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REVIEW OF LITERATURE
3. REVIEW OF LITERATURE
3.1. Discovery of IRF -2:
REVIEW OF LITERATURE
The induction of the IFN-~ gene by a virus is due primarily to transcriptional
activation that requires virus inducible enhancer-like elements of its gene promoter
(Taniguchi et al., 1988). During the study on the regulation of the IFN~ gene, a factor
was found to bind to these elements and was tentatively termed IRF-1 (Taniguchi et
al., 1988). Subsequently, eDNA encoding mouse IRF-1 was cloned and its structure
elucidated (Taniguchi et al., 1988). Subsequently, a eDNA encoding a molecule
structurally related to IRF-1 was isolated by cross hybridization with IRF-1 eDNA
and the molecule was termed IRF-2 (Taniguchi et al., 1989). Infact, the deduced
primary structure of IRF-1 and IRF-2 showed 62% homology in the amino terminal
region, spanning the 1st 154 residues, where as the rest of the molecules showed only
25% homology (Taniguchi et al., 1989). DNA binding site selection studies revealed
that these two factors bind to the same DNA element, termed IRF-E (consensus
sequence: G(A)AAA G/C TIC GAAA G/C T/C)(Taniguchi et al., 1993) which is
almost indistinguishable from the interferon stimulated response element (ISRE;
consensus sequence: A/G NGAAA NNGAAACT) (Stark et al., 1998) activated by
IFN signaling.
3.2. The IRF family of transcription factors
IFNs activate a group of transcription factors called Interferon Regulatory Factors
(IRFs) (Kroger et al., 2002). IRFs have helix-tum-helix (HTH) motif and tryptophan
(w) repeats in their DNA binding Domain (DBD). They regulate transcription of
many mammalian genes induced by cytokines and other agents (Taniguchi et al.,
1999). So, far ten members of the IRF family have been reported, they are IRF-1,
IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, ICSBP (IRF-8), IRF-9/p48/ISGF3y and
IRF-10 and viRF (coded by human herpes virus).
3.2.1. IRF-1 and IRF-2:
The first IRFs, IRF-1 and IRF-2, were identified through their ability to bind to the
positive regulatory domain 1 (PRDI) in the VRE of the IFN-~ gene and were assumed
to function as an activator and repressor of the IFN-~ gene, respectively (Fujita et. al.,
1989). However, homozygous deletion of IRF-1 in mice did not impair activation of
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IFN-a or IFN-P gene in infected MEFs, while dsRNA-mediated induction of Type I
IFN was down regulated (Matsuyama et. al., 1993 and Reis et. al., 1994). Subsequent
studies have revealed that IRF-1 is involved in a broad spectrum of the antiviral
defense mediated by IFN-y. The induction of nitric-oxide synthetase (iNOS),
guanylate binding protein and 2'5'-0AS was impaired in IFN-y treated IRF-1
deficient MEFs (Kamijo et. al., 1994 and Kimura et. al., 1994). Induction of iNOS
and IL-12p35 was also inhibited in IRF-1 null myeloid DC. It was then shown that
IRF-1 is effectively induced by IFN-y and IFN-y stimulated expression of NO
synthetase genes mediated by IRF-1 (Saura et. al., 1999). While IRF-1 does not have
a critical role in the virus stimulation of type I IFN genes, the presence of IRF-1 was
detected in the IFN-P enhanceosome binding to the IFN-P promoter region (Thanos
et. al., 1995) as well as in the IFN-a enhanceosome (Au et. al., 2001). Furthermore,
analysis of the repertoire of lymphoid cells from IRF-1 null mice has shown defects in
the maturation of CDS+ T cells as well as a defective Th1 response, impaired
production of IL-12 in macrophages and defective NK cell development (Duncan et.
al., 1996). These data indicate that IRF-1 has essential functions in the development
and activation of various immune cells. In addition, IRF-1 also plays a critical role in
the inducible expression of MHC class I and apoptosis; cells form IRF-1 deficient
mice are resistant to UV-and drug-induced apoptosis (Reis et. al., 1994).
While both IRF-1 and IRF-2 bind to the PRDI domain in the VRE of the IFN
p gene, this region also binds a protein named B limp-1, which has an important role
in the late stages of the B-cell differentiation (Keller et. al., 1991). It was later shown
that Blimp-1 recognizes the same DNA binding domain as IRF-1 and IRF-2, but not
of IRF-4 and IRF-8 (Kuo et. al., 2004). It would therefore be interesting to examine
whether IRF-1 or IRF-2 have any role in the final stages of B cell differentiation. In
humans, polymorphisms in IRF-1 were shown to be associated with a predisposition
to asthma the pediatric population (Wang et. al., 2006) and deletion of IRF-1 has been
observed in myelodisplastic syndrome and leukemia (Willman et. al., 1993).
L1
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3.2.2. IRF -3 and IRF -7:
While ectopic over-expression of IRF-1 in undifferentiated embryonic stem
(ES) cells stimulated the expression of Type I IFN genes, IRF-1 failed to bind the
VRE of IFN-a. However, mutations that disrupted the IRF binding site in the IFN-a
promoter abolished its inducibility (MacDonald et. al., 1990). The search for a new
IRF, which could activate the promoters of IFN-a and IFN-~ genes led to
identification of IRF-3 and IRF-7. The identification of these two IRFs and their role
in the transcriptional activation of Type IFN genes had a major impact on the
understanding of the molecular mechanism of the pathogen induced innate antiviral
response (Au et. al., 1995, Ronco et. al., 1998, Au et. al., 1998 and Marie et. al.,
1998). It became quite obvious that although pathogen recognition may be mediated
by distinct cellular receptors and signaling pathways, they all lead to the activation of
IRF-3 or IRF-7, which are critical for the transcriptional activation of Type I IFN
genes (Akira et. al., 2006 and Honda et. al., 2006).
IRF-3 and IRF-7 are closely related to each other in terms of their primary
structures (Nguyen et. al., 1997; Mamane et al., 1999 and Taniguchi et al., 2000).
IRF-3 was identified through a search of an EST database for IRF-1 and IRF-2
homologs (Au et al., 1995). IRF-3 was also identified as the component of the virus
inducible dsRNA-activated factor (DRAF1) complex (Weaver et al., 1998).
Subsequently, IRF-7 eDNA was cloned as a factor bind to the Epstein-barr virus QP
promoter region using the yeast one hybrid system (Zhang et al., 1997). The
expression of IRF-7 is ubiquitous (Zhang et al., 1997); however, it is totally
dependent on type 1 IFN signaling. IRF-3 contains an activation domain that includes
the Nuclear Export Signal (NES) and IRF association domain (lAD) (Lin et al.,
1999). Evidence has been provided that this domain is flanked by two autoinhibitory
domain that interact with each other; in virally infected ce!ls, this interaction relieved
by virus induce phosphorylation, thereby unmasking both lAD and DBD (Lin et al.,
1999).
3.2.3. IRF-4 and IRF-8:
IRF-4 and IRF-8 show a high degree of homology. They are expressed
primarily in lymphocytes, macrophages, B cells and DC (Eisenbeis et. al., 1995).
These two proteins demonstrate only a weak DNA binding affinity, which can be
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increased by association with other transcription factors (Marecki et. al., 1999 and
Tailor et. al., 2006). IRF-4 binding is stabilized upon heterodimerisation with the
transcription factor PU .1, and this heterodimer was shown to bind to the lgG enhancer
and activate expression of the immunoglobulin (lg) light chain in B cells (Eisenbeis
et. al., 1995). IRF-4 has also been shown to be a natural antagonist of both IRF-1 and
IRF-5 transactivation. The dominant negative action of IRF-4 was observed on IRF-1-
mediated transactivation of the TRAIL promoter, while the inhibition of IRF-5
activation was due to the completion for binding to MyD88 (Yoshida et. al., 2005).
IRF-8 shares a number of properties with IRF-4; it binds DNA after
interaction with the transcription factors of the IRF family, including IRF-1 and IRF-2
as well as PU.1 and E47 (Politis et. al., 1992). While the IRF-8/IRF-1 complex
generally functions as a suppressor of transcription, the IRF-8/IRF-4 heterodimer
activates transcription of ISG 15 (Meraro et. al., 2002). The IRF-8/IRF-1 complex also
induces numerous genes that are important for macrophage differentiation and
macrophage-induced inflammation (Dror et. al., 2006 and Liu et. al., 2004). IRF-8
null mice develop chronic immunodeficiency and a myelogenous-like syndrome
(Holtschke et. al., 1996). IRF-8 null mice also show major defects in CD8+nC and
pDC and the Flt3L-induced differentiation of mouse bone cells to pDC-like cells is
dependant on IRF-8 (Tamura et. al., 2002 and Tamura et. al., 2005). IRF-8 mice also
displayed increased susceptibility to infection, which was shown to be due to a defect
in Th1immune response and inability to express IL-12 and IRF-8 was consequently
shown to stimulate the transcription of IL-12p40 (Meraro et. al., 1999). The fact that
IRF-8 affects differentiation of the high IFN producing pDC indicates its importance
in the innate antiviral response as well as in TLR-MyD88 antiviral signaling pathway.
3.2.4. IRF -5 and IRF -6:
IRF-5 and IRF-6 is a pair of IRF that show completely different functions.
While IRF-5 seems to have a role in apoptosis and immune response to pathogens,
IRF-6 is a key regulator of the switch from keratinocytes proliferation to
differentiation (Richardson et. al., 2006). IRF-6 null mice are embryonic lethal and
the embryos showed abnormal external morphology; with abnormal skin, short
forelimbs and a lack of ears, hind limbs and tails. These mice have also had abnormal
craniofacial morphogenesis and skeletal defects. The major histological change
detected in these mice was the absence of a normal stratified epidermis as in IRF-6
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null epidermis the keratinocytes did not stop proliferation and failed to differentiate
(Ingraham et. al., 2006). In humans the IRF-6 gene is localized in the critical region
of Van der Woulde syndrome locus. This disorder is associated with an autosomal
dominant form of cleft lip and palate and a nonsense mutation in IRF-6 (Mostowska
et. al., 2005). IRF-6 mutations are also associated with popliteal pterygium syndrome
(PPS) that is characterized by similar orofacial phenotype skin lesions and genital
abnormalities (Kondo et. al., 2002). These unexpected properties of IRF-6 indicate
that IRF may also have a basic role unrelated to the immune response and that IRF
functions may be not limited to the cells of the immune system. Furthermore, the fact
that none of the other IRF null mice are embryonic lethal indicates that there is a
redundancy of some of the IRF functions.
In contrast to IRF-6, IRF-5 has been implicated in the innate inflammatory
response, although its role in the antiviral response has been recently challenged
(Takaoka et. al., 2005). Human IRF-5 eDNA (A Y 504946), cloned from DC, shows
some properties that are distinct from IRF-3 and IRF-7. The IRF-5 polypeptide
contains two nuclear localization signals, whereas IRF-3 and IRF-7 have only one,
and consequently nuclear IRF-5 could be detected in uninfected cells (Barnes et. al.,
2002). The activation and phosphorylation of IRF-5 by viral infection could be
detected in cells infected with NDV or VSV but not in cells infected sendai virus or
treated with poly I:C, indicating that the activation may be virus specific (Barnes et.
al., 2002). While both type I IFN and viral infection stimulate expression of IRF-5
gene (Mancl et. al., 2005), IRF-5 can also induced by the tumor suppressor p53 (Mori
et. al., 2002) suggesting a connection between IRF-5 and p53 induced proapoptotic
pathways (Mori et. al., 2002). Like p53, IRF-5 stimulates the cyclin-dependent kinase
inhibitor p21 while repressing cyclinBl, stimulates the expression of the proapoptotic
genes Bakl, Bax, caspase 8 and DAP kinase 2 (Barnes et. al., 2003), and promotes
cell cycle arrest and apoptosis independently of p53 (Hu et. al., 2006).
3.2.5. IRF -9 (P48/ISGF3y):
IRF-9 plays a major role in the antiviral effect of type I IFN. It is a component
of tertiary complex ISGF3 that is formed in IFN-treated cells and binds to the ISRE
elements of ISG, stimulating their transcription (Veals et. al., 1992, Darnell et. al.,
1994 and Improta et. al., 1994). In this complex, which also contains STAT1 and
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STAT2, IRF-9 is the major DNA binding component. IRF-9 can also form a DNA
binding complex with STAT1 homodimer and with STAT2 alone, with these
complexes binding to DNA with the same specificity as ISGF3 (Kraus et. al., 2003).
MEFs from IRF-9 null mice are deficient in both Type I and Type II responses
(Harada et. al., 1996 and Kimura et. al., 1996). Furthermore the MEFs from these
mice show an impaired induction of IRF-7 expression and inhibition of TNF-a gene
transcription. However, the phenotype of the null mice has not been characterized yet
in detail.
3.2.6. IRF-10:
IRF-10 is most closely related to IRF-4 but differs in both its constitutive and
inducible expression. The expression of IRF-10 is inducible by interferons (IFNs) and
by concanavalin A. In contrast to that of other IRFs, the inducible expression of IRF-
10 is characterized by delayed kinetics and requires protein synthesis, suggesting a
unique role in the later stages of an antiviral defense. Accordingly, IRF-1 0 is involved
in the upregulation of two primary IFN-7 target genes (major histocompatibility
complex [MHC] class I and guanylate-binding protein) and interferes with the
induction of the type I IFNtarget gene for 2', 5'-oligo(A) synthetase. IRF-10 binds the
interferon-stimulated response element site of the MHC class I promoter. In contrast
to that of IRF-1, which has some ofthe same functional characteristics, the expression
of IRF-1 0 is not cytotoxic for fibroblasts or B cells. The expression of IRF-1 0 is
induced by the oncogene v-rel, the proto-oncogene c-rel, and IRF-4 in a tissue
specific manner. Moreover, v-Rel and IRF-4 synergistically cooperate in the induction
of IRF-10 in fibroblasts. The level of IRF-1 0 induction in lymphoid cell lines by Rei
proteins correlates with Rei transformation potential. These results suggest that IRF-
10 plays a role in the late stages of an immune defense by regulating the expression
some of the IFN-7 target genes in the absence of a cytotoxic effect. Furthermore, IRF-
1 0 expression is regulated, at least in part, by members of the Rel!NF-rfB and IRF
families (Nehyba et. al., 2002).
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3.2.7. viRFs:
Kaposi's sarcoma-associated herpes virus (KSHV) is a member of the y herpes
virus family and is genetically similar to EBV and monkey Herpes Virus Saimiri
(HVS) (Moore et. al., 1998). Sequence analysis reveal the presence of about 80 open
reading frames (ORFs) and a number of ORFs showing homology to cellular genes
that regulate cell growth, immune function, inflammation and apoptosis (Moore et.
al., 1998). These include a cluster of four ORFs with homology to the cellular
transcription factors of the IRF family (Cunningham et. al., 2003).
Three v IRFs have been cloned and characterized. The K9-encoded v IRF-1,
expressed in PEL cells treated with TPA, has been studied most extensively and was
shown to inhibit both the virus-mediated induction of Type I IFN genes and ISG, and
its over expression in NIH/3T3 confers tumorigenecity when injected into nude mice
(Burysek et. al., 1999, Flowers et. al., 1998 and Gao et. al., 1997). However targeted
expression of viRF-1 in B cells or endothelial cells failed to induce tumor formation
in transgenic mice (Lubyova and pitha unpublished results).
viRF-2 (ORF K11.1) encodes a small nuclear protein (163 a.a.) that is
constitutively expressed in PEL cells (Burysek et. al., 1999). Unlike the cellular IRF,
viRF-2 binds to an oligodeoxynucleotide corresponding to the NFKB site and
specifically associates with several cellular IRF and with p300 (Burysek et. al., 1999).
In addition, viRF-2 also binds dsRNA-activated protein kinase (PKR), inhibits its
kinase activity and blocks the phosphorylation of the PKR substrate, eukaryotic
translation initiation factor 2a (Burysek et. al., 2001).
viRF-3/LANA2, encoded by ORFs K10.5 and K 10.6 (Lubyova et. al., 2004)
is a multifunctional nuclear protein constitutively expressed in KSHV -positive PEL
and Castleman's disease tumors, but not in kaposi's sarcoma spindle cells (Jenner et.
al., 2001 and Lubyova et. al., 2000). The viRF-3 protein binds to IRF-3, IRF-7 and
CBP/p300 and stimulates IRF-3/IRF-7 mediated transcription of Type I IFN genes
(Lubyova et. al., 2004). Furthermore, interaction of viRF-3 with p53 results in
inhibition of p53- mediated transcriptional activation and p53 induced apoptosis
(Rivas et. al., 2001).
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Table 2. IRF family members: summary of the properties and functions of IRFs.
IRF Expression Inducers of Transcriptional Physiological members Pattern expression role role
Most cell types Type I IFN
IRF-1 Low-level Type II IFN
Activator Tumor
constitutive Viral infection suppressor dsRNA
Oncogene,
Most cell types Type I IFN Repressor/ Antiviral
IRF-2 defense, constitutive Viral infection. Activator
Immune regulation
IRF-3 Most cell types Type I IFN
Activator Antiviral
constitutive Viral infection defense IRF-4/ Pip/ Activated T- PMA Activator/ Immune LSIRF/ Cells HTLV-1 Tax Repressor regulation ICSAT
Type I IFN Activator/repress
IRF-5 Inducible (Taniguchi et Immune system al., 2001)
or
Keratinocytes
IRF-6 Tissue specific
Unknown Unknown proliferation
pattern and differentiation
B-cells and Transcriptional IRF-7 lymphoid Type II IFN Activator activator of
lineages viral promoter Cells of Macrophages Antiviral
IRF-and lymphoid
defense 8/ICSBP
lineages Type II IFN Repressor Immune
Low level regulation
Constitutive inducible
IRF- Most cell types Type II IFN Activator
Antiviral 9/ISGF3y constitutive defense
Induced by v-Later stage of antiviral
Rei defense IRF-10 Tissue specific protoonco gene Activator
Upregulates c-Rel IRF-1
IFNy related genes
(Ref. Adapted from Pitha et. al. 2007)
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3.3. Structure ofiRF-2:
Mutational analysis of IRF-2 revealed that DNA binding activity resides in the
amino-terminal region with strictly conserved amino acids, particularly the 5 times
tryptophan (W) repeats (Taniguchi et al., 1989). The c-terminal of IRF-2 is basic in
nature where as IRF-1 is acidic. Suggesting the distinct function of these factors. IRF-
1 and IRF-2 rnRNA are both expressed in a variety of cell types, and their expression
levels, particularly IRF-1 rnRNA are dramatically unregulated upon viral infection or
IFN stimulation (Taniguchi et al., 1989). A series of eDNA transfection experiment
revealed that IRF-1 can activate IFN ex/~ promoters (Taniguchi et al., 1989). In fact, a
high-level expression of IRF-1 eDNA resulted in the induction of endogenous IFN
cx/~ genes in a variety of cell lines, albeit at low efficiency (Harada et al., 1990).
Unlike IRF-1, IRF-2 had no such effect; rather, it repressed IRF-1 induced
transcriptional activation (Taniguchi et al., 2000). IRF-1 protein is very unstable
(half-life -30 minutes) where as IRF-2 is apparently stable (half life-8 hrs.)
(Watanabe et al., 1991). These initial observations were suggestive that IRF-1 and
IRF-2 function as transcriptional activator and repressor, respectively, for the 1FN-cx/~
genes. Interestingly, evidence has also been provided that IRF-2 functions as a
transcription activator for vascular adhesion molecule -1(VCAM). (Jesse et al., 1998)
and cell cycle regulated Histone H4 genes (Stein et al., 1995).
Crystal structure of IRF-2 DNA binding domain and DNA has been
determined at 2.2 A o resolution. The IRF-2 DNA binding domain consists of four
stranded anti parallel ~ sheets (~1-~4) with three a helices (al-a3) and three long
loops (L1-L3 ). This architecture resembles that of winged helix -tum- helix (HTH)
motif. Compared with those of IRF-1, the residues forming helix a1 of IRF-2 are
::1 shifted by four residues toward the N-terminus. In addition, the residue of strand ~1 ~
are shifted toward the N terminus by one residue and the residue of strand ~4 are ~ "........._ shifted toward the C terminus by two residues. These shift cause differences in the
~ residues of the hydrogen bonded pairs of the~ sheet in addition to differences in loop
structures between the secondary structure element. It is unlikely that these structural
differences between the IRF-1 and IRF-2 DNA binding domains are induced by
crystal packing.
616.079 P8848 Fu
Ill II lllllllll,l/111111111111111 TH15128 21
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All the six IRF-2 domains interact with DNA in a similar manner. One of the
most striking features of the DNA binding is the recognition of a 5' -flanking AA
sequence, which is located 2 bp upstream from the GAAA core sequence that is
recognized by recognition helix a3. This AA sequence is part of another upstream
core sequence, which is also recognized by another DNA binding domain at the major
groove. The recognition is due to the His-40 residue located at loop Ll, which reaches
into the minor groove of the upstream GAAA sequence. His 40 residue forms a
hydrogen bond with a bridging water molecule (WI) between the AA base pair steps
by hydrogen bonding to both the 02 atom of the paired thymine and the N3 atom of
the adenine of the next step. These contacts have not been observed in the crystal
structure of the IRF-1-DNA complex, although His 40 of IRF-1 is located at a
position where it may be form water mediated hydrogen bonds like that in the current
structure. Since His 40 is completely conserved in the IRF family, we believe that a
similar recognition would occur in all members of this family and propose
AANNGAAA as the consensus IRF recognition sequence (IRS) that is physically
recognized by the IRF DNA binding domains.
Contacts with the major groove of the GAAA sequence are localized at the C
terminal half of the recognition helix and are mediated by four residues (Asn-80, Arg-
82, Cys-83 and Asn 86). In the IRF-1- DNA complex, Ser 87, which is variant in the
IRF family, was used to recognize the GAAA core sequence but was missing in the
current complex. Several significant differences are found in the frameworks of
hydrogen bonds and van der Waals contacts between these residues and the core
sequence. These differences are due primarily to an extensive network of water
molecules located within the interface between the recognition helix and DNA. No
significant changes in the position and orientation of the recognition helices were
observed. However, a few side chains in the recognition helix have different
conformations from those of IRF-1, resulting in the differences in DNA recognition.
The side chain of the completely conserved Arg 82 sticks into the major groove to
form two hydrogen bonds with the first guanine of core sequence (GAAA) in a
manner similar to that with IRF-1, but with one of the hydrogen bonds mediated by
water molecule (W2). The recognition of second the adenine (GAAA) is mediated by
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a direct hydrogen bond with the side chain of conserved Cys 83 and water (W3)
mediated hydrogen bonds involving the side chain of conserved Asn 86 (Figure 1).
s' 3'
G 11 L--1 __ -.~II'---------' A1 .__I -----'
i1.__1 ---~~~=--~~-~ H40
R82 L2
N86 K64
3' s'
Figure. 1. Base recognition with water-mediated hydrogen bonds. Schematic
diagram of the protein- DNA contacts in the IRF-2-DNA complexes. Hydrogen
bonding and van der Waals contacts participating in the base recognition are
represented by thin black lines. Hydrogen bond and /or ion pairs to phosphate group
of the DNA backbones are represented by light green lines. The residues marked with
asterisks do not participate in the DNA interactions in some six molecules in crystal.
Similarly, W6 is missing at some of the complex interfaces (Drawn from Ref. Fujii et.
a/., 1999).
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L3
Ll
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Specificity for the third adenine (GAAA) is conferred by a direct hydrogen bond
between Cys 83 and the paired thymine, together with a van der Waals contact
between methyl group of thymine and the side chain of Asp 80. In addition, the
adenine base is hydrated with two water molecules (W4 and W5); this hydration is
part of the hydrogen-bonding network linked to Asn 86. Among the water molecules,
W3 is observed in the IRF-1-DNA complex, but the others are missing. The fourth
adenine (GAA.A) is recognized through a van der Waals contact with the paired
thymine in the IRF-1-DNA complex, although the methyl group contacts with the
main chain carbonyl of Cys 83, instead of the side chain of Ser 87 of IRF-1. The
positions of these side chains (His 40, Arg 82, Ser 87) and the water molecules (W1-
W5) were verified with an omit map (figure 1). It is noteworthy that the recognition
of the fourth AT base pair is poor in comparison with the other base pairs of the core
sequence. We note that direct hydrogen bonds involving the side chains of Arg 82 and
Cys 83 are common in the IRF-2-DNA and IRF-1- DNA complexes.
Several amino acids residues located at all three a helices and three loops plus
strand ~4 contacts with the negatively charged DNA backbone via hydrogen bonds
and ion pairs. The contacts cover the DNA backbones of the 12 bp stretch and involve
the main chain amide groups and the side chain indole rings of tryptophan, as well as
the positively charged lysines and arginines. Interestingly, the conserved Trp 11
located at helix a 1 is buried into the hydrophobic core, whereas in the IRF-1-DNA
complex this residue interacted with phosphate. Arg 82, which is a key residue for the
base recognition, Asn 86, also interact with the 5' -phosphate group of the guanine
nucleotide of the core sequence to form water mediated hydrogen bond together with
Arg 107 of strand ~4. The protein phosphate interactions are localized in two sugar
phosphate backbones forming major groove of the core sequence. These localized
interactions results in a narrowing of the major groove with a local 20° bending of the
DNA helix towards the protein, as observed in the IRF-1-DNA complex.
Superposition of the DNA oligomers in the two complexes gives a relatively small
averaged r.m.s. deviation (0.94 A 0 ) for the corresponding backbone atoms. It is of
particular interest that all six DNA binding domains induce similar DNA distortions at
every GAAA core sequence. Consequently, the helical axis of the continuously
stacked DNA duplexes writhes, due to the tandem binding with each of the 6 bp,
wherein every two neighboring DNA binding Domains induce local bending towards
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the nearly opposite sides. The bending towards the protein enables loop Ll to
approach the minor groove of the 5'-flanking AA sequence.
A metal ion-binding site was found at the C-terminus of the recognition helix
a3 that is connected to a ~ hairpin structure followed by strand ~3. It was assumed
that the ion was potassium ion, since potassium is the only metal cation in
crystallization solution. The average B-factor of the potassium ion was 28.2 A 0 ,
which is comparable with mean B factor of the crystal. The coordination shell is
formed by four main chain carbonyl groups of two residues (Met 85, Asn86) of the
recognition helix and two from the~ hairpin (Leu 88, lie 91). On the current electron
density map, two of the octahedral coordinations are invisible. Surprisingly, a similar
metal ion binding site was found by Clark et. al., (1993) in the DNA binding domain
of HNF-3y, which possesses one of the closely related winged HTH domains with
IRF. In this case, the metal ion was assigned as a magnesium ion. These ions may
play the role of a C-terminal cap that neutralizes the helix dipole by the positive
charge, while also contributing to electrostatic interaction with phosphate group of
DNA. This 'helix-hairpin-strand (HhS) motif, found in the DNA binding sites of
endonuclease III and the related DNA repair enzymes (Thayer et. al. 1995) as well as
human DNA polymerase ~ (Pelletier and Sawaya, 1996). The motif of DNA
polymerase ~ has an affinity for biologically prevalent metal ions in the order K+ >
Na+ > Ca2+ > Mg2+, with the K+ ion displaying the strongest binding. A K+ ion bound
to the site of DNA polymerase ~ has been shown to interact with a phosphate group of
DNA (Pelletier et. al., 1996). The amino acid sequences of the HhH motifs are well
conserved, but are somewhat different from those of the HhS motif. Key residues in
the formation of a hydrophobic core by the HhS motif of IRF-2 are Met, Leu an lie. In
HNF-3y, this core is formed by Leu and a larger side chain of Phe. Remarkably, these
residues are fairly conserved in the HhH motif, indicating that these two motifs are
derivatives of a common 'helix-hairpin motif'.
The proposed IRF recognition sequence (IRS), AANNGAAA, provides a
rational interpretation of various sequences for IRF family members, including IRF-E
and ISRE consensus sequences. Some of the binding sequences that contain the PRDI
of the IFN-~ promoter have a single GAAA core sequences, but all these sequences
are completely endowed with the 5' flanking AA sequence that produces one
complete IRS. This AA sequences is essential for the recognition of a single repeat.
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For instance, IRFs do not bind to the NFKB binding site (PRDII) of the IFN-~
promoter, which contains a GTGGGAAA sequence containing the GAAA core
sequence, but no 5' -flanking AA sequence. Mutation studies also support the role of
5'-flanking AA sequence (Eisenbeis et. al, 1995 and Yee et. al., 1998).
In the IFN-~ enhancer, PRD IV is adjacent to PRD III, whereas no significant
binding cooperativity has been observed between IRF-1 and ATF-2 (Thanos and
Maniatis, 1995). To address this issue, a model of the IRF-PRDIII complex was built
alongside that of the Jun-fos heterodimer (Glover and Harrison, 1995) bound to PRD
IV. Interestingly, the model reveals slight contacts of the bZIP basic region with
flexible loop L3 and the N-terminal region of helix a3 of IRFs, but no contact or
steric clash between these DNA binding domains. This is sharp contrast to the NFAT
Fos-Jun-DNA complex, in which the ZIP regions of both Fos and Jun are in several
contacts with NFAT (Chen et. al., 1998). Recently, it has been shown that HMGI(Y),
architectural protein, binds to the minor groove to bend the DNA toward the major
groove (Huth et. al., 1997). Remarkably, one of the HMGI (Y) binding sites is
overlapped with the 5' repeat of PRD III, at a point where one of the IRF molecules
contacts the minor groove of the 5' -flanking AA sequence of IRS. This dual binding
to the minor groove explains well the results that HMG I(Y) alone slightly inhibits
binding of IRF-1 (Thanos and Maniatis, 1995). Furthermore, we point out that this
HMG I(Y) binding site is tightly sandwiched between the ZIP region and the IRF
DNA binding domain, suggesting that HMG I(Y) may induce binding synergy of the
IRF and ATF-2-c-Jun through contacts with both molecules and DNA distortions. We
also tested the interferences between IRF and NFKB at the PRD I and PRD II sites
using the recently solved crystal structure of p50-p65 heterodimer-DNA (Chen et. al.,
1998). Interestingly, there is no steric clash between IRF-2 and p50-p65 heterodimer
in our model, whereas the N-terminal residues of IRF stick into the interspace
between DNA and the dimerization domain of the p65-p50 heterodimer. These
contacts may result m strong interferences on the DNA binding of these two
transcription factors. It is an interesting question where these transcription factors
positions the activation domains that interact with each other and/or with the other
general transcription factors. Based on the present model, the locations of the C
termini of IRFs, theN termini of ATF-2-c-Jun and the C terminus of p65 suggests that
all regulatory domains of these transcription factors are positioned at the same surface
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REVIEW OF LITERATURE
of the DNA double helix. We note that all the minor grooves of the HMG I(Y)
binding sites are faced toward the solvent region of the opposite DNA surface.
Therefore, the DNA bending by HMG I(Y), towards the major groove of the sites,
might help in juxtaposing the regulatory domains closer together within the IFN-~
enhanceosome. This seems to be consistent with the previous in vitro experiments
(Falvo et. al., 1995).
It has been demonstrated that the endogenous IRFs are complexed with
multiple Histone acetylases, PCAF, GCN5 and CBP/p300 in vivo to bind to the ISRE
and that the Histone acetylases activities are accumulated on the IRF-ISRE complexes
(Masumi et. al., 1999). However, it is only a subset of IRF proteins that have been
shown to interact with histone acetylases, and in tum affect their ability to alter the
chromatin structure of IFN-responsive promoters. PCAF for example, interact only
with IRF-1 and IRF-2 primarily through its C-terminal half lodging the catalytic
domain and the bromodomain. Contact with PCAF by IRF-1 and IRF-2 is brought
about through the first seven amino acids in the conserved DBD. Thus, the DBD of
IRF-1 and IRF-2 shows a dual binding activity in that it could interface ISRE DNA on
one hand and PCAF on the other (Masumi et. al., 1999). Histone acetylases recruited
to the IRF-ISRE complex may bring additional factors to the promoter, since
acetylases appear to occur as a large complex in the cell (Grant et. al., 1997). IRF-2
interacting partners and their function are given in Table 3.
Table 3. IRF-2: its interaction with IRFs and other transcription factors.
S.No. IRF family Interacting factor(s) Target gene References
member
1. IRF-2 IRF-1 CIITA (MHC Xi and Blanck,
class II gene 2003
promoter)
2. IRF-2 IRF-8 and IRF-1 IL-20, p40 Review: R.
pine 2002
3. IRF-2 IRF-1 and IRF-8 Cell mediated Meraro et. al.,
immunity 1999
4. IRF-2 Celtix-1 Cell cycle Staal et. al.,
regulation 2000
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REVIEW OF LITERATURE
5. IRF-2 IRF-2BP1 and IRF- Cell cycle Childs et. al.,
2BP2 regulation 2003
6. IRF-2 P300/CBP Cell mediated Masumi et. al.,
immunity 1999
7. IRF-2 TFIIB Cell mediated Wang et. al.,
immunity 1996
8. IRF-2 IRF-1, viRF-2, p65, Down regulation Burysek et. al.,
p300 of IRF induced 1999
genes Ill HHV-8
infected cells
9. IRF-2 PCAF Type I IFN gene Masumi et.
regulation al., 1999
10. IRF-2 NFKB Regulation of Drew et. al.,
MHC class I and 1995
~-microglobulin
gene regulation
3.4 Functions of IRF -2:
IRF-2 plays diverse roles in vertebrates can be categorized as Cell cycle growth
regulation, immunomodulation, oncogenesis, host defense etc.
3.4.1.Transcriptional repressor: IRF-2 is a potential transcriptional repressor. It
represses the activity of IRF-l.Transcriptional repression by IRF-2 is localized to the
C-terminal. Fusion of this region to the C-terminal end of IRF-1 inhibits
transactivation by IRF-1. A latent activation domain exists in the central region of
IRF-2, which is considered to be silenced by C- terminal repression domain. These
result indicate that IRF-2 is a "mosaic" transcription factor possessing both activation
and repression activity (Yamamoto et al., 1994 ). The exact mechanism by which it
causes repression is not known. One mechanism of repression by IRF-2 involves
inhibition of IRF-1 transactivation by occupying ISRE sequence and subsequently
preventing DNA binding by IRF-1. (Harada et al., 1990; Nguyen et al., 1995).
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REVIEW OF LITERATURE
3.4.2. Transcriptional activator: There is also report of transcription activation by
IRF-2. Transcriptional activation domain is resided in between DNA Binding Domain
and Repression domain (154 -324 amino acids residue). There are at least three
reports that IRF-2 acts as a transcriptional activator. Firstly, human H4 gene found to
be directly activated by IRF-2 through binding to cell cycle element (CCE) present in
the H4 gene promoter (Vaughan et. al., 1995). This histone gene may play a role in
IRF-2 mediated oncogenesis, since it is functionally coupled to DNA replication and
cell cycle progression at G 1 to S transition. Secondly, QP promoter region of the
Epstein Bar Virus encodes EBNA-1 gene has been shown to be activated by IRF-2
and IRF-1 (Nonkwelo et al., 1997). Moreover, IRF-2 found to be involved in the
activation of Vascular Cell Adhesion Molecule -1 (VCAM-1) (Jesse et al., 1998).
Thus, IRF-2 is a dual transcription factor possess both activation as well as repression
property (Yamamoto et al., 1994).
3.4.3. Oncogenic potential of IRF-2: Unlike IRF-1, IRF-2 functions as a
transcriptional attenuator, critical in balancing IFN action in vivo (Hida et al., 2000).
In NIH3T3 cells, the overexpression of IRF-2 causes oncogenic transformation and
concomitant constitutive expression of IRF-1 causes these cells to revert to the non
transformed phenotype (Harada et al., 1993). Although the exact mechanism
underlying this cell transformation is still unknown, it is possible that IRF-2 exerts its
oncogenic function via the mediation of IRF-1 and other IRF family members that
bind to the same IRF-E. This possibility is supported by the finding that NIH 3T3
cells expressing only the DNA-binding domain of IRF-2 were also transformed
(Nguyen et al., 1995). On the other hand, IRF-2 activates gene (s) involved m
oncogenesis, such as Histone 4 (Vughan et. al., 1995 ; Vughan, et. al., 1998).
3.5.Physiological Role of IRF -2
3.5.1. Role of IRF -2 transcription factor in signaling:
Ligand induced activation of type I and type II receptors, IFNGR, results in the
activation or induction of similar transcription factors the IRFs and STAT families
IFNAR stimulation results the activation of Janus family protein tyrosine kinases,
29
c y
T 0 p
L A s M
N u c L E u s
REVIEW OF LITERATURE
IFN-J3 ~ IFN-y ~
l GAF/AAF
>
ISRE IRF-E
v ~AS IRF-1 gene
I
Fig. 2. IFN signaling and IRF-2 (Ref. Modified from Taniguchi et. a/. , 2001 )
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REVIEW OF LITERATURE
Tyk2 and Jakl, which are associated with sub components of IFNARl and IFNAR2,
respectively. This activation of followed by the site specific tyrosine phosphorylations
of STATl [residue 701 for mouse STATl and STAT2 (residue 688 for mouse STAT-
2)] (Darnell et.al., 1994 ; Thle et al., 1995; Taniguchi et al., 1995 and Stark et al.,
1998). These two phosphorylated STATs, in combination with IRF-9, form a
heterotrimeric complex, ISGF3, which translocates into nucleus and binds to ISRE to
activate IFN inducible genes (Darnell et al., 1994). Additionally, the phosphorylated
STAT-1 also undergoes homodimerisation and is converted into its transcriptionally
active form, termed IFN-y activated factor/IFN-a-activated factor (GAF/AAF), which
binds to the IFN-y activated site (GAS; consensus sequence TTCCNNGAA) and
activates its target genes (Darnell et al., 1994). In the case of IFNGR signaling,
IFNGRl, the Ligand binding subunit that interact with Jakl, IFNGR2, which interacts
with Jak2, associate upon of the dimeric form the ligand, resulting into the activation
of these Jak PTKs (Thle et al., 1995). Like IFNAR stimulation IFNGR stimulation
also results in the efficient activation STAT-1 (Darnell et.a/.,1994). Furthermore,
STAT-2 is also tyrosine phosphorylated by IFN-y stimulation, albeit at much lower
levels than IFN-a/~ stimulation, leading to the formation of ISGF3 (Matsumoto et al.,
1999). The formation of trimeric complex formed STAT1-p48; which consists of
STATl dimer and IRF-9, was also reported in a monkey kidney cell line, vero
(Bluyssen et al., 1995) (figure 2).
The activation of the ST A Ts during IFN signaling reqmres their
recruitment to the receptors (Greenlund et al., 1995) recent study offers a mechanism
by which IFNy stimulation results IFN the activation of ISGF3. In fact, a novel form
of cross talk occurs between IFN a/~ signaling is dependent on a weak IFNAR
stimulation by spontaneously produced IFN-a/~ (Takoka et al., 2000). Further
evidence was provided for the physical association between IFNAR1 and IFNGR2
subunits. In view of the report demonstrating the STAT-2 -docking site with in the
intracellular domain IFNARl (Yan et al., 1996), which is physically associated with
IFNGR2 at the caveolar membrane domain (Takaoka et al., 2000) this docking site
may be utilized for IFN-y induced activation of the ISGF3 complex, IRF-1 is
expressed at low levels in unstimulated cells but is induce by many cytokines such as
IFNs (-a, -~, -y), TNF-a, IL-l, IL-6 and by viral infection (Miyamoto et al., 1988;
Abdollahi et al.. 1991). The analysis of the IRF-1 promoter region revealed that this
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REVIEW OF LITERATURE
induction is mediated by STAT and NFKB transcription factors, the binding sites for
which are found within this region (Pine et al., 1994 and Harada et al., 1994). Infact,
IFN induced expression of IRF-1 mRNA was completely abolished in STAT -1
deficient (STAT-1-1) cells (Meraz et. al., 1996). As described above, the recognition
sequence of IRF-1 (IRF-E) overlaps with that of ISRE, which binds ISGF3. These
observations suggest that IRF- 1 function as a regulator of cellular response to IFNs
by affecting a set of IFN-inducible genes. In contrast, IRF 2 is inducible by IFN aJI3, albeit with slower kinetics of induction than IRF-1 (Harada et. al., 1989). This
induction is mediated by ISGF3, the binding site for which found with in the IRF-2
promoter region (Harada et al., 1994).
3.6. IRF-2 in regulation of Immune Responses:
(a) NK cell development
Role of IRF 2 has been investigated in NK cell devlopment. IRF-2_,_ mice
were shown to carry defects in NK cell development (Lohoff et al., 2000). IRF-2_,_
splenocytes displayed a larger decrease in NK cell cytotoxicity than did IRF 2+/
splenocytes stimulated with poly (I): (C) in vivo. A notable defect in NK cell activity
was also noted in vivo in a tumor rejection model using the NK sensitive cell line.
Furthermore, the number of NK cells (NK1.1 + TCRa/13-) was dramatically decreased
in IRF-2_,_ mice (Lohoff et al., 2000). Unlike IRF-r'- BM cells could not generate NK
Cells when cultured with IL-15 (Lohoff et al., 2000). Interesting} y, ·although the
number of NK-T cells was reduced in IRF-1_,_ mice, that in IRF-2_,_ mice was normal
(Lohoff et al., 2000). It is currently unknown how IRF-2 contributes to these
phenomena.
(b) Macrophage function
The inducible nitric oxide synthetase (iNOS) gene is induced by IFN-13 and
lor lipopolysaccharide (LPS) in WT macrophages, but not in IRF-r'- macrophages
(Kamijo et. al., 1994). The enzyme encoded by the iNOS gene catalyzes the
production of nitric oxide, a short lived volatile gas that plays a major role in the
effector phase of Th1 immune response; i.e. macrophage cytotoxicity against tumor
cells, bacteria and other targets. Indeed, this may explain the observation that IRF-1_,_
mice develop severe symptoms resembling military cutaneous tuberoculosis when
infected with Mycobacterium bovis (Kamijo et. al., 1994). In addition, the induction
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REVIEW OF LITERATURE
of the gene encoding the p40 subunit of IL-12, the cytokine essential for Th1 type
differentiation of the immune system, is totally dependent on IRF-1 (Taki et. al., 1997
and Lohoff et al., 1997).
Neutrophils and macrophages play an important role in restricting bacterial
replication (e.g. Listeria monocytogenes infection) in the early phase of primary
infection in mice, and cytokines IFN-y and TNF-a are essential for protection. IRF-2_,_
and IRF-8-/- mice are highly susceptible to Listeria infection compared with IRF-1_,_
mice. Therefore, IRF-8 and IRF-2 are critical for IFN-y production of reactive oxygen
intermediates (ROis) and possibly others in macrophages. (Fehr et al., 1997).
(c) Regulation of MHC expression
1FN-a/~ and IFN-y are known to enhance the expression of MHC class I and
class II molecules. CDS+ T cell population is significantly reduced in IRF1- mice,
which may be due to lower level ofMHC class I molecules (Matsuyama et. al., 1993).
MHCs are membrane-spanning protein, which present the antigen for recognition by
T -cell receptors. The na"ive T -cell on association with the antigen presented by the
MHC molecule undergoes proliferation and differentiates into memory T cell and
effector T -cells. This may be a consequence of reduced expression of transporter
associated with antigen processing -1(TAP-1) and the low molecular weight protein-2
(LMP-2) (white et. al., 1996). This expression ofTAP-1 and LMP-2 induced by IFN
'Y is via IRF-1 (White et. al., 1996). Tapasin, which plays a significant role in peptide
loading of MHC class I molecules, has been described to possess recognition motifs
cytokine inducible transcription factors in the promoter region of the mouse tapasin
gene. Experiments using fibroblast either lacking IRF-1 or inhibited in protein
synthesis reveal that secondary transcription factors are necessary for its upregulation
on stimulation with IFN-y (Abarca-Heidemann et. al., 2002). IRF-1 may also be
involved in the IFN-y-induced expression PA28a, PA28~ and ~2 microglubulin gene
in MEFs (Taniguchi et. al., 2000). IRF-1 gene knock out studies have conclusively
demonstrated a major role of IRF-1/MHC-IRF-E interactions in controlling MHC
class I gene expression in astrocytes in response to IFN-y (Jarosinski and Massa,
2002).
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(d) Regulation of development and function ofT and B cells
IRF-1_,_ mice carry lineage-specific defects in thymocyte development;
immature T cells (i.e. double positive TCRaJ~+CD4+CD8) were able to develop into
mature CD4+ but not CD8+ T cells. Flow cytometric analysis shows a marked
reduction i.e. -10-fold in the number of CD8+ T cells in peripheral blood, spleen and
lymph node. IRF-1 has been shown to control positive and negative selection of CD8+
thymocytes. In fact positive and negative selection ofT cells is impaired in IRF-1_,_ H
y and IRF- r'- transgenic mice (Penninger et. al., 1997). IRF-1_,_ thymocytes
displayed impaired TCR mediated signal transduction. The induction of negative
selection in TCR transgenic thymocytes from IRF-1_,_ mice required a 1000 fold
higher level of the selecting peptide than that in the IRF-1 +I+. Thus, IRF-1 may
regulate expression of gene(s) in developing thymocytes (Penninger et. al., 1997).
The cytotoxic T lymphocytes (CTL) response to LCMV (Lymphocytic
Choriomeningitis Virus)- infected target cells was significantly reduced in IRF-r'
mice (Matsuyama et. al., 1993).
CD8+ T cells have cytotoxic effector functions. Cytotoxic T Lymphocyte (CTL)
response to LCMV (lymphocytic choriomeningitis virus)-infected target cell was
significantly reduced in IRF-1_,_ mice. In contrast, IRF-T1- mice showed normal CTL
activity against LCMV -infected target cells, although some nonspecific cytotoxicity
was detected (Matsuyama et al., 1993).
(e) Regulation of CDS+ T cell response
Cytokines are not always beneficial to the host because many cytokines are
multifunctional and often invoke antigen non-specific response of the cell. In this
context, regulatory mechanisms have been reported for other cytokine systems, down
regulating their signaling events (Ihle 1995). Recently, IRF 2-t- mice in C57BL/6
background spontaneously were found to have developed an inflammatory skin
disease resembling psoriasis (Hida et al., 2000 and Matsuyama et al., 1993). The
pathogenic development is suppressed upon the selective depletion of these T cells.
CD8+ T cells exhibit in vitro hyper-responsiveness to antigen stimulation,
accompanied with a notable upregulation of the expression of genes induced by IFN
aJ~ (Hida et al., 2000). Furthermore, introduction of nullizygosity to the genes that
positively regulate the IFN- aJ~ signaling pathway. (Hida et. al., 2000). Thus, IRF-2
may represent a unique negative regulator, attenuating IFN-aJ~ induced gene
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transcription, which is necessary for balancing the beneficial and harmful effects of
IFN-a/~ signaling in the immune system.
(f) Regulation of Thl/Th2 differentiation
IRF-2 was originally described as an antagonist of the IRF-1 mediated
transcriptional regulation of IFN-inducible genes (Harada et al., 1990). Since Th1 cell
development and NK cell development are impaired in IRF1_,_ mice, one would have
expected that IRF-2_,_ macrophages (Lohoff et al., 2000) manifest opposite
phenotypes. However, IL-12 production is suppressed in IRF-2_,_ macrophages
(Lohoff et al., 2000) and IRF-2_,_ mice are susceptible to Leishmania major infection
due to defect in Thl cell differentiation (Lohoff et al., 2000). Thus, rather than
functioning as negative regulator, IRF 2 may function to IL-2 gene expression in
cooperation with other factors, such as IRF-8, in activated macrophage.
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