro/Thr
(SPT)-rich domain (Hollenbach et al.,
1999). The tethering of hDaxx to DNA by fusing it to the GAL4
DNA-binding domain (GAL4-DBD) resulted in an 85% repression of transcriptional
activity from the constitutively active thymidine kinase promoter that
contained a GAL4-binding site (Hollenbach
et al., 1999
; Li et al., 2000;
Torii et al., 1999
).
Furthermore, hDaxx exists as three distinctly migrating forms with apparent
molecular weights of 70 kDa, 97 kDa and 120 kDa. The 120 kDa form was shown to
result, in part, from a post-translational phosphorylation
(Hollenbach et al., 1999
).
However, only the non-phosphorylated 70 kDa form of hDaxx interacts with the
DNA-bound transcription factor Pax3, which represses the transcriptional
activity of Pax3 by nearly 80% (Hollenbach
et al., 1999
). In a later report, hDaxx was also shown to repress
the transcriptional activity of ETS-1, a member of the ets family of
transcription factors (Li et al.,
2000b
), thus supporting the role of hDaxx as a transcriptional
co-repressor.
To date, the mechanism by which hDaxx exerts its repression activity is
poorly understood. Histone deacetylation, which plays a critical role in
transcriptional silencing of actively transcribed chromatin by inducing
chromatin condensation onto naked DNA
(Pazin and Kadonaga, 1997;
Wolffe and Hayes, 1999
), has
been implicated in hDaxx-mediated transcriptional repression
(Li et al., 2000a
). However,
an exact mechanism describing the repression activity of hDaxx has yet to be
determined. Therefore, to better understand the mechanism of hDaxx repression,
we have created a series of hDaxx deletion mutants fused to the GAL4
DNA-binding domain (DBD). In the present work, we demonstrate that multiple
domains of hDaxx exert transcriptional repression, which suggests that hDaxx
may associate with multiple proteins. Using standard chromatography and
co-immunoprecipitation analyses, we demonstrate that hDaxx associates with the
core histones H2A, H2B, H3 and H4; histone deacetylase II (HDAC II), which is
critical for transcriptional repression; and Dek, a protein that alters the
superhelical density of DNA in vitro
(Alexiadis et al., 2000
) and
associates with chromatin in vivo (Kappes
et al., 2001
). Finally, consistent with the requirement of
multiple domains for the repression activity of hDaxx, we demonstrate that
both the SPT domain and the first paired amphipathic helix of hDaxx are
necessary for its association with HDAC II and acetylated histone H4,
respectively.
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Materials and Methods |
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To determine protein expression, NIH3T3 cells (5x105) were plated on 100 mm dishes and transfected the following day with 10 µg of the pM2-hDaxx deletion constructs using the Fugene6 method (Boehringer Mannheim, Germany) according to the manufacturer's specifications. After 48 hours, the cells were harvested, resuspended in PBS containing both protease and phosphatase inhibitors (0.1 mM PMSF, 10 µg/ml leupeptin, 2 µg/ml Aprotinin, 1 mM ß-glycerophosphate, 1 mM NaF and 0.1 mM NaVO4) and lysed by four rounds of freezing and thawing. Cell lysates (30 µg) were resolved by either 15% or 8% SDS-PAGE, blotted to Immobilon-P membrane (Millipore, Bedford, MA), and the protein was detected by western analysis using the mouse anti-GAL4 DBD monoclonal antibody (Santa Cruz Biotechnology, CA).
Immunofluorescence
To determine the cellular localization of the individual deletion mutants,
NIH3T3 cells (5x104) were plated on one-chamber polystyrene
vessel tissue-culture-treated glass slides (Becton Dickinson, NJ) and
transfected the following day with 2 µg of each of the pM2-hDaxx deletion
constructs. After 48 hours, the cells were fixed and permeablized as
previously described (Lam et al.,
1999) and incubated for 1.5 hours with a 1:2000 dilution of the
anti-GAL4 DBD antibody. After extensively washing with PBS, the cells were
incubated with FITC-conjugated goat anti-mouse antibody (1:250 dilution) for
45 minutes, washed with PBS and mounted with Vectashield® mounting medium
(Vector Laboratories, Burlingame, CA) that contained 3 µM
4',6-diamindino-2phenylindole (DAPI, Sigma, MO). Slides were examined
using an Olympus BX50 fluorescent scope.
Transcriptional assays
To determine the repression activity of the hDaxx deletion mutants, NIH3T3
cells (3x105) were plated on 60 mm dishes and transfected the
following day using the Fugene6 method. The Fugene6-DNA complex consisted of
10 fmol of the indicated pM2-hDaxx deletion constructs, 500 ng of the secreted
alkaline phosphatase (SEAP) control plasmid under control of the MAP1 promoter
and either 1 µg of the 1X-GAL4-TK-CAT reporter plasmid containing one
GAL4-DNA-binding site or 0X-GAL4-TK-CAT reporter plasmid containing no
GAL4-DNA-binding sites. This reporter contained the chloramphenicol acetyl
transferase (CAT) gene under control of the constitutively active thymidine
kinase promoter containing either one or no GAL4-DNA-binding sites. After 48
hours, the medium was assayed for SEAP activity as previously described
(Bram et al., 1993), and the
cells were harvested and assayed for CAT activity as previously described
(Ausubel et al., 1996
). The
percentage of [14C]chloramphenicol acetylation was quantified from
thin-layer chromatography plates using a Molecular Dynamics Phosphorimager
(Amersham Pharmacia Biotech, Piscataway, NJ). The transfection efficiency was
normalized relative to the SEAP activity, and values represent the average and
standard deviation from four independent determinations.
Co-fractionation of hDaxx, HDAC II, core histones and Dek
To determine whether hDaxx, HDACII, histones and Dek co-fractionated, U937T
cells (1.2x108) (Boer et
al., 1998) stably expressing FLAG-epitope tagged Dek were
harvested and resuspended in 10 ml of a buffer containing 20 mM HEPES (pH
7.9), 150 mM KCl and all protease and phosphatase inhibitors as described
above. The cells were sonicated two times for 20 seconds on power level three
with a 550 Sonic Dismembrator (Fisher, Pittsburgh, PA), separated into
10x1 ml aliquots, and each aliquot was sonicated for an additional three
rounds. The debris was removed by centrifugation in a Eppendorf Centrifuge
5415D microfuge at 16,100 g for 10 minutes. The supernatant
was removed, and glycerol and Tween 20 were added to final concentrations of
20% and 0.1%, respectively (Buffer PLB). The total cell lysate (70 mg) was
loaded onto a HiPrep® Sephacryl S-300 size exclusion column (26x600
mm, Pharmacia, Belgium) pre-equilibrated with buffer PLB. Proteins were eluted
with a flow rate of 1 ml/minute into 5 ml fractions. The presence of hDaxx was
determined by Deoxycholate/Trichloroacetic acid (DOC/TCA) precipitating 20
microliters of each individual fraction followed by acetone precipitation and
separating the precipitated proteins on a 4-20% SDS-PAGE gradient gel. The gel
was blotted to Immobilon-P membrane (Millipore, Bedford, MA), and hDaxx was
detected by western analysis using a rabbit polyclonal antibody described
previously (Hollenbach et al.,
1999
). Fractions were also analyzed for the presence of HDACII
using a mouse monoclonal anti-HDACII antibody (Santa Cruz sc-9959, CA),
acetylated histone H4 using a rabbit polyclonal anti-acetylated histone H4
antibody (Upstate Biotechnology #06-866, Lake Placid, NY) and Dek-FLAG using a
mouse monoclonal anti-FLAG antibody (Sigma, St. Louis, MO). The apparent
native molecular weight of endogenous hDaxx was determined from a standard
curve of proteins of known molecular weights.
The fractions containing hDaxx were combined, and the protein (20 mg, 3.5-fold purification over crude lysate) was loaded onto a Resource-Q® anion exchange column (Pharmacia, Belgium) 6 ml bed volume) previously equilibrated with buffer PLB. The column was washed with 10 column volumes of buffer PLB (60 ml) at a flow rate of 1 ml/minute into 5 ml fractions, and proteins were eluted with a linear gradient of 0.15-1 M KCl in buffer PLB at the same flow rate. The presence of hDaxx, HDACII, acetylated histone H4 and Dek-FLAG in each fraction was determined as described above. The fractions containing hDaxx were combined, concentrated to 2 ml, and the protein (2 mg, 35-fold purification over crude lysate) was loaded onto a SuperdexTM HR-200 gel filtration column (20x320 mm, Pharmacia, Belgium) previously equilibrated with buffer PLB. Proteins were eluted with a flow rate of 1 ml/minute into 2.5 ml fractions, and the presence of all proteins was determined as described above. The apparent native molecular weight of hDaxx was determined from a standard curve of proteins of known molecular weights. Approximately 120 µg of hDaxx and its associated proteins were obtained from 140 mg of total cellular extract, resulting in an approximately 1150-fold purification.
Mass spectral analysis of histones
We determined the presence of histones by mass spectral analysis because
commercially available antibodies did not recognize non-acetylated histones by
western analysis in our hands. 20 µg of protein from the final fraction of
the purification were separated by 4-20% SDS-PAGE, and the proteins were
visualized by Coomassie staining. Following electrophoresis and visualization,
the gel was dried and the proteins corresponding to the molecular weights of
histones H2A, H2B, H3 and H4 were excised from the gel. The protein in the
excised gel piece was reduced, alkylated with iodoacetamide and digested with
trypsin. Tryptic peptides were extracted and subjected to combined capillary
liquid chromatography/tandem mass spectrometry. Mass spectrometry was
performed using a ThermoQuest LCQ-DECA ion-trap mass spectrometer with an
electrospray ion source. Fragment ion (MS2) spectra were subjected
to search of the NCBI non-redundant protein database using the SEQUEST program
of Eng and Yates marketed by ThermoQuest.
Co-immunoprecipitation of hDaxx, HDAC II, Dek and acetylated histone
H4
To demonstrate a physical association of hDaxx, HDAC II, Dek and acetylated
histone H4 and to map the domains of hDaxx responsible for its association
with HDACII and acetylated histone H4, co-immunoprecipitation experiments were
performed. Because U937T cells did not transfect well by the Fugene6 method,
293T cells were used. 293T cells (1x106) were plated in 100
mm dishes and transfected the following day with 5 µg of the mammalian
expression vectors encoding either GAL4-hDaxx, GAL4-hDaxx1-132,
GAL4-hDaxx
1-352 or GAL4-hDaxx-SPT using the Fugene6 method according to
the manufacturer's specifications. After 48 hours, the cells were lysed on ice
in NP40 lysis buffer (0.4 M NaCl, 0.2 mM EGTA, 10% glycerol, 1% NP40 and all
protease and phosphatase inhibitors as described above), the cell debris was
removed by centrifugation at maximum speed in a Sorvall RMC 1Y microfuge for
15 minutes at 4°C, and the resulting cell extracts were precleared by
incubating them with Gamma Bind plus Sepharose resin (Pharmacia, Belgium) for
2 hours at 4°C. Total cell lysate (150 µg) was incubated with a mouse
monoclonal anti-GAL4 DBD antibody (clone RK5C1, Santa Cruz, CA) overnight at
4°C on a rotary shaker; immune complexes were collected with Gamma Bind
Plus Sepharose beads; and the beads were washed four times with ice-cold NP40
lysis buffer. The pellets were resuspended in 30 µl of SDS-PAGE buffer and
boiled for 5 minutes. Supernatants were resolved by 4-20% gradient SDS-PAGE,
blotted to Immobilon-P membrane and either HDAC II or acetylated histone H4
were detected by western analysis using a mouse monoclonal anti-HDAC II or
rabbit polyclonal anti-acetylated histone H4 antibody, respectively.
Unfortunately, we were unable to analyze the presence of endogenous Dek owing
to the non-specific interaction of Dek with the gamma-bind plus resin. To
confirm overexpression of all deletion constructs, 20 µg of total cell
lysate was resolved by 4-20% SDS-PAGE, blotted to Immobilon-P membrane, and
protein was detected by western analysis using the mouse monoclonal anti-GAL4
DBD antibody as described above.
Alternatively, associated proteins were also analyzed by immunoprecipitation of Dek-FLAG. Fractions from the Sephacryl S-300 column that contained hDaxx, Dek, acetylated histone H4 and HDAC II (fractions 9-13) were combined and passed over an anti-FLAG antibody affinity column (Sigma, St. Louis, MO). The column was washed extensively with buffer PLB, and proteins that were retained by the column were competitively eluted with PLB buffer containing 100 µg/ml FLAG peptide. The eluted proteins were DOC/TCA/acetone precipitated, separated by 4-20% SDS-PAGE, transferred to Immobilon-P membrane, and hDaxx, HDAC II and acetylated histone H4 were detected by western analysis as described above, or the core-histones H2A, H2B, and H3 were visualized by silver stain.
Histone deacetylase activity assays
To determine the ability of GAL4-hDaxx to immunoprecipitate histone
deacetylase activity, 293T cells (1x106) were plated in 100
mm dishes and transfected the following day with 5 µg of the mammalian
expression vectors encoding either GAL4-hDaxx or GAL4 by the Fugene6 method
according to the manufacturer's specifications. After 48 hours, the cells were
lysed with RIPA buffer containing all protease and phosphatase inhibitors and
precleared, as described above. Total cell lysate (100 µg) was
immunoprecipitated with the anti-GAL4 antibody, and after extensive washing
with lysis buffer, the beads containing the immune complexes were resuspended
in 200 µl of HDAC assay buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10%
glycerol and all protease). [3H]acetyl histone H4 (730,000
cpm/µl), previously prepared according to the manufacturer's specifications
(Upstate Biotechnology, Lake Placid, NY), was added to the immune complexes,
and the reactions were incubated for 36 hours at room temperature. The
reactions were stopped by the addition of 50 µl of quenching solution (1.0
M HCl, 160 mM acetic acid), and the released [3H]acetate was
extracted and quantified according to the manufacturer's specifications
(Upstate Biotechnology, Lake Placid, NY). As a positive control for the
histone deacetylase reaction, 10 µg of HeLa nuclear extract was incubated
with the HDAC assay buffer and [3H]acetyl histone H4, and released
[3H]acetate was analyzed as described above. The specificity of the
histone deactylase reaction was determined by parallel reactions that were
incubated with 50 mM sodium butyrate, a specific histone deacetylase
inhibitor.
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Results |
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Transcriptional repression activity of GAL4-hDaxx deletion
mutants
Before we could use the deletion constructs in transcriptional repression
assays, several controls needed to be performed. First, to demonstrate that
all deletion mutants were expressed at similar levels, each of the constructs
was individually transfected into NIH3T3 fibroblasts, and the presence of
mutant protein was detected by western analysis using a mouse anti-GAL4 DBD
monoclonal antibody. The majority of the deletion constructs expressed similar
levels of protein (Fig. 1)
except for 1-352, which in addition to expressing protein of the
correct molecular weight also demonstrated a considerable amount of protein
degradation (Fig. 2B).
Interestingly, we noted barely detectable levels of protein with the majority
of constructs in which the acid-rich domain had been removed (
AD,
PAH1-AD,
AD-SPT,
PAH1-2-AD,
PAH1-AD-SPT,
PAH1-2-AD-SPT, hDaxx
334-740, and hDaxx133-333)
(Fig. 1). In addition, the
deletion constructs that lacked the acid-rich domain but still retained the
N-terminal paired amphipathic helix (
PAH2-AD,
PAH2-AD-SPT)
expressed protein of the correct molecular weight at a lower level than
full-length hDaxx (Fig. 1). A
pulse-chase analysis of select deletion mutants confirmed that the proteins
were being expressed and that the observed reduction in the steady-state level
of protein expression was caused by a decrease in the stability of deletion
mutants lacking the acid-rich domain. The specific removal of the acid-rich
domain decreased the normal half-life of GAL4-hDaxx by greater than four-fold
(data not shown), suggesting that the acid-rich domain is essential for the
stable expression of hDaxx.
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Second, hDaxx contains two predicted nuclear localization signals (NLS):
one present in the SPT domain (NLS2, K630KSRK634) and
one immediately N-terminal to the acid-rich domain (NLS1,
R391KKRR395)
(Kiriakidou et al., 1997;
Li et al., 2000a
). Therefore,
to confirm that the hDaxx deletion mutants maintained a nuclear localization,
we transiently transfected NIH3T3 cells with each individual construct, and
the localization of the protein was visualized by immunofluorescence using the
anti-GAL4 DBD monoclonal antibody. Consistent with previous reports
(Everett et al., 1999
;
Hollenbach et al., 1999
;
Ishov et al., 1999
;
Kiriakidou et al., 1997
;
Pluta et al., 1998
),
GAL4-hDaxx demonstrated a strict nuclear staining
(Fig. 1). In addition, nuclear
staining was also observed for each of the deletion mutants containing an
intact SPT domain (Fig. 1). By
contrast, despite the presence of a predicted NLS in hDaxx immediately
N-terminal to the acid-rich domain, hDaxx deletion mutants missing all or part
of the SPT domain demonstrated a diffuse staining throughout the cell
(Fig. 1). The loss of strict
nuclear staining upon removal of the SPT domain indicated that NLS2 is
critical for the nuclear localization of hDaxx. Consistent with this
observation, we found that the specific deletion of NLS2 was sufficient to
remove the strict nuclear localization of GAL4-hDaxx. However, the removal of
NLS1 had no effect on the nuclear localization of GAL4-hDaxx (data not shown).
This observation provided direct evidence that the NLS2 is necessary and
sufficient for the localization of hDaxx to the nucleus.
Finally, the deletion constructs that localized strictly to the nucleus and
produced stable protein (hDaxx, hDaxx1-573, hDaxx
1-132,
PAH1-2,
PAH2-AD, hDaxx
1-352,
PAH2, and
PAH1) were tested for their ability to repress transcription. Deletion
mutants that expressed stable protein but displayed a diffuse cellular
localization were cloned in frame with the SV40 NLS between the GAL4 DBD and
the hDaxx mutant construct [(NLS)
PAH1-SPT, (NLS)
SPT,
(NLS)
PAH2-SPT, (NLS)
PAH1-2-SPT, and (NLS)hDaxx353-573]. The
presence of the SV40 NLS did not affect protein stability
(Fig. 2B) nor did it affect the
transcriptional repression activity of GAL4-hDaxx (data not shown). It was,
however, sufficient to restore the strict nuclear localization of the deletion
mutants (data not shown). Consistent with previous reports
(Hollenbach et al., 1999
;
Li et al., 2000a
), expression
of full-length GAL4-hDaxx resulted in an 85% reduction of transcriptional
activity (Fig. 2A). This
reduction was dependent on GAL4-hDaxx binding to DNA and was not caused by
titration of other essential factors by hDaxx as minimal repression (
15%)
was observed when either the GAL4 DBD or the GAL4-DNA-binding sites were
removed (data not shown) (Hollenbach et
al., 1999
). In the same manner as with full-length GAL4-hDaxx, the
presence of the SPT domain in the deletion mutants was sufficient to provide
an 85% reduction of transcriptional activity
(Fig. 2A). However, the SPT
domain is not solely responsible for the repression activity of hDaxx, as the
construct that had the SPT domain deleted ((NLS)
SPT) retained
transcriptional repression activity similar to that of wild-type hDaxx
(Fig. 2A). Removal of PAH1
(hDaxx
1-132 and
PAH1) or PAH2 (
PAH2) independently was
insufficient to alter the repression activity of hDaxx
(Fig. 2A). Analysis of the
transcriptional repression activity of several of the combined deletion
mutants [(NLS)
PAH2-SPT and (NLS)
PAH1-2-SPT] or the deletion
mutant that retained only the region surrounding the acid-rich domain
[(NLS)hDaxx353-573] demonstrated that although there was a significant
increase in transcriptional activity relative to full-length hDaxx
(Fig. 2A), these constructs
still retained transcriptional repression activity. The observed differences
in activity were not caused by different levels of protein expression as all
deletion constructs expressed protein to similar levels, with only
1-352 showing any considerable degradation
(Fig. 2B). Overall, therefore,
the results of this deletion analysis indicate that multiple domains of hDaxx
are required for transcriptional repression.
Co-fractionation of hDaxx, Dek, HDAC II and core histones
Our data suggest that multiple domains of hDaxx are required for
repression, which implies that hDaxx may associate with multiple proteins in
order to exert its repression activity. Therefore, we investigated this
hypothesis by using conventional chromatography to isolate endogenous hDaxx.
We wanted to determine whether hDaxx associated with HDAC II, a protein that
deacetylates acetylated histones, which results in transcriptional repression.
In addition, we wanted to see if core histones and the protein Dek, a protein
that associates with chromatin in vivo
(Kappes et al., 2001) and
induces alterations of the superhelical density of DNA in chromatin in vitro
(Alexiadis et al., 2000
), also
associate with hDaxx. Daxx is a ubiquitously expressed protein that has
transcriptional repression activity in a variety of cell lines
(Hollenbach et al., 1999
;
Lehembre et al., 2001
;
Li et al., 2000a
;
Li et al., 2000b
). Therefore,
in order to facilitate the detection and immunoprecipitation of Dek using a
FLAG affinity resin, we utilized U937T cells
(Boer et al., 1998
) that
express endogenous hDaxx and stably express FLAG-epitope-tagged Dek
(Dek-FLAG). Total cellular lysates from these cells were fractionated with a
Sephacryl S-300 size exclusion column, and the presence of hDaxx, HDACII,
acetylated histone H4, and Dek-FLAG was detected by western analysis.
Examination of the individual fractions demonstrated that hDaxx eluted with an
apparent molecular weight between 500 kDa and 700 kDa
(Fig. 3A). This result
indicated that hDaxx may associate with multiple proteins, because hDaxx
eluted with a native molecular weight that is significantly larger than its
denatured molecular weights of 70 kDa, 97 kDa, and 120 kDa
(Hollenbach et al., 1999
). In
addition, HDACII, acetylated histone H4 and Dek-FLAG were also present in the
fractions that contained hDaxx, indicating that these constituents
co-fractionated with hDaxx from the Sephacryl S-300 column
(Fig. 3A). The fractions that
contained all four proteins (fractions 9-13,
Fig. 3A) were then passed over
a Resource Q® anion exchange column and the proteins were eluted with a
linear gradient from 0.15-1 M KCl. A western analysis demonstrated that the 70
kDa, nonphosphorylated form of hDaxx was eluted from the column 0.4-0.5 M KCl
(Fig. 3B, closed circles),
whereas the 120 kDa phosphorylated form was eluted at higher salt
concentrations (Fig. 3B, open
circles). Once again, HDACII and acetylated histone H4 were eluted with
0.4-0.5 M KCl, consistent with a co-fractionation of these proteins with the
70 kDa form of hDaxx (Fig. 3B).
In addition, Dek-FLAG was found to elute to the same extent over a broad range
of salt concentrations (Fig.
3B), which includes the fractions that contain hDaxx, HDAC II and
acetylated histone H4. Therefore, because a fraction of Dek is present in the
same fractions as hDaxx, HDAC II and acetylated histone H4, we believe that
only a sub-population of Dek co-fractionates with these proteins.
|
The fractions that contained all four proteins (fractions 16-19) were pooled, concentrated and resolved on a Superdex HR-200 high-resolution gelfiltration column. As observed for the Sephacryl S-300 column, the majority of hDaxx eluted as a complex with an apparent molecular weight between 230-500 kDa with an average molecular weight of 360 kDa (Fig. 3C). In addition, both HDACII and Dek-FLAG, but not acetylated histone H4, eluted with hDaxx, confirming a co-fractionation of HDAC II and a sub-population of Dek-FLAG with hDaxx (Fig. 3C, lanes 12 and 13). To confirm that the observed molecular weight of 360 kDa is not due to the presence of oligo-nucleosomes, an aliquot of the peak fraction containing hDaxx (Fig. 3C, fraction 13) was deproteinized, and the presence of DNA was determined by 2% agarose gel electrophoresis. This analysis demonstrated that a small amount of DNA was present in this fraction and consisted of a fragment of approximately 180 bp, a size consistent with the DNA present in a mono-nucleosome (data not shown). In addition to the elution of hDaxx with an apparent molecular weight of 360 kDa, a small amount of hDaxx eluted with an apparent molecular weight of 670 kDa and, in addition to HDACII and Dek-FLAG, co-fractionated with acetylated histone H4 (Fig. 3C, lanes 10 and 11). The co-fractionation of acetylated histone H4 with only a small subset of hDaxx suggests that if hDaxx associates with acetylated histone H4, the association is of a transient nature, potentially because of the presence of HDAC II in these fractions. The co-fractionation of HDAC II with hDaxx and acetylated histone H4 would potentially bring HDAC II and acetylated histone H4 into close proximity, allowing the deacetylation of histone H4. Alternatively, co-fractionation may be fortuitous, with acetylated histone H4 and hDaxx being part of different large complexes.
The potential deacetylation of histones by HDAC II present in the complex suggests that non-acetylated histones may also co-fractionate with hDaxx, Dek and HDAC II in the 360 kDa fraction. Because commercially available antibodies did not recognize non-acetylated histones by western analysis in our hands, we determined the presence of non-acetylated histones in the 360 kDa hDaxx fractions by mass spectral analysis. The Coomassie-stained gel of proteins present in the peak hDaxx fraction from the Superdex HR-200 column demonstrated that in addition to several unidentified proteins of higher molecular weight, there was also the presence of bands consistent with the molecular weights of all histones (Fig. 3D). To confirm the identity of these proteins as the core histones H2A, H2B, H3 and H4, the bands corresponding to these proteins were excised from the gel, digested with trypsin, and the resulting peptide fragments were sequenced and identified by liquid chromatography and tandem mass spectral analysis. This analysis identified fragments corresponding to non-acetylated histones H2A, H2B, H3 and H4. A similar analysis of the band corresponding to histone H1 did not identify any peptide fragments corresponding to histone H1. Therefore, this result demonstrated that the core histones H2A, H2B, H3 and H4 co-fractionate with hDaxx, Dek and HDAC II in the 360 kDa hDaxx fraction.
Daxx, Dek, HDAC II and core histones physically associate
Our results demonstrate that hDaxx, acetylated and non-acetylated core
histones, HDAC II and a subpopulation of Dek-FLAG co-fractionate through a
series of chromatographic separations. However, it is possible that their
co-fractionation is merely fortuitous and that these proteins do not
physically associate in vivo. Therefore we performed a series of
co-immunoprecipitation experiments, which unlike co-fractionation depend on
physical associations, using two independent components to confirm the
association of hDaxx, Dek, HDAC II and core histones. First, we
immunoprecipitated Dek-FLAG from the fractions of the Sephacryl S-300 column
that contained all of these proteins using an anti-FLAG affinity column. After
extensive washing of the precipitate and elution of the proteins from the FLAG
antibody with a FLAG-specific peptide, a silver stain analysis of the bound
proteins demonstrated that in addition to Dek-FLAG
(Fig. 4A, right lane, top
panel), the core histones H2A and H2B, and to a lesser extent H3 and H4, were
efficiently co-immunoprecipitated by Dek
(Fig. 4A, right lane, bottom
panel). The co-immunoprecipitation of core histones was specific for Dek as
they were only precipitated in the presence of Dek-FLAG
(Fig. 4A), demonstrating an
association between Dek and the core histones. The observed association of an
abundance of H2A/H2B over H3/H4 with Dek-FLAG is identical to a previous
report that demonstrated a stronger affinity of Dek for H2A/H2B
(Alexiadis et al., 2000). A
western analysis of the same proteins using either anti-hDaxx, anti-HDAC II or
anti-acetylated histone H4 antibodies demonstrated that the immunoprecipitate
also contained primarily the 70 kDa non-phosphorylated isoform of hDaxx, HDAC
II and acetylated histone H4 (Fig.
4B, left lane). By contrast, the supernatant contained the 120 kDa
phosphorylated isoform of hDaxx and HDAC II
(Fig. 4B, right lane). Because
the amount of protein loaded from the immunoprecipitate was five times more
than the amount of protein loaded from the supernatant, we conclude that most
of the 120 kDa isoform of hDaxx and a fraction of HDAC II were not retained on
the column. The ability of Dek-FLAG to co-immunoprecipitate the 70 kDa form of
hDaxx, HDAC II and core histones therefore confirms that there is a direct
association between Dek and these constituents.
|
Next we performed independent co-immunoprecipitation experiments using
GAL4-hDaxx, as our hDaxx antibodies were raised against the SPT domain of
hDaxx, the region demonstrated to interact with a variety of proteins
(Chang et al., 1998;
Hollenbach et al., 1999
;
Ishov et al., 1999
;
Kiriakidou et al., 1997
;
Li et al., 2000a
;
Pluta et al., 1998
;
Torii et al., 1999
; Yang et
al., 1999). 293T cells were transfected with full-length GAL4-hDaxx or GAL4
DBD alone and equivalent amounts of total cell lysate immunoprecipitated with
the anti-GAL4 antibody. Consistent with the co-fractionation of hDaxx with
both acetylated histone H4 and HDACII (Fig.
3), both proteins were co-immunoprecipitated with full-length
GAL4-hDaxx (Fig. 5C,D, lane 2).
This association was specific for hDaxx since neither acetylated histone H4
nor HDACII were detected when the identical co-immunoprecipitations were
carried out with cells overexpressing GAL4 DBD alone
(Fig. 5C and 5D, lane 1). This
association was also independent of the presence of DNA since the same trace
amount of low molecular weight DNA (
180 bp) was present regardless of
whether the immunoprecipitation was performed with the anti-hDaxx or control
antiserum (data not shown).
|
To determine which domains of hDaxx are required for the inclusion of
chromatin-related proteins in the hDaxx complex, 293T cells were individually
transfected with full-length GAL4-hDaxx or three N-terminal hDaxx deletion
constructs (GAL4-hDaxx1-132, GAL4-hDaxx
1-352, and GAL4-SPT)
followed by immunoprecipitation of the total cell lysate. We observed an
equivalent level of overexpression (Fig.
5A) and an equivalent level of immunoprecipitation
(Fig. 5B) for all proteins. The
GAL4-DBD immunoprecipitated protein signal was masked by the presence of the
IgG light chain (Fig. 5B, lane
1); however, independent experiments using [35S]-Met metabolic
labeling demonstrated an equivalent level of immunoprecipitation (data not
shown). Despite the equivalent expression and immunoprecipitation of all hDaxx
constructs, only full-length GAL4-hDaxx was able to co-precipitate acetylated
histone H4 (Fig. 5C, lanes
2-5). This result demonstrated that the region surrounding the first paired
amphipathic helix of hDaxx is required for the association of acetylated
histone H4 with hDaxx. By contrast, it was found that all deletion constructs
were able to co-immunoprecipitate HDAC II
(Fig. 5D, lanes 2-5).
Therefore, these results are consistent with the SPT domain being necessary
for the association of HDAC II with hDaxx. Whether these interactions are
mediated by a third protein is not known at present. However, despite this
uncertainty, our results from the co-fractionation and two independent
immunoprecipitation experiments demonstrate an association between the 70 kDa
isoform of hDaxx, Dek-FLAG, HDAC II, acetylated histone H4 and the core
histones H2A, H2B, H3 and H4.
Daxx associates with active histone deacetylases
Finally, we investigated the ability of hDaxx to immunoprecipitate active
histone deacetylases. 293T cells were transfected with GAL4-hDaxx or GAL4
alone; equivalent amounts of total cell lysates were immunoprecipitated with
the anti-GAL4 monoclonal antibody, and the immune complexes were tested for
their ability to deacetylate [3H]acetyl histone H4.
Immunoprecipitates from cell lysates overexpressing GAL4-hDaxx contained
two-fold higher levels of histone deacetylase activity compared with that of
the positive control (Fig. 6,
gray bars). This result is consistent with the association of hDaxx with
histone deacetylase I (Li et al.,
2000a) and II (Figs
3,4,5).
This activity was specific for hDaxx as immunoprecipitates from cell lysates
overexpressing GAL4 alone contained no deacetylase activity above background.
The activity was also specific for histone deacetylases as the addition of the
HDAC inhibitor sodium butyrate completely inhibited the release of
[3H]acetate (Fig. 6,
crosshatched bars).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We present a minimal model to explain the transcriptional repression
activity of hDaxx (Fig. 7) that
is consistent with the data presented here and is based on previous models
describing the repression activities of both Sin3
(Kadosh and Struhl, 1997;
Nagy et al., 1997
) and the
retinoblastoma protein (Brehm and
Kouzarides, 1999
). In this model the post-translational
modification status of the SPT-domain of hDaxx regulates its association with
transcription factors such as Pax3
(Hollenbach et al., 1999
) and
ETS-1 (Li et al., 2000b
),
effectively bringing hDaxx to sites of active transcription. Through its
presence at the site of active transcription, hDaxx would then be able to
associate with acetylated histones present in the nucleosomes and Dek that is
associated with chromatin (Kappes et al.,
2001
). Through its association with the SPT-domain of hDaxx,
histone deacetylases would also be brought to the site of active
transcription. As a consequence, nucleosomes in the vicinity of the site of
active transcription will have the histone tails deacetylated, allowing the
deactylated tail to bind to DNA, thereby leading to an inactive chromatin
structure and transcriptional repression.
|
Our proposed working model of transcriptional repression by hDaxx is based
on the following evidence. First, we have previously demonstrated that hDaxx
exists in at least three distinct isoforms, with apparent molecular weights of
70 kDa, 97 kDa and 120 kDa (Hollenbach et
al., 1999). Of these three isoforms, we have demonstrated that the
120 kDa isoform is phosphorylated
(Hollenbach et al., 1999
). The
phosphorylation status of hDaxx is responsible for regulating its association
with different proteins. For example, the transcription factor Pax3
specifically interacts with the non-phosphorylated 70 kDa isoform of hDaxx,
resulting in the repression of Pax3 transcriptional activity
(Hollenbach et al., 1999
).
Conversely, PML, a protein present in PODs, interacts specifically with the
phosphorylated 120 kDa isoform of hDaxx, resulting in an enhancement of
apoptosis potentially by functioning as a transcriptional modulator that
enhances Fas-induced apoptosis through effects on gene expression
(Ishov et al., 1999
;
Li et al., 2000a
;
Torii et al., 1999
;
Zhong et al., 2000
). In both
of these cases the SPT domain of hDaxx, one of the primary regions of hDaxx
phosphorylation (A.D.H. and G.G., unpublished), mediates the interaction
between hDaxx and Pax3 (Hollenbach et al.,
1999
) and PML (Ishov et al.,
1999
). In addition, we demonstrate that only the 70 kDa
non-phosphorylated isoform of hDaxx associates with HDAC II, Dek and core
histones (Figs 3 and
4). Therefore, taken together,
these results support the hypothesis that the phosphorylation status of hDaxx
regulates its association with chromatin, thereby regulating the repression
activity of hDaxx.
Second, we have demonstrated that endogenous hDaxx elutes from size
exclusion chromatography columns with an apparent molecular weight of
approximately 360 kDa (Fig.
3A,C), suggesting that it associates with multiple proteins.
Through a series of chromatographic separations we have demonstrated that the
70 kDa isoform of hDaxx co-fractionates with components of chromatin such as
core histones, proteins that associate with chromatin such as Dek and proteins
that are critical for transcriptional repression such as HDAC II
(Fig. 3). We have also
demonstrated that hDaxx physically associates with HDAC II, Dek and acetylated
core histones, as seen by the co-immunoprecipitation of these components by
either hDaxx (Fig. 5) or
Dek-FLAG (Fig. 4). It is
possible that hDaxx interacts indirectly with HDAC II and core histones
through non-specific association with DNA. However the ability to
co-immnoprecipitate acetylated histone H4 and HDAC II independently of the
presence of DNA supports the conclusion that the association occurs through
protein-protein interactions. Finally, we have demonstrated that hDaxx is
capable of immunoprecipitating histone deacetylase activity
(Fig. 6). The association of
hDaxx with histone deacetylase activity is consistent with the
co-fractionation of HDAC II with hDaxx
(Fig. 3) and the ability of
hDaxx to co-immunoprecipitate HDAC I (Li
et al., 2000a) and HDAC II (Figs
4 and
5). Therefore, by bringing
histone deacetylase activity to sites of active transcription, hDaxx
facilitates the deacetylation of histone tails in nucleosomes, allowing the
otherwise extended and acetylated histone tails to bind to DNA. Through the
binding of the histone tails to DNA, they would then prevent the access of
elements required for transcription, which would result in repression of
transcriptional activity.
At present the exact function of Dek in hDaxx-mediated repression is not
clear. Our data demonstrating the co-immunoprecipitation of hDaxx by Dek
(Fig. 4) would suggest that a
direct association exists between these two proteins. However we cannot
exclude the possibility that this association is secondary in nature, being
mediated through a mutual association with the core histones present in
chromatin. Regardless of whether the association of Dek with hDaxx is direct
or secondary, our results suggest that in addition to its reported effects on
DNA replication (Alexiadis et al.,
2000) and its presence in the exon-exon junction complex
(Le Hir et al., 2000
;
Le Hir et al., 2001
), Dek may
also have a function in transcriptional repression. Dek has been reported to
associate with chromatin in vivo (Kappes
et al., 2001
) and to alter the superhelical density of DNA in
chromatin in vitro, inhibiting the access of proteins and replication factors
to the DNA template (Alexiadis et al.,
2000
). Therefore it is conceivable that in a similar manner Dek
may also function during transcription by altering the structure of chromatin
once histone tails have been deacetylated and preventing the access of
transcription factors to the DNA template.
It is unlikely that hDaxx-mediated deacetylation and subsequent repression of transcription is a continuous process in vivo. Rather, it is likely that the deacetylation and transcriptional repression are regulated via additional signals. Although we have no data to indicate the identity of these potential signals, one possibility is that additional post-translational modifications that regulate the interactions of hDaxx with specific transcription factors, such as Pax3 or Ets1, could trigger the deacetylase activity. Alternatively, its association with transcription factors within a particular promoter context may regulate the activity of hDaxx. For example, although no reports have been published that describe hDaxx-mediated transcriptional activation, the association of acetylated histones with the hDaxx complex could indicate a role for hDaxx in transcriptional activation in certain promoter settings. However, regardless of whether the transcriptional regulatory activity of hDaxx is modulated by interactions with transcription factors or by its presence within a specific promoter context, our results demonstrate that transcriptional repression by hDaxx is mediated through its association with core histones, HDAC II and Dek. Our present efforts are aimed at identifying potential signals that regulate hDaxx transcriptional repression activity.
![]() |
Acknowledgments |
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References |
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