MOLECULAR AND CELLULAR BIOLOGY, Feb. 2005, p. 1113–1123
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 3
Negative Regulation of NF-?B Signaling by PIAS1†
Bin Liu,1Randy Yang,2Kelly A. Wong,3,4,5Crescent Getman,1Natalie Stein,1Michael A. Teitell,6
Genhong Cheng,3,7Hong Wu,3,4,5and Ke Shuai1,2,3*
Division of Hematology-Oncology, Department of Medicine,1Department of Biological Chemistry,2Molecular Biology Institute,3
Department of Molecular and Medical Pharmacology,4Department of Pathology and Pediatrics,6Department of
Microbiology, Immunology, and Molecular Genetics,7and Howard Hughes Medical Institute,5
University of California Los Angeles, Los Angeles, California
Received 17 September 2004/Returned for modification 18 October 2004/Accepted 10 November 2004
The NF-?B family of transcription factors is activated by a wide variety of signals to regulate a spectrum of
cellular processes. The proper regulation of NF-?B activity is critical, since abnormal NF-?B signaling is asso-
ciated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here
that PIAS1 (protein inhibitor of activated STAT1) is an important negative regulator of NF-?B. Upon cytokine
stimulation, the p65 subunit of NF-?B translocates into the nucleus, where it interacts with PIAS1. The binding
of PIAS1 to p65 inhibits cytokine-induced NF-?B-dependent gene activation. PIAS1 blocks the DNA binding
activity of p65 both in vitro and in vivo. Consistently, chromatin immunoprecipitation assays indicate that the
binding of p65 to the promoters of NF-?B-regulated genes is significantly enhanced in Pias1?/?cells. Micro-
array analysis indicates that the removal of PIAS1 results in an increased expression of a subset of NF-?B-mediat-
ed genes in response to tumor necrosis factor alpha and lipopolysaccharide. Consistently, Pias1 null mice
showed elevated proinflammatory cytokines. Our results identify PIAS1 as a novel negative regulator of NF-?B.
A large variety of signals, such as proinflammatory cytokines
(tumor necrosis factor alpha [TNF-?] and interleukin-1 [IL-1])
and bacterial lipopolysaccharide (LPS), activate the NF-?B
signaling pathway. NF-?B is a family of dimeric transcription
factors composed of members of the Rel family of DNA bind-
ing proteins, including NF-?B1 (p50 and its precursor p105),
NF-?B2 (p52 and its precursor p100), c-Rel, RelA (p65), and
RelB (11, 18). Upon stimulation, NF-?B translocates into the
nucleus, where it binds to specific DNA sequences and regu-
lates transcription. NF-?B is involved in mediating a wide
spectrum of cellular responses, including infections, inflamma-
tion, and apoptosis (2, 27). Inappropriate regulation of NF-?B
is involved in a wide range of human diseases, including cancer,
neurodegenerative disorders, arthritis, asthma, and chronic in-
flammation (3, 4, 10, 12). The NF-?B signaling pathway is
tightly modulated at various levels by distinct regulatory pro-
teins. For example, the binding of the I?B family of proteins
prevents the nuclear translocation of NF-?B (16). However, a
protein factor that can regulate the DNA binding activity of
NF-?B has not been documented.
The PIAS (protein inhibitor of activated STAT) family of
proteins consists of four members: PIAS1, PIAS3, PIASx, and
PIASy (33). Members of the PIAS family have been suggested
to regulate STAT-mediated transcription. Upon cytokine stim-
ulation, PIAS binds to STAT and inhibits STAT-mediated
gene activation (1, 8, 21, 22). Among the PIAS family, PIAS1
and PIASy have been shown to inhibit STAT1-dependent tran-
scription through distinct mechanisms. PIAS1 inhibits the tran-
scriptional activity of STAT1 by blocking the DNA binding
activity of STAT1. In contrast, PIASy does not affect the DNA
binding activity of STAT1. It has been suggested that PIASy
may act as a transcriptional corepressor of STAT1. The PIAS
family of proteins has also been suggested to regulate a num-
ber of other transcription factors, including nuclear hormone
receptors (13, 28, 36, 37), LEF1 (31), and p53 (15, 26, 32).
To understand the physiological role of PIAS1, we have
recently generated Pias1 null mice (23). Detailed gene activa-
tion analysis indicates that PIAS1 selectively regulates a subset
of interferon (IFN)-inducible genes. The antiviral activity of
IFNs is significantly enhanced by Pias1 disruption. In addition,
Pias1 null mice show enhanced protection against pathogenic
infection. These results support a physiological role of PIAS1
in the negative regulation of IFN-activated STAT1-mediated
gene activation and demonstrate an important role of PIAS1 in
innate immune responses.
Since STAT1 and the Rel family of proteins share structural
similarity in their DNA binding domains (6), we explored the
possible involvement of PIAS1 in the regulation of NF-?B.
Here we report that PIAS1 interacts with the p65 subunit of
NF-?B and represses its transcriptional activity. In vitro and in
vivo studies indicate that PIAS1 inhibits the DNA binding ac-
tivity of p65. Microarray analysis indicates that the disruption
of Pias1 results in elevated expression of a subset of NF-?B-de-
the negative regulation of the NF-?B signaling pathway.
MATERIALS AND METHODS
Materials. Flag-PIAS1, glutathione S-transferase (GST)–PIAS1, and Gal4-
p65 plasmids have been described (20, 22). Flag-p65, Flag-p65(1-313), Flag-
p65(299-551), Flag-PIAS1(1-415), Flag-PIAS1(416-650), Flag-PIAS1(1-344),
Flag-PIAS1(89-344), Myc-PIAS1, and Myc-p65 were cloned by PCR amplifica-
tion of the corresponding coding regions followed by subcloning into pCMV-
Flag or pMyc expression vectors. The following antibodies were utilized in the
coimmunoprecipitation and Western blotting analyses: anti-p65 (C-20; Santa
Cruz Biotechnology, Santa Cruz, Calif.), anti-p50 (H-119; Santa Cruz Biotech-
nology), anti-E2F-1 (C-20; Santa Cruz Biotechnology), anti-I?B? (C-20; Santa
* Corresponding author. Mailing address: Division of Hematology-
Oncology, 11-934 Factor Bldg., 10833 Le Conte Ave., Los Angeles, CA
90095-1678. Phone: (310) 206-9168. Fax: (310) 825-2493. E-mail: kshuai
† Supplemental material for this article may be found at http://mcb
Cruz Biotechnology), antiactin (C-11; Santa Cruz Biotechnology), anti-Flag (M2;
Sigma), anti-Myc (Cell Signaling), and antitubulin (Sigma). The anti-PIAS1
antibody was raised against a GST fusion protein containing the C-terminal
region of human PIAS1 (amino acids 551 to 650).
Coimmunoprecipitation assays. Coimmunoprecipitation assays were per-
formed as described previously (8). Briefly, whole-cell lysates were prepared 30 h
post-transient transfection in a lysis buffer containing 50 mM Tris (pH 8), 150
mM NaCl, 1% Brij, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 ?g of leupeptin/ml, and 3 ?g of aprotinin/ml. The mixture was incubated on
ice for 30 min and centrifuged at 13,000 ? g for 5 min. The supernatant was used
in coimmunoprecipitation assays with various antibodies.
Transient transfection and luciferase assays. Human 293T cells were trans-
fected by a calcium-phosphate procedure as described previously (34), and cell
lysates were collected for luciferase assays (Promega) 30 h posttransfection. The
relative luciferase units were corrected for relative expression of ?-galactosidase.
Human A549 cells were transfected with Lipofectamine reagent (Invitrogen) and
assayed for luciferase activities with a dual-luciferase system (Promega) using the
cotransfected pRLTK to correct for the differences in transfection efficiency.
Northern blot analysis. Northern blot analysis was performed essentially as
described previously (24).
Microarray analysis. Microarray analysis was performed essentially following
the manufacturer’s instructions (Affymetrix). Briefly, bone marrow-derived mac-
rophages (BMMs) from wild-type or Pias1?/?littermates were either untreated
or treated with TNF-? (20 ng/ml) for 30 min or LPS (10 ng/ml) for 1 h. Total
RNA was prepared with RNA-STAT60 (TEL-TEST) and purified with the
RNeasy kit (QIAGEN). Double-stranded cDNA was synthesized from 20 ?g of
total RNA according to Affymetrix’s methodology and purified with Phase Lock
gels (Eppendorf). Biotin-labeled RNA was synthesized with the BioArray High
Yield RNA transcript labeling kit (Enzo). Samples were cleaned, fragmentated,
and hybridized to murine genome (MGU74Av2) GeneChips (Affymetrix) as
instructed. GeneChips were stained with phycoerythrin-streptavidin (Molecular
Probes) and scanned with a GeneChip scanner (Affymetrix).
Bone marrow-derived macrophages. BMMs were differentiated from marrow
cells from 4- to 8-week-old Pias1 null mice and their wild-type littermates as
described previously (7). BMMs were maintained in 1? Dulbecco’s modified
Eagle medium containing 10% fetal bovine serum, 1% penicillin-streptomycin,
and 30% L929-conditioned medium containing macrophage colony-stimulating
factor for 7 days before they were either untreated or treated with TNF-? (20 ng/
ml) or LPS (10 ng/ml) for various times. Total RNA was prepared and subjected
to real-time PCR analyses.
Immunofluorescence. Immunofluorescence analysis was performed as de-
scribed previously (21). A mouse monoclonal anti-p65 (1:100; F-6; Santa Cruz
Biotechnology) and a rabbit polyclonal anti-PIAS1 (1:400) were added to the
cells simultaneously as primary antibodies. A mixture of anti-rabbit immuno-
globulin G (IgG) Fluor 488 (1:200; Molecular Probes), anti-mouse IgG Cy3
(1:200, Jackson Labs), and Hoechst (1 ?g/ml; Sigma) was added to the cells
during the secondary antibody incubation.
Electrophoretic mobility shift assay. The electrophoretic mobility shift assay
(EMSA) was performed as described previously (8). The sequence of the NF-?B
oligonucleotide is 5?-GATCCGAGAGGGGATTCCCCGATCG-3?. The se-
quence of the SP-1 oligonucleotide is 5?-ATTCGATCGGGGCGGGGCGAG-3?.
Quantitative real-time PCR. Quantitative real-time PCR (Q-PCR) was per-
formed as described previously (9). Briefly, first-strand cDNA was produced by
reverse transcription of 3 ?g of total RNA using SuperScript II (Invitrogen).
Q-PCR was carried out using the iCycler thermocycler (Bio-Rad) in a final
volume of 25 ?l containing the following: Taq polymerase, 1? Taq buffer (Strat-
agene), 125 ?M deoxynucleoside triphosphates, SYBR Green I (Molecular
Probes), and fluorescein (Bio-Rad). Amplification conditions were 95°C (3 min)
and 40 cycles of 95°C (30 s), 55°C (30 s), and 72°C (30 s). Actin was used to
standardize the levels of cDNA. The specific primers used in Q-PCR analyses are
given in Table S2 of the supplemental material.
Chromatin immunoprecipitation assay. Chromatin immunoprecipitation
(ChIP) assays were performed using the ChIP assay kit (Upstate Biotech) as
instructed by the manufacturer. Wild-type or Pias1?/?BMMs (107) were either
untreated or treated with LPS (10 ng/ml) for 20 min or 1 h. Cell extracts were
prepared, and chromatin was sheared by sonication (six 10-s pulses at 30% of the
maximum strength). ChIP assays were performed with an anti-p65 antibody or
rabbit IgG as a negative control. Bound DNA was quantified by Q-PCR and
normalized with the input DNA. Approximately 10% of the immunoprecipitated
samples were analyzed by Western blotting with anti-p65 to reveal that similar
amounts of NF-?B p65 were present in each sample. Similar ChIP assays were
performed with the tetracycline (TET)-off PIAS1 cell line, except that cells
grown in the presence or absence of doxycycline (DOX) for 12 h were either
untreated or treated with TNF-? (20 ng/ml) for 20 min or 1 h. The sequences of
the primers used are given in Table S2 of the supplemental material.
Flow cytometric analysis. Flow cytometric analysis was carried out as de-
scribed previously (19, 25). Briefly, single-cell suspensions from spleens or thy-
muses were depleted of red blood cells by hypotonic lysis and stained with com-
binations of the following antibodies (Pharmingen): anti-B220-phycoerythrin
(PE), anti-IgM-fluorescein isothiocyanate, anti-CD4-fluorescein isothiocyanate,
anti-CD8-PE, and anti-Gr-1-PE. Data were acquired on a FACScan (Becton
Dickinson) and analyzed with CellQuest software.
Measurement of serum cytokines. Serum samples were collected from 4- to 15-
week-old Pias1 null mice and their wild-type littermates, and serum cytokine levels
(TNF-?, IL-1?, IFN-?, and IL-4) were measured by enzyme-linked immunosor-
bent assay (ELISA) as instructed by the manufacturer (Biosources, Camarillo,
PIAS1 specifically interacts with the p65 subunit of NF-?B
in vivo. STAT and NF-?B are two important families of tran-
scription factors activated by cytokines. Upon ligand stimula-
tion, both STAT and NF-?B translocate from the cytoplasm
into the nucleus, where they bind DNA and activate transcrip-
tion of specific genes. We explored the possible involvement of
PIAS1 in the regulation of NF-?B signaling.
To test whether PIAS1 can interact with NF-?B in vivo,
human 293T cells were transiently transfected with expression
constructs encoding Flag-PIAS1 and the p65 subunit of NF-
?B, alone or together. Thirty hours posttransfection, cells were
either left untreated or treated with TNF-? for 15 min, and
whole-cell lysates were utilized in coimmunoprecipitation as-
says using an anti-p65 antibody. After extensive washing, the
immunoprecipitates were subjected to sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis followed by West-
ern blotting using an anti-Flag antibody. When both p65 and
Flag-PIAS1 were overexpressed in 293T cells, PIAS1 was
coimmunoprecipitated by anti-p65, indicating that PIAS1
and p65 interact in vivo (Fig. 1A, top panel, lane 5). This
interaction was not affected by TNF-? treatment. The proper
expression of Flag-PIAS1 and p65 was confirmed by Western
blot analysis of the same lysates (Fig. 1A, middle and bottom
panels). To validate the specific p65-PIAS1 interaction, coim-
munoprecipitation assays were carried out with 293T lysates
overexpressing both Flag-PIAS1 and p65, using rabbit IgG,
anti-p65, or an antibody against E2F-1, an irrelevant transcrip-
tion factor, as a negative control. As shown in Fig. 1B, Flag-
PIAS1 was immunoprecipitated only by anti-p65 but not by
anti-E2F-1 or rabbit IgG. These results indicate that PIAS1
interacts with NF-?B p65 in vivo.
To test whether PIAS1 interacts with other subunits of NF-
?B, 293T cells were transiently transfected with Flag-PIAS1,
NF-?B p65, or NF-?B p50 alone or Flag-PIAS1 together with
one of the NF-?B subunits. Coimmunoprecipitation assays
were performed as described above using the antibodies spe-
cifically recognizing p65 or p50. PIAS1 interacts with p65 but
not the p50 subunit of NF-?B (Fig. 1C, top panel, lanes 3 and
6). The proper expression of each component was confirmed
by Western blot analysis (Fig. 1C, middle and bottom panels).
These results revealed that PIAS1 specifically interacts with
the p65 subunit of NF-?B in vivo.
p65 contains a transcriptional activation domain and a Rel
homology domain. To examine the PIAS1 interaction region of
p65, 293T cells were transiently transfected with Myc-PIAS1
together with Flag-p65(1–313) or Flag-p65(299–551) followed
1114 LIU ET AL.MOL. CELL. BIOL.
FIG. 1. PIAS1 specifically interacts with the p65 subunit of NF-?B in vivo. (A, top panel) Human 293T cells were transiently transfected with
either Flag-PIAS1 or p65 alone or together, as indicated. Thirty hours posttransfection, cells were either untreated or treated with TNF-? for 15
min. The whole-cell lysates were subjected to coimmunoprecipitation (IP) with anti-p65 (Santa Cruz Biotechnology) followed by Western blotting
(WB) with anti-Flag (Sigma). (Middle and bottom panels) The same lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and probed with anti-Flag or anti-p65 as indicated. (B) The same procedure described for panel A was performed except that 293T
cells were transiently transfected with both Flag-PIAS1 and NF-?B p65, and coimmunoprecipitation assays were performed with rabbit IgG,
anti-p65, or anti-E2F-1 (Santa Cruz Biotechnology) followed by Western blotting with anti-Flag (top panel). The same filter was reprobed with
anti-p65 or anti-E2F-1 (middle two panels). The same lysates were also analyzed by Western blotting with anti-Flag to show equal amounts of
Flag-PIAS1 present in each sample (bottom). (C) The same procedure described for panel A was performed except that 293T cells were transfected
with either Flag-PIAS1, p65, or p50 alone or together, as indicated, and coimmunoprecipitation assays were performed with anti-p50 (lanes 1 to
3) or anti-p65 (lanes 4 to 6) (top). The same lysates were probed with anti-p50, anti-p65, or anti-Flag, as indicated (middle and bottom panels).
(D) The same procedure described for panel A was performed except that Myc-PIAS1 and Flag-p65 wt, Flag-p65(1-313), or Flag-p65(299-551) was
used as indicated. (E) The same procedure described for panel A was performed except that Myc-p65 and Flag-PIAS1 wt or different mutants were
used as indicated.
by coimmunoprecipitation analysis with anti-Flag. PIAS1 was
found to interact with Flag-p65(299–551) but not Flag-p65(1–
313). Thus, the C-terminal region of p65 containing the tran-
scriptional activation domain is responsible for binding to
PIAS1 (Fig. 1D).
Similar coimmunoprecipitation analysis was performed to
identify the p65 interaction region of PIAS1. 293T cells were
transiently transfected with Myc-p65 together with various de-
letion mutants of PIAS1. The region encoding amino acid
residues 89 to 344, which is located between the PIAS1 SAP
(SAF-A, Acinus, PIAS) domain and the RING domain, was
found to be sufficient for interacting with p65 (Fig. 1E).
PIAS1 inhibits NF-?B-mediated gene activation. To study
the effect of PIAS1 on NF-?B-mediated gene activation, lucif-
erase reporter assays were carried out in human 293T cells. Cells
were transiently transfected with a luciferase reporter construct
containing two copies of the NF-?B binding site together with
increasing amounts of Flag-PIAS1. Twenty-four hours posttrans-
fection, cells were either left untreated or treated with TNF-? for
6 h, followed by luciferase assays. As shown in Fig. 2A, TNF-?
treatment leads to an approximately 100-fold increase in the en-
dogenous NF-?B-mediated gene activation, which was inhibited
by PIAS1 in a dose-dependent manner (Fig. 2A, left panel). Sim-
ilar results were observed in human A549 lung cancer cells (Fig.
2A, right panel). The proper expression of Flag-PIAS1 in both
cell lines was confirmed by Western blot analysis (Fig. 2A, bottom
panels). These results suggest that PIAS1 is an inhibitor of the
NF-?B signaling pathway.
To further confirm the role of PIAS1 in the regulation of the
NF-?B signaling pathway, we examined the effect of PIAS1 on
the activation of the endogenous NF-?B downstream genes in
response to TNF-? stimulation. We established a TET-off
PIAS1 cell line (Utf-PIAS1) from human osteosarcoma U2OS
cells, where the expression of PIAS1 is induced in the absence
of DOX, an analog of TET (24) (Fig. 2B). To examine the
effect of PIAS1 on the transcriptional activation of endogenous
NF-?B-dependent genes, Utf-PIAS1 cells growing in the pres-
ence or absence of DOX for 12 h were either left untreated or
treated with TNF-? for 1 or 3 h. Total RNA was collected and
subjected to Northern blot analysis using a cDNA probe of
FIG. 2. PIAS1 inhibits TNF-?-induced NF-?B-dependent gene activation. (A) Luciferase (Luc.) reporter assays. Human 293T cells (left) or
lung cancer A549 cells (right) were transiently transfected with luciferase reporter construct 2?NF-?B together with increasing amounts of
Flag-PIAS1. Twenty-four hours posttransfection, cells were either untreated (gray bar) or treated with TNF-? for 6 h (black bar), and cell lysates
were subjected to luciferase assays. Shown is the average of results from three independent experiments. (B) Inducible expression of Flag-PIAS1
upon removal of DOX in Utf-PIAS1 cells. Lysates (10 ?g of protein/lane) from Utf-PIAS1 cells grown in the presence or absence of DOX for 12 h
were subjected to Western blot analysis using anti-Flag and antitubulin antibodies. (C) PIAS1 blocks the endogenous NF-?B-inducible genes in
response to TNF-? stimulation. Utf and Utf-PIAS1 cells growing in the presence or absence of DOX for 12 h were either untreated or treated
with TNF-? for 1 or 3 h. Total RNA (10 ?g/lane) was subjected to Northern analysis using a cDNA probe of Bfl1, I?B?, or GAPDH. Shown is
a representative of three independent experiments.
1116LIU ET AL.MOL. CELL. BIOL.
Bfl-1 or I?B?, two known NF-?B downstream genes (17, 20).
Quantitative analysis indicated that the induction of PIAS1 by
DOX removal in Utf-PIAS1 cells inhibited the transcriptional
activation of Bfl-1 by approximately 60% in response to 3 h of
TNF-? treatment, while DOX treatment had no significant ef-
fect on Bfl-1 induction in Utf control cells (Fig. 2C). Similarly,
the TNF-?-mediated transcriptional activation of I?B? was
significantly repressed by PIAS1. These results are consistent
with those of the luciferase reporter assays and suggest that
PIAS1 acts as an inhibitor of NF-?B-mediated gene activation.
Enhanced NF-?B-mediated gene activation in Pias1 null
cells. To validate a role of PIAS1 in the regulation of NF-?B,
we examined whether NF-?B-mediated gene activation is al-
tered in Pias1 null cells. BMMs from Pias1 null mice and their
wild-type littermates were untreated or treated with TNF-? for
various time periods (0 h, 30 min, 1 h, and 3 h). Total RNA
isolated from these cells was used for the analysis of transcrip-
tional activation of several known NF-?B-dependent genes by
Q-PCR (38). The induction of I?B?, IL-1?, Mip2 (macrophage
inflammatory protein 2), Irf1 (interferon regulatory factor 1),
FIG. 3. Enhanced expression of NF-?B-regulated genes in response to TNF-? and LPS in Pias1 null cells. (A) Q-PCR analyses of BMMs from
wild-type (black bars, ?/?) or Pias1 null (gray bars, ?/?) littermates, untreated or treated with TNF-? for various times. Genes are indicated at
the top left of each panel. Shown is a representative of results from three independent experiments. (B) The procedure described for panel A was
carried out except that wild-type and Pias1 null primary MEF cells were used. Shown is the average of results from three independent pairs of MEF
cells. (C) Microarray analyses of genes affected by PIAS1. Total RNA from wild-type or Pias1 null BMMs untreated or treated with TNF-? for
30 min (left) or LPS for 1 h (right) was subjected to microarray analyses. TNF- or LPS-induced genes are defined as having at least twofold in-
duction over untreated samples. PIAS1-affected genes are defined as having at least 1.3-fold higher induction in Pias1 null cells than in wild-type cells.
VOL. 25, 2005INHIBITION OF NF-?B BY PIAS1 1117
and Junb by TNF-? was significantly increased in Pias1 null
cells compared with wild-type controls (Fig. 3A). Interestingly,
the induction of other NF-?B target genes, including Cxcl10,
Nos2, and Mcp1, was not significantly affected in the absence of
PIAS1 (Fig. 3A). These data suggest that PIAS1 displays spec-
ificity in the regulation of NF-?B target genes. Similarly, the
TNF-?-induced activation of the IL-1? or TNF-? genes, but
not Mcp1, was significantly enhanced in Pias1 null primary
embryonic fibroblasts (Fig. 3B).
To further examine the specificity of PIAS1 in the regulation
of NF-?B signaling, BMMs from Pias1 null cells and their
wild-type control cells were either untreated or treated with
TNF-? for 30 min or LPS for 1 h. Total RNA isolated from
these cells was subjected to microarray analysis. Under these
conditions, 65 genes were induced at least twofold after TNF-?
stimulation for 30 min, 48% of which showed at least 1.3-fold
more induction in Pias1 null cells than wild-type cells. Simi-
larly, 98 genes were induced at least twofold after 1 h of LPS
stimulation, but only 22% of them were affected by PIAS1 (Fig.
3C and Table S1 in the supplemental material). These data
indicate that PIAS1 selectively affects the induction of a sub-
group of TNF-? or LPS-induced genes.
Elevated proinflammatory cytokine production in Pias1 null
mice. The induction of IL-1? and TNF-? genes by TNF-?
treatment was significantly enhanced in Pias1 null cells (Fig. 3),
consistent with a known important role of NF-?B in the reg-
ulation of proinflammatory cytokine gene expression. To di-
rectly determine the cytokine levels in mice under physiologi-
cal conditions, serum samples were prepared from Pias1 null
mice and their wild-type littermates and subjected to ELISAs.
The levels of two proinflammatory cytokines, IL-1? and TNF-
?, were elevated in Pias1 null mice 6-fold and 3.5-fold, respec-
tively (Fig. 4). In contrast, serum levels of IFN-? and IL-4, two
cytokines not directly regulated by the NF-?B pathway, were
not significantly altered in Pias1 null mice.
Normal T- and B-lymphocyte development and enhanced
granulopoiesis in Pias1 null mice. Given a potential role of
PIAS1 in cytokine-activated signaling pathways, we analyzed
whether Pias1 null mice have defects in lymphocyte develop-
ment. PIAS1 protein is normally expressed in the spleen and
thymus. Flow cytometric analyses were carried out with cells
from spleens and thymuses of 4- to 8-week-old Pias1 null mice
and their wild-type littermates. The CD4/CD8 profiles of the
thymocytes and the B220/IgM profiles of the splenocytes were
similar for these mice (Fig. 5A), suggesting that both T and B
lymphocytes developed normally in the absence of PIAS1.
The NF-?B pathway regulates the proliferation and differ-
entiation of granulocytes. In fact, I?B? null mice exhibited
enhanced granulopoiesis (5). When splenocytes were stained
with the granulocyte-specific marker Gr-1, an increased pop-
ulation of mature granulocytes was reproducibly observed in
Pias1 null mice compared to that in the wild-type controls (Fig.
5B). It has been implicated that granulocyte colony-stimulating
factor (G-CSF), a key cytokine regulated by the NF-?B path-
way, plays an important role in the production of granulocytes
(5). Therefore, we determined the RNA levels of G-CSF in
wild-type and Pias1 null mice. Total RNA isolated from the
thymus of the wild-type and Pias1 null mice was subjected to
Q-PCR analysis using specific primers for murine G-CSF. As
shown in Fig. 5C, the relative expression of G-CSF was in-
creased in Pias1 null thymus compared to that of the wild-type
littermates. In contrast, the RNA levels of GM-CSF were not
altered in Pias1 null thymus. Similar observations have been
described for I?B? null mice (5). These results further support
the role of PIAS1 in the negative regulation of NF-?B in vivo.
PIAS1 has no effect on the activation and nuclear translo-
cation of NF-?B. To understand how PIAS1 regulates NF-?B
signaling, we examined whether PIAS1 affects the activation
and nuclear translocation of NF-?B p65. The localization of
the endogenous PIAS1 and p65 was examined by immunoflu-
orescence analyses. Wild-type (6?/?) and Pias1 null (7?/?)
mouse embryo fibroblasts (MEFs) were either untreated or
treated with TNF-? for 15 min and then stained with anti-p65
and anti-PIAS1 antibodies simultaneously. In wild-type MEFs,
NF-?B p65 resided in the cytoplasm of untreated cells and
translocated into the nucleus upon TNF-? stimulation, which is
consistent with published results. In contrast, PIAS1 remained
in the nucleus with or without TNF-? stimulation. The colo-
calization of p65 and PIAS1 was observed in the nucleus after
TNF-? stimulation (Fig. 6A). In Pias1 null MEFs, anti-PIAS1
did not reveal specific nuclear staining as observed in Pias1?/?
cells, which validates the specificity of the anti-PIAS1 antibody
(Fig. 6A). Most importantly, the nuclear translocation of p65
appeared normal in Pias1 null MEFs, suggesting that PIAS1
does not affect the nuclear translocation of NF-?B p65 in
response to TNF-?. Similar results were also obtained in wild-
FIG. 4. Elevated serum cytokine levels in Pias1 null mice compared to their wild-type littermates. Serum samples were collected from 4- to
15-week-old Pias1 null mice (open circles) and their wild-type littermates (filled circles). Serum IL-1? (n ? 9), TNF-? (n ? 6), IFN-? (n ? 6), and
IL-4 (n ? 5) levels were determined by ELISA (Biosources). P values were determined by paired t test. The average of cytokine levels in each group
is indicated by a dash.
1118LIU ET AL.MOL. CELL. BIOL.
type and Pias1 null BMMs untreated or treated with LPS (data
To further examine the effect of PIAS1 on the activation and
nuclear translocation of NF-?B, cytoplasmic and nuclear ex-
tracts of Pias1?/?and Pias1?/?BMMs untreated or treated
with TNF-? or LPS for various times were prepared, followed
by Western blot analysis using anti-I?B? or anti-p65. In
Pias1?/?BMMs, I?B? was degraded in the cytoplasm with the
concurrent translocation of p65 into the nucleus upon stimu-
lation (Fig. 6B). In Pias1?/?BMMs, I?B? degradation and p65
translocation were normal compared to the wild-type controls
(Fig. 6B, compare lanes 2 to 6 and 8 to 12). Thus, PIAS1 does
not affect the signaling events leading to the nuclear translo-
cation of p65.
FIG. 5. Flow cytometric analysis of the hematopoietic cells from Pias1 null mice and their wild-type littermates. (A) Normal T- and B-lymphoid
development in Pias1 null mice. Cells from the thymus and spleen of 6-week-old mice were stained with anti-CD4 and anti-CD8 (thymus) or
anti-B220 and anti-IgM (spleen) and analyzed by flow cytometry. The percentages of cells within defined regions are indicated. Data are
representative of three separate analyses. (B) Enhanced granulopoiesis in Pias1 null mice. The same splenocytes as in panel A were stained with
the granulocyte-specific marker Gr-1 followed by flow cytometric analysis. Data are representative of three separate analyses. (C) Increased
expression of G-CSF in Pias1 null thymus. Total RNA from the thymuses of Pias1 null mice and their wild-type littermates (n ? 6) was subjected
to Q-PCR analyses.
VOL. 25, 2005INHIBITION OF NF-?B BY PIAS11119
PIAS1 blocks the DNA binding activity of NF-?B. We ex-
amined the effect of PIAS1 on the DNA binding activity of
NF-?B in vitro by EMSA. Nuclear extracts from MCF-7 cells
untreated or treated with TNF-? were analyzed by EMSA
using an NF-?B binding site as the probe. TNF-? treatment
induced the formation of a specific shift band, which represents
the NF-?B p50-p65 heterodimer, since it was specifically su-
pershifted by anti-p50 or anti-p65 antibody but not by anti-E2F
or rabbit IgG (Fig. 7A). To test the effect of PIAS1 on the
DNA binding activity of NF-?B, GST-PIAS1 protein was pre-
pared and used in EMSA. The addition of purified GST-PIAS1
protein inhibited the DNA binding activity of NF-?B p50-p65
in a dose-dependent manner (Fig. 7B, lanes 6 to 8). As a
control, the addition of the same amounts of GST protein had
no effect on the DNA binding activity of NF-?B (Fig. 7B, lanes
3 to 5). Under similar conditions, GST-PIAS1 does not affect
the DNA binding activity of SP-1 (Fig. 7C), supporting the
conclusion that the effect of GST-PIAS1 on the DNA binding
of NF-?B is specific.
To test the hypothesis that PIAS1 inhibits the NF-?B-medi-
ated gene activation by blocking the DNA binding activity of
p65 in vivo, we used a Gal4-p65 fusion protein, which can
activate the luciferase reporter constructs carrying either the
Gal4-binding site or the NF-?B binding site. When human
293T cells were transiently transfected with the Gal4-p65 ex-
pression construct, the 5?Gal4 reporter containing five copies
of the Gal4 binding site and increasing amounts of PIAS1,
PIAS1 showed no inhibition on Gal4-p65-mediated gene acti-
vation (Fig. 7D). In contrast, when the 2?NF-?B reporter
construct was used in the luciferase assays, PIAS1 inhibited the
transcriptional activity of the Gal4-p65 fusion protein (Fig.
7D). These results support the conclusion that PIAS1 inhibits
NF-?B-mediated transcription by blocking the DNA binding
activity of NF-?B p65.
To further examine whether PIAS1 has the ability to regu-
late the DNA binding activity of p65 in vivo, we performed
ChIP assays to examine the binding of p65 to the promoters of
endogenous NF-?B-regulated genes. Protein extracts from
wild-type and Pias1 null BMMs untreated or treated with LPS
for 20 min or 1 h were immunoprecipitated with anti-p65 or
anti-IgG. The bound DNA was quantified by Q-PCR analysis
using specific primers (Fig. 8A). The binding of p65 to the
endogenous I?B? promoter upon LPS stimulation was signif-
icantly enhanced in Pias1 null cells. Similar analysis was also
performed with protein extracts prepared from the TET-off
PIAS1 cell line (Utf-PIAS1). The induction of PIAS1 in the
absence of DOX significantly repressed the binding of p65 to
the promoter of I?B? in response to TNF-? treatment (Fig.
8B). These results further support the conclusion that PIAS1
inhibits the activity of NF-?B by interfering with the recruit-
ment of p65 to the promoters of NF-?B-regulated genes.
In this paper, we examined the role of PIAS1 in the regu-
lation of NF-?B signaling. We showed that PIAS1 interacts
with the p65 subunit of NF-?B by coimmunoprecipitation as-
says. We demonstrated that the ectopic expression of PIAS1
inhibits NF-?B-mediated gene activation. Consistently, the in-
duction of a subset of NF-?B-mediated genes by TNF-? and
LPS is significantly enhanced in Pias1?/?cells. Taking together
the biochemical and genetic results, we suggest that PIAS1 is a
novel negative regulator of NF-?B. We further explored the
molecular mechanism of PIAS1-mediated inhibition on NF-
?B. We showed that PIAS1, which is expressed mainly in the
nucleus, does not affect the activation or the nuclear translo-
cation of NF-?B p65. PIAS1 can block the DNA binding ac-
tivity of p65 both in vitro and in vivo. Through analysis of the
Gal4-p65 fusion protein, we showed that the inhibition by
PIAS1 of the transcriptional activity of Gal4-p65 specifically
occurs on the NF-?B binding site but not the Gal4-binding site.
Consistently, ChIP assays indicate that the recruitment of p65
to the promoters of NF-?B-regulated genes is significantly
enhanced in Pias1?/?cells but repressed in PIAS1-overex-
pressing cells. These data suggest that PIAS1 is a physiologi-
cally important negative regulator of NF-?B. PIAS1 inhibits
the transcriptional activity of NF-?B by blocking the DNA
binding activity of NF-?B p65.
In addition to regulating the DNA binding activity of a
FIG. 6. PIAS1 does not affect the activation and nuclear translo-
cation of NF-?B. (A) Immunofluorescence analysis of PIAS1 and
NF-?B p65 in wild-type (6?/?) and Pias1 null (7?/?) MEFs. Cells
were untreated or treated with TNF-? for 30 min followed by costain-
ing with anti-p65 and anti-PIAS1 antibodies. The nucleus is visualized
by the Hoechst DNA dye. (B) I?B? degradation and p65 transloca-
tion were not altered in Pias1 null cells. BMMs from Pias1?/?and
Pias1?/?mice were either untreated or treated with TNF-? (20 ng/ml)
or LPS (10 ng/ml) for various times. Cytoplasmic and nuclear fractions
were prepared and subjected to Western blot analyses using anti-I?B?
or anti-p65 as indicated. The same filters were probed with antiactin to
reveal equal loadings in each lane.
1120LIU ET AL.MOL. CELL. BIOL.
transcription factor, PIAS proteins have also been suggested to
regulate transcription through other molecular mechanisms
(33). For example, PIAS proteins possess SUMO E3 ligase
activity. It has been suggested that PIASy may repress the
transcriptional activity of LEF1 by targeting LEF1 to nuclear
bodies (31). Experiments described in this paper indicate that
the lack of PIAS1 has no effect on the cellular localization of
p65. Similarly, the nuclear translocation of STAT1 in response
to IFNs is not affected in the absence of PIAS1 (23). The
physiological significance of PIAS1 SUMO E3 ligase activity in
the regulation of NF-?B or STAT1 remains to be determined.
NF-?B plays an important role in immune and inflammatory
responses. The dysregulation of NF-?B activity is associated
with a number of human diseases (3, 4, 10, 12). Thus, the
activity of NF-?B must be properly regulated. In the cytoplasm,
the binding of the I?B family of proteins prevents the nuclear
translocation of NF-?B (16). In the nucleus, it has been shown
that Twist-2 can directly bind and repress the transcriptional
FIG. 7. PIAS1 can block the DNA binding activity of NF-?B. (A) Nuclear extracts from MCF-7 cells untreated or treated with TNF-? for 15
min were analyzed by EMSA using an oligonucleotide containing the NF-?B binding site as a probe. The identity of the shift band was examined
by incubating with anti-p65, anti-p50, anti-E2F, or rabbit IgG as indicated. (B) The same extracts from panel A were mixed with increasing amounts
of purified GST (lanes 3 to 5) or GST-PIAS1 proteins (lanes 6 to 8) followed by EMSA analysis. (C) The same procedure as described for panel
B was carried out except that an SP-1 oligonucleotide was used as a probe. (D) Luciferase assays were performed with 293T cells with an expression
construct encoding Gal4 (gray bar) or a Gal4-p65 fusion protein (black bar), increasing amounts of Flag-PIAS1, and either 5?Gal4 reporter (left)
or 2?NF-?B reporter (right). Shown is the average of results from three independent experiments.
VOL. 25, 2005 INHIBITION OF NF-?B BY PIAS11121
activity of NF-?B (35). Interestingly, both I?B and Twist pro-
teins are cytokine inducible, suggesting that they act as nega-
tive feedback loops of NF-?B signaling. In this paper, we showed
that the activity of NF-?B is tightly regulated by PIAS1: either
the increased or the decreased expression of PIAS1 results in
abnormal NF-?B activity. PIAS1 is constitutively expressed in
the nucleus. It is possible that PIAS1 may act as a threshold to
control the strength of NF-?B signaling. Alternatively, the ac-
tivity of PIAS1 may be regulated by TNF-? through an as-yet-
Our results suggest that the endogenous PIAS1 and p65
reside in different compartments in unstimulated cells, and
they become colocalized to the nucleus upon stimulation.
However, we have not been able to detect the endogenous
PIAS1-p65 interaction by coimmunoprecipitation analysis. It is
possible that the PIAS1-p65 complex is not stable under the
coimmunoprecipitation conditions used. Alternatively, only a
small portion of p65 may interact with PIAS1 in vivo. Ligand-
independent interaction between PIAS1 and p65 was observed
when PIAS1 and p65 were coexpressed in 293T cells. This
ligand-independent PIAS1-p65 interaction may result from the
constitutive nuclear localization of p65 when it is overex-
pressed. Under physiological conditions, ligand stimulation is
required to cause the translocation of p65 into the nucleus,
where it interacts with PIAS1. Our results indicate that PIAS1
does not affect the signaling events leading to the nuclear
translocation of p65. Instead, PIAS1 acts in the nucleus to
repress the DNA binding activity of p65. The effect of PIAS1
on the DNA binding activity of p65 in Pias1 null cells was
validated by in vivo ChIP assays. However, an enhanced DNA
binding activity of p65 was not observed in Pias1 null cell
extracts when examined by in vitro EMSA (unpublished ob-
servation). It is possible that under physiological conditions,
the local concentration of PIAS1 and/or the native chromatin
structure of the promoter regions may affect the inhibitory
activity of PIAS1 on NF-?B.
Studies using PIAS1-deficient cells indicate that PIAS1 af-
fects only a subset of TNF-?-mediated gene expression. The
selective effect of PIAS1 on NF-?B-dependent gene expression
is similar to its specific role in STAT1 signaling (23). Interest-
ingly, the transcriptional activation of certain genes requires
the participation of both NF-?B and STAT1. For example,
STAT1 is involved in the induction of Irf1 and Cxcl10 by
IFN-?, while the induction of Irf1 and Cxcl10 by TNF-? re-
quires NF-?B (29, 30). However, Irf1 is a PIAS1-insensitive
gene in response to IFNs (23), whereas the induction of Irf1 by
TNF-? is significantly enhanced in the absence of PIAS1. In
contrast, the induction of Cxcl10 by IFNs, but not by TNF-?, is
PIAS1 sensitive (23). These results support the conclusion that
PIAS1 can independently regulate the transcriptional activity
of STAT1 and NF-?B. The selective effect of PIAS1 on the
regulation of TNF-?-responsive genes is not fully understood
yet. One possible explanation is the redundant role of other
PIAS family members in NF-?B signaling. Interestingly, a po-
tential role of PIAS3 in regulating NF-?B activity has been
proposed (14). Further studies are required to understand
whether PIAS3 or other PIAS members are indeed involved in
the transcriptional regulation of endogenous NF-?B-depen-
We thank J. Black and W. Xie for help on generating constructs.
This work was supported by grants from the NIH (M.A.T., G.C.,
H.W., and K.S.) and the Howard Hughes Medical Institute (H.W.).
B.L. is a special fellow of the Leukemia and Lymphoma Society.
K.A.W. was supported by a Dr. Norman Sprague, Jr., Fellowship and
U.S. Public Health Service National Research Service award GM07185.
FIG. 8. PIAS1 affects the binding of p65 to the endogenous pro-
moters revealed by ChIP assays. (A) The binding of p65 to the I?B?
promoter is increased in Pias1 null cells. ChIP assays were performed
with BMMs from wild-type (?/?) or Pias1 null (?/?) littermates
untreated or treated with LPS (10 ng/ml). p65-coprecipitated DNA
was analyzed by Q-PCR with specific primers against the ?B site in the
murine I?B? promoter (mI?B?). Rabbit IgG was used as a negative
control. (B) The procedure described for panel A was performed
except that extracts used were from PIAS1 TET-off cells in the pres-
ence or absence of DOX for 12 h. Q-PCR was performed with specific
primers against the ?B site in the human I?B? promoter (hI?B?).
1122 LIU ET AL.MOL. CELL. BIOL.
1. Arora, T., B. Liu, H. He, J. Kim, T. L. Murphy, K. M. Murphy, R. L. Modlin,
and K. Shuai. 2003. PIASx is a transcriptional co-repressor of signal trans-
ducer and activator of transcription 4. J. Biol. Chem. 278:21327–21330.
2. Baeuerle, P. A., and V. R. Baichwal. 1997. NF-kappa B as a frequent target
for immunosuppressive and anti-inflammatory molecules. Adv. Immunol. 65:
3. Barnes, P. J., and I. M. Adcock. 1997. NF-kappa B: a pivotal role in asthma
and a new target for therapy. Trends Pharmacol. Sci. 18:46–50.
4. Barnes, P. J., and M. Karin. 1997. Nuclear factor-kappaB: a pivotal tran-
scription factor in chronic inflammatory diseases. N. Engl. J. Med. 336:
5. Beg, A. A., W. C. Sha, R. T. Bronson, and D. Baltimore. 1995. Constitutive
NF-kappa B activation, enhanced granulopoiesis, and neonatal lethality in I
kappa B alpha-deficient mice. Genes Dev. 9:2736–2746.
6. Chen, X., U. Vinkemeier, Y. Zhao, D. Jeruzalmi, J. E. Darnell, Jr., and J.
Kuriyan. 1998. Crystal structure of a tyrosine phosphorylated STAT-1 dimer
bound to DNA. Cell 93:827–839.
7. Chin, A. I., P. W. Dempsey, K. Bruhn, J. F. Miller, Y. Xu, and G. Cheng.
2002. Involvement of receptor-interacting protein 2 in innate and adaptive
immune responses. Nature 416:190–194.
8. Chung, C. D., J. Liao, B. Liu, X. Rao, P. Jay, P. Berta, and K. Shuai. 1997.
Specific inhibition of Stat3 signal transduction by PIAS3. Science 278:1803–
9. Doyle, S., S. Vaidya, R. O’Connell, H. Dadgostar, P. Dempsey, T. Wu, G.
Rao, R. Sun, M. Haberland, R. Modlin, and G. Cheng. 2002. IRF3 mediates
a TLR3/TLR4-specific antiviral gene program. Immunity 17:251–263.
10. Foxwell, B., K. Browne, J. Bondeson, C. Clarke, R. de Martin, F. Brennan,
and M. Feldmann. 1998. Efficient adenoviral infection with IkappaB alpha
reveals that macrophage tumor necrosis factor alpha production in rheuma-
toid arthritis is NF-kappaB dependent. Proc. Natl. Acad. Sci. USA 95:
11. Ghosh, S., and M. Karin. 2002. Missing pieces in the NF-kappaB puzzle. Cell
12. Gilmore, T. D., M. Koedood, K. A. Piffat, and D. W. White. 1996. Rel/NF-
kappaB/IkappaB proteins and cancer. Oncogene 13:1367–1378.
13. Gross, M., B. Liu, J. Tan, F. S. French, M. Carey, and K. Shuai. 2001.
Distinct effects of PIAS proteins on androgen-mediated gene activation in
prostate cancer cells. Oncogene 20:3880–3887.
14. Jang, H. D., K. Yoon, Y. J. Shin, J. Kim, and S. Y. Lee. 2004. PIAS3
suppresses NF-kappaB-mediated transcription by interacting with the p65/
RelA subunit. J. Biol. Chem. 279:24873–24880.
15. Kahyo, T., T. Nishida, and H. Yasuda. 2001. Involvement of PIAS1 in the
sumoylation of tumor suppressor p53. Mol. Cell 8:713–718.
16. Karin, M. 1999. How NF-kappaB is activated: the role of the IkappaB kinase
(IKK) complex. Oncogene 18:6867–6874.
17. Karin, M., and Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination:
the control of NF-?B activity. Annu. Rev. Immunol. 18:621–663.
18. Karin, M., and A. Lin. 2002. NF-kappaB at the crossroads of life and death.
Nat. Immunol. 3:221–227.
19. Le, L. Q., J. H. Kabarowski, Z. Weng, A. B. Satterthwaite, E. T. Harvill, E. R.
Jensen, J. F. Miller, and O. N. Witte. 2001. Mice lacking the orphan G
protein-coupled receptor G2A develop a late-onset autoimmune syndrome.
20. Lee, H. H., H. Dadgostar, Q. Cheng, J. Shu, and G. Cheng. 1999. NF-
kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40
survival signaling in B lymphocytes. Proc. Natl. Acad. Sci. USA 96:9136–
21. Liu, B., M. Gross, J. ten Hoeve, and K. Shuai. 2001. A transcriptional
corepressor of Stat1 with an essential LXXLL signature motif. Proc. Natl.
Acad. Sci. USA 98:3203–3207.
22. Liu, B., J. Liao, X. Rao, S. A. Kushner, C. D. Chung, D. D. Chang, and K.
Shuai. 1998. Inhibition of Stat1-mediated gene activation by PIAS1. Proc.
Natl. Acad. Sci. USA 95:10626–10631.
23. Liu, B., S. Mink, K. A. Wong, N. Stein, C. Getman, P. W. Dempsey, H. Wu,
and K. Shuai. 2004. PIAS1 selectively inhibits interferon-inducible genes and
is important in innate immunity. Nat. Immunol. 5:891–898.
24. Liu, B., and K. Shuai. 2001. Induction of apoptosis by protein inhibitor of
activated Stat1 through c-Jun NH2-terminal kinase activation. J. Biol. Chem.
25. Majeti, R., Z. Xu, T. G. Parslow, J. L. Olson, D. I. Daikh, N. Killeen, and A.
Weiss. 2000. An inactivating point mutation in the inhibitory wedge of CD45
causes lymphoproliferation and autoimmunity. Cell 103:1059–1070.
26. Megidish, T., J. H. Xu, and C. W. Xu. 2002. Activation of p53 by protein
inhibitor of activated Stat1 (PIAS1). J. Biol. Chem. 277:8255–8259.
27. Mercurio, F., and A. M. Manning. 1999. NF-kappaB as a primary regulator
of the stress response. Oncogene 18:6163–6171.
28. Moilanen, A. M., U. Karvonen, H. Poukka, W. Yan, J. Toppari, O. A. Jeanne,
and J. J. Palvimo. 1999. A testis-specific androgen receptor coregulator that
belongs to a novel family of nuclear proteins. J. Biol. Chem. 274:3700–3704.
29. Nazar, A. S., G. Cheng, H. S. Shin, P. N. Brothers, S. Dhib-Jalbut, M. L.
Shin, and P. Vanguri. 1997. Induction of IP-10 chemokine promoter by
measles virus: comparison with interferon-gamma shows the use of the same
response element but with differential DNA-protein binding profiles. J. Neu-
30. Pine, R. 1997. Convergence of TNFalpha and IFNgamma signalling path-
ways through synergistic induction of IRF-1/ISGF-2 is mediated by a com-
posite GAS/kappaB promoter element. Nucleic Acids Res. 25:4346–4354.
31. Sachdev, S., L. Bruhn, H. Sieber, A. Pichler, F. Melchior, and R. Grosschedl.
2001. PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1
activity by sequestration into nuclear bodies. Genes Dev. 15:3088–3103.
32. Schmidt, D., and S. Muller. 2002. Members of the PIAS family act as SUMO
ligases for c-Jun and p53 and repress p53 activity. Proc. Natl. Acad. Sci. USA
33. Shuai, K., and B. Liu. 2003. Regulation of JAK-STAT signalling in the
immune system. Nat. Rev. Immunol. 3:900–911.
34. Shuai, K., G. R. Stark, I. M. Kerr, and J. E. Darnell, Jr. 1993. A single
phosphotyrosine residue of Stat91 required for gene activation by interferon-
gamma. Science 261:1744–1746.
35. Sosic, D., J. A. Richardson, K. Yu, D. M. Ornitz, and E. N. Olson. 2003.
Twist regulates cytokine gene expression through a negative feedback loop
that represses NF-kappaB activity. Cell 112:169–180.
36. Tan, J., S. H. Hall, K. G. Hamil, G. Grossman, P. Petrusz, J. Liao, K. Shuai,
and F. S. French. 2000. Protein inhibitor of activated Stat-1 is a nuclear
receptor co-regulator expressed in human testis. Mol. Endocrinol. 14:14–26.
37. Tan, J. A., S. H. Hall, K. G. Hamil, G. Grossman, P. Petrusz, and F. S.
French. 2002. Protein inhibitors of activated STAT resemble scaffold attach-
ment factors and function as interacting nuclear receptor coregulators.
J. Biol. Chem. 277:16993–17001.
38. Zhou, A., S. Scoggin, R. B. Gaynor, and N. S. Williams. 2003. Identification
of NF-kappa B-regulated genes induced by TNFalpha utilizing expression
profiling and RNA interference. Oncogene 22:2054–2064.
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