Identification of GAS-dependent interferon-sensitive target
genes whose transcription is STAT2-dependent but
ISGF3-independent
Melissa M. Brierley
1
, Katie L. Marchington
1
, Igor Jurisica
2
and Eleanor N. Fish
1
1 Department of Cell and Molecular Biology, Toronto General Research Institute, University Health Network, and Department of
Immunology, University of Toronto, ON, Canada
2 Division of Signaling Biology, Ontario Cancer Institute, University Health Network, and Department of Medical Biophysics,
University of Toronto, ON, Canada
The type I interferons (IFN)-a ⁄ b are multifunctional
cytokines that mediate host defense against microbial
challenges, influence both normal and neoplastic pro-
liferation, and modulate innate and adaptive immune
responses [1,2]. The binding of type I IFNs to their
shared cognate receptor, type I interferon receptor
(IFNAR), activates multiple intracellular signaling
cascades that coordinate to trigger both the tran-
scriptional activation and translational modifications
necessary to invoke various biological responses [3,4].
Arguably the most notable of these cascades is the
Janus kinase (Jak)-signal transducer and activator of
transcription (STAT) pathway that regulates the
transcription of numerous IFN-sensitive genes (ISGs).
Upon IFN binding to IFNAR, the receptor-associated
kinases tyrosine kinase 2 (Tyk2) and Jak1 phos-

and independent of ISGF3 but highly dependent on the STAT2 DNA
binding domain. This report is the first analysis of the contribution of the
STAT2 DNA binding domain to IFN responses on a global basis, and
shows that STAT2 is required for the IFN-inducible activation of the full
spectrum of GAS target genes.
Abbreviations
IFN, interferon; ISG, IFN-sensitive gene; ISGF3, IFN-stimulated gene factor 3; ISRE, IFN-stimulated response element; IFNAR, type I
interferon receptor; GAS, gamma-activated sequence; IRF, IFN regulatory factor; BSTVQ, binary tree-structured vector quantization; OPHID,
online predicted human protein interaction database; SOM, self-organizing map; STAT2, signal transducer and activator of transcription 2;
TSS, transcriptional start site.
FEBS Journal 273 (2006) 1569–1581 ª 2006 The Authors Journal compilation ª 2006 FEBS 1569
phosphorylated residues act as recruitment sites for
STAT proteins, whereupon activated Jaks phosphory-
late a single tyrosine residue within the carboxy termi-
nus of the STATs [6,7]. The phosphorylated and
activated STATs form both homodimeric and hetero-
dimeric complexes that translocate to the nucleus and
bind specific DNA sequences within the promoter
regions of ISGs to initiate transcription [8].
An important IFN-inducible complex is IFN-stimu-
lated gene factor 3 (ISGF3), comprised of STAT1,
STAT2 and IRF-9 (a member of the IFN regulatory
factor [IRF] family) [9]. Upon nuclear import, ISGF3
binds to the IFN-stimulated response element (ISRE)
present in the promoter regionsof a subset of IFN-
inducible genes and triggers transcription. As well as
ISGF3, type I IFNs induce the formation of additional
STAT-containing complexes, including STAT1–1,
STAT3–3 and STAT5–5 homodimers as well as
STAT3–1 and STAT2–1 heterodimers [10–12]. Rather

STAT2 molecules. In that study, cells bearing the V453I,
V454I (VV-II) mutation (U6A-2VV-II cells) that com-
promises the STAT2 DNA binding domain, exhibited
intact ISRE-mediated transcriptional activation but
impaired GAS-mediated transcription [14]. To precisely
determine the transcriptional target genes of ISGF3-
independent STAT2-containing complexes, cDNAs
from IFN-treated cells overexpressing either intact
STAT2 (U6A-2 cells) or the VV-II mutant form of
STAT2 (U6A-2VV-II) were hybridized to an Affymetrix
DNA microarray containing over 22 000 unique tran-
scripts. By comparing the IFN-inducible gene expres-
sion profiles of these cells, we identified a subset of
GAS-dependent ISGs whose activation is exclusively
regulated by ISGF3-independent STAT2-containing
complexes.
Results
ISG expression in the absence of the STAT2 DNA
binding domain as revealed by DNA microarray
We used DNA microarray analysis to compare the
gene expression profiles of U6A (STAT2-deficient),
U6A-2 (intact STAT2), and U6A-2VV-II (mutant
STAT2 lacking the DNA binding domain) cells treated
with 5 ngÆmL
)1
IFN-alfacon-1 for 6 h. Differences in
mRNA expression among these groups (normalized to
untreated controls) were evaluated using the Affyme-
trix U-133A GeneChip microarray and genespringÒ
software. As expected, IFN treatment of U6A cells

To more quantitatively examine the expression of IFN-
regulated genes in the absence of the STAT2 DNA
binding domain, we treated U6A, U6A-2 and U6A-
2VV-II cells with IFN-alfacon-1 for 6 h and analyzed
gene expression using relative quantitative real-time
PCR. We also carried out MatInspector analyses (see
below) of the upstream promoter regions of IFN-regu-
lated genes to determine the presence of ISRE, GAS
and additional regulatory sequences. Among the genes
selected for examination were PKR, 2¢5¢OAS and Mx1;
genes whose products are known mediators of the
IFN-inducible antiviral response [16]. Comparable
transcriptional activation of the PKR, 2¢5¢OAS and Mx
genes was observed in IFN-stimulated U6A-2 and
U6A-2VV-II cells, and the promoters of all three genes
contained the expected ISRE elements (Fig. S1A–C).
These data support our previous findings that ISGF3
activation is intact in U6A-2VV-II cells (above and
[14]). Moreover, while the promoter regions of PKR
and 2¢5¢OAS also contain potential GAS-like elements,
ISGF3-independent STAT2-containing complexes
dependent on a functional STAT2 DNA binding
domain do not appear to play an important role in
mediating the transcription of these genes.
The c-fos gene was examined in this system as an
example of a GAS-driven gene whose IFN-inducibility
is independent of both ISRE and STAT2. Our analysis
confirmed previous findings [21] that the c-fos
promoter contains a single GAS-like element but not
an ISRE. As well, we found that STAT2 expression

35
20
8
351
120
51
35
18
8
1 10 100 1000
Number of Genes Activated upon IFN Stimulation
U6A
VV-II
U6A-2
> 20.0 fold
> 10.0 fold
> 6.0 fold
> 4.0 fold
> 2.0 fold
> 1.5 fold
Fig. 1. IFN-inducible transcriptional activation in the absence of the STAT2 DNA binding domain as determined by Affymetrix DNA microarray
analysis. Total mRNA samples from U6A-2, U6A-2VV-II and U6A cells either left untreated or treated with IFNa for 6 h were applied to
Affymetrix U-133A microarray gene chips. Hybridization data from the IFN-treated samples were normalized to the data from the correspond-
ing untreated samples. The normalized gene expression profiles for each cell category were analyzed as described in Experimental proce-
dures to determine the number and expression level of genes up-regulated following stimulation with IFN. A total of 286 genes were
induced by IFN to a greater than two-fold increase over untreated controls in the absence of the STAT2 DNA binding domain (VV-II).
M. M. Brierley et al. ISGF3-independent STAT2-dependent GAS genes
FEBS Journal 273 (2006) 1569–1581 ª 2006 The Authors Journal compilation ª 2006 FEBS 1571
results indicate that, although STAT2 is required for
IFN-inducible GBP1 expression, ISGF3-independent

217502_at IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) – 56.1 84.8
204747_at IFN-induced protein with tetratricopeptide repeats 4 (IFIT4) – 41.1 58.6
219211_at Ubiquitin specific protease 18 (USP18) – 40.3 17.7
206553_at 2¢5¢ oligoadenylate synthetase 2 (OAS2) – 31.8 9.0
213797_at Viperin – 30.7 50.6
218943_s_at RNA helicase (RIG-I) – 22.4 23.1
205483_s_at IFN-stimulated protein, 15 kDa (ISG15) – 19.0 38.6
202869_at 2¢5¢ oligoadenylate synthetase 1 (40–46 kDa) (OAS1) – 15.2 13.9
203882_at IFN-stimulated transcription factor 3 gamma (ISGF3G ⁄ IRF9) – 13.5 14.9
213293_s_at Tripartite motif-containing 22 (TRIM22) – 13.3 7.1
214453_s_at IFN-induced, hepatitis C-associated microtubular aggregate
protein (44 kDa) (MTAP44)
– 12.6 14.1
204994_at Myxovirus (influenza) resistance 2 (MX2) – 11.1 12.3
218986_s_at Hypothetical protein FLJ20035 – 10.8 7.0
206271_at Toll-like receptor 3 (TLR3) – 10.2 –
214022_s_at IFN induced transmembrane protein 1 (9–27) – 9.5 7.4
202411_at IFN alpha-inducible protein 27 (IFI27) – 8.5 16.8
204415_at IFN alpha-inducible protein (6–16 or G1P3) – 7.5 22.4
219691_at Hypothetical protein FLJ20073 – 7.0 8.1
208747_s_at Complement subcomponent C1s, alpha- and beta-chains – 6.1 9.5
202270_at Guanylate binding protein 1, interferon-inducible (GBP1) – 5.7 10.0
204533_at Small inducible cytokine subfamily B (Cys-X-Cys) 1.9 5.2 4.5
219209_at Melanoma differentiation associated protein-5 (MDA5) – 5.1 5.5
208392_x_at IFN-induced protein 75, 52 kDa (IFI75) – 5.0 5.3
220104_at Hypothetical protein FLB6421 – 5.0 3.9
207571_x_at Basement membrane-induced gene (ICB-1) – 4.2 11.7
219417_s_at Similar to IFN-induced protein 35 – 4.1 6.1
210797_s_at 2¢5¢ oligoadenylate synthetase-related protein p30 (OASL) – 3.8 5.4
206767_at RNA binding motif, single stranded interacting protein 3 (RBMS3) – 3.8 –

complexes. The BTSVQ algorithm sorts data into binary
trees based on equality of expression of each mRNA
target [26]. Samples having progressively dissimilar
levels of target gene expression are placed further down
the tree. The data are then visualized by the means of
self-organizing maps (SOMs; see below) to cluster genes
into distinct units having similar expression levels.
When the total gene expression profiles of untreated
and IFN-treated U6A, U6A-2 and U6A-2VV-II cells
were analyzed using BTSVQ, the resulting binary tree
showed that cells expressing intact STAT2 segregated
from the U6A and U6A-2VV-II cells at the first level
(Fig. 2). Interestingly, the data suggested that the gene
U6A UT U6A T VV-II UT U6A-2 TU6A-2 UTVV-II T
Level 1
Child 1 Child 2
Level 2 U6A UT U6A T VV-II T VV-II UT U6A-2 TU6A-2 UT
Child 3 Child 4Child 1Child 2
Level 3 U6A UT U6A T VV-II T
Child 4Child 3
Level 4
U6A UT U6A T
Child 6Child 5
U6A UT U6A T VV-II UT U6A-2 UTVV-II T
Samples
Areas representing
genes not expressed
Areas representing
expressed genes
Index

Identification and characterization of a subset of
ISGF3-independent STAT2-dependent target
genes
To identify those genes whose expression was
exclusively regulated by ISGF3-independent STAT2-
containing complexes, we directly compared the gene
expression profiles of the IFN-treated U6A-2 and
U6A-2VV-II cells shown in Fig. 3. Using SOM exam-
ination and the BTSVQ program, we were able to
extract a list of 19 differentially expressed transcripts
that were highly expressed in the IFN-treated U6A-2
sample but absent from the IFN-treated U6A-2VV-II
sample (Table 2). Nine of these transcripts represented
genes encoding well-characterized proteins with defined
functions. The remaining 10 transcripts represented
either hypothetical proteins or proteins with unknown
functions. When genespringÒ was employed to calcu-
late the fold-increase in expression of these genes upon
IFN treatment, we found that each of these mRNAs
was up-regulated about 20–60-fold in IFN-treated
U6A-2 cells compared to IFN-treated U6A-2VV-II
cells (Table 2).
To investigate the promoters of the nine known dif-
ferentially expressed ISGs, we sequenced a region 1000
bases upstream from the transcriptional start site
(TSS) of each gene and searched for the presence
of various STAT-binding GAS elements and ISGF3-
binding ISREs. While three of the genes under study
contained potential ISREs, all exhibited potential
STAT-binding elements with the GAS-like palindromic

4
6
8
10
12
14
16
BF CLDN4 DGKE MSR1
Relative fold induction U6A-2T vs VV-IIT
Fig. 3. Characterization of the induction levels of a subset of
ISGF3-independent STAT2-dependent ISGs identified by BSTVQ.
The differential expression of four of the ISGs examined in Fig. 4
was assessed in IFN-stimulated U6A, U6A-2 and VV-II cells using
relative quantitative real-time PCR as for Fig. 2. For each sample,
b-actin was evaluated as a reference gene and used for normaliza-
tion. For each gene, data are presented as the fold-increase in
expression in IFN-treated U6A-2 cells compared to IFN-treated
U6A-2VV-II cells. Values ± SE were calculated using
RELATIVE QUANTI-
FICATION
software (Roche) and are the mean of three separate react-
ions, each performed in triplicate.
ISGF3-independent STAT2-dependent GAS genes M. M. Brierley et al.
1574 FEBS Journal 273 (2006) 1569–1581 ª 2006 The Authors Journal compilation ª 2006 FEBS
CLDN4, BF and DGKE contain GAS elements and no
ISREs (Fig. S2), both elements are present in the
upstream region of MSR1. This result suggests that
IFN-inducible, ISGF3-dependent (as well as ISGF3-
independent) STAT2-containing complexes make a con-
tribution to the regulation of MSR1 gene expression.

Claudin 4 (CLDN4) Integral membrane protein and component of tight strand junctions 52.2
Macrophage scavenger receptor 1 (MSR1) Macrophage-specific trimeric integral membrane glycoprotein 42.7
Jun D proto-oncogene (JUND)
a
Component of the AP1 transcription factor complex, role in regulation
of transcription from Pol II promoter
35.0
Desmin (DES) Muscle-specific class II intermediate filament, implicated in
cytoskeleton organization and biogenesis
24.8
Interleukin 20 receptor, alpha (IL-20RA) Receptor for interleukin 20 (IL-20), a cytokine that may be involved
in epidermal function.
24.6
Peptidyl-prolyl cistrans isomerase
NIMA-interacting 1-like (PIN1L)
May be involved in organization of the synaptic cell–cell junction
through interaction with the delta-catenin ⁄ NPRAP-N-cadherin complex
24.3
Neuromedin B receptor (NMBR) Binds neuromedin B, a potent mitogen and growth factor for normal
and neoplastic lung and for gastrointestinal epithelial tissue. Involved
in G-protein signalling
24.3
Diacylglycerol kinase, epsilon (64 kDa) May be involved mainly in the regeneration of phosphatidylinositol (PI)
from diacylglycerol in the PI-cycle during cell signal transduction.
Role in ATP binding and diacylglycerol kinase activity (DGKE)
24.0
B-factor, properdin (BF) Complement factor B, a component of the alternative pathway of
complement activation
22.5
Hypothetical ⁄ unknown (10)

STAT2 with that of cells expressing the mutated
STAT2, we have identified a subset of GAS-driven
target genes that are selectively regulated by ISGF3-
independent STAT2-containing complexes. We are
confident that our stepwise approach to analyzing our
microarray data has produced results in which bias
has been minimized, ensuring that our results are bio-
logically relevant. We first established full gene expres-
sion profiles of main subgroups of individual cells
responding to IFN treatment. This unsupervised clus-
tering step was followed by identification of the most
differentially regulated genes. Finally, these genes were
validated by real-time PCR and placed into the context
of IFN-related biological pathways.
Target gene binding by STAT complexes is
determined by DNA motif sequence specificity. STAT
homo- or heterodimeric complexes recognize and bind
promoter sequences containing the GAS-like palin-
dromic core motif, TTNNNNN(N)AA. Although the
preferential GAS element for the STAT2–1 heterodi-
mer is ATTTCCCGGAAA [18], the STAT2–1 complex
can also bind to the GAS elements within the promo-
ters of both IRF1 (ATTTCCCCGAAA) and FccRI
(ATTTCCCAGAAA) [28]. While the close conserva-
tion of these three elements suggests a highly con-
served binding motif, other binding site studies have
suggested that STAT2-containing complexes can bind
to sequences that are distinct from canonical ISRE
and GAS elements [18,29]. Thus, there is a degree of
promiscuity in binding to various DNA motifs that

PROLIFERATION
MSR1
lipoprotein uptake
MAPKs
IL-20Rα
DES
mitochondrial
structural integrity,
intracellular signaling
DES
nuclear shape
DES
gene expression
regulation
TLR3
PI3K
NMBR
PIN1L
proliferation
regulator of mitosis
extracellular
cytoplasm
nucleus
Fig. 4. ISGF3-independent STAT2-dependent ISGs in a signaling context. Schematic representation of potential pathway interactions
between known IFN signaling effectors and factors whose expression was found to be regulated by ISGF3-independent STAT2-containing
complexes.
ISGF3-independent STAT2-dependent GAS genes M. M. Brierley et al.
1576 FEBS Journal 273 (2006) 1569–1581 ª 2006 The Authors Journal compilation ª 2006 FEBS
study, the 5¢ flanking regions of the identified ISGF3-
independent STAT2-dependent ISGs contained GAS-

proliferation [38].
It is less obvious how other genes identified in Table 2
are related to IFN-mediated activities, as none has been
previously characterized as an ISG. Nevertheless, the
case can be made for several of these genes to be linked
to different aspects of IFN biology. For example,
although its precise function remains unknown, the
alpha chain of the IL-20 receptor, IL-20RA, mediates
the signaling of cytokines that are involved in immune
regulation and inflammatory responses, namely IL-19,
IL-20, IL-24 and IL-26 [39–43]. Therefore, IFN regula-
tion of IL-20RA will affect various aspects of the innate
and adaptive immune response. DGKE encodes a diacyl-
glycerol kinase that influences the diacyglycerol-protein
kinase C pathway [44], associated with CLDN4 assem-
bly and membrane integrity [45]. As suggested above,
IFN regulation of CLDN4 may be associated with
antiproiferative activity. DES encodes a filamentous
protein involved in cytoskeletal organization and the
control of nuclear shape and has also been implicated
in intracellular signaling and the regulation of gene
expression [46,47]. How other genes, such as PIN1L,
MSR1 and NMBR, might function as ISGs is currently
a matter of speculation.
As well as the nine known genes cited above, our
BSTVQ comparison of the gene expression profiles of
IFN-treated U6A-2 and U6A-2VV-II cells revealed an
additional 10 differentially expressed transcripts that
encode proteins with unknown functions. It remains to
be determined how these transcripts influence the bio-

) was provided by L Blatt
(Intermune, Brisbane, CA).
RNA preparation for Affymetrix microarray
analysis
To prepare total cellular RNA, cells were either left untreated
or treated with 5 ngÆmL
)1
IFN-alfacon-1 for 6 h at 37 °C.
Cell pellets were lysed and homogenized using Qiagen (Mis-
sissauga, ON, Canada) QIA-shredder columns and RNA
isolation was performed using the Qiagen RNeasy mini-kit
according to the manufacturer’s protocol. The preparation
of cDNAs, sample hybridization and scanning of HG-U-133
A GeneChip
Ò
Arrays (Affymetrix, Santa Clara, CA, USA)
was performed at the Centre for Applied Genomics Micro-
array Facility (Hospital for Sick Children, Toronto, ON) in
accordance with procedures established by Affymetrix.
Microarray data analysis
Raw microarray data were normalized and analyzed using
both the genespringÒ version 6.1 (Silicon Genetics, Santa
M. M. Brierley et al. ISGF3-independent STAT2-dependent GAS genes
FEBS Journal 273 (2006) 1569–1581 ª 2006 The Authors Journal compilation ª 2006 FEBS 1577
Clara, CA, USA) and binary tree-structured vector quanti-
zation (BTSVQ) software programs [26,48]. Analysis using
the genespringÒ program was performed as follows: (a)
raw microarray data were first normalized according to
default settings to ensure per chip normalization; (b) data
were filtered to exclude raw data readings lower than 80;

PCR
Cells were either left untreated or treated with 5 ngÆmL
)1
IFN-alfacon-1 for 6 h at 37 °C. Cells were lysed and homo-
genized using Qiagen QIA-shredder columns and RNA
isolation was performed as described above. cDNAs were
synthesized using 1 lg RNA in the presence of random
primers and AMV Reverse Transcriptase (Promega, Madi-
son, WI, USA) for 1 h at 42 °C.
Components for real-time PCR were obtained from the
LightCycler
Ò
FastStart Plus DNA Master SYBR Green I
kit (Roche). The LightCycler
Ò
instrument (Roche, Missis-
sauga, ON, Canada) and relative quantification soft-
ware were used for all reactions. PCR reactions were
performed in a final volume of 20 l L containing 0.5 lm of
each primer and 5 lL template cDNA (concentration
100 ngÆlL
)1
). The primer sets used are listed in Table S1.
Standard curves were established for each primer set and
reference (b-actin) and target reactions were performed in
triplicate for each sample.
Promoter analysis
The 5¢ flanking sequences were obtained from the NCBI
Entrez Gene database ( />query.fcgi?db ¼ gene). Promoters were assessed for poten-
tial STAT-binding sites using the gene2promoter and

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Supplementary material
The following material is available for this article
online:
Table S1. Primers used for real-time PCR.
Fig. S1. Characterization of the promoter sequences
and induction levels of a subset of ISGF3-independent
STAT2-dependent ISGs identified by microarray. Left
panels: The indicated genes were differentially induced
in U6A-2, U6A-2VV-II and U6A in response to IFN
and were selected for promoter analysis. Sequenced
1000 bases 5¢ upstream from the transcriptional start
site (TSS) as identified by the gene2promoter pro-

nodes in the graph and edges correspond to interac-
tions. To aid interpretation, we highlight the identified
proteins as triangles, interferon related proteins as
ovals, and hubs (highly connected components within
the network) as rectangles. All other proteins are rep-
resented as small circles. Color of individual nodes cor-
responds to gene ontology [Ashburner M, Ball CA,
Blake JA, Bolstein D, Butler H, Cherry JM, Davis
AP, Dolinski K, Dwight SS, Eppig JT, et al. (2000)
Gene ontology: tool for unification of biology. The
Gene Ontology Consortium. Nat Genet 25 25–29], as
shown on the legend. Most of the proteins fall into cel-
lular fate and organization, followed by uncharacter-
ized proteins.
This material is available as part of the online article
from
M. M. Brierley et al. ISGF3-independent STAT2-dependent GAS genes
FEBS Journal 273 (2006) 1569–1581 ª 2006 The Authors Journal compilation ª 2006 FEBS 1581