review of literatureshodhganga.inflibnet.ac.in/bitstream/10603/14402/9/09_chapter 2.pdf · in the...

REVIEW OF LITERATURE

3. REVIEW OF LITERATURE

3.1. Discovery of IRF -2:


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


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

16


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

17


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).

10


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


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

22


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).

23

L3

Ll


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

24


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.

25


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

26


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

27


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).

28


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


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 )

30


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

31


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

32


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).

33


(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

34


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.

35

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