Acetylation of Stat1 modulates
Oliver H. Krämer,1,4Daniela Baus,1,3Shirley K. Knauer,1,3Stefan Stein,1Elke Jäger,2
Roland H. Stauber,1Manuel Grez,1Edith Pfitzner,1and Thorsten Heinzel1,4,5
1Georg-Speyer-Haus, D-60596 Frankfurt, Germany;2Medizinische Klinik II-Onkologie, Krankenhaus Nordwest,
D-60488 Frankfurt, Germany
Acetylation of signaling molecules can lead to apoptosis or differentiation of carcinoma cells. The molecular
mechanisms underlying these processes and the biological role of enzymes mediating the transfer or removal
of an acetyl-group are currently under intense investigation. Our study shows that Stat1 is an acetylated
protein. Stat1 acetylation depends on the balance between Stat1-associated histone deacetylases (HDACs) and
histone acetyltransferases (HATs) such as CBP. Remarkably both inhibitors of HDACs and the cytokine
interferon ? alter this equilibrium and induce Stat1 acetylation. The analysis of Stat1 mutants reveals Lys 410
and Lys 413 as acetylation sites. Experiments with Stat1 mutants mimicking either constitutively acetylated
or nonacetylated states show that only acetylated Stat1 is able to interact with NF-?B p65. As a consequence,
p65 DNA binding, nuclear localization, and expression of anti-apoptotic NF-?B target genes decrease. These
findings show how the acetylation of Stat1 regulates NF-?B activity and thus ultimately apoptosis.
[Keywords: Stat1; NF-?B; acetylation; histone deacetylase; Interferon ?; HDAC inhibitor]
Received August 26, 2005; revised version accepted December 19, 2005.
Many signal transduction pathways ultimately result in
the post-translational modification of histones, which
determines the expression of genes important for cell
growth, differentiation, and apoptosis (Wolffe and Hayes
1999; Schreiber and Bernstein 2002). Acetylation of the
N-terminal tails of histones correlates with gene activa-
tion, while histone deacetylation mediates transcrip-
tional repression (Strahl and Allis 2000). It has also be-
come clear that regulated acetylation of nonhistone pro-
teins determines cellular fate and survival (Blobel 2000;
Kouzarides 2000; Cohen et al. 2004). The fine-tuned
equilibrium of protein acetylation and deacetylation is
maintained by histone acetyltransferases (HATs) and
histone deacetylases (HDACs) (Kouzarides 1999). This
constitutes a precise and dynamic regulatory system
modulating protein assemblies in response to external
and internal signals. Protein acetylation therefore ap-
pears to play a role that is comparable to phosphoryla-
tion (Schreiber and Bernstein 2002).
HDAC inhibitors (HDACi) have been shown to change
the expression pattern of genes involved in differentia-
tion, cell cycle arrest, and apoptosis, and they are con-
sidered as candidate drugs for cancer therapy (Krämer et
al. 2001; Kelly et al. 2002; Melnick and Licht 2002).
However, the molecular mechanisms underlying cell-
specific modulation of signaling pathways by HDACi
and factors determining sensitivity toward these com-
pounds are still subject to intense investigation (Mayo et
al. 2003). Several recent reports suggest that HDACi-in-
duced apoptosis depends on the expression of Jun, Bcl-2
proteins, p21WAF/CIP1, p53, NF-?B, and Akt (Vrana et al.
1999; Henderson et al. 2003; Mayo et al. 2003). For some
of these proteins the association with HDACs and/or
acetylation of lysine residues has been shown (Kouza-
rides 2000; Chen and Greene 2003; Kiernan et al. 2003;
Weiss et al. 2003).
In the case of NF-?B, which contributes significantly
to anti-apoptotic signaling (Perkins 2004), HDACi were
shown to repress NF-?B signaling and expression of sev-
eral NF-?B target genes (Huang et al. 1997; Inan et al.
2000; Krämer et al. 2001). Given the critical role of NF-
?B in tumorigenesis, several studies were undertaken to
identify factors influencing this tumor promoter (Per-
kins 2004). It became clear that NF-?B signaling is con-
trolled at several levels by regulatory proteins, such as
the I-?B protein family. Furthermore, Stat1 has been sug-
gested to repress NF-?B-mediated signaling (Wang et al.
2000; Suk et al. 2001; Shen and Lentsch 2004), and sev-
eral stimuli induce apoptosis to a significantly greater
extent in a Stat1-positive cellular background (Kumar et
al. 1997; Meyer et al. 2002). Similar to NF-?B, Stat1 as-
3These authors contributed equally to this work.
4Present address: Institute of Biochemistry and Biophysics, Friedrich-
Schiller-University of Jena, D-07743 Jena, Germany
E-MAIL T.Heinzel@uni-jena.de; FAX 49-3641-949352.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/
GENES & DEVELOPMENT 20:473–485 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org 473
sociates with HATs and HDACs (Korzus et al. 1998;
Nusinzon and Horvath 2003). Stat1 regulates the expres-
sion of gene products mediating various cellular pro-
cesses constitutively or inducibly by growth factors and
cytokines such as interferons in a phosphorylation-de-
pendent manner (Chatterjee-Kishore et al. 2000; Ihle
2001). Induction of interferon signaling has been shown
to activate the apoptotic program, which indicates that
these cytokines could be useful in cancer therapy (de
Vries et al. 2003). Remarkably, tyrosine phosphorylation
of Stat1 and its transcriptional activity appear to be dis-
pensable for NF-?B inhibition and apoptosis induction in
response to certain stimuli (Wang et al. 2000; Meyer et
al. 2002). However, it is still unclear which other post-
translational modifications are involved in this process
and whether conditions exist in which Stat1 and NF-?B
can interact with each other.
We investigated the molecular mechanisms underly-
ing the induction of apoptosis and the modulation of
signaling pathways by HDACi and interferon ? in hu-
man melanoma cell lines. Our results show that cells
undergoing apoptosis in response to such substances in-
crease expression and acetylation of Stat1. This leads to
altered interactions of Stat1 with HDACs, CBP, and NF-
?B. Our results indicate that the acetylation of Stat1
functions as a molecular switch, which permits binding
of Stat1 to NF-?B and thus reduces NF-?B signaling. This
mechanism appears to be critical for the induction of cell
death by pharmacological and physiological stimuli.
Response of human melanoma cells to HDACi
HDACi can induce growth arrest and apoptosis in tumor
cells of different origin (Krämer et al. 2001; Kelly et al.
2002). We found significant differences in the sensitivity
of various melanoma cell lines toward these compounds.
SK-37 cells showed strong growth reduction in the MTT
assay upon HDACi treatment, whereas NW-1539 cells
were not affected significantly at the inhibitor concen-
trations used (Fig. 1A). These cell lines are prototypical
examples and have characteristics similar to other mela-
noma cell lines that are either HDACi sensitive (e.g.,
MZ-19) or resistant (e.g., NW-450).
To investigate the molecular mechanisms underlying
this differential response, we evaluated whether the re-
duced proliferation due to HDACi relies on proapoptotic
properties and effects on caspases (Thornberry and Lazeb-
nik 1998). In the HDACi-sensitive SK-37 cell line, we
detected activation of the initiator caspases 8 and 9 after
MTT test after exposure to VPA (0.5–5 mM) or TSA (100 nM) for 48 h (SK-37) or 72 h (NW-1539); (0) untreated cells. (B) Induction of
activated caspase 9 (Casp 9, activation denoted by an asterisk) and cleavage of full-length caspase 8 (Casp 8 fl) into the active subunits
p43/41/18 were detected by Western blot after treatment of SK-37 cells with VPA (V, 1.5 mM) or TSA (T, 100 nM) for 48 h. Caspase
3 activity was measured by conversion of Ac-DEVD-pNA to pNA, which has an absorption peak at 405 nm. This increase is given
relative to the activity of lysates from untreated cells (Ctl). HDACi-induced conversion of full-length caspase 3 (Casp 3 fl) to the active
p17/19 subunits was analyzed in SK-37 and NW-450 cells. (C) Proteolytic cleavage of PARP and apoptotic chromatin fragmentation
induced by VPA (1.5 mM) or TSA (100 nM) after 48 h were detected by Western blot and PI FACS analysis. Cotreatment of SK-37 cells
with Z-VAD-FMK (Z, 100 µM) blocks HDACi-induced apoptosis.
HDACi induce apoptosis in SK-37 cells. (A) The proliferation of SK-37 and NW-1539 melanoma cells was determined by
Krämer et al.
474GENES & DEVELOPMENT
treatment (Fig. 1B). Furthermore, we measured caspase 3
activity in extracts from these cells by colorimetric as-
say. Conversion of the proenzyme form of the execu-
tioner caspase 3 (p32) to the catalytically active proteases
p17 and p19 was detectable in SK-37 but not in HDACi-
resistant NW-450 cells (Fig. 1B).
Activation of caspase 3 during HDACi-mediated apo-
ptosis of SK-37 cells was also verified by examining the
cleavage of PARP (116 kDa) into 85- and 28-kDa frag-
ments (Thornberry and Lazebnik 1998). The pan-caspase
inhibitor Z-VAD-FMK inhibited PARP cleavage as well
as the occurrence of a hypodiploid (sub-G1) fraction re-
sulting from DNA fragmentation in SK-37 cells (Fig. 1C).
Analysis of nuclei stained with Hoechst dye gave similar
results (data not shown). These observations confirm
that HDACi trigger apoptotic, caspase-dependent path-
ways in SK-37 melanoma cells (Fig. 1C). Furthermore, no
signs of nonspecific cell permeabilization and necrotic
cell death were found in a PI/Hoechst staining assay
(data not shown).
Alteration of Stat1 gene expression after HDAC
We employed microarray and Western blot analyses to
define alterations in gene expression patterns after incu-
bation with HDACi. These assays revealed a time- and
dose-dependent increase in Stat1 expression at the
mRNA and protein level in SK-37 (Fig. 2A) and several
other HDACi-sensitive cell lines. Treatment of SK-37
cells with HDACi and cycloheximide showed that the
HDACi-induced increase in Stat1 expression was depen-
dent on de novo protein synthesis (data not shown).
Hence, an increase in Stat1 stability due to reduced
HDAC activity cannot account for higher Stat1 expres-
sion levels. Intriguingly, HDACi-resistant cell lines,
such as NW-450 and NW-1539, did not undergo HDACi-
induced caspase 3 cleavage and apoptosis (Fig. 1A,B) and
expressed very low levels of Stat1, which were not in-
duced by HDACi (Fig. 2B,C). Since no significant differ-
ence in HDACi-induced histone hyperacetylation was
detected between NW-1539 and SK-37 cells, HDACi
were equally effective in blocking HDAC activity in
both cell lines (Fig. 2C). This result indicates that not
only inhibition of HDACs but also the presence of Stat1
is crucial for HDACi-mediated apoptosis in melanoma
cells. Consistent with previous reports (Wong et al.
2002), we detected a strong increase in Stat1 expression
in the Stat1-positive SK-37 cell line, but not in NW-1539
cells treated with interferon ?. Moreover, cotreatment
with HDACi further increased Stat1 expression in SK-37
cells (data not shown). This correlated with enhanced
induction of apoptosis as assessed by MTT and FACS
analysis (Fig. 2D).
These results prompted us to investigate whether
Stat1? is required for HDACi-induced apoptosis in NW-
1539 cells. We transduced these cells with a lentiviral
vector expressing Stat1?. Indeed, sensitivity toward
HDACi was conferred to Stat1?-transduced NW-1539
cells but not to cells which received only the vector en-
coding GFP (Fig. 3A,B; data not shown). Hence, Stat1
expression levels appear to determine the response of
this cell line to HDACi. Furthermore, introduction of
Stat1? renders these cells susceptible to enhanced apo-
ptosis induction by VPA and interferon ? (Fig. 3B). How-
ever, interferon ? alone did not induce apoptosis and
consistently did not induce Stat1 in this cell line. Similar
results were obtained with the Stat1-negative cell line
U3A (Müller et al. 1993) reconstituted with Stat1?, al-
beit with less pronounced apoptosis induction (data not
Our results clearly show that HDACi induce Stat1 in
a cell type-specific manner. However, microarray analy-
ses gave no evidence for increased expression of Stat1
target genes as a result of HDAC inhibition in SK-37
cells. Therefore, the activity of Stat1 in HDACi-induced
tion. (A) The time- and dose-dependent increase of Stat1 expres-
sion was investigated by Western blot. SK-37 cells were exposed
to 1.5 mM VPA or 100 nM TSA for the indicated periods of time.
Alternatively, cells were treated for 24 h with different concen-
trations of VPA (0.1–1.5 mM) or TSA (10–300 nM) as indicated
or left untreated (0). (B) Expression of Stat1? in SK-37 and NW-
450 melanoma cells treated with 1.5 mM VPA (V) or 100 nM
TSA (T) for 24 h or left untreated (C) was analyzed by Western
blot. (C) Expression of Stat1 and accumulation of hyperacety-
lated histone H4 (AcH4) in SK-37 and NW-1539 cells were ana-
lyzed after 24 h by Western blot. Cells were treated with VPA
(V, 1.5 mM) or TSA (T, 30 nM) or left untreated (C). (D) Sensi-
tivity of melanoma cell lines to VPA (V, 1.5 mM) and interferon
? (?, 103U/mL) was determined by MTT assay. Enhanced in-
duction of apoptosis after treatment of SK-37 cells with VPA
and interferon ? (?/V) was detected by PI FACS analysis.
Correlation of Stat1 expression and apoptosis induc-
Acetylation of Stat1
GENES & DEVELOPMENT 475
apoptosis is likely to involve nongenomic effects, such
as cross-talk with other signaling pathways.
HDACi modulate NF-?B activity
To identify such signaling pathways, gene expression
analyses can provide valuable information. Since a criti-
cal role of NF-?B for HDACi-induced apoptosis has been
described, we analyzed the expression of NF-?B-depen-
dent genes in the HDACi-sensitive cell line SK-37. Our
data indicate HDACi-dependent repression of NF-?B tar-
get genes such as Bcl-XL, survivin, and Stat5 (Fig. 4A).
These results confirm several reports describing effects
of HDACi on these genes (Eickhoff et al. 2000; Krämer et
al. 2001; Hinz et al. 2002; De Schepper et al. 2003). On
the other hand, expression of NF-?B-regulated genes re-
mained unaltered in HDACi-resistant NW-1539 cells,
which expressed hardly any Stat1 (Fig. 4A). Treatment
with the cytokine interferon ? similarly led to repression
of these genes in SK37 but not in NW-1539 cells (Fig.
Data shown in Figures 2C and 4A indicate that Stat1?
expression inversely correlates with the activity of NF-
?B after HDACi treatment or interferon ? stimulation.
Therefore, we employed a binding assay with a biotinyl-
ated NF-?B consensus site oligonucleotide to investigate
whether DNA binding of NF-?B might be affected in
cells expressing different amounts of Stat1. Decreased
affinity of NF-?B for a cognate DNA sequence was ob-
served after treatment with HDACi or interferon ? only
in cells expressing Stat1 (Fig. 4B). EMSAs confirmed
functional impairment of NF-?B p65/p50 binding to its
DNA sequence in extracts from HDACi-treated Stat1-
positive SK-37 cells (Fig. 4C, left). EMSAs performed
with the same lysates and an oligonucleotide specific for
Stat3 showed no decrease in DNA binding, which rules
out nonspecific effects of HDACi treatment on the DNA
binding of transcription factors (data not shown). Re-
duced affinity of NF-?B for DNA after HDAC inhibition
was also observed with Stat1?-transduced NW-1539
cells, but not in parental or vector-transduced Stat1-
negative NW-1539 cells (Fig. 4C, cf. lanes 5,9,13 and
6,10,14). Hence, DNA binding of NF-?B after treatment
with HDACi is only reduced in the presence of Stat1?,
suggesting a link of both signaling pathways.
We challenged the proposed role of Stat1 in regulating
NF-?B function by transduction of SK-37 cells with len-
tiviral vectors encoding small interfering RNA (siRNA)
directed against Stat1 or a nonrelevant sequence. We ob-
served that basal and induced Stat1 levels were reduced
only in cells that received the siStat1 vector. In control-
transduced SK-37 cells, HDACi or interferon ? still de-
creased the expression of NF-?B target genes as well as
NF-?B DNA binding. However, NF-?B functions were no
longer affected under identical conditions in SK-37 cells
subjected to a constitutive siRNA-mediated knock-
down of Stat1 levels (Fig. 4D,E). In a rescue experiment
we transfected a mutated siRNA-resistant Stat1 vector
into the Stat1 knock-down SK-37 cells. Consequently, a
strong repression of NF-?B functions by HDACi and in-
terferon ? could again be induced. Moreover, high levels
of Stat1 expression caused a reduction of NF-?B activity
independent of these substances. These data demon-
strate that Stat1 is a crucial regulator of NF-?B in vivo
The localization of nuclear p65 is modulated by Stat1
We gained further insights into the mechanism underly-
ing the regulation of NF-?B in SK-37 cells by analyzing
its subcellular distribution. In situ immunofluorescence
analysis showed a shift of NF-?B p65 from the nucleus to
the cytosol and an increased colocalization with Stat1?
in the cytosol when HDACs were inhibited or cells were
treated with interferon ? (Fig. 5A; data not shown).
Treatment of cells with the nuclear export inhibitor
LMB prevented basal and HDACi-induced export of NF-
?B p65 and caused nuclear accumulation of Stat1? and
p65. Again, colocalization of these proteins was en-
hanced (Fig. 5A).
and interferon ?. (A) Western blot analysis was employed to
detect Stat1 expression and induction of apoptosis in NW-1539
cells transduced with SIEW (vector) or S-Stat1?-IEW (Stat1) and
treated with VPA (1.5 mM) for 48 h. Asterisks denote activated
forms of full-length caspase 3 or caspase 8. (B) DNA fragmen-
tation was analyzed by PI FACS analysis after treatment with
VPA (1.5 mM) or VPA and interferon ? (103U/mL) for 60 h. (Ctl)
Stat1 sensitizes resistant melanoma cells to HDACi
Krämer et al.
476GENES & DEVELOPMENT
The analysis of cytosolic and nuclear fractions of SK-
37 cells by Western blot indicates that Stat1 expression
increased both in the cytosol and in the nucleus (Fig. 5B).
In these cells, as in many other cancer cell lines, low
constitutive amounts of NF-?B p65 reside in the nucleus
and help to attenuate proapoptotic signaling. For p65 a
clear reduction in the nuclear compartment was ob-
served after treatment with HDACi as well as interferon
?, which confirms our microscopy results. These data
suggest that removal of p65 from the nucleus and inter-
action with Stat1 might be key steps in the HDACi-
induced repression of NF-?B target genes and apoptosis
induction. Another possible explanation, changes in NF-
?B p65 expression, can be ruled out since p65 levels were
not altered significantly (Fig. 5B). Western blot analysis
of 2fTGH cells and their derived Stat1-negative cell line
U3A confirmed the dependence on Stat1 for nuclear ex-
port of p65 upon HDAC inhibition (Fig. 5C). Hence, it
appears plausible that HDACi and interferon ? inhibit
nuclear localization of p65 only in cells expressing Stat1.
HDACi induce the interaction of Stat1? and NF-?B
Having established a role of Stat1? in NF-?B signaling,
we investigated whether these transcription factors
could interact physically. First, we tested if this interac-
tion is mediated by TRADD, which was shown to asso-
ciate with Stat1? under certain conditions (Wang et al.
2000). However, in several immunoprecipitation (IP) ex-
periments, we could not detect an HDACi-dependent in-
teraction of Stat1? with TRADD in SK-37 cells (data not
shown). On the other hand, precipitation of Stat1? or
NF-?B p65 revealed an association of these proteins upon
HDAC inhibition or interferon stimulation (Fig. 5D). Co-
with cytosolic and nuclear fractions confirmed our mi-
croscopy data showing that such complexes are located
in the cytosol. Moreover, Stat1–NF-?B complex forma-
tion is likely to be DNA independent, since addition of
ethidium bromide did not alter its stability (data not
shown). Next, we analyzed Stat1 complexes by Superose
6 column fractionation of SK-37 cell extracts and found
that in high molecular weight fractions the amount of
Stat1 increased together with NF-?B p65 after VPA treat-
ment (Fig. 5E). Notably, this complex accumulated in a
time-dependent manner that paralleled apoptosis induc-
tion after HDAC inhibition. IP of Stat1? from these frac-
tions followed by Western blotting against p65 showed
that a weak basal interaction of these proteins increased
upon HDAC inhibition (Fig. 5E).
We also analyzed the presence of HDACs in the Stat1
complex before and after HDAC inhibition or treatment
with interferon ? by IP and Western blot. The addition of
HDACi to SK-37 cells led to reduced association of Stat1
Expression of NF-?B target genes after HDAC inhi-
bition and treatment with interferon ? was investi-
gated by Western blot analysis of SK-37 and NW-
1539 cell lysates. Cells were incubated with 1.5 mM
VPA (V), 30 nM TSA (T), or 103U interferon ? (IFN),
or left untreated (C) for 48 h. (B) ABCD-assay with a
biotinylated NF-?B consensus oligo was used to de-
tect NF-?B–DNA binding under conditions de-
scribed in A. (N) Nonrelevant biotinylated oligos.
(C) NF-?B DNA binding was analyzed by EMSA of
lysates from SK-37, NW-1539, and transduced NW-
1539 cells (vector or Stat1) that were either un-
treated or treated with VPA (1.5 mM) for 48 h. Iden-
tity of the NF-?B–DNA complex was verified by p65
and p50 antibody supershifts (SS-AB). (D) NF-?B tar-
get gene expression was investigated in lysates of
SK-37 cells transduced with an siRNA vector encod-
ing scrambled RNA or an si-sequence against Stat1.
Experimental conditions are as described in A. (E)
NF-?B–DNA binding was investigated in lysates of
SK-37 cells by ABCD-assay. Cells and conditions are
as described in A. (F) NF-?B target gene expression
was investigated in lysates of SK-37 cells transduced
as stated in D and transfected with empty vector or
pc3 HA-Stat1 containing mutations conferring resis-
tance against the siRNA. Experimental conditions
are as described in A. (G) NF-?B–DNA binding was
investigated in lysates of SK-37 cells by ABCD-as-
say. Cells and conditions are as described in E.
Stat1 interferes with NF-?B function. (A)
Acetylation of Stat1
GENES & DEVELOPMENT 477
with HDAC1 and HDAC3 (Fig. 5F). No binding of Stat1
to other class I HDACs (2 and 8) was observed (data not
shown). Superose 6 fractionation substantiated these re-
sults and also revealed decreased comigration of Stat1?
complexes with the corepressor mSin3 (Fig. 5E). Our ob-
servations not only confirm that Stat1 can interact with
repressive cofactors but also indicate that HDACs disso-
ciate upon interferon ? or HDACi treatment.
Acetylation of Stat1
Recent publications show that the acetylation of pro-
teins regulates multiple cellular processes (Kouzarides
2000; Cohen et al. 2004). We therefore tested whether
Stat1 undergoes acetylation. Stat1? was immunoprecipi-
tated from SK-37 whole-cell extracts with a monoclonal
antibody under stringent lysis conditions in RIPA buffer.
A pan-acetyl-lysine antibody recognized a band corre-
sponding to the molecular weight of Stat1? (Fig. 6A).
Reprobing with the monoclonal Stat1? antibody con-
firmed the acetylation signal as Stat1?. As expected, the
basal acetylation level of endogenous Stat1? was in-
creased after HDAC inhibition. Remarkably, long-term
stimulation with interferon ? also enhanced Stat1 acety-
lation (Fig. 6A). To obtain further evidence for the acety-
lation of Stat1, an anti-acetyl-lysine antibody was used
for IP and Western blots were probed with an antibody
against Stat1. In this experiment, the pan-acetyl-lysine
antibody precipitated Stat1. Again, the acetylation level
of Stat1 increased after HDACi or interferon ? treatment
and allowed recovery of increasing amounts of Stat1
from treated cells (Fig. 6A, right panel). These findings
indicate that endogenous Stat1 is acetylated in vivo and
that this modification can be increased by HDACi or
interferon ? (Fig. 6A). No acetylation of endogenous NF-
?B p65 was detectable under these conditions, and p65
could not be precipitated with the pan-acetyl-lysine an-
tibody (Fig. 6A; data not shown). This is consistent with
reports stating the need for overexpression of a HAT to
detect NF-?B acetylation in vivo (Chen and Greene
Transfection experiments with 293T cells revealed
that increased expression of CBP, though not of p300,
enhances Stat1 acetylation (Fig. 6B, left panel; data not
Stat1? and NF-?B p65. (A) Interaction and colocaliza-
tion of Stat1? and NF-?B p65 in SK-37 cells treated with
VPA (1.5 mM, 24 h) and/or LMB (10 nM) were analyzed
by immunofluorescence microscopy. (Ctl) Untreated.
(B) Exclusion of p65 from the nuclear compartment af-
ter treatment of SK-37 cells with VPA (V, 1.5 mM), TSA
(T, 100 nM), or interferon ? (?, 103U) for 24 h was
confirmed by cellular fractionation and p65 Western
blot. (C) Untreated. Reprobing was done with antibod-
ies against Stat1. The affinity of the Stat1? antibody is
not sufficient to detect nuclear Stat1. All other proteins
detected serve as loading and fractionation controls. (C)
U3A and 2fTGH cells were analyzed for cytoplasmic
retention of p65 after incubation with 1.5 mM VPA (V)
for 24 h by Western blot of cytosolic and nuclear frac-
tions. (D) Interaction of Stat1? and NF-?B p65 in SK-37
cell lysates was investigated by Western blot of specific
IPs as described in B (IP, Pre [preimmune serum], Input).
(E) The composition of Stat1 complexes in SK-37 cells
after treatment with VPA (1.5 mM, 48 h) was investi-
gated by Western blot analysis of Superose 6 column
fractions. (Lower panel) IP was used to verify the inter-
action of Stat1? with NF-?B. (F) HDAC1 and HDAC3
were precipitated from whole-cell extracts (IP). Western
blot analysis was performed with an antibody against
Stat1?/?. Treatment conditions are as described in D.
Complex formation and localization of
Krämer et al.
478 GENES & DEVELOPMENT
shown). Moreover, we were able to acetylate Stat1? in
vitro using immunoprecipitated CBP as an acetyltrans-
ferase (Fig. 6B, right panel). Ectopically expressed Stat1
was also found to be acetylated upon cotransfection of
CBP (Fig. 6C). Considering that CBP can mediate the
transfer of an acetyl-group to Stat1, we analyzed poten-
tial interactions of these proteins by microscopy and IP
analysis. In untreated cells, CBP resided in the nucleus.
However, upon treatment with HDACi and especially
with interferon ?, CBP translocated from the nucleus to
the cytosol and displayed increased association with
Stat1 (Fig. 6C).
A deletion mutant of Stat1 (?XbaI) lacking the C ter-
minus including the Ser727phosphorylation site retained
acetylation, as indicated by a corresponding smaller band
detected with an anti-acetyl-lysine antibody (Fig. 6D).
Hence, Stat1 serine phosphorylation (Ihle 2001), which
can be induced by VPA (Gurvich et al. 2004), is not criti-
cal for Stat1 acetylation. Considering that the Stat1
?XbaI mutant resembles Stat1? and the fact that
or left untreated for 48 h. Endogenous Stat1? was immunoprecipitated from RIPA lysates and analyzed by Western blot with an
antibody recognizing acetylated lysines (anti-AcLys, left). Reprobing of the same membrane confirms that the acetylation signal
corresponds to Stat1? and shows efficacy and specificity of the IP. Anti-AcLys immunoprecipitates from RIPA lysates were probed
with antibodies recognizing Stat1?/? or NF-?B p65. Preimmune serum was used as a control. Input lanes show 2% of the extract used
for IP. (B) Increasing amounts of a CBP expression vector (1, 5, or 10 µg) were transfected into 293T cells. Stat1? was precipitated from
RIPA lysates and probed with anti-AcLys. IPs with preimmune serum and IPs from cells transfected with the empty vector pc3.1 (10
µg) are controls. TNT-translated HA-Stat1 was acetylated in vitro as described (Gu and Roeder 1997) using immunoprecipitated CBP.
(C) Interaction and colocalization of Stat1 and CBP in SK-37 cells were analyzed by immunofluorescence microscopy and IP of CBP.
Cells were either treated with VPA (V, 1.5 mM), TSA (T, 30 nM), or interferon ? (?, 103U), or left untreated (C) for 48 h. (D) Acetylation
levels of HA-Stat1?XbaI compared with full-length HA-Stat1? were determined by IP from 293T cell lysates as described in A. Cells
were transfected with recombinant Stat1 and CBP vectors at a ratio of 5:1. (E) The experiment was performed as in D, except that
HA-Stat1? or GFP-Stat1 410,413K → Ewere transfected. (F) NF-?B p65 was immunoprecipitated from 2fTGH or U3A cell extracts. The
presence and acetylation of Stat1? were detected by Western blot as described in A. Cells were treated with 1.5 mM VPA for 24 h or
left untreated. (G) Schematic representation of Stat1? showing positions of acetylated lysines and mutants generated. (NTD) N-
terminal domain; (CC) coiled coil; (DBD) DNA-binding domain; (LD) linker domain; (TAD) transcriptional activation domain. Mu-
tants are designated QQ (mutation of both K410 and K413 to Q) and RR (mutation of both K410 and K413 to R).
Acetylation of Stat1. (A) SK-37 cells were either treated with VPA (V, 1.5 mM), TSA (T, 30 nM), or interferon ? (?, 103U),
Acetylation of Stat1
GENES & DEVELOPMENT479
Stat1?/? are precipitated with the anti-acetyl-lysine an-
tibody (Fig. 6A, left panel), it is evident that both Stat1
splice variants can be acetylated.
To identify lysine residues in Stat1? that are subject to
acetylation, several Stat1 lysine mutants (Horvath et al.
1996; Yang et al. 1999, 2002; Meyer et al. 2002) were
expressed in 293T cells and immunoprecipitated. West-
ern blot analysis with an antibody against acetylated ly-
sine showed that only the Stat1 410,413K → Emutant
(Meyer et al. 2002) was not acetylated under conditions
in which wild-type Stat1 became strongly acetylated
(Fig. 6E). Since this was not due to decreased interaction
of this mutant with CBP (data not shown), Lys 410 and
Lys 413 located within the Stat1 DNA-binding domain
(DBD) are likely to be the major sites of acetylation.
Acetylation of Stat1? mediates p65-binding and
confers susceptibility to apoptosis
Given that the acetylation of Stat1 correlates with the
induction of apoptosis, interaction with p65, and repres-
sion of NF-?B signaling, we tested whether p65-associ-
ated Stat1? is acetylated in vivo. Indeed, an acetylated
protein corresponding in size to Stat1? coprecipitated
with NF-?B p65 from 2fTGH cell lysates after VPA treat-
ment. We confirmed that this acetylated protein was in-
deed Stat1 by reprobing the membrane with a Stat1 an-
tibody. Additionally, both the acetylation signal and the
signal for Stat1 were not detectable in the Stat1-negative
U3A cell line (Fig. 6F). Thus, we conclude that at least a
fraction of p65-associated Stat1? is acetylated in vivo.
We hypothesized that acetylation of Stat1? might ren-
der cells sensitive to HDACi-induced apoptosis. There-
fore, we tested whether induction of apoptosis is specifi-
cally due to the acetylation of lysines within the DBD of
Stat1?. Lys 410 and Lys 413 were replaced either with
glutamine (K → Q) or arginine (K → R) resembling con-
stitutively acetylated or nonacetylated states, respec-
tively (Fig. 6G). These experiments were conducted in
NW-1539 and U3A cells and treatment with VPA was
included as an additional control.
Proliferation and apoptosis of NW-1539 cells trans-
fected with wild-type and mutant Stat1? expression vec-
tors were scored by MTT and FACS analysis (Fig. 7A).
Overexpression of wild-type Stat1 together with VPA
treatment led to reduced proliferation and apoptosis in-
duction (Fig. 7A). Transfection of the Stat1 mutant, in
which K410 and K413 were replaced by glutamine
(410,413K → Q) also reduced proliferation and even en-
hanced the rate of apoptosis of NW-1539 cells in re-
sponse to HDACi treatment. In contrast, substitution of
K410 and K413 with arginine (410,413K → R) could not
render cells sensitive to apoptosis induction by HDACi
(Fig. 7A). These data indicate that the acetylation of
Stat1 and inhibition of NF-?B are required, though not
sufficient, for the induction of apoptosis.
Next, we examined whether ectopically expressed
wild-type and mutant Stat1 proteins interact differen-
tially with endogenous NF-?B p65. Equal expression of
all Stat1 constructs was verified by Western blot (Fig.
7B). Immunoprecipitates of p65 were analyzed for the
presence of Stat1 by Western blot. Results shown in Fig-
ure 7B confirm that treatment with VPA significantly
enhances the association of wild-type Stat1? and p65
(see Fig. 5). Furthermore, the pseudo-acetylated Stat1?
mutant 410,413K → Qconstitutively bound p65 in vivo,
whereas Stat1? 410,413K → Rdid not associate with p65
even after treatment with VPA (Fig. 7B).
When we analyzed the effect of Stat1 lysine mutations
on the expression of the NF-?B target gene survivin, we
found that Stat1? 410,413K → Qcaused a significant de-
crease. In contrast, Stat1? 410,413K → Rfailed to reduce
survivin expression under identical conditions (Fig. 7C).
Figures 5A,B and 7E show that NF-?B p65 is depleted
from the nucleus upon acetylation of Stat1 in vivo. We
could confirm this result in vitro by demonstrating the
efficient depletion of p65 from nuclear lysates by a
pseudo-acetylated mutant of Stat1 (Fig. 7D).
Furthermore, immunofluorescence analysis showed
that nuclear p65 was no longer detectable in NW-1539
cells if wild-type Stat1 was expressed ectopically and
cells were treated with HDACi. Stat1? 410,413K → Q
mimicked these effects, whereas expression of Stat1?
410,413K → Rfailed to increase cytoplasmic localization
of p65 (Fig. 7E, cf. transfected and untransfected cells
within each field). Moreover, expression of Stat1?
410,413K → Qin NW-1539 cells reduced DNA binding of
NF-?B similar to overexpressed wild-type Stat1 in cells
treated withVPA, whereas
410,413K → Rdid not (Fig. 7F). Thus, neither treatment of
cells with an HDACi nor mere expression of Stat1 ex-
erted unspecific effects on NF-?B. Based on these results,
we propose a model in which acetylated Stat1? binds to
and sequesters NF-?B p65 in the cytoplasm, thereby in-
terfering with NF-?B function (Fig. 7G). Consequently,
the susceptibility of cells to apoptosis induction depends
on the presence and acetylation status of Stat1.
Currently, the specific roles of acetyltransferases and
deacetylases as well as their substrates are under intense
investigation. Results based on these studies allow in-
sights into the molecular mechanisms determining
whether and how particular cell lines and types respond
to changes in protein acetylation. Here, we show that in
melanoma cell lines, resistance toward HDACi and in-
terferon ?-induced apoptosis inversely correlates with
Stat1 expression and acetylation. The acetylation of Lys
410 and Lys 413 within the DNA-binding domain of
Stat1 triggers its interaction with NF-?B p65. As a con-
sequence, the level of nuclear p65 decreases significantly
and DNA binding of NF-?B is inhibited. This leads to the
down-regulation of anti-apoptotic NF-?B target genes,
thus shifting the balance toward cell death. Based on our
results, we propose a model of altered cross-talk between
Stat1 and NF-?B signal transduction pathways that pro-
vides an explanation of how HDACi and interferon ?
down-regulate NF-?B activity.
Krämer et al.
480GENES & DEVELOPMENT
with Stat1? (WT, wild type), lysine mutants, or equal amounts of empty vector (pc3.1). Proliferation and apoptosis were scored 72 h
later by MTT and PI FACS analysis, respectively. (WT) Wild type; (QQ) 410,413K → Q; (RR) 410,413K → R; (−) untreated; (V) 1.5 mM VPA.
(B) Interaction of overexpressed wild-type (WT) and mutant Stat1? (QQ, RR) with NF-?B p65 in U3A cells was analyzed by IP and
Western blot. Cells were incubated with 1.5 mM VPA for 48 h or left untreated. Input lanes are 2% of the lysate used for IP and are
shown at expositons allowing signal comparison. (C) U3A cells were transfected and treated as described in B. Survivin expression was
analyzed by Western blot. Detection of actin and AcH4 serve as loading and treatment controls, respectively. (D) SK-37 nuclear lysates
were incubated with HA-Stat1 (QQ, 410,413K → Q; RR, 410,413K → R) immunoprecipitated in RIPA buffer or a precipitate formed with
a control antibody (pre). Ten microliters of input and 20 µL of depleted nuclear extract were loaded (upper panel). The lower panel
shows equal Stat1 IP efficiencies. (E) NF-?B p65 localization was analyzed by immunofluorescence microscopy of NW-1539 cells
transfected and treated as described in B. Note: Compare transfected and nontransfected cells within each field. (F) DNA binding of
NF-?B was investigated by EMSA with cell lysates of NW-1539 cells transfected and treated as described in B. (G) Model for
acetylation-dependent Stat1–NF-?B cross-talk.
Identification of Stat1? acetylation as critical regulator of HDACi-induced apoptosis. (A) NW-1539 cells were transfected
Acetylation of Stat1
GENES & DEVELOPMENT481
HDACi resistant and sensitive melanoma cell lines
When we started to study the effects of HDACi on mela-
noma cell lines, we realized that they can be divided into
sensitive and resistant subclasses. This allowed us to
investigate the underlying molecular mechanisms in a
set of cell lines derived from the same type of tumor. Our
data indicate that sensitive cell lines (e.g., SK-37) un-
dergo programmed cell death via both the extrinsic and
the intrinsic apoptotic pathways (Fig. 1). In sensitive cell
lines HDACi treatment significantly decreases the ex-
pression of anti-apoptotic genes such as Bcl-XL, Stat5,
and survivin, which are bona fide target genes of NF-?B
(Eickhoff et al. 2000; Krämer et al. 2001; Hinz et al. 2002;
De Schepper et al. 2003). In resistant cell lines, on the
other hand, neither changes in expression levels of these
genes nor apoptosis induction are detectable, although
hyperacetylation of histones is readily apparent (Figs.
A microarray analysis revealed that Stat1 is among
those genes that are significantly up-regulated in sensi-
tive melanoma cell lines in response to the HDACi VPA
and TSA. These findings were confirmed at the protein
level (Fig. 2A,B). Interestingly, Stat1 expression was very
low (close to the detection limit) and not inducible in the
HDACi-resistant cell lines NW-450 and NW-1539. Fur-
thermore these cell lines, in contrast to HDACi-sensi-
tive cells, were not affected by interferon ? (Fig. 2C,D).
To investigate whether Stat1 plays indeed a causative
role in the induction of apoptosis in response to HDACi,
we introduced Stat1? into NW-1539 cells by lentiviral
transduction. Our results show that expression of Stat1
in NW-1539 restored sensitivity of this cell line toward
HDACi and interferon ? (Fig. 3A,B). Further experiments
are required to establish whether this Stat1-dependent
mechanism determining resistance or sensitivity toward
HDACi represents a general principle relevant to many
types of tumor cells. The HDACi-resistant cell lines
were originally established from patients who had un-
dergone immunotherapy including interferon ? treat-
ment. We speculate that during this process interferon ?
resistant cells with defects in Stat1 signaling were se-
lected. In principle, both mutations within the Stat1
gene as well as epigenetic silencing could shut down
Stat1 expression. Our observation that the resistant cell
lines NW-450 and NW-1539 re-express Stat1 when
treated with 5-aza-cytidine highlights the relevance of
DNA methylation in this context (O.H. Krämer and T.
Heinzel, unpubl.). HDACi and interferon ? are being
considered as candidate drugs for cancer therapy (Krämer
et al. 2001; Kelly et al. 2002; de Vries et al. 2003). Ac-
cording to our data, the combination of HDACi and in-
terferon ? or demethylating agents could be particularly
effective in the treatment of melanomas. If this were the
case, Stat1 expression might serve as a useful marker for
the prediction of clinical response.
We observed that the expression of a subset of NF-?B
target genes after treatment with HDACi or interferon ?
inversely correlates with Stat1 levels. This prompted us
to analyze the affinity of NF-?B for DNA in cells with
different Stat1 expression statuses. A reduction of NF-?B
DNA binding was only observed with HDACi-treated or
interferon ?-stimulated Stat1-positive but not with
Stat1-negative cell extracts (Fig. 4). This is consistent
with the cell type-specific repression of NF-?B-depen-
dent genes by this cytokine and HDACi (Fig. 4A,B).
Moreover, in cells expressing Stat1, the amount of p65 in
the nucleus drops significantly in response to such treat-
ment, and this effect can be inhibited by the nuclear
export inhibitor LMB (Fig. 5A).
Since these results could be due to a physical interac-
tion of Stat1 and p65, we performed co-IP experiments.
Indeed, we detected the formation of a Stat1–NF-?B com-
plex upon treatment with HDACi and interferon ? (Fig.
5D). Gel filtration experiments indicate that the molecu-
lar weight of this complex is in the mega-Dalton range.
Therefore, the complex is likely to contain several addi-
tional proteins (Fig. 5). Although a potential cross-talk of
Stat1 and NF-?B signaling pathways has been discussed
in several reports (Chatterjee-Kishore et al. 2000; Suk et
al. 2001; Shen and Lentsch 2004; Sizemore et al. 2004),
evidence for the physical association of these factors has
not been published. We speculate that this is due to the
fact that we observed their interaction only upon treat-
ment of cells with HDACi or extended stimulation with
interferon ? (Fig. 5D).
Regulation of Stat1 acetylation status
HDACi and the cytokine interferon ? points out com-
mon actions of these drugs, and indeed both induce
acetylation of Stat1. The dynamic control of Stat1 acety-
lation is reflected by its association with acetyltransfer-
ases and deacetylases. Increased expression of the acet-
yltransferase CBP promotes Stat1 acetylation in vivo and
this HAT can also acetylate Stat1 in vitro (Fig. 6B). Fur-
thermore, in response to treatment with interferon ?,
CBP can translocate to the cytosol, which permits physi-
cal interaction with the major cellular pool of Stat1 (Fig.
6C). This correlates with the occurrence of acetylated
Stat1 (Fig. 6A,C). Therefore, CBP is likely to mediate
Stat1 acetylation. Moreover, since the CBP bromodo-
main could dock directly to acetylated Stat1, this could
impose a positive feedback on the acetylation of Stat1.
This would be consistent with the notion that N?-acety-
lated proteins stably associate with acetyltransferases
and deacetylases in a signal-dependent manner (Yang
Based on interactions of endogenous proteins in co-IP
experiments, HDAC1 and HDAC3, but not HDAC2 and
HDAC8, presumably deacetylate Stat1. Inhibition of
these enzymes by HDACi is an obvious mechanism by
which these compounds could induce Stat1 acetylation
(Fig. 6A). However, acetylation of Stat1 also correlates
with the dissociation of HDACs from Stat1 (Fig. 5F) and
an increased interaction with CBP (Fig. 6C), which
would both favor Stat1 acetylation. This implies that we
coincidenceofcellular susceptibility against
Krämer et al.
482 GENES & DEVELOPMENT
identified a regulatory system that allows convergence of
multiple inputs on protein acetylation and deacetyla-
tion. Moreover, cell type-specific differences are likely to
affect the dynamic equilibrium of HDACs and HATs
controlling Stat1 acetylation.
Acetylated Stat1 mediates suppression of
anti-apoptotic NF-?B target genes
Acetylation is considered as a covalent modification that
could, similar to phosphorylation, affect the activity of a
wide range of proteins by altering intermolecular inter-
actions. Transcription factors such as NF-?B and p53
were shown to be acetylated in their DNA-binding do-
mains (Yang 2004). Here, we show that acetylation of
Stat1 lysine residues 410 and 413 within its DBD regu-
lates its interaction with NF-?B p65. Since the cells used
in our analysis are characterized by a low constitutive
activity of NF-?B, the majority of p65 molecules should
exist in cytoplasmic I–?B complexes. The formation of
Stat1–NF-?B complexes correlates with the depletion of
residual transcriptionally active p65 from the nucleus.
This interferes with NF-?B function and renders cells
permissive to apoptosis induction (Figs. 4, 7). Our data
indicate that acetylation of Stat1 and inhibition of NF-?B
is required, though not sufficient, for this process. Such
an observation is consistent with data describing in-
creased susceptibility to apoptosis upon introduction of
dominant-negative I-?Bs, yet no apoptosis induction
solely by expression of these proteins in solid tumors
(Mayo et al. 2003). Additional actions of HDACi such as
the modulation of other signaling pathways and altered
cell cycle regulation appear to be necessary. Consistent
with this hypothesis, low levels of acetylated Stat1 are
detectable in untreated cells but do not lead to sponta-
Interestingly, microarray experiments revealed that in
cells exposed to HDACi about one-third of significant
changes in gene expression consist of repression rather
than activation events (Van Lint et al. 1996; Mitsiades et
al. 2004). This initially unexpected observation could be
due to the induction of transcriptional repressors that do
not require HDAC activity to function. Such an indirect
mechanism would require de novo protein synthesis. On
the other hand, the modulation of cross-talk between
Stat1 and NF-?B signaling pathways we discovered is
independent of protein synthesis and provides a plau-
sible explanation for the suppression of NF-?B target
genes by both interferons and the inhibition of HDAC
activity. The resulting down-regulation of anti-apoptotic
genes could be a prototypical example for the HDACi-
and cytokine-mediated inhibition of gene expression.
Our data generated with HDACi, experiments with
pseudo-acetylated and nonacetylated Stat1 mutant mol-
ecules, ectopic expression, siRNA, and rescue ap-
proaches show how acetyl-lysine moieties may contrib-
ute to the temporal and spatial regulation of protein
function. An interesting future challenge will be to un-
derstand which other signaling networks rely on protein
acetylation events and how they generate in vivo re-
sponses to external and internal signals.
Materials and methods
Drugs and chemicals
Valproic acid, TSA, prodidium iodide, LMB, Hoechst 33258,
trypan blue, and MTT were purchased from Sigma, interferon ?
was purchased from Roche, and Z-VAD-FMK and Ac-DEVD-
pNA were purchased from Alexis.
Cell lines, transfections, and microscopy
SK-Mel-37, Mz-Mel-19, NW-Mel-1539, NW-Mel-450 (Jäger et
al. 2002) (abbreviated as SK-37, Mz-19, NW-1539, or NW-450),
2fTGH, and U3A cells were maintained in DMEM supple-
mented with 10% FCS, 1% penicillin/streptomycin, and 5%
L-glutamine at 37°C in a 5% CO2atmosphere. SK-Mel-28 and
Malme-3-M cell lines were grown in RPMI containing the same
additives. Cells were transfected using PEI (Sigma) or lipofect-
amine (Invitrogen). Preparation and image analysis of cells were
performed as described (Heger et al. 2001). Cy3- and FITC-la-
beled secondary antibodies were used for immunofluorescence.
Preparation of cell lysates and immunoblotting
Lysate preparation and Western blot procedures were carried
out as described (Standke et al. 1994; Krämer et al. 2003). An-
tibodies were obtained from Santa Cruz Biotechnology (Stat1,
p65, p50, HDAC1, HDAC3, CBP, survivin, caspase 3, Tradd,
TBP, HA, GFP, mSin3), Sigma (actin), Pharmingen (Bcl-XL,
PARP), Transduction labs (Stat5), and NEB (caspase 8, caspase 9,
AcLys). The AcH4 antibody has been described (Göttlicher et al.
2001). Co-IP experiments were performed as described (Heinzel
et al. 1997). For direct IP of Stat1? and NF-?B, cells were lysed
in RIPA buffer. To detect interactions, NETN buffer containing
0.1% NP-40 was used. TSA (1 µM) was added to preserve acety-
Measurement of proliferation and apoptosis
MTT assays were performed as described (Denizot and Lang
1986). The cellular DNA content was determined by PI flow
cytometry (Göttlicher et al. 2001). Cell viability was also deter-
mined by trypan blue exclusion and a PI/Hoechst staining assay
(Suk et al. 2001). Caspase 3 assays were performed with 200 µL
of caspase 3 cleavage buffer (100 mM Tris at pH 8.0, 10% su-
crose, 150 mM NaCl, 0.1% CHAPS, 10 mM DTT), 2.5 µL of 2
mM Ac-DEVD-pNA, and 50 µg protein.
Production of lentiviral particles
HA-Stat1? was cloned into the SacII site of the pHR?cPPT SIEW
Sin vector to yield pS-Stat1?-IEW. Lentiviral vector stocks were
produced as described (Zufferey et al. 1997; Baus and Pfitzner
2005). Effective transduction was confirmed by fluorescence mi-
croscopy, FACS, and Western blot.
EMSA (gel retardation assay) and ABCD-Assay
(Avidin–Biotin-coupled DNA assay)
Radioactive DNA-binding assays were performed as described
(Garcia et al. 1997). The NF-?B oligonucleotide (sc-2505) and
supershift antibodies were purchased from Santa Cruz. ABCD-
assays were performed as described (Baumann et al. 2005) with
the NF-?B oligonucleotides Bio 5?-GGAATTTCCGGGAA
Acetylation of Stat1
GENES & DEVELOPMENT483
TTTCCGGGAATTTCCGGGAATTTCCC-3? and 5?-GGGAA
or biotinylated nonrelevant oligos.
Stat1? was mutagenized by overlap extension PCR (Ho et al.
1989). The primers used were KK, 5?-AAAAGATCTATGTCT
ACTGTGTTCATCATACTGTCGAACTCTAC-3?; QQ, 5?-G
TTTGCTGTTCTTGCAATTGC-3?; and RR, 5?-GCAATTGC
PCR products were cloned into pc3.1 TOPO (Invitrogen). Al-
ternatively, HA-Stat1 was mutagenized using the Quick change
site-directed mutagenesis kit (Stratagene). The siRNA se-
quences for Stat1 were described (Higashi et al. 2003). Mutagen-
esis of this site toward RNA interference (RNAi)-resistance was
carried out by creating silent mutations with the QuickChange
kit using the primers 5?-GAGAAGCTTCTTGGTCCGAATG
CCAGCCCCGATGG-3? and 5?-CCATCGGGGCTGGCATTC
We thank G. Carra, A. Schimpf, S. Reichardt, and H. Kunkel for
excellent technical assistance; D. Zimmermann, I. Oehme, B.
Dälken, N. Novac, M. Landersz, U. Dietrich (GSH), B. Schlott,
and K.H. Gührs (FLI Jena, Dept. F. Grosse) for experimental
support; and M. Göttlicher for critical reading of the manu-
script. M. Zörnig and S. Hövelmann were invaluable discussion
partners throughout the project. G. Stark, M. Müller, and P.
Heinrich generously provided cell lines; M. Scherr, K. Brocke-
Heidrich, and F. Horn provided the lentiviral expression vector;
and C. Glass, J. Darnell, and U. Vinkemeier provided Stat1 ex-
pression vectors. This work was supported by NGFN grants to
T.H. (N1KR-S31T30), E.P. (N1KR-S31T21), R.S. (N1KR-S31T24),
and M.G. (N1KR-S31T19), in addition to funding through DFG
SFB 604 (T.H.).
Baumann, S., Dostert, A., Novac, N., Bauer, A., Schmid, W., Fas,
S.C., Krueger, A., Heinzel, T., Kirchhoff, S., Schu ¨tz, G., et al.
2005. Glucocorticoids inhibit activation-induced cell death
(AICD) via direct DNA-dependent repression of the CD95
ligand gene by a glucocorticoid receptor dimer. Blood
Baus, D. and Pfitzner, E. 2005. Specific function of STAT3,
SOCS1 and SOCS3 in the regulation of proliferation and sur-
vival of classical Hodgkin lymphoma cells. Int. J. Can. (Epub
ahead of print October 4, 2005. PMID: 16206268]
Blobel, G.A. 2000. CREB-binding protein and p300: Molecular in-
tegrators of hematopoietic transcription. Blood 95: 745–755.
Chatterjee-Kishore, M., Wright, K.L., Ting, J.P., and Stark, G.R.
2000. How Stat1 mediates constitutive gene expression: A
complex of unphosphorylated Stat1 and IRF1 supports tran-
scription of the LMP2 gene. EMBO J. J 19: 4111–4122.
Chen, L.F. and Greene, W.C. 2003. Regulation of distinct bio-
logical activities of the NF-?B transcription factor complex
by acetylation. J. Mol. Med. 81: 549–557.
Cohen, H.Y., Lavu, S., Bitterman, K.J., Hekking, B., Imahi-
yerobo, T.A., Miller, C., Frye, R., Ploegh, H., Kessler, B.M.,
and Sinclair, D.A. 2004. Acetylation of the C terminus of
Ku70 by CBP and PCAF controls Bax-mediated apoptosis.
Mol. Cell 13: 627–638.
De Schepper, S., Bruwiere, H., Verhulst, T., Steller, U., Andries,
L., Wouters, W., Janicot, M., Arts, J., and Van Heusden, J.
2003. Inhibition of histone deacetylases by chlamydocin in-
duces apoptosis and proteasome-mediated degradation of
survivin. J. Pharmacol. Exp. Ther. 304: 881–888.
de Vries, E.G., Timmer, T., Mulder, N.H., van Geelen, C.M.,
van der Graaf, W.T., Spierings, D.C., de Hooge, M.N., Gi-
etema, J.A., and de Jong, S. 2003. Modulation of death recep-
tor pathways in oncology. Drugs Today (Barc) 39, Suppl
Denizot, F. and Lang, R. 1986. Rapid colorimetric assay for cell
growth and survival. Modifications to the tetrazolium dye
procedure giving improved sensitivity and reliability. J. Im-
munol. Methods 89: 271–277.
Eickhoff, B., Ruller, S., Laue, T., Kohler, G., Stahl, C., Schlaak,
M., and van der Bosch, J. 2000. Trichostatin A modulates
expression of p21waf1/cip1, Bcl-xL, ID1, ID2, ID3, CRAB2,
GATA-2, hsp86 and TFIID/TAFII31 mRNA in human lung
adenocarcinoma cells. Biol. Chem. 381: 107–112.
Garcia, R., Yu, C.L., Hudnall, A., Catlett, R., Nelson, K.L.,
Smithgall, T., Fujita, D.J., Ethier, S.P., and Jove, R. 1997.
Constitutive activation of Stat3 in fibroblasts transformed
by diverse oncoproteins and in breast carcinoma cells. Cell
Growth Differ. 8: 1267–1276.
Göttlicher, M., Minucci, S., Zhu, P., Krämer, O.H., Schimpf, A.,
Giavara, S., Sleeman, J.P., Lo, C.F., Nervi, C., Pelicci, P.G., et
al. 2001. Valproic acid defines a novel class of HDAC inhibi-
tors inducing differentiation of transformed cells. EMBO J.
Gu, W. and Roeder, R.G. 1997. Activation of p53 sequence-
specific DNA binding by acetylation of the p53 C-terminal
domain. Cell 90: 595–606.
Gurvich, N., Tsygankova, O.M., Meinkoth, J.L., and Klein, P.S.
2004. Histone deacetylase is a target of valproic acid-medi-
ated cellular differentiation. Cancer Res. 64: 1079–1086.
Heger, P., Lohmaier, J., Schneider, G., Schweimer, K., and
Stauber, R.H. 2001. Qualitative highly divergent nuclear ex-
port signals can regulate export by the competition for trans-
port cofactors in vivo. Traffic 2: 544–555.
Heinzel, T., Lavinsky, R.M., Mullen, T.M., Söderström, M., La-
herty, C.D., Torchia, J., Yang, W.M., Brard, G., Ngo, S.D.,
Davie, J.R., et al. 1997. A complex containing N-CoR, mSin3
and histone deacetylase mediates transcriptional repression.
Nature 387: 43–48.
Henderson, C., Mizzau, M., Paroni, G., Maestro, R., Schneider,
C., and Brancolini, C. 2003. Role of caspases, Bid, and p53 in
the apoptotic response triggered by histone deacetylase in-
hibitors trichostatin-A (TSA) and suberoylanilide hydrox-
amic acid (SAHA). J. Biol. Chem. 278: 12579–12589.
Higashi, K., Inagaki, Y., Fujimori, K., Nakao, A., Kaneko, H.,
and Nakatsuka, I. 2003. Interferon-? interferes with trans-
forming growth factor-? signaling through direct interaction
of YB-1 with Smad3. J. Biol. Chem. 278: 43470–43479.
Hinz, M., Lemke, P., Anagnostopoulos, I., Hacker, C., Krap-
pmann, D., Mathas, S., Dorken, B., Zenke, M., Stein, H., and
Scheidereit, C. 2002. Nuclear factor ?B-dependent gene ex-
pression profiling of Hodgkin’s disease tumor cells, pathoge-
netic significance, and link to constitutive signal transducer
and activator of transcription 5a activity. J. Exp. Med.
Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R.
1989. Site-directed mutagenesis by overlap extension using
the polymerase chain reaction. Gene 77: 51–59.
Horvath, C.M., Stark, G.R., Kerr, I.M., and Darnell Jr., J.E. 1996.
Interactions between STAT and non-STAT proteins in the
interferon-stimulated gene factor 3 transcription complex.
Krämer et al.
484 GENES & DEVELOPMENT
Mol. Cell. Biol. 16: 6957–6964.
Huang, N., Katz, J.P., Martin, D.R., and Wu, G.D. 1997. Inhibi-
tion of IL-8 gene expression in Caco-2 cells by compounds
which induce histone hyperacetylation. Cytokine 9: 27–36.
Ihle, J.N. 2001. The Stat family in cytokine signaling. Curr.
Opin. Cell Biol. 13: 211–217.
Inan, M.S., Rasoulpour, R.J., Yin, L., Hubbard, A.K., Rosenberg,
D.W., and Giardina, C. 2000. The luminal short-chain fatty
acid butyrate modulates NF-?B activity in a human colonic
epithelial cell line. Gastroenterology 118: 724–734.
Jäger, E., Karbach, J., Gnjatic, S., Jäger, D., Maeurer, M., Atmaca,
A., Arand, M., Skipper, J., Stockert, E., Chen, Y.T., et al.
2002. Identification of a naturally processed NY-ESO-1 pep-
tide recognized by CD8+ T cells in the context of HLA-B51.
Cancer Immun. 2: 12.
Kelly, W.K., O’Connor, O.A., and Marks, P.A. 2002. Histone
deacetylase inhibitors: From target to clinical trials. Expert
Opin. Investig. Drugs 11: 1695–1713.
Kiernan, R., Bres, V., Ng, R.W., Coudart, M.P., El Messaoudi, S.,
Sardet, C., Jin, D.Y., Emiliani, S., and Benkirane, M. 2003.
Post-activation turn-off of NF-?B-dependent transcription is
regulated by acetylation of p65. J. Biol. Chem. 278: 2758–2766.
Korzus, E., Torchia, J., Rose, D.W., Xu, L., Kurokawa, R., Mc-
Inerney, E.M., Mullen, T.M., Glass, C.K., and Rosenfeld,
M.G. 1998. Transcription factor-specific requirements for
coactivators and their acetyltransferase functions. Science
Kouzarides, T. 1999. Histone acetylases and deacetylases in cell
proliferation. Curr. Opin. Genet. Dev. 9: 40–48.
———. 2000. Acetylation: A regulatory modification to rival
phosphorylation? EMBO J. 19: 1176–1179.
Krämer, O.H., Göttlicher, M., and Heinzel, T. 2001. Histone
deacetylase as a therapeutic target. Trends Endocrinol.
Metab. 12: 294–300.
Krämer, O.H., Zhu, P., Ostendorff, H.P., Golebiewski, M., Tie-
fenbach, J., Peters, M.A., Brill, B., Groner, B., Bach, I., Hein-
zel, T., et al. 2003. The histone deacetylase inhibitor valproic
acid selectively induces proteasomal degradation of HDAC2.
EMBO J. 22: 3411–3420.
Kumar, A., Commane, M., Flickinger, T.W., Horvath, C.M., and
Stark, G.R. 1997. Defective TNF-?-induced apoptosis in
STAT1-null cells due to low constitutive levels of caspases.
Science 278: 1630–1632.
Mayo, M.W., Denlinger, C.E., Broad, R.M., Yeung, F., Reilly,
E.T., Shi, Y., and Jones, D.R. 2003. Ineffectiveness of histone
deacetylase inhibitors to induce apoptosis involves the tran-
scriptional activation of NF-?B through the Akt pathway. J.
Biol. Chem. 278: 18980–18989.
Melnick, A. and Licht, J.D. 2002. Histone deacetylases as thera-
peutic targets in hematologic malignancies. Curr. Opin. He-
matol. 9: 322–332.
Meyer, T., Begitt, A., Lodige, I., van Rossum, M., and Vinke-
meier, U. 2002. Constitutive and IFN-?-induced nuclear im-
port of STAT1 proceed through independent pathways.
EMBO J. 21: 344–354.
Mitsiades, C.S., Mitsiades, N.S., McMullan, C.J., Poulaki, V.,
Shringarpure, R., Hideshima, T., Akiyama, M., Chauhan, D.,
Munshi, N., Gu, X., et al. 2004. Transcriptional signature of
histone deacetylase inhibition in multiple myeloma: Bio-
logical and clinical implications. Proc. Natl. Acad. Sci.
Müller, M., Laxton, C., Briscoe, J., Schindler, C., Improta, T.,
Darnell Jr., J.E., Stark, G.R., and Kerr, I.M. 1993. Comple-
mentation of a mutant cell line: Central role of the 91 kDa
polypeptide of ISGF3 in the interferon-? and -? signal trans-
duction pathways. EMBO J. 12: 4221–4228.
Nusinzon, I. and Horvath, C.M. 2003. Interferon-stimulated
transcription and innate antiviral immunity require deacety-
lase activity and histone deacetylase 1. Proc. Natl. Acad. Sci.
Perkins, N.D. 2004. NF-?B: Tumor promoter or suppressor?
Trends Cell Biol. 14: 64–69.
Schreiber, S.L. and Bernstein, B.E. 2002. Signaling network
model of chromatin. Cell 111: 771–778.
Shen, H. and Lentsch, A.B. 2004. Progressive dysregulation of
transcription factors NF-?B and STAT1 in prostate cancer
cells causes proangiogenic production of CXC chemokines.
Am. J. Physiol. Cell Physiol. 286: C840–C847.
Sizemore, N., Agarwal, A., Das, K., Lerner, N., Sulak, M., Rani,
S., Ransohoff, R., Shultz, D., and Stark, G.R. 2004. Inhibitor
of ?B kinase is required to activate a subset of interferon
?-stimulated genes. Proc. Natl. Acad. Sci. 101: 7994–7998.
Standke, G.J., Meier, V.S., and Groner, B. 1994. Mammary gland
factor activated by prolactin on mammary epithelial cells
and acute-phase response factor activated by interleukin-6 in
liver cells share DNA binding and transactivation potential.
Mol. Endocrinol. 8: 469–477.
Strahl, B.D. and Allis, C.D. 2000. The language of covalent his-
tone modifications. Nature 403: 41–45.
Suk, K., Chang, I., Kim, Y.H., Kim, S., Kim, J.Y., Kim, H., and
Lee, M.S. 2001. Interferon ? (IFN?) and tumor necrosis factor
? synergism in ME-180 cervical cancer cell apoptosis and
necrosis. IFN? inhibits cytoprotective NF-?B through
STAT1/IRF-1 pathways. J. Biol. Chem. 276: 13153–13159.
Thornberry, N.A. and Lazebnik, Y. 1998. Caspases: Enemies
within. Science 281: 1312–1316.
Van Lint, C., Emiliani, S., and Verdin, E. 1996. The expression
of a small fraction of cellular genes is changed in response to
histone hyperacetylation. Gene Expr. 5: 245–253.
Vrana, J.A., Decker, R.H., Johnson, C.R., Wang, Z., Jarvis, W.D.,
Richon, V.M., Ehinger, M., Fisher, P.B., and Grant, S. 1999.
Induction of apoptosis in U937 human leukemia cells by
suberoylanilide hydroxamic acid (SAHA) proceeds through
pathways that are regulated by Bcl-2/Bcl-XL, c-Jun, and
p21CIP1, but independent of p53. Oncogene 18: 7016–7025.
Wang, Y., Wu, T.R., Cai, S., Welte, T., and Chin, Y.E. 2000.
Stat1 as a component of tumor necrosis factor ? receptor
1–TRADD signaling complex to inhibit NF-?B activation.
Mol. Cell. Biol. 20: 4505–4512.
Weiss, C., Schneider, S., Wagner, E.F., Zhang, X., Seto, E., and
Bohmann, D. 2003. JNK phosphorylation relieves HDAC3-
dependent suppression of the transcriptional activity of c-
Jun. EMBO J. 22: 3686–3695.
Wolffe, A.P. and Hayes, J.J. 1999. Chromatin disruption and
modification. Nucleic Acids Res. 27: 711–720.
Wong, L.H., Sim, H., Chatterjee-Kishore, M., Hatzinisiriou, I.,
Devenish, R.J., Stark, G., and Ralph, S.J. 2002. Isolation and
characterization of a human STAT1 gene regulatory ele-
ment. Inducibility by interferon (IFN) types I and II and role
of IFN regulatory factor-1. J. Biol. Chem. 277: 19408–19417.
Yang, X.J. 2004. Lysine acetylation and the bromodomain: A
new partnership for signaling. Bioessays 26: 1076–1087.
Yang, E., Wen, Z., Haspel, R.L., Zhang, J.J., and Darnell Jr., J.E.
1999. The linker domain of Stat1 is required for ? interferon-
driven transcription. Mol. Cell. Biol. 19: 5106–5112.
Yang, E., Henriksen, M.A., Schaefer, O., Zakharova, N., and
Darnell Jr., J.E. 2002. Dissociation time from DNA deter-
mines transcriptional function in a STAT1 linker mutant. J.
Biol. Chem. 277: 13455–13462.
Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L., and Trono, D.
1997. Multiply attenuated lentiviral vector achieves effi-
cient gene delivery in vivo. Nat. Biotechnol. 15: 871–875.
Acetylation of Stat1
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