A potential novel genetic etiology of autoimmune encephalopathy

The Nb2 PRL receptor (PRL-R) is known to mediate PRL signaling to the interferon (IFN) regulatory factor-1 (IRF-1) gene via the family of signal transducers and activators of transcription or Stats. To analyze the components of the PRL-R/Stat/IRF-1 signaling pathway, various PRL-R, Stat, and IRF-1-CAT reporter constructs were transiently cotransfected into COS cells. First, mutations in the IFNγ-activated sequence (GAS), either multimerized or in the context of the 1.7-kb IRF-1 promoter, failed to mediate a PRL response, showing that the IRF-1 GAS is a target of PRL signaling. Next, pairwise alanine substitutions into conserved residues in the proline-rich motif or Box 1 region and two tyrosine mutations, Y308F and Y382F, in the PRL-R intracellular domain all impaired PRL signaling to multimerized GAS or to the 1.7-kb IRF-1 promoter. Furthermore, these PRL-R mutants mediated reduced Stat1 binding to the IRF-1 GAS. Transfection of Stat1 further enhanced PRL signaling to the IRF-1 promoter, suggesting that Stat1 is a positive mediator of PRL action. These studies show that both membrane proximal and distal residues of the PRL-R are involved in signaling to the IRF-1 gene. Further, Stat1 and the GAS element are important for PRL activation of the IRF-1 gene.

The Nb2 PRL receptor (PRL-R) is known to mediate PRL signaling to the interferon (IFN) regulatory factor-1 (IRF-1) gene via the family of signal transducers and activators of transcription or Stats. To analyze the components of the PRL-R/Stat/IRF-1 signaling pathway, various PRL-R, Stat, and IRF-1-CAT reporter constructs were transiently cotransfected into COS cells. First, mutations in the IFNgamma-activated sequence (GAS), either multimerized or in the context of the 1.7-kb IRF-1 promoter, failed to mediate a PRL response, showing that the IRF-1 GAS is a target of PRL signaling. Next, pairwise alanine substitutions into conserved residues in the proline-rich motif or Box 1 region and two tyrosine mutations, Y308F and Y382F, in the PRL-R intracellular domain all impaired PRL signaling to multimerized GAS or to the 1.7-kb IRF-1 promoter. Furthermore, these PRL-R mutants mediated reduced Stat1 binding to the IRF-1 GAS. Transfection of Stat1 further enhanced PRL signaling to the IRF-1 promoter, suggesting that Stat1 is a positive mediator of PRL action. These studies show that both membrane proximal and distal residues of the PRL-R are involved in signaling to the IRF-1 gene. Further, Stat1 and the GAS element are important for PRL activation of the IRF-1 gene.

The interferon regulatory factor-1 (IRF1) gene, localized on chromosome 5q31.1, is mutated or rearranged in several cancers including some hematopoietic and gastric cancers. However, whether loss of IRF1 occurs in sporadic breast cancer is unknown. Loss of 5q12-31 is reported in 11% of sporadic breast cancers, and high-resolution array-CGH studies have shown loss at 5q31.1 in 50% of breast cancers with a mutated BRCA1 gene. Functionally, overexpression of IRF1 reduces, and a dominant negative IRF1 construct increases, tumorigenesis of human breast cancer xenografts. Taken together, these observations indicate that the IRF1 gene may play a potentially important role as a breast cancer tumor suppressor gene. In this study, we investigated allelic loss of the IRF1 gene in breast tumor specimens from 52 women with invasive breast cancer using an IRF1 intragenic dinucleotide polymorphic marker. Thirty-seven cases were informative. LOH at the IRF1 locus was detected in 32% of these informative cases (12/37). There was a significant association between IRF1 loss and both older age (P = 0.0167) and earlier stage (Stages 1 and 2) (P = 0.0165). To assess the association of IRF1 mRNA expression with clinical outcomes in breast cancer, we studied data from two published gene expression microarray datasets. In breast cancer patients, low IRF1 mRNA expression is strongly correlated with both risk of recurrence (OR = 3.00; P = 0.003; n = 273 cases) and risk of death (OR = 4.18; P = 0.004; n = 191 cases). Our findings strongly imply a tumor suppressor role for the IRF1 gene in breast cancer.

Interferon regulatory factor 1 (IRF-1) and IRF-2 are structurally similar DNA-binding factors which were originally identified as regulators of the type I interferon (IFN) system; the former functions as a transcriptional activator, and the latter represses IRF-1 function by competing for the same cis elements. More recent studies have revealed new roles of the two factors in the regulation of cell growth; IRF-1 and IRF-2 manifest antioncogenic and oncogenic activities, respectively. In this study, we determined the structures and chromosomal locations of the human IRF-1 and IRF-2 genes and further characterized the promoters of the respective genes. Comparison of exon-intron organization of the two genes revealed a common evolutionary structure, notably within the exons encoding the N-terminal portions of the two factors. We confirmed the chromosomal mapping of the human IRF-1 gene to 5q31.1 and newly assigned the IRF-2 gene to 4q35.1, using fluorescence in situ hybridization. The 5' regulatory regions of both genes contain highly GC-rich sequences and consensus binding sequences for several known transcription factors, including NF-kappa B. Interestingly, one IRF binding site was found within the IRF-2 promoter, and expression of the IRF-2 gene was affected by both transient and stable IRF-1 expression. In addition, one potential IFN-gamma-activated sequence was found within the IRF-1 promoter. Thus, these results may shed light on the complex gene network involved in regulation of the IFN system.

Interferon regulatory factor 1 (IRF-1) and IRF-2 are structurally similar DNA-binding factors which were originally identified as regulators of the type I interferon (IFN) system; the former functions as a transcriptional activator, and the latter represses IRF-1 function by competing for the same cis elements. More recent studies have revealed new roles of the two factors in the regulation of cell growth; IRF-1 and IRF-2 manifest antioncogenic and oncogenic activities, respectively. In this study, we determined the structures and chromosomal locations of the human IRF-1 and IRF-2 genes and further characterized the promoters of the respective genes. Comparison of exon-intron organization of the two genes revealed a common evolutionary structure, notably within the exons encoding the N-terminal portions of the two factors. We confirmed the chromosomal mapping of the human IRF-1 gene to 5q31.1 and newly assigned the IRF-2 gene to 4q35.1, using fluorescence in situ hybridization. The 5' regulatory regions of both genes contain highly GC-rich sequences and consensus binding sequences for several known transcription factors, including NF-kappa B. Interestingly, one IRF binding site was found within the IRF-2 promoter, and expression of the IRF-2 gene was affected by both transient and stable IRF-1 expression. In addition, one potential IFN-gamma-activated sequence was found within the IRF-1 promoter. Thus, these results may shed light on the complex gene network involved in regulation of the IFN system.

Heart failure (HF) is a leading cause of morbidity and mortality in the western world. Although optimal medical care and treatment is widely available, the prognosis of patients with HF is still poor.Toll-like receptors (TLRs) are important compartments of the innate immunity. Current studies have identified TLRs as critical mediators in cardiovascular diseases. In the present study, we investigated the involvement of TLRs and interferon (IFN) regulatory factors (IRFs) in different experimental HF models including viral myocarditis, myocardial ischemia, diabetes mellitus, and cardiac hypertrophy. In addition, we investigated for the first time comprehensive TLR and IRF gene and protein expression under basal conditions in murine and human cardiac tissue. We found that Tlr4, Tlr9 and Irf7 displayed highest gene expression under basal conditions, indicating their significant role in first-line defense in the murine and human heart. Moreover, induction of TLRs and IRFs clearly differs between the various experimental HF models of diverse etiology and the concomitant inflammatory status. In the HF model of acute viral-induced myocarditis, TLR and IRF activation displayed the uppermost gene expression in comparison to the remaining experimental HF models, indicating the highest amount of myocardial inflammation in myocarditis. In detail, Irf7 displayed by far the highest gene expression during acute viral infection. Interestingly, post myocardial infarction TLR and IRF gene expression was almost exclusively increased in the infarct zone after myocardial ischemia (Tlr2, Tlr3, Tlr6, Tlr7, Tlr9, Irf3, Irf7). With one exception, Irf3 showed a decreased gene expression in the remote zone post infarction. Finally, we identified Irf7 as novel cardiovascular stress-inducible factor in the pathologically stressed heart. These findings on TLR and IRF function in the inflamed heart highlight the complexity of inflammatory immune response and raise more interesting questions for future investigation.

We have studied 35 single nucleotide polymorphisms (SNPs) in the interferon (IFN) pathway to determine their contribution to multiple sclerosis (MS) and hepatitis C virus (HCV) infection. A total of 182 patients with MS, 103 patients with chronic hepatitis C, and 118 control subjects were enrolled in the study. Of the 35 SNPs studied, 3 were in IFN-alpha receptor (IFNAR-1), 10 in IFN-alpha/beta receptor (IFNAR-2), 9 in Stat1, 5 in Stat2, and 8 in IFN regulatory factor-1 (IRF-1). Compared to controls, Stat1 gene polymorphisms were significantly more frequent in MS patients (rs# 2066802 OR = 7.46, 95% CI = 2.22-25.10; rs# 1547550 OR = 1.69, 95% CI = 1.01-2.81) and in HCV patients (rs# 2066802 OR = 5.95, 95% CI = 1.55-22.81; rs# 1547550 OR = 2.30, 95% CI = 1.24-4.24). Also one IRF-1 gene SNP was associated with MS (rs# 2070721 OR = 2.05, 95% CI = 1.03-4.09), and four IRF-1 gene SNPs were associated with HCV infection (rs# 2070721 OR = 2.59, 95% CI = 1.23-5.43; rs# 2070723 OR = 4.8, 95% CI = 1.26-18.20; rs# 2070728 OR = 9.81, 95% CI = 1.21-79.4; rs# 2070729 OR = 3.6, 95% CI = 1.23-10.48; rs# 839 OR = 4.67, 95%CI = 1.29-16.87). Characteristic nucleotide combinations on single chromosomes (haplotype) generated block structures, including SNPs, that differed between patients and controls. Using a permutation test to detect differences in haplotype distribution between groups, the CCATTGA and the CCGAA haplotypes in the IRF-1 gene were more frequent in MS (p = 0.03) and in HCV patients (p = 0.001) than in controls. In conclusion, our data show that genetic variants in the IRF-1 and Stat1 genes of the IFN pathway are associated with MS and HCV infection.

Interferon (IFN) regulatory factor 1 (IRF-1) is a transcriptional regulatory protein that mediates the transcriptional activation of the IFN-alpha and IFN-beta genes by viruses and IFNs. To characterize the mechanisms that govern the level of IRF-1 in cells, we isolated the IRF-1 gene and characterized the structure of its intronic and exonic domains and of its regulatory promoter region. A human placental genomic library was screened with an IRF-1 cDNA probe, and two clones that contained the IRF-1 gene and its 5' regulatory region were obtained. We used these clones to determine the complete nucleotide sequence for the IRF-1 gene, finding that the IRF-1 gene spanned 7.72 kb of DNA and included 10 exons and 9 introns. When the deduced amino acid sequences were compared among different species, the most conserved exons were exons 2, 3, and 4, in which the putative DNA binding domain for the IRF-1 protein is located.

Interferon regulatory factor 7 (IRF-7) plays an important role in innate immunity, where, together with IRF-3, it controls the expression of interferon A/B genes as well as chemokine RANTES (regulated on activation normal T cell expressed and secreted). Previously, we characterized human IRF-7 promoter and showed that an interferon-stimulated response element site in the first intron binds interferon-stimulated gene factor 3 (ISGF3) and confers the response to interferon. Here we report the stimulation of IRF-7 expression by 12-O-tetradecanoylphorbol-13-acetate (TPA) and tumor necrosis factor alpha (TNFalpha) in human peripheral blood monocytes. Using promoter analysis in combination with electrophoretic mobility shift assays, we have demonstrated that an NFkappaB site located next to the TATA box, binds p50 and p65 heterodimer and is required for the induction of the IRF-7 gene by TPA and TNFalpha. In addition, we report stimulation of IRF-7 gene expression by topoisomerase II (TOPII) inhibitors. We show by chromatin immunoprecipitation assay that treatment with the TOPII inhibitor etoposide induces association of acetylated histone 3 with the promoter of IRF-7 gene, indicating that TOPII-mediated changes in chromatin structure could be responsible for the induction. This suggests that the IRF-7 gene is localized in the condensed area of the chromosome where it is inaccessible to transcription factors that would promote its constitutive expression.

Genes containing an interferon (IFN)-stimulated response element (ISRE) can be divided into two groups according to their inducibility by IFN and virus infection: one induced only by IFN and the other induced by both IFN and virus infection. Although it is now clear that IFN regulatory factor 7 (IRF7) is a multifunctional gene essential for induction of type I IFNs, regulation of the IRF7 promoter (IRF7p) is poorly understood. The IRF7 gene includes two IFN responsive elements, an IRF-binding element (IRFE) in the promoter region and an ISRE in the first intron, and is induced by the IFN-triggered Jak-STAT pathway by binding of the IFN-stimulated gene factor 3 (ISGF3) complex to the ISRE. In this study, we demonstrate that IRF3 and IRF7, which with the coactivators CREB-binding protein and P300 form the virus-activated factor (VAF) complex upon Sendai virus infection, bind to the IRF7 ISRE and IRFE and can directly activate IRF7 transcription. Promoter reporter assays show that both the ISRE and IRFE are responsive to activation by IRF7 and IRF3. In cells transiently expressing IRF7 or/and IRF3, the VAF level and binding of VAF are clearly increased after Sendai virus infection. Studies with Jak1 kinase inactive 293 cells that were stably transfected with a Jak1 kinase dead dominant negative construct, and the mutant cell lines SAN (IFNalpha-/beta-), U2A (IRF9-), U4A (Jak1-), and DKO (IRF1-/IRF2-) show that the IRF7 transcription activated directly by VAF is distinct from and independent of the IFN signaling pathway. Thus, IRF7 transcription is autoregulated by binding of the IRF7-containing VAF to its own ISRE and IRFE. The results show two distinct mechanisms for the activation of the IRF7 promoter, by IFN and by virus infection. A regulatory network between type I IFNs and IRF7 is proposed. The distinct pathways may reflect special roles for an efficient antiviral response at different stages of virus infection.

IL-4 gene cluster on chromosome 5 contains several candidate genes for atopy and asthma. Several independent studies have shown evidence for linkage between the markers flanking IL-4 gene cluster and asthma and/or asthma-related traits. Interferon regulatory factor 1 (IRF-1) is located approximately 300 kb telomeric to IL-4 and recent study reveals that IRF-1 deficiency results in an elevated production of Th2-related cytokines and a compensatory decrease in the expression of native cell- and Th1-related cytokines. To determine if there are any mutations associated with the development of atopy and asthma present in the coding exons and 5' flanking region of the IRF-1 gene. We have screened the promoter and coding regions of the IRF-1 gene in atopic asthmatics and controls by SSCP method. We found three novel nuclear variants (the -300G/T and 4396 A/G polymorphisms and the 6355G > A rare variant) in the IRF-1 gene. No variants causing amino acid alterations of IRF-1 were detected. The -300G/T polymorphism was in nearly complete linkage disequilibrium with the 4396 A/G polymorphism. An association between the 4396 A > G polymorphism and atopy/asthma was examined by transmission disequilibrium test in 81 asthmatic families. Either of 4396 A or 4396G alleles was not significantly preferentially transmitted to atopy- or asthma-affected children. The IRF-1 gene is less likely to play a substantial role in the development of atopy and asthma in the Japanese population.

Imiquimod is an oral inducer of interferon (IFN) and several other proinflammatory cytokines and has been successfully used topically as an antiviral agent for the treatment of genital warts. We have investigated the molecular mechanisms by which imiquimod induces the expression of IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines in vivo, using mice deficient in various components of the IFN signaling system. Mice deficient in the transcription factor interferon regulatory factor 1 (IRF-1) or in the serine/threonine protein kinase PKR responded normally to imiquimod, producing high levels of circulating IFN and induction of several ISGs. On the other hand, when mice deficient in STAT-1 were treated, a 32-fold reduction in the level of circulating IFN was observed, together with a lack of induction of 2-5 oligo adenylate synthetase (2-5 OAS) and IRF-1 genes. Interestingly, there was also a lack of induction of interleukin-6 (IL-6) gene expression, although tumor necrosis factor was induced and readily detected in serum. In mice deficient in the type I IFN receptor, imiquimod induced levels of IFN similar to those in control mice, but again, neither 2-5 OAS, IRF-1, nor IL-6 genes were induced in mutant mice. Our results suggest that STAT-1 plays a critical role in the mechanism of gene activation by imiquimod. Moreover, induction of IL-6 gene expression appears to be dependent on components of the IFN signaling cascade.

Juvenile idiopathic arthritis (JIA) comprises several clinically distinct subgroups and is the most widespread cause of chronic childhood disability. A significant association between JIA and a polymorphism in the interferon regulatory factor 1 (IRF-1) gene has previously been reported, implicating IRF1 in disease susceptibility. The aim of this study was to replicate the IRF1 association in JIA using single-marker and haplotype analyses in a case-control study, using control subjects different from those in the previous study and a larger cohort of patients (n = 765). DNA from 765 patients with JIA and 508 unaffected control subjects was genotyped for 4 single-nucleotide polymorphisms in the IRF-1 gene. Association of genotypes at the IRF1 loci was assessed using single-marker and haplotype analyses. No significant differences in genotype frequency or allele frequency were observed between patients and control subjects. This replication study used a much larger patient cohort to examine the association of IRF1 in JIA. However, despite the increased statistical power, we observed no significant association for IRF1 markers, either individually or as haplotypes. It is therefore unlikely that this gene is involved in JIA susceptibility.

Interferon regulatory factor 9 plays a dual function in health and disease

Variation in susceptibility to HIV-1 infection depends on numerous factors, and host genetic variation has been well-described as an important component. As a transcriptional regulator, interferon regulatory factor 1 (IRF-1) plays a key role in both innate and adaptive immunity against viral infection. IRF-1 has also been shown to directly interact with HIV-1 5' LTR and efficiently initiate or amplify HIV-1 replication. By complete gene sequencing, we investigated genetic polymorphism of the IRF-1 gene in an HIV-1-endemic Kenyan population. This population displayed extensive genetic diversity at the IRF-1 locus. Fifty-three single nucleotide polymorphisms (SNPs) were identified in this population, including 26 novel SNPs. Two insertion and one deletion polymorphisms in IRF-1 were also identified. Linkage disequilibrium (LD) among these genetic variations was shown to be common in IRF-1. The functional consequences of these mutations in the context of HIV-1/AIDS remain to be determined. We also identified 35 consistent discrepancies between IRF-1 GenBank sequences and our population based sequencing data, suggesting that the previously submitted GenBank data were not representative of the majority of human IRF-1 sequences.