The role of interferon regulatory factors in the interferon system and cell growth control

Biochimie (1998) 80, 641-650 © Soci6t6 franqaise de biochflnic el biok~gic mol6cukfirc / Elscvicr, Paris

The role of interferon regulatory factors in the interferon system and cell growth control Hisashi Harada '~*, Tadatsugu Taniguchi~'~ Nobuyuki Tanaka ~' "Division ¢~J"Mnlecutar Oncolog3; Departments of Medicine and Patholog3; Washington Univer~'itv School o/ Medi~ine, Box 8069. 660 South Euclid Avenue, St. Louis, MO 63110, USA t'Department ¢~f" Immunology l-'ucuhv ~f'Medicine, University of Tokyo, Hongo 7-3-1, Bunkvo-ku, ~kxo 113, Japan

(Received 17 March 1998, accepted 7 May 1998) Abstract - - Complex cellular responses are often coordinated by a genetic regulatory network in which a given transcription factor controls the expression of a diverse set of target genes. Interferon regulatory factor (IRF)-1 and IRF-2 have originally been identilied as a transcriptional activator and repressor, respectively, of the i-lterleron-[:~IIFN-[~) as well as of IFN-inducible genes. However. these factors have since been shown to modulate not only the cellular response to IFNs, but also cell growth, susceptibility to transformation by oncogenes, induction of apoptosis, and development of the T cell immune response. Furthermore. the evidence suggests that deletion and/or inactivation of the IRF-! gcne may be a critical step in the development of some human hematopoietic neoplasms. Subsequently, these factors have been sh~wn to constitute a family of transcription factors, termed the IRF-family. Recent studies indicate that other 1RF family members also involve the regulation of the IFN system and cell translbrmation. The IRF-family may be examples of transcription factors which can se!ectively modulate several sets of genes depending on the cell type and/or nature of the cellular stimuli, so as to evoke host defense mechanisms against infection and oncogenesis. © Socirt6 fran~;aise de biochimie et biologic molrculaire / Elsevier, Paris interferon regulatory factor / transcription / interferon response / cell growth control 1. Introduction Cytokines, soluble mediators involved in cell-to-cell communication, play an essential role in the regulation of cell growth and differentiation. The expression of cytokines is lightly regulated by complex mechanisms. These newly synthesized cytokines subsequently exert their biological effects by inter,l::ting with specllic receptors expressed on the surface of target cells. Interfcrons (1FNs) are a family of multi-functional cytokines that were originally identified as the proteins responsible for the induction of cellular resistance to viral infection. Subsequently, much evidence has been accumulated with regard to their roles in cell growth, differentiation, and immunomodulation. In a variety of cells, type I IFNs (i.e., IFN-~ts and IFN-[5) ate produced by virus induction, whereas type II IFN (IFN-y) is produced by mitogen-activated T cells and natural killer cells. Virus-induced expression of the IFN-~t and IFN-I] genes constitutes an essential part in the initial host defense before cellular immunity comes into play: the expression of these otherwise silent genes is induced promptly and efficiently upon virus infection, at~d the IFNs thus produced transmit signals to the target ceils to elicit the antiviral state [I-41. We have been studying on the regulation of IFN system as a model system to * Correspondence and reprints

elucidate the molecular mechanisms of cytokine-mediated cellular responses. During the course of study, we have identified and cloned two novel transcription factors, termed interferon regulatory factor-i (IRF-1) and IRF2 15-71. Subsequently, these factors have been shown to constitute a family of transcription factors, termed the IRF-family, which now includes IRF-3 181, IRF-4 (LSIRE ICSAT/Pip) 19-111, IRF-5, IRF-6, IRF-7 1121. t348 IISGF3y) Ii31 and ICSBP[141. These members share significal~t homology in the amino-terminal 115 amino acids, which comprise the DNA binding domain. Recently, a viral member of this family, v-IRE encoded by the Kaposi's sarcoma-associated herpes virus (KSI-IV), was also identified [151. Here, we summarize the CUlTent status of how IRFs operate in the regulation of IFN system and cell growth.

2. Structure and function of IRF-I and IRF-2 The induction of type I IFN genes by virus or doublestranded RNA is primarily due to transcriptional activation requiring sequences in the 5' region of IFN-ct and IFN-~ genes. Numerous studies have identified cis-acti,'ag DNA elements within these regions and transcription factors which operate these elements 14. 16-18]. We have identified and cloned two transcription factors, termed

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Table I. The IRF-E and ISRE sequences within the promoters of IFN and IFN-inducible genes. G(A) AAA~ICTICGAAA6/CTIC IRF-E consensus A/o NGAAA N N GAAA C T

ISREconsensus Gene

Sequence

HumanlFN-13 Human IFN-ct

G A AAA C TGAAA G G G A GAA G TGAAA G T G AAA T G GAAA G T

Human 2'-5'A Mouse PKR Human GBP Human 9-27 lk~.~ ~-2D d

A G A A C

GGAAA GGAAA A T GAAA C G GAAA T A GAA- G

C GAAA CCA C GAAA CAG T GAAA GTA A GAAA CTT T GAAA CT

2'-5' A, 2'-5' oligoadenylate synthetase; PKR, double-stranded RNA dependent protein kinase; GBP, guanylate binding protein.

interferon regulatory factor-I (IRF-I) and IRF-2, which bind to the sequences between -92 and -67 (+ I as a transcription initiation site) of the human IFN-~ gene promoter [5-7 I. These two factors are structurally related, particularly in the amino terminal region between amino acids (aa) 1 and aa 113, which contains the DNA binding domain. Both factors recognize the same DNA binding elements, termed IRF-E [G(A)AAAG /c T /cGAAA G /c T /el, and they are found within the promoters of IFN-et, IFN-13, and many IFN-inducible genes (tab&i)Ii91. The NMR spectroscopy and the crystal structure studies revealed that the DNA binding domain of IRF- I and IRF-2 is classified into the ct + 13helix-turn-helix-like structure class 120, 211. This structure is observed in bacterial activator CAP, heat shock transcription factors, and hepatocyte nuclear factor37, however, these members are not related by sequence homology. All the IRF family shows remarkable sequence homology in the DNA binding domain (especially the conserved amino acids in the secondary structure elements), suggesting that the IRF family members have identical three-dimensional structure. Series of expression studies have shown that IRF-I acts as a transcriptional activator, whereas IRF-2 acts as a transcriptional repressor by competing for the binding to the same DNA sequence elements[22, 231. Furthermore, the IRF-I and IRF-2 genes per se are induced by virus infection and IFN treatment. These findings indicate an important role for IRF-I and IRF-2 in the expression of IFN and IFNinducible genes. To gain more insights in to the physiological role of IRF-1 and IRF-2, mice with targeted disruption of IRF-1 or IRF-2 were generated 124, 251. The results using IRF-1 deficient (IRF-I -/-) mice revealed the presence of an IRF-l-independent induction pathway(s) for both the IFN-a and IFN-13 genes. Induction of both IFN-a and IFN-I~ mRNAs remained essentially unchanged between

mutant and wild-type embryonic fibroblasts (EFs) following induction by Newcastle disease virus (NDV) infection, indicating IRF-1 is dispensable for the virus induction of the IFN-ct genes. On the other hand, IRF-1 appears to be required for the induction of these genes by doublestranded RNA such as poly(rI):poly(rC). To investigate the role of IRF-1 in the antiviral action of IFNs, EFs were first treated with either IFN-a or IFN-y, and challenged with encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), or herpes simplex virus (HSV) [261. The inhibition of EMCV replication by IFN-~t and especially by IFN-y was impaired in IRF-l -zEFs. However, after infection with VSV, no difference in virus yield was seen between wild-type and IRF-1 -z- EFs. Moreover, a small difference was observed after HSV infection, particularly after IFN-y treatment. The IRF-I -zmice were less resistant than wild-type mice to EMCV infection, as revealed by accelerated mortality and a larger virus titer in target organs, brain and heart. From the analysis of several IFN-inducible genes mRNA expression, induction of double-stranded RNA dependent protein kinase (PKR), 9-27 and 1-8 genes by either IFN-ct or IFN-y was similar in wild-type and IRF-! -/- EFs. Induction o! guanylate binding protein (GBP) gene by IFN-ct was mildly impaired, and induction by IFN-y was severely impaired in IRF-! -z- EFs. Thus, dependency on IRF-! varies among these genes, with GBP and iNOS gene induction being strongly IRF-1-dependent 126, 27 I. Taken together, IRF-! is necessary for the antiviral action of IFNs against some viruses, but IFNs activate multiple activation pathways through diverse target genes to induce the antiviral state. In addition, IRF-I is a critical transcription factor mediating the antibacterial function of type ! and type I1 IFNs 1281. Production of nitric oxide (NO) by macrophage is important for the killing of intracellular infectious agents. IFN-y and lipopolysaccharide (LPS), which are the most potent activators, stimulate NO production by transcriptional up-regulation of the inducible NO synthase (iNOS). The macrophages from IRF-I -/- mice produced no detectable NO in response to IFN-y or LPS alone, and barely detectable NO after simultaneous treatment with IFN-y and LPS. IFN-a, though less potent than IFN-y, induced NO in the presence of LPS from wild-type macrophages. This stimulatory effect of IFN-~t was decreased in macrophages from IRF-1 -¢- mice. The increase in iNOS mRNA after stimulation with IFN-y and LPS, seen in macrophages from wild-type mice, was virtually absent in IRF-I -/- macrophages. In fact, two adjacent IRF-I response elements were identified in the iNOS promoter. Infection with Mycobacterium bovis (BCG) was more severe in iRF-l -z- mice than in wild-type mice. Thus IRF-! is essential for iNOS activation in murine macrophages, and contributes to antibacterial function.

Role of IRFs in the interferon system and cell growth control 3. The function of p48 (ISGF3?~ and IRF-3 in the IFN system It has been well established that a heterotrimeric transcription factor complex, termed ISGF3, which consists of Stat lot, Stat2 and p48, is lormed upon type I IFN stimulation, and binds to ISRE, whose consensus sequence is A/GNGAAANGAAACT, found in the promoters of various IFN-inducible genes. On the other hand, GAF (IFN-gamma activated factor)/AAF (IFN-alpha activated factor), a homodimer of Statl0t, binds to a distinct cis-element, termed GAS (IFN-gamma activated sequence), within the promoters of many lFN-inducible genes [29, 30]. Interestingly, the sequence of IRF-E overlaps with ISRE (table I), suggesting that these two factors may function redundantly in the regulation of IFNinducible genes. An IRF-fam_ily transcription factor, p48 (ISGF3y), is the main DNA binding component of ISGF3 which is structurally related to IRF-1. To assess the contribution and cooperation of ISGF3 and 1RF-I in mediating IFN action, mice deficient for the p48 gene (p48 -/- mice) were generated by gene targeting 1311. In the mutant p48 -4- cells the induction of the antiviral state by type I or type !I IFNs is dramatically impaired by EMCV, VSV and HSV infection. The DNA binding activity of ISGF3 was absent in nuclear extracts of IFN-~t treated p48 -/- EFs. Moreover, a DNA binding activity which resembles ISGF3 was detected in IFN-y-stimulated EFs from wild-type, but not p48 / mice. We also generated mice deficient for both p48 and IRF-i and found that at least one IFN-inducible gene (GBP) is dependent on both factors. Thus, p48 and IRF-! do not perlbrm redundant functions in the ceil, but rather complement one another in both type 1 and type 11 IFN responses. Our results, together with the previous results, support the following model (.ligtoe !). Both IFNs induce GAF/AAF and IRF-i 1321. In addition to type 1 IFN, type 11 IFN also activates an ISGF3-1ike activity which contains at least p48 and Statl as components 131, 331. Thus, both types of IFNs may induce shared biological activities by activating a common set of transcription factors which act on the GAS or ISRE of the IFN-inducible genes. The functional similarities between IRF-! and p48 prompted us to examine whether p48, which was originally identified as a regulator of the IFN response, is also involved in IFN production. In fact, p48 has previously shown to bind to the IFN-[3 promoter I341. EFs from p48 -/- mice show severe defects in virus-induced IRF-ct gene expression ]35]. A DNA binding factor, which is indistinguishable from the lFN-activated ISGF3, is detected in wild-type but not in p48 -/- EFs upon NDV infection and binds to the virus-inducible DNA elements of the IFN-cx promoters. In addition, EFs from mice deficient in other molecules which are also involved in the IFN signal transduction pathway, i.e., type I IFN receptor (IFNR) [361 or Statl 1371, show a similar defect in IFN-cx

643 gene induction. These results suggest lhat the unique feature of the IFN system regulation involving an "autostimulatory IFN loop' which functions subsequent to the initial triggering of IFN synthesis by an as-yet-unknown mechanism(s) and which may be important to the efficient function of the IFN-mediated host defense against virus intection (figure 2) [35, 38]. IRF-3 was identified by primary sequence homology to the IRF-family and shown to be expressed ubiquitously in all cell types tested [8]. A transient overexpression of IRF-3 augments the virus-induced activation of IFNA4 gene enhancer. Very recently, it has been demonstrated that IRF-3 is present in its inactive form, restricted to the cytoplasm in unstimulated cells. Virus infection induces serine and threonine phosphorylation on IRF-3, thereby allowing it to complex with the co-activator CBP/p300 with nuclear translocation and its specific DNA binding [39, 40]. These findings suggest that 1RF-3 also plays an important role in the virus-induction of type I IFN genes and that IRF-3 may be a candidate transcription factor t~r triggering the initial IFN synthesis.

4. Cell growth control by IRF-I and IRF-2 Although the antiviral activity is a characteristic feature of IFNs, their actions on cell growth are considered to be of greater biological significance. In fact. IFNs were regarded as "negative growth factors', which may be important to be the control of cell growth in a variety of cell types[l-4]. As IRF-I mediates antiviral activity of 1FNs, an interesting question is how IRF-I and IRF-2 involve in cell growth control, in this regard, IRF-1 shows anti-proliferative properties when it is overexpressed il3 viw) and in vitro [41, 42]. First, tile expression of IRF-I and IRF-2 mRNAs in mouse NIII3T3 cells are quantilied tlaroughout the cell cycle. Cells were growth-arrested by serum starvation (G I arrest) and were then induced to transit the cell cycle by serum restoration. IRF-i mRNA expression reached a peak in growth-arrested cells, declined sharply after serum stimulation, and then increased gradually prior to the onset of DNA synthesis. In contrast, IRF-2 mRNA expression remained essentially constant throughout the cell cycle (ligure 3A)[43]. These observations suggest that a transient transition in the IRF-2:IRF-1 ratio may be a critical event in the regulation of cell growth. To examine the effect of perturbing the IRF-2:IRF-i ratio on cell growth, NIH3T3 cell clones that overexpressed IRF-2 were generated. These cells became transformed and displayed enhanced tumorigenicity in nude mice. Furthermore, this transformed phenotype was reverted by concomitant overexpression of the IRF- 1 gene. Thus, restrained cell growth depends on a balance between these two mutually antagonistic transcription factors (figure 3A) [431. This antioncogenic function of IRF-1 is not limited to only IRF-2

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Harada et al.

IFN-(x/p

IFN-y

ISGF3 like factor ~

IRF-1

gene

~

,,.

==

IFN responses

overexpressing cells; c-myc- or fosB-transformed rat embryonic fibroblasts can be reverted by the introduction of the IRF-I gene [441. These results suggest the broad role of IRF-1 as a tumor suppressor. A powerful approach to assess the tumor-suppressive function of a given gene is to create model animals by gene targeting, thus enabling the gene defect to be examined in the context of cell transformation and tumorigenesis. For example, the mice lacking p53 tumor suppressor gene spontaneously develop various types of tumors at relatively young ages [451. In this regard, mice lacking the functional IRF-1 alleles grow without any spontaneous development of tumors. However, in view of the importance of multiple genetic events for the development of tumors in vivo and in vitro, it is conceivable that the IRF-l-deficient cells may manifest properties different from wild-type cells with respect to their susceptibility to transformation by oncogenes.

Figure 1. Network of transcriptional regulation in type I and type !! IFN responses. See text lbr the details.

The EFs from IRF-I '-/- mice can be transformed by expression of an activated c-Ha-ras gene. This property is not observed in EFs from wild-type or IRF-2-/- mice but still observed in EFs from mice deficient in both genes, demonstrating that it is the IRF-I defect, rather than the preponderance of the antagonistic IRF-2, that renders EFs susceptible to this oncogene. The transformed phenotype can be suppressed by the concomitant expression of IRF- 1 eDNA [461. It has been shown that oncogenes, such as c-myc or adenovirus EIA genes, can sensitize cells to undergo programmed cell death (apoptosis), under conditions of low serum concentration or high cell density [471. In this context, the involvement of IRF-I in apoptosis was examined. The expression of the c-Ha-ras oncogene causes wild-type but not IRF-1 -/- EFs to undergo apoptosis under conditions of low serum concentration, high cell density, or following treatment by anticancer drugs or

Role of IRFs in the interfcron system and cell growth control

Figure 2. Two stage activation of the IFN-ot/~ genes. Viral infection activates the initial stage of IFN-ct and IFN-I:I gene (I), and the infected cells produce a small amount of IFNs (2). The produced IFNs then stimulate the lFN-ct receptor in an autocrine fashion, leading to the formation and activation of,the ISGF3 complex (3). The ISGF3 complex brads to the prornoters of the IFNot/[~ genes (or it induces other factor(s) (designated X in the figure), leading to a further induction of the genes (second stage)) (4).

t ' X

645

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ionizing radiation. Hence, IRF-i may be a critical determinant of oncogene-induced cell transformation or apoptosis (figtoe 3B) 1461.

5. The target genes of IRF-I in cell growlli control it is likely that IRF-! exerts its anti-oncogenic function and regulating apoptosis by activating a target gene(s), in this context, at least four genes have been identified as the target genes of IRF-i. i) Lymphocytes are particularly susceptible to DNA damage-induced apoptosis. The tumor suppressor p53 has been shown to regulate this type of apoptosis in thymocyte I48, 491, but a p53-independent pathway(s) appears to mediate the same event in mitogen-activated mature T lymphocyte 150]. DNA damage-induced apoptosis in these T lymphocytes is dependent on IRF-! 1511. Thus, two different anti-oncogenic transcription factors, p53 and IRF-I, are required for distinct apoptotic pathways in T lymphocyte. Moreover, mitogen-induction of the interleukin-l~ converting enzyme (ICE, caspase !) gene, a mammalian homologue of the C. elegans cell death gene ced-3, is dramatically affected by the loss of IRF-1. Ecotopic overexpression of IRF-1 results in the activation of the endogeneous caspase 1 gene and enhances the sensitivity of cells to radiation induced apoptosis. Thus caspase ! (ICE) gene is a target gene of IRF-1 in apoptosis regulation.

)

...... . . "'

m-o<.'l , gens

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j /

00

0

2) We attempted to identify genes that are differentially expressed in wild-type and IRF-! -/ EFs. Using the mRNA differential display system, one gene was isolated that was different;ally expressed only in wild-type EFs but not in IRF- I / EFs. From the sequence analysis, this gene was Ibt, nd to be identical to the mouse lysyl oxidase gcnc 1521. An IRF response clement was identilicd in the promoter of tiffs gene. The transformed phenotypc of ras-exprcssing IRF-I ; EFs could be suppressed by the expression of the lysyl oxidasc eDNA, implicating its potential role in tumor suppression. Thus the regulation of the lysyl oxidase gene by IRF-I could contribute to the multistep process of malignant transformation. 3) To investigate further the role of IRF-1 in the regulation of the cell cycle, EFs fi'om IRF-1 -/ or p53 -4- mice were subjected to cell-cycle analysis 153 !. The IRF-! -~- EFs are deficient in their ability to undergo DNA-damage-induced cell cycle arrest. A similar phenotype has been observed in p534- EFs 1541, although the expression of IRF-I and p53 are independent of one another. Furthermore, transcriptional induction of p21 (WAFi, CIPI) gene by ~,-irradiation is dependent on both p53 and IRF-1, and the p21 promoter is activated, either directly or indirectly, by both in transient cotransfection assay. Therefore, these two tumor suppressor transcription factors converge functionally regulate the cell cycle through the activation of a common target genes.

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Harada et al.

(a)

(B)

Wild-typecells IRF-1"/"cells

NIH3T3

/

/r.,o /

G1arrest

S phase

!

I

[ 'intLroductioof nactivatedHa-rasgeneI

IRF-2overexpression ~

Transformation

~3

~

I

Transformation

I DNAdamages I

; IRF-1overexpression

[

~~Reversion

Apo~ptosis

;

Figure3. The role of IRF-I and IRF-2 in cell growth control. A. IRF-I expression reached a peak in GI arrest, declined sharply after

serum stimulation, and then increased gradually prior to the onset of DNA synthesis. In contrast, IRF-2 expression remained essentially constant throughout the cell cycle. The IRF-2 overexpressing NIH3T3 cell clones became transformed. This transformed phenotype was reverted by concomitant overexpression of the IRF- I gene. B. The EFs from IRF-I ; mice can be transformed by expression of an activated c-Ha-ras gene. This property is not observed in EFs from wild-type mice. The expression of the c-Ha-ras oncogene causes wild-type but not IRF-I; EFs to undergo al:optosis under conditions inducing DNA damage.

4) It has been shown that induction of IRF-i activity induces PKR expression in NIH3T3 cells and that a catalytically inactive dominant negative PKR mutant abolishes the antiproliferative action of IRF-I [55, 561. In IRF-I-;- EFs, PKR expression is reduced relative to wild-type cells. Furthermore, human leukemic U937 cells contain a deletion of one IRF-I gene and express low levels of PKR. Upregulation of IRF- 1 expression in U937 cells by transfection is sufficient to induce PKR expression, Moreover, a marked reduction in the expression of PKR in blood samples from two myelodysplasia patients, carrying a deletion of chromosome 5q, a locus to which tRF-I was mapped (see below) [571. Taken together, PKR is a candidate for a mediator of tumor suppressor activity of IRF- i,

6. IRF-Iin humanneoplasia The human IRF-1 gene has been mapped to 5q31.1 [58, 591. Chromosome band 5q31 was previously determined

to be the mo~t commonly deleted segment, the so-called 'critical region', in human leukemia and myelodysplastic syndrome (MDS) with interstitial deletion of chromosome 5q. Del(5q) is also a hallmark of a unique clinical myelodysplastic disorder with refractory anemia and abnormal megakaryocytes occurring predominantly in elder females, known as the'5q-syndrome' I601. It has been demonstrated that one or both IRF-1 alleles were deleted in each of 13 representative cases of MDS and leukemia with del(5q) or translocation of 5q31. Furthermore, an inactivating gene rearrangement of one IRF-i allele, accompanied by deletion of the residual allele, was found in one case of de novo acute leukemia I591. On the other hand, it has been reported that, in some patients with MDS/AML associated with 5q abnormalities, one allele is deleted, but the other allele remains intact [61 ]. However, it has been shown that loss of one IRF-I allele in a leukemic cell line results in significant reduction of a target gene expression [571. These observations support the idea that IRF-I may be the critical tumor suppressor gene deleted in del(5q); thus loss of one or both IRF-1

Role of IRFs in the interferon system and cell growth control

(A)

647

(B)

(C)

Chromosome 5 IRF-1 gene (5q31.1)

IRF-1 gene ~

-

" IRF-E / overexpressionof NPM

AUG

Production of intact IRF-1 mRNA One allele deletion

~ exon skipping

.,~pM

~'~

-

:~ Inactivation of residual allele

Production of abberantly spliced IRF-1 mRNA

Figure 4. The inactivation of IRF-I in leukemia/myclodysplastic syndrome (MDS). A. The 1RF-1 gent itself is deleted or inactivated by mutation. B. The aberrant spliced IRF-I mRNA is produced by cxon skipping. C. The IRF-I protein is sequestered by NPM, resulting in the inactivation of IRF-1.

alleles may contribute to unrestrained cellular proliferation and thereby promote the development of human leukemia and MDS (figure 4A). In addition, another mechanism of inactivation of IRF-I, accclerated exon-skipping by alternative splicing, has been observed 1621. The exon skipped form of IRF-! lacks DNA-binding activity, and cannot manifest antitumor activity. As a result, approximately 30% of patients with MDS or overt leukemia from MDS showed inactivation of IRF- ! (figure 4B). Recently, an alternative mechanisrn by which IRF-I may be inactivated has been reported 1631. The purilication of an IRF-I association molecule revealed to be identical to a nuclear factor nucleophosmin (NPM)/B23/numatrin. Functional analysis showed that NPM inhibited the DNA-binding and transcriptional activity of IRF-I. Moreover, NPM was overexpressed in several clinical leukemia samples and human-derived leukemia cell lines. Overexpression of NPM in NIH3T3 cells resulted in malignant transformation. These results suggest the possible involvement of NPM i,; inactivating IRF-l-dependent anti-oncogenic surveillance in human cancer development (figure 4C).

7. Dual function of IRF-2

IRF-I and IRF-2 ~hare 62% homology in their amino terminal DNA binding,, domain and 25% homology in their remaining carboxyl regions 171. However, two regions stand out as unique to IRF-2; the carboxyl-terminal end of

IRF-2 is relatively rich in basic amino acids; seven out of the carboxyl-terminal 25 residues (aa 325-349) are basic whereas only three are acidic. The unique central region is relatively acidic; live out of 20 residues (aa 182-201) are acidic and none are basic. Deletion of the carboxyl region, or alanine substitution of five basic amino acids within this region, converts IRF-2 into an activator. Conversely, transplanting the carboxyl end of IRF-2 onto IRF-1 strongly inhibits the latter's ability to activate [64]. Thus IRF-2 is a mosaic transcriptional regulator possessing both potential activation and repression dornains. Then is there any physiological role for this latent activation domain of IRF-2? The histone H4 gene is regulated during the cell cycle and a peak in transcription during S phase. The cell-cycle element (CCE) required for H4 histone activation is a sequence of !! base pair:~ that binds a protein Ihctor that has been designated histone nuclear factor M (HiNFM) I651. The purification of HiNF-M revealed the protein to be identical to IRF-2 1661. In fact, the CCE has high homology to IRF-E. IRF-2 binds the CCE specifically and activates transcription of the H4 histone gene. Therefore, the effects of IRF- ! and IRF-2 vary with promoter context. and IRF-2 joins the ranks of other transcription factors with dual activator/repressor functions (such as YYI, RAP- 1, Dorsal). IRF-2 has been shown to have oncogenic potential, and these results demonstrate a link between IRF-2 and a gene that is functionally coupled to DNA replication and cell-cycle progression at the G I/S phase transition.

648

8. Other IRF-family members in cell growth control Interferon consensus sequence binding protein (ICSBP) is a transcription factor of the IRF family, that is expressed exclusively in cells of the immune system. ICSBP is constitutively expressed throughout B cell ontogeny from early pro-B cells to mature antibody-producing cells. While ICSBP expression is very low in resting T cells and macrophages, its expression is strongly induced in these cells upon immune stimulation and IFN treatment [671. Previous studies have indicated that ICSBP has negative effects on transcription of ISRE-carrying promoters. Furthermore, ICSBP has been shown to complex with IRF-1 and IRF-2 [68]. Mice with a null mutation of ICSBP exhib'~t enhanced susceptibility to virus infections associated with impaired production of IFN. Moreover, hematopoiesis is deregulated in both ICSBP 4- and ICSBP ÷zmice that manifests as a syndrome similar to human chronic myelogenous leukemia. The chronic period of the disease progresses to a fatal blast crisis characterized by a clonal expansion of undifferentiated cells. Normal mice injected with cells from mice in blast crisis developed acute leukemia within 6 weeks of transfer. These results suggest a role for ICSBP in regulating the proliferation and differentiation of hematopoietic progenitor cells [691. IRF-4 also belongs to the IRF-family, but expresses only in lymphocytes. Mice deficient in IRF-4 showed normal distribution of B and T lymphocytes at 4 to 5 weeks of age but developed progressive generalized lymphadenopathy. IRF-4 deficient mice exhibited a profound reduction in serum immunoglobulin concentrations and did not mount detectable antibody responses. T lymphocyte function was also impaired in vivo; these mice could not generate cytotoxic or antitumor responses. Thus, IRF-4 is essential for the function and homeostasis of both mature B and mature T lymphocytes 1701. Intercstingly, by cloning the chromosomal breakpoints in an multiple myeloma (MM) cell line, it has been shown that the 14q+translocation represents a t(6;14)(p25;q32), and the translocation juxtaposes the immunoglobulin heavychain (lgH) locus to MUM I/IRF-4 gene I711. As a result, the MUM I/IRF-4 gene is overexpressed, an event that may contribute to tumorigenesis, as MUM I/IRF-4 has oncogenic activity in vitro, These findings, together with the gene knockout study, suggest that IRF-4 deregulated expression may contribute to the aberrant phenotype ot" MM cells. The KSHV gene ORF K9 encodes vlRF which has significant homology to members of the lRF-family 115!. vlRF inhibits IFN-responsive gene expression in reporter assays. Furthermore, stable transfectant NIH3T3 clones e,~pressing vlRF grew in soft agar and at low serum concentrations, lost contact inhibition and formed tumors after injection into nude mice indicating that vlRF has properties of a viral oncogene I721. Since vlRF is primarily expressed in KSHV-infected B cells, not KS spindle

Harada et ai. cells, this suggests that vlRF is a transforming oncogene active in B cell neoplasias.

Acknowh=dgments The work from Taniguchi's laboratory described in this review was supported in part by grants from the Ministry of Education, Science and Culture of Japan. We thank our colleagues for valuable discussions and suggestions.

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