Daxx represses RelB target promoters
via DNA methyltransferase recruitment
and DNA hypermethylation
Lorena A. Puto1,2and John C. Reed1,3
1Program in Apoptosis and Cell Death Research, Burnham Institute for Medical Research, La Jolla, California 92037, USA;
2Graduate Program in Molecular Medicine, Burnham Institute for Medical Research, La Jolla, California 92037, USA
The apoptosis-modulating protein Daxx functions as a transcriptional repressor that binds to and suppresses
the activity of nuclear factor-?B member RelB, among other transcription factors. The mechanism by which
Daxx represses RelB target genes remains elusive. In this report, we demonstrate that Daxx controls
epigenetic silencing of RelB target genes by DNA methylation. Daxx potently represses the RelB target genes
dapk1, dapk3, c-flip, and birc3 (ciap2) at both the mRNA and protein levels. Recruitment of Daxx to target
gene promoters, and its ability to repress them, is RelB-dependent, as shown by experiments using relB−/−
cells. Importantly, methylation of target promoters is decreased in daxx−/−cells compared with daxx+/+cells,
and stable transfection of daxx−/−cells with Daxx restores DNA methylation. Furthermore, Daxx recruits
DNA methyl transferase 1 (Dnmt1) to target promoters, resulting in synergistic repression. The observation
that Daxx functions to target DNA methyltransferases onto RelB target sites in the genome provides a rare
example of a gene-specific mechanism for epigenetic silencing. Given the documented role of several of the
RelB-regulated genes in diseases, particularly cancer, the findings have implications for developing therapeutic
strategies based on epigenetic-modifying drugs.
[Keywords: Daxx; RelB; DNMT1; transcription; DNA methylation; epigenetics]
Supplemental material is available at http://www.genesdev.org.
Received November 7, 2007; revised version accepted February 29, 2008.
Substantial evidence suggests that epigenetic mecha-
nisms control the genome. The epigenome is responsible
for establishing gene expression profiles in various tis-
sues and cell types in vertebrates (D’Alessio and Szyf
2006). DNA methylation at CpG dinucleotides is the
most intensely investigated epigenetic modification
(Baylin 2005; Fraga and Esteller 2007) and is typically
associated with transcriptional repression (Baylin 2005;
Baylin and Ohm 2006). However, the mechanisms by
which DNA methylation patterns are established are
only partially understood. Identification of DNA methyl
transferases (Dnmts) has provided important initial in-
sights, yet knowledge of how specific CpG methylation
patterns are established by Dnmts in a gene-selective
manner is still limited.
Daxx is predominantly a nuclear protein, often local-
ized within subnuclear compartments called PML onco-
genic domains, or PODs (Torii et al. 1999; Maul et al.
2000; Michaelson 2000; Kawai et al. 2003; Takahashi et
al. 2004). Two recurring themes continue to emerge
throughout the literature on Daxx: (1) regulation of tran-
scription, and (2) regulation of apoptosis. At one level,
Daxx is a repressor of gene expression that binds histone
deacetyl transferases (Hdacs) (Hollenbach et al. 2002; Ec-
sedy et al. 2003), Dnmts (Michaelson et al. 1999; Muro-
moto et al. 2004), and chromatin-modifying proteins
(Hollenbach et al. 2002; Xue et al. 2003; Tang et al. 2004).
On another level, Daxx is an apoptosis-modulating pro-
tein implicated in certain types of cell death pathways
(Torii et al. 1999; Michaelson 2000; Kawai et al. 2003;
Salomoni and Khelifi 2006). How, and if, these two
themes are inextricably linked, however, is unclear.
RelB is a member of the nuclear factor (NF)-?B tran-
scription factor family that plays crucial roles in regulat-
ing innate and adaptive immunity, cell differentiation,
and apoptosis. Generation of mice deficient for each Rel
family protein has revealed that individual NF-?B family
members have distinct functions (Burkly et al. 1995; Al-
camo et al. 2002). RelB is essential for the development
of thymic medullary epithelium, dendritic cell function,
and secondary lymphoid tissue organization (Burkly et
al. 1995). RelB-deficient mice commonly succumb to
multiorgan inflammation early in life (Hoffmann and
Baltimore 2006). Because RelB acts as both a transcrip-
E-MAIL email@example.com; FAX (858) 646-3194.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1632208.
998 GENES & DEVELOPMENT 22:998–1010 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org
tional activator and as a repressor of NF-?B-dependent
gene expression (Burkly et al. 1995; Hoffmann and Bal-
timore 2006), it may have both inflammatory and anti-
inflammatory activities in vivo.
Recent data from our laboratory showed that Daxx in-
teracts with and suppresses the ability of RelB to activate
target genes (Croxton et al. 2006). Based on the ability of
RelB to bind and recruit Daxx to RelB target sites in the
genome, we hypothesized that Daxx may do more than
simply inhibit RelB, but may also actively repress target
genes, effectively converting RelB from a transactivator
to a repressor. Further, based on previous knowledge that
Daxx interacts with Dnmts (Michaelson et al. 1999;
Muromoto et al. 2004), we hypothesized that Daxx ex-
erts its transcriptional-repressive actions, at least in part,
through DNA methylation. In this report, we demon-
strate that Daxx functions to target Dnmt1 onto RelB
target sites in the genome, resulting in their DNA hy-
permethylation and epigenetic silencing. Furthermore,
Daxx’s repressive function is RelB-dependent, with Daxx
failing to target and silence these genes in relB−/−cells.
The RelB/Daxx interaction thus represents a paradigm
for explaining how a single transcription factor (RelB)
can operate as either an activator or repressor of target
genes. Our study may also reconcile some ambiguities
about Daxx’s apoptotic functions by discovering that
Daxx can regulate both pro- and anti-apoptotic genes,
thus suggesting a connection between its transcriptional
and apoptotic activities.
Expression of Daxx-repressed genes is increased
We reported previously that gene transfer-mediated over-
expression of Daxx represses expression of a subset of
apoptosis-modulating genes regulated by NF-?B, includ-
ing the genes encoding Birc3 (cIAP2), Survivin, c-Flip,
and Dapk3 (Croxton et al. 2006). To extend these results,
we used Daxx-deficient mouse embryonic fibroblasts
(MEFs), analyzing by quantitative real-time PCR (Q-
PCR) the expression of some of the candidate target
genes that appeared to be repressed by Daxx overexpres-
sion in various human epithelial cancer cell lines. In
addition to these genes, we also examined the gene en-
coding Dapk1, because preliminary experiments sug-
gested that its expression is suppressed by Daxx overex-
pression in several human tumor cell lines and because
of its close relation to Dapk3. Our a priori expectation
was that expression of the Daxx-repressed genes would
be increased in daxx−/−cells. In agreement with this pre-
diction, we observed approximately fivefold higher lev-
els of Birc3 (cIAP2) mRNA, ∼10-fold higher c-Flip (Cflar)
mRNA, ∼20-fold higher Dapk3 (Zipk) mRNA, and ∼30-
fold higher Dapk1 mRNA levels in daxx−/−compared
with daxx+/+MEFs (Fig. 1A). Survivin (Birc5) mRNA lev-
els were also fivefold higher in daxx−/−compared with
daxx+/+MEFs (Supplemental Fig. S2A). In contrast, Cph
and Gapdh mRNA levels were similar in daxx−/−and
genes is increased in Daxx−/−MEFs. (A)
Analysis of Mail, Bok, Cph, Gapdh, Dapk1,
Dapk3, Birc3, and c-Flip mRNA levels.
daxx+/+and daxx−/−MEFs were used for ex-
pression analysis of RelB candidate target
genes (mail, bok, dapk1, dapk3, birc3, and c-
flip) or control housekeeping genes (cph and
gapdh). RNA (1 µg) was converted to cDNA
by RTase and amplified by Q-PCR (40 cycles)
using SYBR Green fluorescence to quantify
DNA products. Data for daxx+/+cells are ex-
pressed as a fold change (based on 2??Ctval-
ues) relative to daxx−/−cells (mean ± SD;
n = 3). (B) Analysis of Dapk1, Dapk3, Birc3,
c-Flip, and Traf6 protein levels. Lysates from
daxx+/+and daxx−/−MEFs were normalized
for total protein content (60 µg) and analyzed
by SDS-PAGE and immunoblotting using an-
tibodies specific for mouse Dapk1, Dapk3,
Birc3, c-Flip, and ?-Actin, and results were
quantified by densitometry. (C) Analysis of
Daxx, HA-Daxx, and ?-Actin protein levels in
Daxx-reconstituted cells. mDaxx (HA-tagged)
was reconstituted into daxx−/−as described in
the Materials and Methods. Lysates from four
different cell types (daxx+/+, daxx−/−, daxx−/−+ mDaxx-pBabe, and daxx−/−+ pBabe) were processed as described in B and then blotted
with anti-Daxx, anti-HA, or anti-?-Actin antibodies. (D) Analysis of Cph, Gapdh, Dapk1, Dapk3, Birc3, and c-Flip mRNA levels in
Daxx-reconstituted cells. MEF daxx−/−+ pBabe (control) and daxx−/−+ mDaxx-pBabe were used for expression analysis of RelB can-
didate target genes (dapk1, dapk3, birc3, and c-flip) or control housekeeping genes (cph and gapdh) similarly to A. (E) Analysis
of Dapk1, Dapk3, Birc3, c-Flip, and Traf6 protein levels in Daxx-reconstituted cells. The daxx−/−+ pBabe control lysates and
daxx−/−+ mDaxx-pBabe lysates were processed as in B and then immunoblotted with the corresponding antibodies.
Daxx methylates promoters via Dnmt1
GENES & DEVELOPMENT999
daxx+/+MEFs. Protein analysis by immunoblotting cor-
roborated mRNA expression data, showing increases in
c-Flip, Birc3, Dapk1, Dapk3 (Fig. 1B), and Survivin
(Supplemental Fig. S2B) protein levels in Daxx-deficient
cells, but not Traf6 (Fig. 1B) or ?-actin (data not shown).
Because our previous analysis of Daxx-overexpressing
epithelial cancer cell lines indicated increased expres-
sion of the genes encoding Bok and Mail (Croxton et al.
2006), we also evaluated expression of these genes in
daxx−/−and daxx+/+MEFs. Consistent with previous re-
sults, Bok mRNA levels were approximately fourfold
higher in daxx+/+cells. Mail mRNA levels were only
slightly decreased (Fig. 1A). Thus, Daxx also may col-
laborate with other transcription factors to increase ex-
pression of some genes, but these effects could also be
indirect and, due to repression of repressors that control
expression of genes such as bok−, a topic that will not be
addressed further here.
Cell cycle analysis and comparisons of cell cycle pro-
teins showed that the differences in gene expression ob-
served in daxx−/−and daxx+/+cells were not due to varia-
tions in cell proliferation rates (Supplemental Fig. S1).
Altogether, these data obtained using Daxx-deficient
murine cells complement previous results obtained us-
ing Daxx-overexpressing human cells (Croxton et al.
2006) and strengthen evidence that Daxx regulates a sub-
set of apoptosis-relevant genes.
Daxx reconstitution in daxx−/−MEFs restores
wild-type expression of RelB target genes
To confirm that Daxx is directly involved in repression
of NF-?B-regulated target genes in MEFs, we reconsti-
tuted Daxx protein expression in daxx−/−MEFs by retro-
viral infection. Accordingly, daxx−/−MEFs were recon-
stituted with empty vector (pBabe-puro, control) versus
mouse Daxx vector (pBabe-mDaxx, HA-tagged). Immu-
noblot analysis using anti-HA antibody confirmed ex-
pression of Daxx-HA (∼120 kDa) in the stably transfected
cells. Immunoblot analysis with anti-Daxx antibody sug-
gested that the level of Daxx protein in Daxx-reconsti-
tuted daxx−/−MEFs was twofold to threefold higher than
that of endogenous Daxx protein in the wild-type MEFs.
Actin served as a loading control (Fig. 1C), validating
these comparisons of protein levels.
Both mRNA (Fig. 1D) and protein analysis (Fig. 1E)
revealed that Daxx reconstitution into Daxx-deficient
cells caused a significant decrease in expression of
Dapk1, Dapk3, c-Flip, and Birc3. In contrast, expression
of several control genes measured at the mRNA level
(Cph and Gapdh) or at the protein level (Traf6) was un-
changed, demonstrating the specificity of these reconsti-
tution experiments. Thus, these experiments confirm
that Daxx is directly responsible for the repression of
Recruitment of Daxx to target gene promoters
To confirm that expression of the analyzed gene set is
RelB-dependent, relB+/+and relB−/−MEFs were treated
with the cytokine TWEAK, which is known to stimulate
RelB activity (Varfolomeev et al. 2007). TWEAK induced
increases in the expression of the candidate target genes
encoding Dapk1, c-Flip, and Birc3 but not of the Cph
control in wild-type (relB+/+) MEFs, confirming RelB re-
sponsiveness (Fig. 2A). In contrast, TWEAK stimulated
either no increase (c-Flip), only a slight increase (Birc3),
or a decrease (Dapk1) in mRNA expression in relB−/−
cells, demonstrating that RelB modulates the activities
of these genes in TWEAK-stimulated cells (Fig. 2A). Fur-
thermore, evidence that RelB regulates the activity of
these genes was observed at basal conditions, based on
comparisons of unstimulated relB+/+versus relB−/−cells
(Fig. 2A, top panel). In unstimulated cells, levels of
Dapk1, c-Flip, and Birc3 mRNAs were lower in relB+/+
compared with relB−/−cells, suggesting that RelB medi-
ates repression of these genes under usual culture con-
To explore whether Daxx associates directly with RelB
candidate target gene promoters, chromatin immunopre-
cipitation (ChIP) followed by Q-PCR analysis was per-
formed, comparing relB+/+and relB−/−MEFs. The pro-
moter regions amplified for these experiments are pro-
vided as Supplemental Material (Supplemental Fig. S7).
ChIP assays showed that Daxx associates with dapk1,
dapk3, birc3, and c-flip promoters in RelB-containing
cells but not in RelB-deficient cells (Fig. 2B), suggesting
that Daxx associates with these genes through its inter-
action with RelB. Q-PCR analysis, performed to quantify
the extent to which RelB is required for Daxx’s recruit-
ment to the target genes, revealed significant RelB de-
pendence (Fig. 2C). As controls, ChIP assays with traf6,
gapdh, bcl-x, and ?-actin promoters (RelB-unresponsive
genes) revealed specificity of Daxx protein binding to
RelB-responsive genes, in that Daxx was not associated
with the traf6, gapdh, or ?-actin gene promoters. Note,
however, that Daxx association with the bcl-x promoter
was detected in both relB+/+and relB−/−cells, which may
be due to the presence of a HSF1-binding site in the re-
gion of the bcl-x gene promoter assayed (Supplemental
Fig. S7). In this regard, HSF1 has been reported to bind
Daxx (Boellmann et al. 2004).
Daxx repression of target gene promoters
Because we found that the Daxx protein associates with
target genes in a RelB-dependent manner, we next inves-
tigated whether Daxx’s repressional activity is also RelB-
dependent. We performed reporter gene assays using
relB+/+and relB−/−cells. Transient transfection of dapk1,
c-flip, or birc3 promoter–luciferase reporter gene con-
structs together with mouse Daxx (mDaxx) led to potent
suppression of these promoters in RelB-containing but
not in RelB-deficient cells (Fig. 2D). In contrast, mDaxx
did not repress the bcl-x promoter–luciferase reporter
gene regardless of RelB status (Fig. 2D, right-most panel),
confirming the specificity of these results. Note that the
promoter regions cloned into these luciferase reporter
gene plasmids contain the regions assayed for DNA
Puto and Reed
1000GENES & DEVELOPMENT
methylation plus adjacent regions that constitute the en-
tirety of the presumed promoters (see Supplemental Fig.
S8). Together, these data suggest that Daxx associates
with and represses target promoters through RelB.
Daxx associates with Dnmt family members in vivo
To determine whether Daxx’s mechanism of repression
of RelB-responsive genes involves Dnmts, we first ana-
lyzed whether Daxx interacts with Dnmts in vivo in
ments were performed using HA-Daxx-expressing MEFs
(Fig. 3A,B). Using anti-Daxx antibody for immunopre-
cipitations, we detected via immunoblotting that endog-
enous Dnmt1 and Dnmt3a interact with Daxx (Fig. 3A).
No interaction was detected with the Dnmt family
member Dnmt3b (Fig. 3A). Reciprocally, using anti-
Dnmt1, anti-Dnmt3a, and anti-Dnmt3b for immunopre-
cipitations (IPs), Daxx association was detected with en-
dogenous Dnmt1 and Dnmt3a, but not Dnmt3b (Fig. 3B).
These data thus extend a prior report of Dnmt1 associa-
tion with Daxx (Muromoto et al. 2004), demonstrating
that endogenous Dnmt1 and Dnmt3a associate with
Daxx in MEFs.
Methylation of target promoters is decreased
To explore whether DNA methylation participates in
Daxx-mediated repression of target genes, we investi-
gated the methylation status of the RelB target promot-
ers using daxx+/+and daxx−/−MEFs. Two approaches
were utilized—namely, (1) affinity capture of methylated
DNA using the CpG-binding protein MeCP2 (Fig. 3C),
and (2) bisulfite treatment and sequencing (Fig. 4). Using
the MeCP2 capture method, increased methylation of
the RelB-regulated promoters of the dapk1 and c-flip
genes was observed in Daxx-containing relative to Daxx-
deficient cells (Fig. 3C). To confirm specificity for RelB
target genes, we also analyzed ?-actin, bcl-x, and traf6
promoters as controls. These promoters do not bind RelB
and are insensitive to Daxx-mediated repression. No
Daxx-related change in methylation of the ?-actin, bcl-x,
and traf6 promoters was detected (Fig. 3C).
These results were confirmed independently by the
bisulfite sequencing method (Fig. 4), showing extensive
methylation differences between daxx+/+and daxx−/−
MEFs in the RelB target gene promoters. DNA methyl-
ation differences were especially striking in the dapk1
promoter, where the percentage of CpG dinucleotide
methylation was 28% in daxx+/+MEFs versus ∼5% in
daxx−/−MEFs. The promoters of the dapk3, c-flip, and
survivin genes also showed Daxx-dependent differences
in DNA methylation patterns, with significant decreases
in promoter methylation in daxx−/−MEFs (Fig. 4; Supple-
mental Fig. S2C). Although we assayed only a portion of
these promoters where CpG-rich regions are found, we
also performed DNA methylation analysis of adjacent
regions for two of the promoters (dapk1 and c-flip) and
observed similar differences in CpG methylation in
daxx+/+and daxx−/−cells (Supplemental Figs. S4, S5),
consistent with the notion that epigenetic silencing and
DNA hypermethylation commonly span large regions of
DNA (Frigola et al. 2006). In contrast, no methylation
with target gene promoters. (A) Expression analy-
sis of RelB candidate target genes (dapk1, c-flip,
and birc3) or control housekeeping gene (cph) in
relB−/−cells. MEFs (relB+/+and relB−/−) were cul-
tured without or with the cytokine TWEAK (25
ng/mL) for 24 h. Total RNA was then isolated;
RNA (1 µg) was converted to cDNA by RTase
and amplified by Q-PCR (40 cycles) using SYBR
Green fluorescence to quantify DNA products.
PCR products were analyzed by agarose gel elec-
trophoresis. (B) Comparison of relB+/+and relB−/−
MEFs to determine association of Daxx with
RelB candidate target gene promoters (dapk1,
dapk3, birc3, and c-flip) and control gene pro-
moters (bcl-x, ?-actin, traf6, and gapdh) in vivo,
using ChIP assays. Chromatin was immunopre-
cipitated with anti-Daxx or control IgG antibod-
ies. Target genes were amplified by Q-PCR (36
cycles) using primers that encompassed the NF-
?B-binding sites of each promoter (Supplemental
RelB-dependence of Daxx association
Fig. S7), and then was analyzed by gel electrophoresis. Input represents 10% of the chromatin specimen, subjected directly to Q-PCR
without IP. (C) ChIP analysis reveals differences in RelB dependence of Daxx’s recruitment to various promoters. Values represent
averages of two duplicate Q-PCR reactions from two ChIP experiments. (D) Daxx represses RelB target gene promoters. MEFs (relB+/+
and relB−/−; 85%–90% confluency in 12-well plates) were transiently transfected with three different plasmids: mDaxx-pEBB, renilla
luciferase reporter (pRL-TK), and either Dapk1-pLuc-pGIB, Flip-pGL3-1500, Birc3-pGL2-1400, or Bcl-X-pGL2-1200, for a total of 1.6 µg
of DNA per well. The ratio of experimental vector:coreporter vector (renilla luciferase) was 10:1. Luciferase assays were performed at
24 h post-transfection. All assays were performed in triplicate.
Daxx methylates promoters via Dnmt1
GENES & DEVELOPMENT 1001
changes were observed in the bcl-x and traf6 control
gene promoters (Fig. 4; Supplemental Fig. S3). These data
demonstrate that Daxx modulates DNA methylation of
the promoters of RelB target genes.
Daxx reconstitution in daxx−/−MEFs restores DNA
Using retrovirus-mediated stable gene transfer, we re-
constituted Daxx protein expression in daxx−/−MEFs
and then measured DNA methylation patterns by bisul-
fite sequencing. An increase in dapk1, dapk3, and c-flip
promoter methylation was observed in the Daxx-recon-
stituted cells, but not in cells infected with control vec-
tor (Fig. 5). The DNA methylation patterns of Daxx-re-
constituted cells were very similar to wild-type daxx+/+
MEFs in terms of percentage of CpG dinucleotides meth-
ylated within the CpG islands examined (cf. Figs. 4 and
5). In contrast, no difference was seen in traf6 control
gene promoter methylation between Daxx- and control-
infected cells (Fig. 5). Moreover, when the positions of
each methylated CpG within the relevant promoters
were examined (Fig. 6), an almost identical methylation
pattern was observed for wild-type and Daxx-reconsti-
tuted knockout cells for all of the RelB target gene pro-
moters examined (Fig. 6). Specifically, positions 64, 57,
43, 40, 6, and −103 relative to the dapk1 gene transcrip-
tion start site were consistently methylated only in wild-
type and Daxx-reconstituted cells (Fig. 6A); positions
−844, −856, −997, and −1041 relative to the dapk3 gene
transcription start site were similarly methylated only in
wild-type and Daxx-reconstituted cells (Fig. 6B); and po-
sition −57 relative to the c-flip gene transcription start
site was strictly methylated in wild-type and Daxx-re-
constituted cells, but not in Daxx-deficient cells (Fig.
6C). In contrast, the methylation pattern of the traf6
gene promoter showed no consistent differences regard-
less of Daxx status (Supplemental Fig. S3). In conclusion,
these gene reconstitution experiments confirm that the
presence of Daxx is required for DNA methylation of the
examined RelB target gene promoters.
Daxx targets Dnmt1 to repressed promoters
Since we demonstrated that Dnmt1 interacts with Daxx
in MEFs (Fig. 3A,B), we tested whether Dnmt1 could be
recruited to prototypical RelB target gene promoters by
Daxx by performing ChIP assays using daxx+/+and
daxx−/−cells. In daxx+/+cells, Dnmt1 bound to dapk1
and c-flip promoters (Fig. 7A, lane 2). In contrast, in
daxx−/−cells, Dnmt1 binding to dapk1 and c-flip pro-
moters was undetectable (Fig. 7A, lane 5). These differ-
methylation of RelB target genes: analysis by MeCP2 capture.
(A) Daxx-reconstituted MEF lysates were subjected to IP with
anti-Daxx antibody or control IgG. The resulting immune com-
plexes were analyzed by SDS-PAGE, followed by immunoblot-
ting with antibodies specific for Dnmt1, Dnmt3a, or Dnmt3b.
Lysates (same total protein content used for IP) were also run
directly in gels, as a control. (B) Daxx-reconstituted MEF lysates
were subjected to IP with anti-Dnmt1, Dnmt3a, Dnmt3b, or
control IgG. The resulting immune complexes were analyzed by
SDS-PAGE, followed by immunoblotting with antibodies spe-
cific for Daxx. Lysates were also analyzed as a control. Immu-
noblotting with anti-HA was performed to confirm effective IP
of HA-Daxx (reconstituted Daxx). (C) An affinity capture
method employing an immobilized MeCP protein that binds
methylated DNA was used to assess the DNA methylation sta-
tus of RelB candidate target gene (dapk1 and c-flip) and control
promoters (?-actin, bcl-x, and traf6) in daxx+/+versus daxx−/−
MEFs. Genomic DNA (4 µg) was digested with MseI; fragments
of MseI-digested DNA retaining CpG islands were purified
through a DNA purification column and then incubated with
immobilized MeCP2. Captured DNA fragments were amplified
by Q-PCR. PCR products were analyzed by agarose gel electro-
Daxx associates with Dnmts in vivo and regulates
method. Genomic DNA from daxx+/+(circles)
and daxx−/−(squares) MEFs was treated with
bisulfite, and 0.5 µg was amplified by PCR
using primers specific for the indicated gene
promoters. Amplified products were cloned
and 10–16 clones were sequenced per target
promoter. The methylation profile of the pro-
moters was determined by comparing the se-
quence of bisulfite-converted DNA with un-
modified DNA. The percentage of methylated
CpG sites are shown for each promoter exam-
ined. Promoters are listed in order of methyl-
ation change between daxx+/+and daxx−/−
MEFs from highest to lowest. The difference
in percentage of methylation between daxx+/+
and daxx−/−MEFs was as follows: dapk1,
23%; dapk3, 6.3%; c-flip, 5%; survivin, 4.6%;
traf6, 0%; and bcl-x, 0%.
Daxx regulates methylation of
Puto and Reed
1002 GENES & DEVELOPMENT
ences in Dnmt1 association with RelB target gene pro-
moters were specific in that no change was observed be-
tween daxx+/+and daxx−/−cells when ChIP analysis was
performed for bcl-x, ?-actin, and traf6 gene promoters
(Fig. 7A, cf. lanes 2 and 5). Specifically, Dnmt1 bound to
the bcl-x gene promoter regardless of Daxx status (pos-
sibly due to the binding of other Daxx-interacting pro-
teins to this promoter such as Ets and HSF1) (Supple-
mental Fig. S7; Boellmann et al. 2004), whereas no
Dnmt1 binding to the ?-actin or traf6 gene promoters
was detected in either daxx+/+or daxx−/−cells. The
specificity of these results was also confirmed by the IgG
control of ChIP reactions (Fig. 7A, lanes 3,6). Q-PCR
analysis of the ChIP reactions showed approximately
fivefold to eightfold more Dnmt1 association with the
RelB target gene promoters in daxx+/+compared with
daxx−/−cells (dapk1 and c-flip) (Fig. 7B). Collectively,
these data show that Daxx is required for recruitment of
Dnmt1 to RelB target gene promoters.
Daxx and Dnmt1 synergistically repress target
promoters via DNA methylation
We examined the functional significance of Dnmt1 re-
cruitment by Daxx to target gene promoters by perform-
ing reporter gene assays using RelB target gene promot-
er–luciferase contructs (Supplemental Fig. S8). While
Daxx transfection decreased expression of dapk1 and c-
flip reporter gene constructs, Dnmt1 transfection did not
(Fig. 7C). Cotransfection of Daxx together with Dnmt1,
however, resulted in a cooperative repressive effect on
these RelB target gene promoters, significantly reducing
expression beyond Daxx alone (P < 0.05 for dapk1 and
c-flip). Moreover, this repression was relieved by Dnmt
inhibitor 5-azacytidine, restoring gene expression to the
levels of control cells. The Hdac inhibitor TSA had much
less effect (Fig. 7C), suggesting a less prominent role in
the repression compared with Dnmts. In contrast to the
RelB target genes, expression of the bax gene promoter
was not affected by Daxx, Dnmt1, or Daxx/Dnmt1 trans-
fections (Fig. 7C). Likewise, the bcl-x gene promoter
showed no Daxx-dependent repression, although Dnmt1
transfection reduced the activity of this promoter (Fig.
7C, right-most panel). On the basis of this analysis, we
conclude that Dnmt1 collaborates with Daxx to repress
RelB target genes.
Given that the chemical inhibitor of Dnmtases, 5-aza-
cytadine, restored expression of the RelB target gene pro-
moters (dapk1 and c-flip), we hypothesized that DNA
methylation is responsible for the repression of these
promoters. To assess the relative levels of methylation of
the transfected plasmids, we transiently transfected vari-
ous promoter–luciferase constructs either alone or in
combination with Daxx, Dnmt1, or Daxx and Dnmt1
daxx+/+and daxx−/−cells. Genomic DNA from daxx+/+, daxx−/−,
daxx−/−+ mDaxx-pBabe, and daxx−/−+ pBabe MEFs was sub-
jected to sodium bisulphite modification and sequencing, ana-
lyzing dapk1 (A), dapk3 (B), and c-flip (C) proximal promoters.
Methylated sites are indicated by solid circles and unmethyl-
ated sites are indicated by open circles. Predicted or known
transcription factor-binding sites and transcription initiation
sites are indicated in the depictions of the genes studied. Gray
areas indicate the region analyzed.
Comparison of methylation status of the genes in
methylation patterns to RelB target gene promoters. Genom-
ic DNA from mouse daxx−/−+ mDaxx-pBabe (circles) and
daxx−/−+ pBabe (squares) MEFs was treated with bisulfite,
amplified, cloned, and sequenced. The percentage of methyl-
ated CpG sites are shown for each promoter examined. Pro-
moters are listed in the order of methylation change between
daxx−/−+ mDaxx-pBabe and daxx−/−+ pBabe MEFs from highest
to lowest as follows: dapk1, 24.5%; dapk3, 7%; c-flip, 3.5%; and
Daxx protein reconstitution restores normal DNA
Daxx methylates promoters via Dnmt1
GENES & DEVELOPMENT1003
(Fig. 7D). The isolated transiently transfected plasmids
were subjected to DNA methylation analysis using the
MeCP2 affinity capture method. Methylation of the
dapk1 and c-flip promoter constructs was increased
when either Daxx or Daxx/Dnmt1 was cotransfected
with the respective reporter constructs, with the combi-
nation of Daxx and Dnmt1 inducing greater methylation
than Daxx alone (Fig. 7E). Dnmt1 transfection alone had
no effect on methylation (Fig. 7D). In contrast, the bax
promoter–luciferase construct (used here as a control)
showed no change in DNA methylation in response to
Daxx or Daxx/Dnmt1 (Fig. 7D,E). Also, the bcl-x pro-
moter construct showed methylation changes that par-
alleled the promoter activity analysis (Fig. 7C), with in-
creased DNA methylation induced by Dnmt1 but not
Daxx (Fig. 7D,E). Taken together, these experiments di-
rectly demonstrate that Daxx is capable of promoting the
methylation of the RelB target gene promoters to which
In this report, we demonstrate that Daxx is required for
endogenous repression of a variety of RelB target genes,
and we link this repressive effect of Daxx to a mecha-
nism involving recruitment of Dnmt1 and DNA hyper-
methylation. Many studies have shown that Daxx can
function as a transcriptional repressor (Salomoni and
Khelifi 2006), yet the precise mechanism by which Daxx
exerts its suppressive effects on endogenous genes has
remained unclear. In this study, we observed that Daxx
recruits Dnmt1 onto RelB target gene promoters, result-
ing in DNA hypermethylation and epigenetic silencing
of target regions. Repression of target genes was found to
be RelB-dependent (Fig. 2), consistent with data showing
that Daxx physically binds RelB without interfering
with RelB binding to DNA (Croxton et al. 2006). In this
regard, RelB, but not Rel-A or c-Rel, contains a candidate
Daxx-interacting domain motif (DID), a motif identified
previously in the Ets family of transcription factors (Li et
al. 2000), and we reported recently that Daxx physically
interacts with RelB but not the other members of the Rel
family (Croxton et al. 2006). Thus, the picture emerging
(Fig. 8) is that RelB binds to target promoters, resulting in
target gene transactivation when Daxx is absent or un-
derexpressed. In contrast, when both RelB and Daxx are
present, Daxx interacts with RelB bound to its target
sites in the genome, recruiting Dnmtases and resulting
resulting in synergistic repression and increased DNA
methylation. (A) Comparison of daxx+/+and daxx−/−
MEFs to determine association of Dnmt1 with RelB
candidate target gene promoters (dapk1 and c-flip) and
control gene promoters (?-actin, bcl-x, and traf6) in
vivo, using ChIP assays. Chromatin was immunopre-
cipitated with anti-Dnmt1 or control IgG antibodies.
Target genes were amplified by PCR (36 cycles) using
primers that encompassed the NF-?B-binding sites of
each promoter (Supplemental Fig. S7), and then were
analyzed by gel electrophoresis. Input represents 10%
of the chromatin specimen, subjected directly to PCR
without IP. (B) Daxx-dependent Dnmt1 recruitment to
promoters. Values represent averages (±SEM) of two du-
plicate Q-PCR reactions from two ChIP experiments.
(C) Daxx-mediated repression of target gene promoters
is enhanced by Dnmt1. Wild-type MEFs were tran-
siently transfected with various combinations of ex-
pression and reporter gene as indicated (total 1.6 µg of
DNA per well). 5-azacytidine (2 µM) or TSA (100 ng/
mL) were added 4 h after transfections. Luciferase as-
(mean + SD; n = 3). (*) P < 0.05 for dapk1 and c-flip. (D)
DNA methylation analysis of transiently transfected
were transiently transfected for 24 h with or without
plasmids encoding Daxx and/or Dnmt1. Low-molecu-
lar-weight (LMW) DNA was recovered from cells and
incubated with immobilized MeCP2 to capture meth-
ylated DNA, followed by PCR analysis using primers
targeting promoter regions cloned into the luciferase
constructs. PCR products were visualized by ethidium
bromide staining after agarose gel electrophoresis. (E)
Q-PCR analysis of MeCP2-capatured LMW DNA was
performed to quantify relative methylation levels of the
transiently transfected promoter constructs.
Daxx recruits Dnmt1 to target promoters,
Puto and Reed
1004 GENES & DEVELOPMENT
in CpG methylation of RelB target gene promoters and
gene silencing. Because Daxx lacks domains for se-
quence-dependent DNA binding (Salomoni and Khelifi
2006), Daxx is unable to suppress RelB target genes when
RelB is absent. Thus, Daxx effectively converts RelB
from a transactivator to a repressor. Consistent with this
hypothesis, we observed reduced expression of the RelB
target genes dapk1, c-flip, and birc3 in relB+/+compared
with relB−/−MEFs, implying that, under basal condi-
tions, RelB contributes to the repression of these genes
rather than their activation. Upon stimulation with
TWEAK, however, expression of these genes increased in
relB+/+but not relB−/−cells, suggesting that, under con-
ditions of cytokine stimulation, repression is reversed.
The RelB/Daxx interaction may represent a novel
paradigm for explaining how a single transcription fac-
tor—in this case, RelB—can operate as either an activa-
tor or repressor of target genes, depending on cellular
context. Specifically, RelB has been reported to act as
both a transcriptional activator and a repressor of NF-?B-
dependent gene expression (Ruben et al. 1992; Ryseck et
al. 1992; Bours et al. 1994; Bonizzi and Karin 2004). RelB
transactivates through its association with p50 or p52
(Bonizzi and Karin 2004), and it represses when bound to
RelA (Marienfeld et al. 2003; Jacque et al. 2005). In ad-
dition, a switch from p52/RelB complexes to p50/RelB
complexes correlated with repression of skp2 (Schneider
et al. 2006). The finding that Daxx converts RelB from
activator to repressor thus reveals another mode beyond
heterodimerization for flipping the phenotype of this
transcriptional regulator. In addition to dual functional-
ity of RelB, likewise RelA can act as both an activator
and a repressor of NF-?B target genes, depending on the
stimulus for induction (Dong and Goldschmidt-Cler-
mont 2002; Campbell et al. 2004). One class of NF-?B
activators (e.g., TNF and etoposide) induces RelA to act
as an activator of anti-apoptotic gene expression (XIAP
and Bcl-XL) (Campbell et al. 2004), while a second class
of activators (e.g., UV-C, doxorubicin, and ARF tumor
suppressor) induces RelA to act as a repressor of the same
anti-apoptotic genes (Campbell et al. 2004; Perkins and
Gilmore 2006). Thus, our findings for the Daxx/RelB in-
teraction suggest that not only RelB but probably also
other Rel family members may convert from transacti-
vators to repressors, depending on with what other fac-
tors they interact. Interestingly, Daxx is also induced by
doxorubicin (Boehrer et al. 2005), UV-C irradiation (Khe-
lifi et al. 2005), and hydrogen peroxide treatment (Khelifi
et al. 2005).
Relatively few examples exist thus far that account for
selective DNA methylation of specific classes of target
genes. Among the known mechanisms in mammalian
cells are (1) RP58, a DNA-binding transcriptional repres-
sor that recruits Dnmt3a to silence transcription (Fuks et
al. 2001); (2) the PML-RARa fusion protein that results
from t(15;17) chromosomal translocations found in cer-
tain leukemias, in which the DNA-binding domain of
RAR? dictates the genomic target sites to which PML
recruits Dnmt3a (Di Croce et al. 2002); (3) the Kaposi’s
which directly binds to DNA and recruits Dnmt3a to
repress specific promoters (Shamay et al. 2006); and (4)
Myc transcription factor, which activates genes through
direct DNA binding, but represses indirectly through in-
teractions with another transcription factor, inducing si-
lencing via Dnmt3a recruitment (Brenner et al. 2005).
Most of these examples differ from the RelB/Daxx inter-
action in that Dnmtases are directly recruited to sites in
the genome by sequence-specific DNA-binding proteins.
In contrast, Daxx does not directly bind DNA sequences
in the genome (Salomoni and Khelifi 2006), but rather is
indirectly brought to specific sites through its ability to
sion of RelB-binding promoters. A minimalist
model is presented, showing Daxx, RelB, and
Dnmtases interacting with a RelB target gene
promoter. (Top left) In the absence of Daxx,
RelB binds to target promoters, resulting in
light). Promoters are hypomethylated. (Bot-
tom left) In the absence of RelB, Daxx is un-
able to suppress RelB target genes because
Daxx lacks DNA-binding domains (green traf-
fic light). Promoters remain hypomethylated.
(Right) When both RelB and Daxx are present,
Daxx interacts with RelB bound to its target
sites in the genome, recruits Dnmtases, and
induces CpG hypermethylation of RelB target
gene promoters, thus resulting in gene silenc-
ing (red traffic light).
Model for Daxx-mediated repres-
Daxx methylates promoters via Dnmt1
GENES & DEVELOPMENT 1005
bind DNA-binding transcription factor RelB (Croxton et
al. 2006). Myc-mediated gene silencing is similar to
Daxx in that Myc recruits Dnmtases to genomic target
sites when interacting with another DNA-binding pro-
tein, Miz1, but not when directly binding its specific
target DNA sequences in the genome via its canonical
DNA-binding domain (Brenner et al. 2005). However,
Daxx differs from Myc in that it lacks a DNA-binding
domain (Salomoni and Khelifi 2006). Thus, while sharing
similarities with Myc, the mechanism for site-selective
DNA methylation described here for Daxx/RelB consti-
tutes a new paradigm for explaining how DNA methyl-
ation is achieved in a gene-selective manner in mam-
Daxx and Dnmt1 synergistically repressed target gene
promoters in transient transfection reporter gene assays.
The observation that the Dnmt inhibitor 5-azacitydine
completely restored gene expression strongly suggests
that Dnmts are substantially responsible for the repres-
sive action of Daxx in the context of promoters exam-
ined. Plasmid methylation experiments further corrobo-
rated these findings, providing direct evidence that Daxx
is capable of promoting methylation of the RelB target
gene promoters to which it binds, and showing enhance-
ment of DNA methylation by coexpression of Dnmt1
with Daxx but not when expressing Dnmt1 alone. How-
ever, using ChIP assays, we found that Daxx also recruits
Hdac2 to target promoters (Supplemental Fig. S6). We
cannot, therefore, exclude the possibility that Hdacs are
also involved as corepressors, although experiments
with Hdac inhibitor TSA suggested a more minor role
compared with DNA methylation. In this regard, meth-
ylated DNA and Hdacs frequently colocate (Gore 2005).
To the extent that Hdacs contribute to Daxx-mediated
repression, recruitment of Daxx to RelB on genomic sites
is reminiscent of Sin3A corepressor complex recruit-
ment to type II nuclear receptors (NRs) in the absence of
activating ligands, wherein NRs bound to their target
sites in the genome actively repress rather than trans-
activate genes (Mathur et al. 2001). However, while
Sin3A-mediated repression is dependent on Hdacs (Viiri
et al. 2006), we found that Daxx-mediated repression in-
volves Dnmts. Also, other studies have shown that DNA
methylation is often dominant over Hdac activity in
maintaining transcriptional repression (Cameron et al.
Daxx-dependent changes in the DNA methylation of
gene promoters typically involved several CpGs and
showed reproducible patterns of CpG methylation (Fig.
6), suggesting that only certain CpG motifs are presented
to the DNA methylation machinery in a manner condu-
cive to their modification. In the case of the c-flip gene
promoter, a single CpG site (at position −57) was identi-
fied that displayed Daxx-dependent methylation, at least
within the small region of the promoter that we exam-
ined (−248 to 88, relative to the transcription start site).
Because only a small portion of the c-flip promoter was
analyzed for DNA methylation here, other CpG motifs
are likely to be regulated by methylation elsewhere in
the c-flip promoter. Also, while we cannot forecast the
functional importance of this Daxx-dependent difference
in c-flip promoter methylation, it should be noted that a
single methylated site per 300 bases within a gene’s pro-
moter has been found, on several occasions, to be suffi-
cient to dramatically repress expression (Hsieh 1994;
Robertson et al. 1995; Carvin et al. 2003). It will be in-
teresting to explore how DNA methylation patterns in
promoters of the c-flip, dapk1, birc3, and other RelB tar-
get genes are impacted by agents that affect the activity
of Daxx, such as interferons and arsenical, which induce
Daxx recruitment to PML-oncogenic domains within
Daxx reportedly has proapoptotic activity in many
contexts but also has been implicated in apoptosis sup-
pression, including in vivo in daxx−/−embryos (Mi-
chaelson et al. 1999; Chen and Chen 2003; Salomoni and
Khelifi 2006). By analyzing the effects of Daxx on a panel
of ∼260 apoptosis-relevant genes, we previously found
examples where Daxx represses both anti- and proapo-
ptotic genes (Croxton et al. 2006). For example, Daxx-
dependent repression of genes encoding anti-apoptotic
proteins Birc3 (cIAP2), Survivin, and c-Flip was observed
simultaneously with repression of the gene encoding the
proapoptotic protein Dapk3 (Croxton et al. 2006). In the
current study, we also included another proapoptotic
protein—namely, Dapk1, which is the founding member
of the Dapk family of Ser/Thr kinases (Bialik and Kimchi
2006). We observed that Daxx down-regulated expres-
sion of both anti-apoptotic genes (c-flip, birc3, and sur-
vivin) and proapoptotic genes (dapk1 and dapk3). Thus,
perhaps Daxx association with RelB and its regulation of
sets of anti- and proapoptotic RelB target genes are at
least partly responsible for the reported contradictory
roles for Daxx (proapoptotic vs. anti-apoptotic). Our re-
port thus may reconcile longstanding ambiguities about
Daxx’s functions, and also points to RelB as a likely link
between its transcriptional and apoptotic activities.
Given evidence that Daxx protein levels vary among
certain types of tumors, correlating with sensitivity or
resistance to chemotherapeutic agents (Lindsay et al.
2007), the role of Daxx as an epigenetic regulator has
potential ramifications for cancer biology and cancer
therapy. In this regard, drugs that directly inhibit
Dnmtases (e.g., 5-azacytidine [Vidaza] and 5-aza-2?-de-
oxycytidine [Decitabine]) are currently in clinical use for
selected types of malignancies, with exploratory clinical
trials under way for a wide variety of types of cancers and
several oncological contexts. An underlying assump-
tion of these epigenetic therapies is that inhibition of
Dnmtases will demethylate genes in dividing cells, re-
storing the activity of epigenetically silenced tumor sup-
pressor genes (Baylin 2005). Interestingly, however, our
study shows that while Daxx induces DNA methylation
and inhibits expression of certain genes that operate as
tumor suppressors, such as Dapk1 (Bialik and Kimchi
2006; Raval et al. 2007), it also represses expression of
several anti-apoptotic genes whose reactivation would
presumably be counterproductive in a cancer context
(e.g., c-Flip and Survivin), the expression of which has
been associated with poor prognostic features of tumors
Puto and Reed
1006GENES & DEVELOPMENT
(Valnet-Rabier et al. 2005; Hinnis et al. 2007). Therefore,
knowledge of the status of Daxx in tumors may be help-
ful for identifying those cancer patients for whom
Dnmtase inhibitory drugs are most likely to be benefi-
cial (counteracting hypermethylation of tumor suppres-
sors), as well as for possibly avoiding use of these agents
in patients for whom Dnmtase inhibition may be coun-
terproductive (counteracting hypermethylation of sur-
In addition to RelB, Daxx has been reported to repress
a variety of transcription factors including Ets-1 (Li et al.
2000), p73 (Kim et al. 2003), glucocorticoid receptors
(GR) (Lin et al. 2003), Tcf4 (Tzeng et al. 2006), Pax-3
(Hollenbach et al. 1999), and Pax-5 (Emelyanov et al.
2002). It remains to be determined whether Daxx re-
cruits Dnmtases to the target genes where these tran-
scription factors bind and induces epigenetic silencing.
Thus, although we focused here on apoptosis-regulating
RelB target genes, it is likely that the Daxx-dependent
mechanism for epigenetic regulation described here ap-
plies to other categories of Daxx-regulated genes and
other classes of transcription factors that Daxx binds.
The cellular consequences of Daxx-mediated gene meth-
ylation therefore may be broad, with implications be-
yond RelB and apoptosis regulation.
Materials and methods
Antibodies and cytokines
The following antibodies were used for immunoblot and immu-
noprecipitation analyses: rabbit anti-Daxx (Santa Cruz Biotech-
nology), mouse anti-Dapk1 (Abcam), rabbit anti-ZIPK (Dapk3)
(Abcam), goat anti-cIAP2 (Birc3) (Abcam), rat anti-Flip (Alexis
Biochemicals), rabbit anti-Survivin (Santa Cruz Biotechnology),
rabbit anti-Dnmt1 (Santa Cruz Biotechnology), rabbit anti-
Dnmt3a (Santa Cruz Biotechnology), rabbit anti-Dnmt3b (Santa
Cruz Biotechnology), rabbit anti-Traf6 (Santa Cruz Biotechnol-
ogy), mouse anti-?-actin (Sigma), rabbit anti-HA (hemagglutinin
epitope tag) (Covance), and mouse anti-Cyclin-D1 (Santa Cruz
Biotechnology). The following antibodies were used for ChIP
assays: rabbit anti-Daxx (Santa Cruz Biotechnology), mouse
anti-Dnmt1 (Imgenex), and rabbit anti-Hdac2 (Abcam). Immu-
noblotting, following SDS-PAGE, was performed using the en-
hanced SuperSignal West Pico Chemiluminescent protein de-
tection system (Pierce). Data were quantified by scanning den-
sitometry analysisof scanned
processing and analysis program. The cytokine TWEAK was
purchased from Alexis Biochemicals and used at a concentra-
tion of 25 ng/mL to stimulate MEFs for 24 h.
Cell culture conditions and luciferase assays
MEFs, including daxx+/+, daxx−/−, relB+/+, and relB−/−, were
maintained in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% bovine calf serum (BCS) (Hyclone), and 1%
penicillin/streptomycin plus L-glutamine. For luciferase assays,
MEFs (85%–90% confluency in 12-well plates) were transiently
transfected at 24 h post-plating with various plasmids using
LipofectAMINE 2000 reagent (Invitrogen), following the manu-
facturer’s protocol. The amount of DNA was 1.6 µg per well, the
amount of LipofectAMINE was 4 µL per well, and the ratio of
experimental vector:coreporter vector was 10:1. Transfection
efficiency for MEFs was calculated to be ∼15%, using a GFP
plasmid (pGFP2). At 24 h following transfection, cells were
lysed following the Dual-Luciferase Reporter Assay System pro-
tocol (Promega). Firefly and renilla luciferase activities were
measured using a microplate luminometer (BD PharMingen
Monolight 3096), normalizing firefly to renilla values. All as-
says were performed in triplicate. For methylation inhibition
studies, 5-azacytidine (Pharmion) or TSA (US Biological) were
added 4 h following transfections at a final concentration of 2
µM or 100 ng/mL, respectively.
Plasmid methylation assays
Various promoter–luciferase constructs were transiently trans-
fected for 24 h either alone (control) or in combination with
Daxx and/or Dnmt1. Transfection reagents included 4 µg of
DNA per well of a six-well plate and LipofectAMINE 2000 re-
agent (Invitrogen). The transiently transfected plasmids were
recovered by isolation of low-molecular-weight (LMW) DNA
using a modified protocol available from Qiagen. LMW DNA
samples were then subjected to DNA methylation analysis, us-
ing the MeCP2 affinity capture method (described below), but
without performing restriction digestion, followed by PCR
analysis using primers encompassing promoter regions cloned
into the luciferase constructs (Supplemental Fig. 8).
Daxx+/+and Daxx−/−MEFs were grown to 70%–80% confluency
in 15-cm plates. Cells were processed for ChIP assays using the
ChIP-IT kit (Active Motif). DNA was enzymatically sheared to
a size of 300–900 base pairs (bp) prior to performing the IPs.
Sheared chromatin was precleared by incubating with protein G
beads (Active Motif) for 2 h at 4°C to reduce nonspecific back-
ground. Precleared chromatin was then incubated with various
antibodies (3 µg) overnight at 4°C. The kit’s negative control
IgG was also used in parallel reactions. Protein G bead incuba-
tion, washing of ChIP reactions, DNA elution from protein G,
cross-link reversal, RNA removal, and purification of eluted
DNA were performed following the kit’s protocol. Isolated
DNA was subjected to Q-PCR, using promoter-specific primers
as shown in Supplemental Figure 7. Q-PCR products were ana-
lyzed using Stratagene’s MxPro software and running agarose
gels, stained with ethidium bromide, followed by UV visualiza-
pBABEpuro-mDaxx (HA-tagged) was a generous gift of Dr. Gerd
Maul, The Wistar Institute. 293T cells were seeded in 60-mm
dishes (50% confluency) and transfected 24 h later with 3 µg of
pCL-ECO (retroviral packaging vector) and 9 µg of pBABEpuro-
mDaxx-HA (or pBABEpuro-empty as a control) for a total of 12
µg of DNA per 60-mm dish, using a calcium phosphate precipi-
tation method (Promega ProFection Mammalian Tranfection
System). Twenty-four hours following calcium phosphate trans-
fection, the supernatant of 293T cells was removed and filtered
through a 0.45-µm filter. Filtered supernatants, along with 8
µg/mL polybrene-hexadimethrine bromide (Sigma), were added
to Daxx−/−MEFs. MEFs were then grown to confluency before
splitting and were cultured for 10 d in media containing 1 µg/
Birc3 (pGL2-cIAP2-1400-Luc), Bax (BAX-pGL3), and Bcl-x (BCL-
X-pGL2) promoter constructs have been described previously
Daxx methylates promoters via Dnmt1
GENES & DEVELOPMENT 1007
(Croxton et al. 2006). The following promoter constructs were
kind gifts: mDaxx-pEBB (Dr. David Baltimore, CalTech),
mDnmt1-pBluescript-SK (Dr. Tim Bestor, Columbia Univer-
sity), mSurvivin (1430)-pLuc-pGEM3 (Dr. Dario Altieri, Univer-
sity of Massachusetts), mDapk1-pLuc-pGIB (Dr. Chris Marine,
University of Ghent, Belgium), and pGL3-FLIP-1500 (Dr. Wafik
El-Deiry, University of Pennsylvania School of Medicine).
Total RNA was isolated using RNeasy mini kit (Qiagen). cDNA
was generated using StrataScript First-Strand Synthesis System
(Stratagene). RT–PCR (duplicate) was performed using SYBR
Green PCR master mix (Qiagen) and Mx3000P Q-PCR system
(Stratagene). Values were normalized to the level of CPH or
GAPDH mRNA abundance and analyzed using Stratagene’s
MxPro software. Thermal DNA melting experiments and gel
electrophoresis confirmed generation of a single PCR product of
the expected length for each amplification product shown.
Graphs were constructed based on ??Ct values generated by
Q-PCR [(Ctsample− CtCph)Daxx+/+− (Ctsample− CtCph)Daxx−/−], using
the formula 2??Ct.
Bisulfite conversion and genomic sequencing
Methylation status of various promoters was assessed by bisul-
fite genomic sequencing. Briefly, genomic DNA was isolated
from Daxx+/+and Daxx−/−MEFs using DNeasy kit (Qiagen).
Bisulfite reactions were performed using EZ DNA Methylation-
Gold Kit (Zymo Research) under conditions that allowed for
complete conversion of cytosines, but not 5-methylcytosines,
to uracil. The bisulfite-modified DNA was amplified by PCR,
using the following conditions: 2 min at 94°C; 40 cycles of 30
sec at 94°C, 30 sec at 50°C, and 1.5 min at 68°C; and finally 10
min at 68°C. Amplified products were subcloned into the
pCR2.1 TOPO vector via TA cloning (Invitrogen). Ten to 16
clones were sequenced (per target promoter). The methylation
profile of the promoter of interest was determined by comparing
the sequence of bisulfite-converted DNA with that of unmodi-
To assess the methylation status of target promoters via an
alternative method, an affinity capture method employing an
immobilized protein that binds methylated DNA was used (Pro-
moter Methylation PCR, Panomics). Genomic DNA was di-
gested with MseI restriction enzyme (New England Biolabs),
then incubated with immobilized MeCP2 (methylation-binding
protein), according to the manufacturer’s instructions. Cap-
tured methylated DNA fragments were amplified by Q-PCR,
comparing Daxx+/+versus Daxx−/−samples using Stratagene’s
MxPro software. The products were visualized using agarose gel
We thank Drs. Manuel Perucho, Sergio Alonso, and Fumi Ya-
mamoto (Burnham Institute for Medical Research) for insightful
discussions on DNA methylation studies; Drs. Alexander Hoff-
mann and Soumen Basak (University of California, San Diego)
for helpful advice on retrovirus transduction experiments; Mel-
anie Hanaii and Tessa Siegfried for manuscript submission; and
Drs. David Baltimore, Tim Bestor, Dario Altieri, Chris Marine,
and Wafik El-Deiry for plasmid reagents. This work was sup-
ported by an NIH/National Cancer Institute grant CA69381 to
J.C.R., and a dissertation fellowship from California Breast Can-
cer Research Program (CBCRP) 13GB-0056 to L.A.P.
Alcamo, E., Hacohen, N., Schulte, L.C., Rennert, P.D., Hynes,
R.O., and Baltimore, D. 2002. Requirement for the NF-?B
family member RelA in the development of secondary lym-
phoid organs. J. Exp. Med. 195: 233–244.
Baylin, S.B. 2005. DNA methylation and gene silencing in
cancer. Nat. Clin. Pract. Oncol. 2 (Suppl. 1): S4–S11. doi:
Baylin, S.B. and Ohm, J.E. 2006. Epigenetic gene silencing in
cancer—A mechanism for early oncogenic pathway addic-
tion? Nat. Rev. Cancer 6: 107–116.
Bialik, S. and Kimchi, A. 2006. The death-associated protein
kinases: Structure, function, and beyond. Annu. Rev. Bio-
chem. 75: 189–210.
Boehrer, S., Nowak, D., Hochmuth, S., Kim, S.Z., Trepohl, B.,
Afkir, A., Hoelzer, D., Mitrou, P.S., Weidmann, E., and
Chow, K.U. 2005. Daxx overexpression in T-lymphoblastic
Jurkat cells enhances caspase-dependent death receptor- and
drug-induced apoptosis in distinct ways. Cell. Signal. 17:
Boellmann, F., Guettouche, T., Guo, Y., Fenna, M., Mnayer, L.,
and Voellmy, R. 2004. DAXX interacts with heat shock fac-
tor 1 during stress activation and enhances its transcrip-
tional activity. Proc. Natl. Acad. Sci. 101: 4100–4105.
Bonizzi, G. and Karin, M. 2004. The two NF-?B activation path-
ways and their role in innate and adaptive immunity. Trends
Immunol. 25: 280–288.
Bours, V., Azarenko, V., Dejardin, E., and Siebenlist, U. 1994.
Human RelB (I-Rel) functions as a ?B site-dependent trans-
activating member of the family of Rel-related proteins. On-
cogene 9: 1699–1702.
Brenner, C., Deplus, R., Didelot, C., Loriot, A., Viré, E., De
Smet, C., Gutierrez, A., Danovi, D., Bernard, D., Boon, T., et
al. 2005. Myc represses transcription through recruitment of
DNA methyltransferase corepressor. EMBO J. 24: 336–346.
Burkly, L., Hession, C., Ogata, L., Reilly, C., Marconi, L.A.,
Olson, D., Tizard, R., Cate, R., and Lo, D. 1995. Expression
of relB is required for the development of thymic medulla
and dendritic cells. Nature 373: 531–536.
Cameron, E.E., Bachman, K.E., Myohanen, S., Herman, J.G., and
Baylin, S.B. 1999. Synergy of demethylation and histone
deacetylase inhibition in the re-expression of genes silenced
in cancer. Nat. Genet. 21: 103–107.
Campbell, K.J., Rocha, S., and Perkins, N.D. 2004. Active re-
pression of antiapoptotic gene expression by RelA(p65) NF-
?B. Mol. Cell 13: 853–865.
Carvin, C.D., Parr, R.D., and Kladde, M.P. 2003. Site-selective
in vivo targeting of cytosine-5 DNA methylation by zinc-
finger proteins. Nucleic Acids Res. 31: 6493–6501.
Chen, L.Y. and Chen, J.D. 2003. Daxx silencing sensitizes cells
to multiple apoptotic pathways. Mol. Cell. Biol. 23: 7108–
Croxton, R., Puto, L.A., de Belle, I., Thomas, M., Torii, S., Ha-
naii, F., Cuddy, M., and Reed, J.C. 2006. Daxx represses ex-
pression of a subset of antiapoptotic genes regulated by
nuclear factor-?B. Cancer Res. 66: 9026–9035.
D’Alessio, A.C. and Szyf, M. 2006. Epigenetic tete-a-tete: The
bilateral relationship between chromatin modifications and
DNA methylation. Biochem. Cell Biol. 84: 463–476.
Di Croce, L., Raker, V.A., Corsaro, M., Fazi, F., Fanelli, M.,
Puto and Reed
1008GENES & DEVELOPMENT
Faretta, M., Fuks, F., Lo Coco, F., Kouzarides, T., Nervi, C.,
et al. 2002. Methyltransferase recruitment and DNA hyper-
methylation of target promoters by an oncogenic transcrip-
tion factor. Science 295: 1079–1082.
Dong, C. and Goldschmidt-Clermont, P.J. 2002. E2F1: A magic
bullet for atherosclerosis? Circulation 106: 2640–2641.
Ecsedy, J.A., Michaelson, J.S., and Leder, P. 2003. Homeodo-
main-interacting protein kinase 1 modulates Daxx localiza-
tion, phosphorylation, and transcriptional activity. Mol.
Cell. Biol. 23: 950–960.
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.
Fraga, M.F. and Esteller, M. 2007. Epigenetics and aging: The
targets and the marks. Trends Genet. 23: 413–418.
Frigola, J., Song, J., Stirzaker, C., Hinshelwood, R.A., Peinado,
M.A., and Clark, S.J. 2006. Epigenetic remodeling in colo-
rectal cancer results in coordinate gene suppression across
an entire chromosome band. Nat. Genet. 38: 540–549.
Fuks, F., Burgers, W.A., Godin, N., Kasai, M., and Kouzarides, T.
2001. Dnmt3a binds deacetylases and is recruited by a se-
quence-specific repressor to silence transcription. EMBO J.
Gore, S.D. 2005. Combination therapy with DNA methyl-
transferase inhibitors in hematologic malignancies. Nat.
Clin. Pract. Oncol. 2 (Suppl. 1): S30–S35. doi: 10.1038/
Hinnis, A.R., Luckett, J.C., and Walker, R.A. 2007. Survivin is
an independent predictor of short-term survival in poor prog-
nostic breast cancer patients. Br. J. Cancer 96: 639–645.
Hoffmann, A. and Baltimore, D. 2006. Circuitry of nuclear fac-
tor ?B signaling. Immunol. Rev. 210: 171–186.
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 as-
sociate with chromatin through an interaction with core his-
tones and the chromatin-associated protein Dek. J. Cell Sci.
Hsieh, C.L. 1994. Dependence of transcriptional repression on
CpG methylation density. Mol. Cell. Biol. 14: 5487–5494.
Jacque, E., Tchenio, T., Piton, G., Romeo, P.H., and Baud, V.
2005. RelA repression of RelB activity induces selective gene
activation downstream of TNF receptors. Proc. Natl. Acad.
Sci. 102: 14635–14640.
Kawai, T., Akira, S., and Reed, J.C. 2003. ZIP kinase triggers
apoptosis from nuclear PML oncogenic domains (PODs).
Mol. Cell. Biol. 23: 6174–6186.
Khelifi, A.F., D’Alcontres, M.S., and Salomoni, P. 2005. Daxx is
required for stress-induced cell death and JNK activation.
Cell Death Differ. 12: 724–733.
Kim, E.J., Park, J.S., and Um, S.J. 2003. Identification of Daxx
interacting with p73, one of the p53 family, and its regula-
tion of p53 activity by competitive interaction with PML.
Nucleic Acids Res. 31: 5356–5367.
Li, R., Pei, H., Watson, D.K., and Papas, T.S. 2000. EAP1/Daxx
interacts with ETS1 and represses transcriptional activation
of ETS1 target genes. Oncogene 19: 745–753.
Lin, D.Y., Lai, M.Z., Ann, D.K., and Shih, H.M. 2003. Promy-
elocytic leukemia protein (PML) functions as a glucocorti-
coid receptor co-activator by sequestering Daxx to the PML
oncogenic domains (PODs) to enhance its transactivation
potential. J. Biol. Chem. 278: 15958–15965.
Lindsay, C.R., Scholz, A., Morozov, V.M., and Ishov, A.M. 2007.
Daxx shortens mitotic arrest caused by paclitaxel. Cell
Cycle 6: 1200–1204.
Marienfeld, R., May, M.J., Berberich, I., Serfling, E., Ghosh, S.,
and Neumann, M. 2003. RelB forms transcriptionally inac-
tive complexes with RelA/p65. J. Biol. Chem. 278: 19852–
Mathur, M., Tucker, P.W., and Samuels, H.H. 2001. PSF is a
novel corepressor that mediates its effect through Sin3A and
the DNA binding domain of nuclear hormone receptors.
Mol. Cell. Biol. 21: 2298–2311.
Maul, G.G., Negorev, D., Bell, P., and Ishov, A.M. 2000. Review:
Properties and assembly mechanisms of ND10, PML bodies,
or PODs. J. Struct. Biol. 129: 278–287.
Michaelson, J.S. 2000. The Daxx enigma. Apoptosis 5: 217–220.
Michaelson, J.S., Bader, D., Kuo, F., Kozak, C., and Leder, P.
1999. Loss of Daxx, a promiscuously interactive protein, re-
sults in extensive apoptosis in early mouse development.
Genes & Dev. 13: 1918–1923.
Muromoto, R., Sugiyama, K., Takachi, A., Imoto, S., Sato, N.,
Yamamoto, T., Oritani, K., Shimoda, K., and Matsuda, T.
2004. Physical and functional interactions between Daxx
and DNA methyltransferase 1-associated protein, DMAP1. J.
Immunol. 172: 2985–2993.
Perkins, N.D. and Gilmore, T.D. 2006. Good cop, bad cop: The
different faces of NF-?B. Cell Death Differ. 13: 759–772.
Raval, A., Tanner, S.M., Byrd, J.C., Angerman, E.B., Perko, J.D.,
Chen, S.S., Hackanson, B., Grever, M.R., Lucas, D.M., Mat-
kovic, J.J., et al. 2007. Downregulation of death-associated
protein kinase 1 (DAPK1) in chronic lymphocytic leukemia.
Cell 129: 879–890.
Robertson, K.D., Hayward, S.D., Ling, P.D., Samid, D., and
Ambinder, R.F. 1995. Transcriptional activation of the Ep-
stein-Barr virus latency C promoter after 5-azacytidine treat-
ment: Evidence that demethylation at a single CpG site is
crucial. Mol. Cell. Biol. 15: 6150–6159.
Ruben, S.M., Klement, J.F., Coleman, T.A., Maher, M., Chen,
C.H., and Rosen, C.A. 1992. I-Rel: A novel rel-related protein
that inhibits NF-?B transcriptional activity. Genes & Dev. 6:
Ryseck, R.P., Bull, P., Takamiya, M., Bours, V., Siebenlist, U.,
Dobrzanski, P., and Bravo, R. 1992. RelB, a new Rel family
transcription activator that can interact with p50–NF-?B.
Mol. Cell. Biol. 12: 674–684.
Salomoni, P. and Khelifi, A.F. 2006. Daxx: Death or survival
protein? Trends Cell Biol. 16: 97–104.
Schneider, G., Saur, D., Siveke, J.T., Fritsch, R., Greten, F.R.,
and Schmid, R.M. 2006. IKK? controls p52/RelB at the skp2
gene promoter to regulate G1- to S-phase progression. EMBO
J. 25: 3801–3812.
Shamay, M., Krithivas, A., Zhang, J., and Hayward, S.D. 2006.
Recruitment of the de novo DNA methyltransferase
Dnmt3a by Kaposi’s sarcoma-associated herpesvirus LANA.
Proc. Natl. Acad. Sci. 103: 14554–14559.
Takahashi, Y., Lallemand-Breitenbach, V., Zhu, J., and De The,
H. 2004. PML nuclear bodies and apoptosis. Oncogene 23:
Tang, J., Wu, S., Liu, H., Stratt, R., Barak, O.G., Shiekhattar, R.,
Picketts, D.J., and Yang, X. 2004. A novel transcription regu-
latory complex containing Daxx and the ATR-X syndrome
protein. J. Biol. Chem. 279: 20369–20377.
Torii, S., Egan, D.A., Evans, R.A., and Reed, J.C. 1999. Human
Daxx regulates Fas-induced apoptosis from nuclear PML on-
cogenic domains (PODs). EMBO J. 18: 6037–6049.
Tzeng, S.L., Cheng, Y.W., Li, C.H., Lin, Y.S., Hsu, H.C., and
Kang, J.J. 2006. Physiological and functional interactions be-
tween TCF4 and Daxx in colon cancer cells. J. Biol. Chem.
Daxx methylates promoters via Dnmt1
GENES & DEVELOPMENT1009
Valnet-Rabier, M.B., Challier, B., Thiebault, S., Angonin, R.,
Margueritte, G., Mougin, C., Kantelip, B., Deconinck, E.,
Cahn, J.-Y., Fest, T. 2005. c-Flip protein expression in Bur-
kitt’s lymphomas is associated with a poor clinical outcome.
Br. J. Haematol. 128: 767–773.
Varfolomeev, E., Blankenship, J.W., Wayson, S.M., Fedorova,
A.V., Kayagaki, N., Garg, P., Zobel, K., Dynek, J.N., Elliott,
L.O., Wallweber, H.J., et al. 2007. IAP antagonists induce
autoubiquitination of c-IAPs, NF-?B activation, and TNF?-
dependent apoptosis. Cell 131: 669–681.
Viiri, K.M., Korkeamäki, H., Kukkonen, M.K., Nieminen, L.K.,
Lindfors, K., Peterson, P., Mäki, M., Kainulainen, H., and
Lohi, O. 2006. SAP30L interacts with members of the Sin3A
corepressor complex and targets Sin3A to the nucleolus.
Nucleic Acids Res. 34: 3288–3298.
Xue, Y., Gibbons, R., Yan, Z., Yang, D., McDowell, T.L., Sechi,
S., Qin, J., Zhou, S., Higgs, D., and Wang, W. 2003. The
ATRX syndrome protein forms a chromatin-remodeling
complex with Daxx and localizes in promyelocytic leukemia
nuclear bodies. Proc. Natl. Acad. Sci. 100: 10635–10640.
Puto and Reed
1010 GENES & DEVELOPMENT