of June 13, 2013.
This information is current as
cis-Acting Enhancer in Macrophages
Induction via a
Stress to Augmented IFN-
XBP-1 Couples Endoplasmic Reticulum
Judith A. Smith
Ling Zeng, Yi-Ping Liu, Haibo Sha, Hui Chen, Ling Qi and
2010; 185:2324-2330; Prepublished online 21
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cites 46 articles
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The Journal of Immunology
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The Journal of Immunology
XBP-1 Couples Endoplasmic Reticulum Stress to Augmented
IFN-b Induction via a cis-Acting Enhancer in Macrophages
Ling Zeng,* Yi-Ping Liu,* Haibo Sha,†Hui Chen,†Ling Qi,†and Judith A. Smith*
Perturbation of the endoplasmic reticulum (ER) results in a conserved stress response called the unfolded protein response (UPR).
Macrophages undergoing a UPR respond to LPS with log-fold increased production of IFN-b, a cytokine with diverse roles in
innate and adaptive immunity. In this study, we found that thapsigargin-induced ER stress augmented recruitment of IFN
regulatory factor-3, CREB binding protein/p300, and transcriptional machinery to the murine ifnb1 promoter during LPS
stimulation. Although full synergistic IFN-b production requires X-box binding protein 1 (XBP-1), this UPR-regulated transcrip-
tion factor did not appreciably bind the ifnb1 promoter. However, XBP-1 bound a conserved site 6.1 kb downstream of ifnb1, along
with IFN regulatory factor-3 and CREB binding protein only during concomitant UPR and LPS stimulation. XBP-1 physically
associates with p300, suggesting a mechanism of multimolecular assembly at the +6.1 kb site. Luciferase reporter assays provide
evidence this +6 kb region functions as an XBP-1–dependent enhancer of ifnb1 promoter activity. Thus, this study identifies
a novel role for a UPR-dependent transcription factor in the regulation of an inflammatory cytokine. Our findings have broader
mechanistic implications for the pathogenesis of diseases involving ER stress and type I IFN, including viral infection, ischemia-
reperfusion injury, protein misfolding, and inflammatory diseases.
macrophages and NK cells, promote T cell survival and dendritic
kines (1). Cells of the innate immune system, such as macrophages
and dendritic cells, produce type I IFNs upon detection of patho-
gens through pattern recognition receptors that include the TLRs
(2). These pattern recognition receptors bind conserved motifs
found on pathogens, such as LPS (TLR4), dsRNA (TLR3 and
RIG-I), and hypomethylated CpG DNA (TLR9). TLRs may also
mediate responses to “endogenous” products released during tis-
sue necrosis, such as hyaluronic acid, heparin sulfate, fibrinogen,
and heat shock proteins (3).
IFN-b appears to be the primary cytokine that mediates
macrophage type I IFN responses to the TLR4 agonist LPS (4).
IFN-b-deficient animals were shown to be much more susceptible
to lethal sepsis from several strains of pathogenic bacteria,
The Journal of Immunology, 2010, 185: 2324–2330.
ype I IFNs (IFN-a/b) play diverse roles in adaptive and
innate immune responses. Although they were first noted
for their antiviral properties, type I IFNs also activate
presumably through weakened host inflammatory responses (5).
Mice deficient in IFN-b are also more susceptible to particular
viral infections, have lower numbers of macrophages and mature
B cells, and exhibit reduced bone mass (6–8).
The regulation of IFN-b transcription in the setting of viral
infection has been well studied. Briefly, in the uninfected cell,
a nucleosome obstructs the 1+ start site, preventing transcription.
During infection, a group of transcription factors, including NF-kB,
AP-1, IFN regulatory factor (IRF)-7 and IRF-3 cooperatively as-
semble over a 55-bp stretch of DNA, between 2102 and 247 bp
upstream of the transcriptional start site (9). This grouping, termed
the “enhanceosome,” recruits histone acetylases, such as CREB
binding protein (CBP/p300), a large flexible transcription coactiva-
tor that may interact simultaneously with multiple transcription
factors (activating transcription factor [ATF]-2, c-Jun, p65, and
IRF-3/7) (10, 11). CBP/p300 thus acts as a signal integrator. Histone
acetylation facilitates the recruitment of chromatin modifiers that
slide the nucleosome off the TATA box start site, thus enabling
transcription (12, 13). Less is known about the induction of IFN-b
transcription following LPS stimulation, although it appears slightly
different. For instance, although viral infection induces recruitment
of IRF-7 to the enhanceosome, LPS-induced IFN-b appears to
depend on IRF-3 rather than IRF-7 (14–16).
Our previous studies have shown that macrophages undergoing
an intracellularstress response called the unfolded proteinresponse
(UPR) respond to LPS and dsRNA with greatly enhanced IFN-b
production (17). The UPR is an adaptive response initiated by
environmental stressors (hypoxia, nutrient deprivation, hypo-
glycemia) or internal derangements (increased protein load, mis-
folding proteins, calcium gradient deregulation) that disrupt endo-
plasmic reticulum (ER) function. When ER function is perturbed,
excess unfolded protein competes with the ER resident proteins,
inositol-requiring enzyme (IRE)-1, protein kinase receptor–like
ER kinase (PERK), and ATF-6, for binding of the folding chap-
erone Ig binding protein (BiP/GRP78). IRE-1 is an endonuclease
that is activated after release of BiP and that cleaves a 26-bp intron
from the X-box binding protein 1 (XBP-1) transcription factor
mRNA. This unusual splicing event removes a premature stop
*Department of Pediatrics, University of Wisconsin School of Medicine and Public
Health, Madison, WI 53792; and†Division of Nutritional Sciences, Cornell Univer-
sity, Ithaca, NY 14853
Received for publication September 16, 2009. Accepted for publication June 10,
This work was supported in part by the Department of Pediatrics at the University of
Wisconsin–Madison, National Institutes of Health KL2 Grants UL1RR025011 and
K08-AI081045, and the American Federation for Aging Research Grant RAG08061.
Address correspondence and reprint requests to Dr. Judith A. Smith, Department of
Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison,
WI 53792. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
reticulum; GCN5, general control nonderepressible 5; HA, hemagglutinin; HMGI,
NT, no ER stress inducer; PERK, protein kinase receptor–like ER kinase; Pol II, RNA
ing RNA; TBP, TATA box binding protein; Tpg, thapsigargin; Tu, tunicamycin; UPR,
unfolded protein response; XBP-1, X-box binding protein-1.
by guest on June 13, 2013
codon through frame shifting the open reading frame, thus allow-
ing for the translation of the full-length XBP-1 transcription fac-
tor. Upon release of BiP, PERK transiently inhibits global protein
translation apart from selected transcripts (e.g., ATF-4). Finally,
ATF-6 leaves the ER and traffics to the Golgi, where it is pro-
cessed to an active form. UPR target genes aimed at resolving ER
stress include folding chaperones and proteins that aid in ER-
associated protein degradation. If these and other adaptations fail,
the UPR results in apoptosis (18).
The UPR appears to play a physiologic role in highly secretory
cells, such as pancreatic acinar cells, hepatocytes, and plasma
cells (19). However, the UPR has also been implicated in such di-
verse pathologic processes as cardiovascular disease, ischemia-
reperfusion injury, neurodegenerative diseases, diabetes, viral infec-
tions, and cancer (20). It is becoming increasingly apparent that the
UPR also plays a role in immune function. For example, the dif-
ferentiation of B cells into plasma cells requires splicing of
XBP-1 (21). XBP-1 deficiency in intestinal epithelial cells leads
to spontaneous enteritis and increased susceptibility to Listeria
(22). Cholesterol-loaded macrophages undergoing a UPR secrete
the inflammatory cytokines TNF-a and IL-6 (23). ER stress leads
to the proteolytic activation of CREBH (processed similarly to
ATF-6), a transcription factor that induces the production of serum
amyloid and C-reactive proteins (24).
light on disease processes in which both UPR and type I IFNs have
been implicated, such as ischemia-reperfusion injury and viral
infections, as well as diseases where they may be related (HLA-
B27–associated spondyloarthritis and inflammatory myopathies)
(25–29). Previous work has supported a critical role for the
UPR-regulated transcription factor XBP-1 in mediating synergistic
molecular mechanism behind the synergy was not clear. We
hypothesized that XBP-1, as a transcription factor, may regulate
IFN-b induction by either a direct or epigenetic mechanism during
ERstress. Inthis study, we demonstrate binding of XBP-1, CBP, and
IRF-3 to a DNA region 6.1 kb downstream of the ifnb1 gene during
these factors at this +6 kb site correlated temporally with increased
recruitment of CBPand IRF-3 to the ifnb1 promoter. Finally, the pres-
ence of the +6 kb site significantly enhanced ifnb1 promoter activity.
Collectively, these data suggest that this newly described region 6 kb
downstream of the ifnb1 gene is a cis-acting XBP-1–dependent
enhancer of IFN-b production that provides a mechanistic link
between ER stress and augmented IFN-b induction. As a broader
consideration, these findings provide an explanation for how ER
stress may drive the pathogenesis of type I IFN-related diseases.
Materials and Methods
Cells, reagents, and stimulations
The RAW 264.7 macrophage cell line (American Type Culture Collection)
was maintained in DMEM/high glucose with 4 mM L-glutamine, sodium
pyruvate (HyClone Laboratories, Logan, UT) and supplemented with 10%
FBS (HyClone Laboratories), 100 U/ml penicillin, and 100 mg/ml strep-
tomycin. C57BL/6 bone marrow macrophages were isolated as previously
described (17); briefly, low-density bone marrow cells from C57BL/6 femurs
were isolated on Histopaque 1083 (Sigma-Aldrich, St. Louis, MO) and
plated for 3 d in non–tissue culture petri dishes in DMEM (as above)
supplemented with 5% M-CSF–containing conditioned supernatant from
CMG-14-12 cells (30). Adherent cells were detached by 10 mM EDTA
and replated in tissue culture dishes with the 5% conditioned supernatant
3 more days prior to stimulation. The University of Wisconsin is accredited
by the American Association of Laboratory Animal Care, and mouse experi-
ments were performed with Institutional Animal Care and Use Committee
oversight and approval. To induce ER stress, cells were pretreated with
10 mg/ml tunicamycin for 6 h, 20 mM 2-deoxyglucose for 6 h, 10 mM
A23187 for 4 h, 1 mM DTT for 2 h, or 1 mM thapsigargin (Tpg) for 1 h,
depending on time required for maximal XBP-1 mRNA splicing. Splicing
was determined by OD of PCR products separated on a 3–4% agarose gel.
Salmonella enteritidis LPS (Sigma-Aldrich) was used at 100 ng/ml. ER
stress agents and LPS were from Sigma-Aldrich, except for DTT (Thermo
Fisher Scientific, Waltham MA). The DMSO vehicle for Tpg and A23187
had no effect on IFN-b mRNA induction (data not shown). Supernatant
IFN-b was quantified by ELISA (PBL InterferonSource, Piscataway, NJ)
after 1 h of Tpg treatment followed by 6 h of LPS treatment.
XBP-1 knockdown and immunoblotting
RAW cells were transfected with 200–300 nM mXBP-1 stealth small in-
terfering RNA (siRNA) or control medium GC content RNA interference
(RNAi) (Invitrogen, Carlsbad, CA; catalog no. 12935-300) by Amaxa
nucleofection (kit V; Lonza, Walkersville, MD). The sequences of the
XBP-1–specific sense and antisense strands were 59-CAGCGCAGACUG-
CUCGAGAUAGAAA-39 and 59-UUUCUAUCUCGAGCAGUCUGCGC-
UG-39. Twenty-four hours posttransfection, cells were stimulated, lysed
with RIPA buffer, and whole cell lysates were resolved by 4–12%
SDS-PAGE (Invitrogen). Nitrocellulose blots (Whatman, Piscataway, NJ)
were probed with anti–XBP-1 (Santa Cruz Biotechnology, Santa Cruz,
CA) or b-actin (Santa Cruz Biotechnology), followed by HRP-conjugated
secondary Ab (Bio-Rad, Hercules, CA), and proteins were visualized by
ECL (Amersham Biosciences, Piscataway, NJ)/film.
Quantitative PCR (qPCR)
RNA was purified with TRIzol (Invitrogen), treated with DNaseI (Invi-
trogen), and then reverse transcribed using random primers (Promega,
Madison, WI). Relative cDNA was quantified by SYBR Green (Bio-Rad),
detected by MyiQ (Bio-Rad), and normalized to 18S rRNA. Primers were
designed using Beacon Design software (Premier Biosoft, Palo Alto, CA)
and are as follows: 18S rRNA: forward, 59-GGACACGGACAGGATTGA-
CAG-39 and reverse, 59-ATCGCTCCACCAACTAAGAACG-39. IFN-b:
forward, 59-ACTAGAGGAAAAGCAAGAGGAAAG-39 and reverse, 59-CC-
ACCATCCAGGCGTAGC-39. ERdj4: forward, 59-GGCAAAGGACAAAG-
AGGCAATGG-39 and reverse, 59-CCTGGCGTGTGTGGAAGTGG-39. IL-
1b: forward, 59-CTCGCAGCAGCACATCAAC-39 and reverse, 59-ACGG-
GAAAGACACAGGTAGC-39. IL-6: forward, 59-CTTCCATCCAGTTGCC-
TTC-39 and reverse, 59-ATTTCCACGATTTCCCAGAG-39. ifnb1 promoter:
forward, 59-AACTGAAAGGGAGAACTGAAAG-39 and reverse, 59-GCA-
AGATGAGGCAAAGGC-39. ERdj4 promoter: forward, 59-AGGGAA-
GGATGAGGAAATCG-39 and reverse, 59-ACTGTTGTTGCCGTTTGG-39.
+6.0 kb site: forward, 59-CGAAGGGAAAGAGAAATGTG-39 and reverse,
59-CTGGAGGTAACTGGTTGC-39. XBP-1: forward, 59-ACACGCTTGGG-
AATGGACAC-39 and reverse, 59-CCATGGGAAGATGTTCTGGG-39.
Chromatin immunoprecipitation (ChIP) was performed as previously de-
scribed (31); briefly, RAW cells were fixed with 1% formaldehyde, lysed,
and sonicated to generate chromatin fragments. After preclearing with
normal rabbit serum (Covance Research Products, Princeton, NJ), immu-
noprecipitations were performed with protein A-Sepharose (Sigma-
Aldrich) coupled to anti-IRF-3, CBP/p300, XBP-1, TFIID/TATA box bind-
ing protein (TBP), and RNA polymerase II (Pol II; Santa Cruz Biotech-
nology). DNA–protein complexes were eluted with 0.1 M NaHCO3, 1%
SDS, cross-links were reversed with 0.3 M NaCl, and protein was degraded
with proteinase K (Promega). Phenol chloroform–extracted DNA samples
were analyzed by qPCR as above. Percentage occupancy was derived by
comparison with input chromatin.
HEK293 T cells were transfected with expression vectors for Flag-tagged
ofectamine 2000 (Invitrogen) (32, 33). Eighteen to 24 h later, cells were
lysed (150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 50 mM Tris [pH
7.5], DTT, protease inhibitor mixture), and lysates were immunoprecipi-
tated with agarose coupled anti-HA or anti-Flag (Sigma-Aldrich). After
washing (20 mM Tris, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100,
10% glycerol, 0.5 mM DTT), samples were boiled and proteins were re-
solved on SDS 6–8% gels. Western blots were probed with anti-HA-HRP
or anti-Flag-HRP (Sigma-Aldrich), developed with ECL substrate (Pierce,
Rockford, IL), and exposed to film.
IFN-b promoter (bp 2330 to +9) and +6 kb sequences (305 bp, 27790207–
27790507 [GenBank]/88161592–88161890 [FASTA]) were cloned from
The Journal of Immunology 2325
by guest on June 13, 2013
RAW 264.7 genomic DNA isolated by a DNeasy kit (Qiagen, Valencia,
CA). XBP-1 binding sites (IRF-proximal TGCAC [D1] or distal TGCA
[D2]) were deleted by PCR with Pfu DNA polymerase (Stratagene, La
Jolla, Ca). These segments were inserted into the pGL3 basic luciferase
reporter plasmid (Promega) using the KpnI/Xho1 and XhoI/BglII sites (re-
spectively) upstream of the luciferase gene. To identify required XBP-1 bind-
ing sites, RAW 264.7 macrophages were transiently transfected with 2 mg of
luciferase reporter and 0.2 mg of Renilla TK (Invitrogen) by Amaxa. Twenty
hours later, cells were stimulated for 8 h. Luciferase activity was detected by
the Dual-Luciferase reporter assay system (Promega) and read on a Synergy
plate reader (BioTek Instruments, Winooski, VT). Transfection efficiency was
normalized to Renilla activity. For comparison of XBP-1s and XBP-1u,
RAW cells were transfected with 1 mg of pCDNA3.1 vector, XBP-1s, or
XBP-1u (provided by Dr. Laurie Glimcher, Harvard School of Public Health,
Boston, MA) plus 1 mg of luciferase reporters using TransIT-Neural trans-
fection reagent (Mirus, Madison, WI) (21). Sixteen hours later, cells were
stimulated with LPS for 7 h. Luciferase activity was detected using luciferase
assay reagent II (Promega) read on a TR717 luminometer (PE Applied Bio-
systems, Foster City, CA). Results were normalized to total protein (BCA
assay; Pierce). All luciferase assay samples were run in duplicate.
Statistical differences between groups of data were determined by a two-
tailed Student t test. All error bars from combined experiments represent
SE of the mean. For representative experiments, error bars represent devi-
ations of duplicate determinations.
ER stress augments LPS-induced IFN-b in macrophages
Our previous studies had shown that pretreatment of primary
macrophages with a commonly used pharmacologic inducer of ER
IFN-b transcription. Similarly, macrophages expressing a mis-
folding protein (HLA-B27) and undergoing a UPR produced more
IFN-b mRNA in response to LPS (17). In this study, Tpg-primed
bone marrow macrophages responded to LPS with log-fold syner-
gistic induction of IFN-b protein (Fig. 1A). Tpg inhibits the
SERCA Ca2+pump, which potently and rapidly induces a UPR
through disruption of the ER–cytosol calcium gradient (34). Be-
cause Tpg could have multiple effects on cell signaling beyond
disruption of the ER, we examined the effect of pretreatment with
other pharmacologic UPR inducers on LPS-induced IFN-b
transcription (Fig. 1B). Pretreatment with these other UPR-
inducing agents also significantly augmented LPS-stimulated
IFN-b gene expression in primary bone marrow macrophages.
IFN-b induction in the absence of LPS was insignificant. Tpg
remained the most potent potentiator of IFN-b transcription. The
∼2 log increase in transcript observed with Tpg pretreatment
correlated well with the observed increase in protein secretion.
In RAW 264.7 macrophages, Tpg rapidly induces XBP-1 splicing
(Fig. 1C), and pretreatment for 1 h maximally increased LPS-
stimulated IFN-b mRNA by 3 h with a return to baseline by 8 h
Tpg amplifies factor recruitment to the ifnb1 promoter
The log-fold increase in IFN-b mRNA and kinetics of synergy
suggested that regulation of IFN-b induction by ER stress occurs
at the transcriptional level. Preliminary studies with actinomycin D
also suggested that ER stress did not prolong IFN-b mRNA half-life
(data not shown). To determine how ER stress affected LPS-induced
recruitment of transcriptional and other regulatory factors to the
ifnb1 gene promoter, occupancy of the ifnb1 promoter was exam-
ined by ChIP. Guided by the kinetics of IFN-b transcriptional
synergy (Fig. 1D), we examined factor occupancy during the first
4 h following LPS stimulation. To ensure maximal sensitivity in
RAW 264.7 macrophages, we used the most potent and rapid
inducer of ER stress, Tpg. Tpg pretreatment resulted in a 2- to
3-fold increase in IRF-3 and CBP occupancy and a 7- to 10-fold
increase in transcriptional machinery recruitment (Pol II and TBP)
compared with stimulation with LPS alone (Fig. 2). Preliminary
evidence suggests a 2- to 3-fold increase in NF-kB (p65) occu-
pancy as well at the 2 h time point. Maximum occupancy occurred
for most factors around 2 h, with a significant decrease in transcrip-
tional machinery occupancy by 4 h, correlating with peak IFN-b
mRNA kinetics. The enhanced transcriptional machinery recruit-
ment also correlated well with the degree of synergistic mRNA in-
duction typically observed in RAW cells (Fig. 1D).
The UPR transcription factor XBP-1 regulates the synergistic
induction of IFN-b in macrophages
Our previous studies examining the different signaling pathways
initiated by the UPR (originating from PERK, IRE-1, and ATF-6)
ER stress. A, Murine bone marrow macrophages were pretreated for 1 h
with Tpg and then stimulated for 6 h with LPS. Results were combined
from two independent experiments. pp = 0.001 vs LPS. B, Murine marrow
macrophages were pretreated with ER stress inducers calcium ionophore
(A23187), tunicamycin, DTT, 2-DG, Tpg, or NT prior to 3 h of LPS
treatment. Relative mRNA expression was determined by qPCR. Bars rep-
resent fold induction of IFN-b mRNA by ER stress pretreatment plus LPS
compared with LPS without ER-stress pretreatment (NT = 1). Results were
combined from three independent experiments. p # 0.05 for other ER stress
inducers versus NT. C and D, RAW 264.7 macrophages were pretreated with
Tpg for 1 h and then with LPS for up to 8 h. Filled boxes indicate LPS only
and open boxes indicate Tpg plus LPS. Percentage of XBP-1 splicing (C)
represents ratio of spliced and spliced plus unspliced PCR products. D, For
fold induction of IFN-b mRNA, results were combined from two
independent experiments by normalizing to LPS-induced IFN-b mRNA at
4 h (=1). Representative experiments for B and D are shown in Supplemental
Fig. 1. pp = 0.003 vs LPS. 2-DG, 2-deoxyglucose; NT, no ER stress inducer;
Synergistic induction of IFN-b in macrophages by LPS and
cupancy of the ifnb1 promoter. RAW 264.7 macrophages were pretreated
with Tpg for 1 h and then stimulated with LPS for the times indicated.
Binding of IRF-3, CBP, TBP, and Pol II to the ifnb1 promoter was detected
by ChIP. Relative factor occupancy compared with input chromatin was
determined by qPCR. Control IgG results were combined for all stimula-
tion conditions (IgG, no symbol). Results were combined from four
(IRF-3, TBP), five (CBP), and two (Pol II) independent experiments.
pp , 0.001 for LPS vs Tpg plus LPS.
ER stress increases transcription factor and machinery oc-
2326 XBP-1 BINDING ENHANCER OF IFN-b TRANSCRIPTION IN MACROPHAGES
by guest on June 13, 2013
had suggested a critical role for the IRE-1–dependent transcription
factor XBP-1: synergy was abrogated in XBP-1 knockout mouse
embryonic fibroblasts and by XBP-1 RNAi knockdown in LPS
receptor expressing 293 cells (17). In this study, to determine
whether XBP-1 was required for synergy in macrophages, we used
two approaches: we initially transfected the RAW cells with
a dominant-negative XBP-1 containing the DNA binding region,
but not the trans-activating region (35). Interestingly, we were
unable to expand macrophages containing this construct, suggest-
ing a role for XBP-1 in macrophage survival. We then knocked
down XBP-1 with siRNA. Transiently transfecting RNAi to
achieve a 4- to 5-fold knockdown of XBP-1 mRNA during stim-
ulation did not have an obvious impact on viability or expression
of the 18S rRNA housekeeping gene (Fig. 3C). XBP-1 RNAi de-
creased both baseline and Tpg-induced XBP-1 protein (Fig. 3A).
As can be seen in Fig. 3B, XBP-1 RNAi decreased synergistic
induction of IFN-b mRNA by an average of ∼70%. The decrease
in LPS-induced IFN-b in the absence of Tpg was not statistically
significant. In comparison, the induction of ERdj4, a known
XBP-1–regulated chaperone, was reduced 80% by XBP-1 RNAi
during combined Tpg and LPS stimulation (35). In our transient
transfection system, XBP-1 knockdown did not impair induction
of IL-1b or IL-6 mRNA (Fig. 3C), suggesting relative specificity
of XBP-1 regulation for the IFN-b cytokine.
XBP-1 binds a site 6.1 kb downstream of the ifnb1 gene during
concomitant Tpg and LPS stimulation
Analysis of the ifnb1 promoter region using TFSEARCH and
TRANSFAC online databases did not reveal any XBP-1 consensus
sites. However, it was possible that XBP-1 recognized a DNA se-
quence that had not yet been described. By ChIP, XBP-1 did not
appear to bind directly to the ifnb1 promoter, although we could
detect strong binding of XBP-1 to the ERdj4 promoter (Fig. 4).
An alternative hypothesis was that XBP-1 binds a regulatory
DNA segment near the ifnb1 gene. Therefore, we analyzed the
175 kb between the single exon ifnb1 gene and its nearest neigh-
bors, ifna14 and Ptplad2 (Fig. 5A). Mouse and human sequences
were submitted to zPicture (zpicture.dcode.org) to search for con-
served regions. We then used TFSEARCH (www.cbrc.jp/research/
db/TFSEARCH) to find described XBP-1 consensus binding sites
within conserved regions: Using an abbreviated UPRE consensus
site (CACG) and core-binding site (ACGT), several candidate
regions were identified at 6.1, 11.5, 18.3, 21, 30.6, 43, and
70 kb downstream of ifnb1 (36, 37). Two sites were predicted to
bind multiple other factors relevant to IFN-b transcriptional at 6.1
kb (AP-1, IRF, NF-kB) and 18.3 kb (IRF, B lymphocyte-induced
maturation protein-1, high-mobility group I protein [HMGI]-Y)
away. By ChIP, only the +6.1 kb site bound XBP-1 unequivocally
(5-fold, p = 1 3 1027) over the IgG immunoprecipitation control.
The conserved DNA sequence of this site, predicted transcription
factor binding sites, and XBP-1 core sites (lower strand is TGCA)
are represented in Fig. 5B. The DNA sequence surrounding
AP-1, IRF, and XBP-1 predicted binding sites was relatively highly
conserved (78% identity over the first 117 bp) compared with the
region as a whole (71% over the whole 234 bp). Interestingly, at the
same time the +6 kb site bound XBP-1, we observed binding of key
ifnb1 enhanceosome components IRF-3 and CBP (Fig. 5C). We
were unable to detect significant binding of NF-kB (p65) or
AP-1 (ATF-2) to the +6.1 kb site during concomitant Tpg and
LPS stimulation (data not shown).
The +6 kb XBP-1 binding site enhances ifnb1 promoter activity
CBP/p300 occupancy has been proposed as a “gene enhancer
signature” (38). Finding CBP bound to the site during concurrent
Tpg and LPS stimulation raised the possibility that the +6 kb site
may be an ER stress-sensitive enhancer of IFN-b induction. To
determine whether this putative enhancer site had any functional
relevance for ifnb1 promoter activity, the ifnb1 promoter and
+6 kb site were cloned into a vector bearing a luciferase
reporter gene. The putative enhancer alone did not induce any
luciferase activity over the vector control in the absence of the
promoter (data not shown). In the absence of Tpg (LPS only), the
+6 kb site augmented promoter activity, consistent with baseline
presence of spliced XBP-1 in RAW macrophages (Figs. 1C, 5D).
Tpg treatment augmented promoter activity in the absence of the
enhancer, consistent with described induction of NF-kB and
MAPK signaling by ER stress (23). However, Tpg treatment
further increased promoter activity in the presence of the
enhancer. When compared with LPS driven promoter activity
alone (no ER stress or enhancer), the presence of the enhancer
and addition of Tpg pretreatment augmented activity by ∼4-fold
in these assays. To determine which conserved XBP-1 binding
in macrophages. A, RAW 264.7 macrophages transfected with 300 nM
control or XBP-1 siRNA (XBP-1i) were treated for 1 h with Tpg and then
for 3 h with LPS. XBP-1 (top) or actin (bottom) was detected by Western
blot. Results are representative of two separate experiments. B, RAW cells
transfected with 300 nM control RNAi or XBP-1i were stimulated as in A,
and relative IFN-b (top) and ERdj4 (bottom) mRNA was determined by
qPCR. Results were combined from two (ERdj4) and three (IFN-b)
independent experiments. pp = 0.029; ppp = 0.021. C, RAW cells
transfected with 200 nM control RNAi (black) or XBP-1i (gray) were
stimulated as in A, and relative expressions of IFN-b, 18S rRNA, IL-1b,
and IL-6 mRNA were determined by qPCR. Results are representative of
three independent experiments.
XBP-1 knockdown decreases synergistic induction of IFN-b
rophages were stimulated as described in Fig. 2, and then ChIP was per-
formed with anti–XBP-1. Relative occupancy of the ifnb1 (left) and ERdj4
(right) promoters was assessed by qPCR by comparison with input sample.
For ERdj4 ChIP, occupancies of control IgG were combined for all stim-
ulation conditions. Results were combined from four (ifnb1 promoter) and
two (Erdj4 promoter) independent experiments.
XBP-1 does not bind the ifnb1 promoter. RAW 264.7 mac-
The Journal of Immunology2327
by guest on June 13, 2013
core site mediated enhancer activity, the IRF consensus-proximal
site and more distal site were deleted (D1, D2, and D1 plus D2,
respectively, Fig. 5D). The D1 deletion reduced activity to the
level seen with the promoter alone, in both single and double
deletion enhancers. Taken together, these data suggest that the
IRF consensus-proximal XBP core sequence is critical for en-
hancer activity of this +6 kb site.
XBP-1s physically associates with CBP/p300 and augments
ifnb1 promoter activity via the +6 kb enhancer
XBP-1 is a CREB family basic leucine zipper transcription factor that
can form heterodimers (e.g., with c-fos and ATF-6) (39, 40). As
a CREB family member, it was possible that XBP-1 interacted with
CREB binding protein CBP/p300. CBP/p300 has been shown to
associate directly with phosphorylated IRF-3 following viral stimu-
lation (41). Thus, an interaction between XBP-1 and CBP/p300
might explain the increased recruitment of both CBP and IRF-3 to
the putative enhancer site in a multimolecular complex during con-
comitant ER stress and LPS stimulation. Spliced XBP-1s encodes the
371-aa ER stress-induced active transcription factor, whereas the
267-aa unspliced XBP-1u has the DNA binding N-terminal domain,
but not the trans-activating C-terminal domain (18). In overexpres-
sion studies (Fig. 6A), XBP-1s, but not XBP-1u, coprecipitated with
p300. Thus, the CBP/p300 coactivator may associate with the active
XBP-1 transcription factor during ER stress. The predicted molecular
mass of the unspliced XBP-1 is ∼30 kDa, so the higher molecular
mass products in lane 2 may represent ubiquitinated protein visual-
ized as a result of the overexpression system (21).
To determine whether XBP-1s or XBP-1u binding regulated en-
hancer activity, RAW 264.7 macrophages were transfected with
XBP-1 expression vectors and the above luciferase reporter con-
structs. XBP-1s (but not XBP-1u) increased ifnb1 promoter activ-
ity in the presence of the +6 kb enhancer element (Fig. 6B).
This increase was abrogated when the IRF-proximal XBP-1
core sequence was deleted (D1 Enh-Pro). In the absence of
XBP-1, deletion of this core sequence also decreased enhancer-
related luciferase activity to the level observed with the promoter
alone, suggesting that the background enhancer activity in the
enhancer site 6.1 kb downstream of ifnb1. A, Genomic
uous genes. Ifnb1 gene: 27796544–27797313 (Gen-
Bank)/88168698–88167929 (FASTA). B, Nucleotide
sequence of the +6 kb site containing base pairs
(FASTA). Conserved nucleotides between mouse and
human are in bold type. Predicted ifnb1 enhanceosome
component binding sites (80–90% consensus identity)
are denotedbygraybox (IRF),dottedline(NF-kB), and
Tpg followed by LPS for the times indicated. Factor
occupancy of the +6.1 kb site was detected by ChIP.
Results were combined from three (XBP-1) and four
(CBP, IRF-3) independent experiments. pp # 0.01.
D, RAW cells were transfected with luciferase reporters
containing the ifnb1 promoter alone or promoter +6 kb
site with no deletions or with deletions of either or
both conserved XBP-1 core binding sites (D1, D2, or
D1 + D2). Cells were stimulated for 1 h with Tpg and/
LPS stimulation (=100%). Results were combined from
two (D1 + D2), three (D1, D2), and six (promoter versus
promoter + 6 kb enhancer) independent experiments.
A sample experiment is shown in Supplemental Fig. 2.
pp = 0.00002; ppp , 0.04.
Identification of an XBP-1–dependent
moter activity. A, Lysates from transfected HEK293 cells were immunopre-
cipitated with anti-Flag and Western blots probed with anti-HA-HRP (top
panel) or anti-Flag-HRP (lower panel). One percent of the input lysate was
probed with HA-HRP (middle panel). Results are representative of three
independent experiments. B, RAW 264.7 macrophages cotransfected with
pCDNA3.1 vector, XBP-1s, or XBP-1u plus luciferase expression vectors
containing either ifnb1 promoter only (Pro), +6 kb site (Enh-Pro), or +D1
(D1 Enh-Pro, Fig. 4D) were stimulated for 7 h with LPS. Results were
combined from four to five independent experiments by normalization to
maximum luciferase activity. p , 0.002 for Pro vs Enh-Pro constructs across
all pCDNA3.1, XBP-1s, and XBP-1u cotransfections. A representative set of
experiments is shown in Supplemental Fig. 3. pp , 0.00001.
XBP-1s associates with CBP/p300 and enhances ifnb1 pro-
2328XBP-1 BINDING ENHANCER OF IFN-b TRANSCRIPTION IN MACROPHAGES
by guest on June 13, 2013
unstimulated conditions reflected baseline ER stress and the
presence of spliced XBP-1 in RAW cells (Fig. 1C). In the LPS-
stimulated conditions, XBP-1s increased enhancer activity by
∼2-fold over baseline ER stress and increased luciferase activity
6- to 7-fold over the promoter alone. Taken together, these data
support a role for the +6.1 kb site as an XBP-1–dependent
enhancer of ifnb1 promoter function.
We have identified an enhancer site 6 kb downstream of the ifnb1
gene that exhibits significantly increased binding of XBP-1 only
during concomitant Tpg and LPS treatment. Furthermore, en-
hancer activity of the +6 kb site was responsive to the active
XBP-1s transcription factor, not XBP-1u, and was dependent on
a predicted IRF-proximal XBP-1 core binding sequence. This +6
kb region also bound the key IFN-b enhanceosome components
IRF-3 and CBP/p300 during concomitant LPS stimulation and ER
stress. The kinetics of XBP-1, CBP, and IRF-3 binding to the
enhancer site showed a striking synchronicity with the increased
recruitment of CBP and IRF-3 to the ifnb1 gene promoter, with
maximal occupancy after 2 h of LPS treatment. The physical in-
teraction between XBP-1 and CBP/p300 (which is known to as-
sociate with phosphorylated IRF-3 following LPS stimulation)
provides a mechanistic explanation for the presence of all of these
factors together at the enhancer site: one could hypothesize the
formation of a multimolecular complex whereby XBP-1 associates
with CREB/p300, which in turn associates with IRF-3, thus allow-
ing for cooperative and synchronous binding of XBP-1 and
IRF-3 to the enhancer. This model reflects the need for both ER
stress and TLR stimulation to promote simultaneous binding. Pre-
cedence for such cooperative assembly may be found at the ifnb1
promoter itself (16).
of the IRF-3–regulated chemokine RANTES (CCL5) by LPS. Also,
XBP-1 RNAi did not affect the induction of the IRF-3–regulated
chemokine IL-8 in TLR4-bearing 293 cells (17). These data would
suggest that Tpg (and by extension the UPR) does not generally
activate all IRF-3–regulated genes and that the effect on IFN-b is
more specific. Indeed, by gene expression microarray in primary
mouse macrophages, the only chemokine or cytokines showing
10-fold or greater synergy during combined Tpg plus LPS stimula-
tion were IFN-b and IL-23 (42).
Looping of chromatin has been proposed as a mechanism that
brings gene promoters and distal regulatory sites into physical ap-
position (43). Following synchronous binding of XBP-1, IRF-3, and
CBP/p300 to the +6 kb region during ER stress and LPS stimula-
tion, the enhancer may then loop around to provide increased CBP
and IRF-3 delivery to the ifnb1 promoter (Fig. 7). Given the co-
operative assembly of factors at the ifnb1 promoter, a small amount
of “extra” IRF-3 (even 2- to 3-fold) early in the sequence could be
greatly magnified during the successive recruitment of histone
modifiers and transcriptional machinery to result in the observed
log-fold synergy. According to this looping enhancer theory, the
enhancer would only come into play during combined ER stress
and LPS stimulation; thus, the comparison between LPS-stimulated
promoter function and enhancer-promoter function during concom-
itant Tpg/XBP-1s plus LPS would be the most relevant. Because
XBP-1 was not detected on the promoter, XBP-1 may dissociate
following looping. The compressed kinetics makes it difficult to
assess this possibility. Otherwise, the cross-linking may have been
insufficient to detect factors indirectly associated with the promoter.
An alternative explanation for the role of XBP-1 in synergy is
that XBP-1 induces an unknown factor that binds the ifnb1 pro-
moter. However, the induction of a negative regulator of IFN-b
transcription by LPS precluded more direct evaluation of this
hypothesis using cyclohexamide (44). The time frame, with 1 h
of Tpg pretreatment sufficient to detect synergy after 2 h of LPS
treatment, would argue against the involvement of a newly
transcribed XBP-1 gene target.
In this study, the kinetics of promoter occupancy following LPS
stimulation was greatly compressed compared with what has been
described for viral infection, with a significant decrease in the
transcriptional machinery by 4 h (12). This decreased promoter
occupancy correlated well with the disappearance of IFN-b mRNA
transcript. Other transcription factors and chromatin modifiers,
besides the ones mentioned in this study, have been reported to
bind the ifnb1 promoter during viral infection. However, we were
unable to detect significant binding (.0.002 occupancy) of IRF-1,
IRF-7, or ATF-2 transcription factors, general control nonderepres-
sible 5 (GCN5) histone acetyltransferase, or the HMGI-Y architec-
tural factor (data not shown) (13, 16). The HMGI-Y DNA binding
protein has been proposed as a chaperone that facilitates and
stabilizes assembly of the enhanceosome, although it is not likely
to be present in the final structure (9, 45). Acetylation of the
HMGI-Y structural protein at Lys71by GCN5 promotes association
of HMGI-Y with enhanceosome components and protects against
destabilization. CBP-mediated acetylation of Lys65decreases the
affinity of HMGI-Y for DNA and destabilizes the enhanceosome
(46). Thus, sequential activity of GCN5 followed by CBP appears to
be critical for sustained transcription. With regard to this study, the
brief duration of transcription machinery occupancy following LPS
stimulation may reflect CBP predominance and insufficient acety-
lation of HMGI-Y by GCN5 activity.
The luciferase results support a functional role for the newly
However, the luciferase assay may greatly underestimate the effect
of the +6 kb enhancer site on promoter activity in situ for the fol-
lowing reasons: (1) The regulation of IFN-b transcription is highly
blocks access of the transcriptional machinery to the TATA box
start site. The orchestrated sequential and cooperative recruitment
of various factors to the ifnb1 promoter culminates in sliding this
nucleosome upstream, thus enabling transcription (13). This event
has been described as a regulatory “on–off” switch for ifnb1
transcription. The luciferase construct would not recapitulate
this nucleosomal sliding event. (2) The 6-kb distance between en-
and optimal orientation of enhancer and promoter. (3) Finally,
there may be a cooperative opening/modification of chromatin by
histone acetylation that is simply not captured in a luciferase-
promoter. In the presence of LPS stimulation alone, IRF-3 and CBP bind to
the ifnb1 promoter (top). When macrophages undergoing ER stress (Tpg
treatment) are stimulated with LPS, XBP-1, IRF-3, and CBP cooperatively
bind the region 6.1 kb downstream of the ifnb1 gene. Through chromatin
looping, the enhancer-bound IRF-3 and CBP factors are delivered to the
multimolecular complex at the ifnb1 promoter, ultimately resulting in
greater recruitment of transcriptional machinery.
Model for XBP-1–enhanced factor recruitment to the ifnb1
The Journal of Immunology2329
by guest on June 13, 2013
regulation would be challenging to recreate in a standard luciferase
The UPR and type I IFN have been separately implicated in
a variety of diseases ranging from viral infections to ischemia-
reperfusion injury, as well as in inflammatory myopathies. There is
evidence linkingthe spondyloarthritis-related
HLA-B27, a molecule shown to misfold and induce a UPR in
a rat model and type I IFN; macrophages derived from the
HLA-B27 transgenic rat show evidence for both an ongoing
UPR and IFN gene signature by microarray (27). Furthermore,
macrophages from the transgenic animals produce enhanced lev-
els of IFN-b in response to LPS compared with wild-type animals
when undergoing a UPR (17). The data presented in this study
provide a mechanistic link between the UPR and augmented
IFN-b. During concomitant ER stress and TLR4 stimulation,
XBP-1 binds a potential enhancer element 6 kb distal to the ifnb1
gene that may enhance recruitment of IRF-3 and CBP/p300 to the
ifnb1 enhanceosome. These findings have significant mechanistic
implications for understanding the pathogenesis of protein mis-
folding and ER stress-related inflammatory diseases.
We thank Dr. Emery Bresnick for helpful advice.
The authors have no financial conflicts of interest.
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2330XBP-1 BINDING ENHANCER OF IFN-b TRANSCRIPTION IN MACROPHAGES
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