Interferon Regulatory Factor-1 Gene Research Articles

Single‐nucleotide polymorphisms of IRF8 gene are associated with systemic lupus erythematosus in Chinese Han population

Interferon regulatory factor 8 (IRF8) has been shown to have diverse roles in the regulation of the immune system. Two recent studies had revealed the association between the single-nucleotide polymorphisms (SNPs; rs11644034 and rs2280381) of IRF8 and systemic lupus erythematosus (SLE) in a multiethnic population. The purpose of this study was to evaluate whether the association could be replicated in a Chinese Han population. Genotypes were determined by a multiplex polymerase chain reaction-ligase detection reaction (PCR-LDR) in 358 patients and 357 geographically matched healthy controls. Significant differences in genotype frequency were found between SLE and control individuals (rs11644034: AA vs. GG, P = 0.014, odds ratio (OR) = 0.980, 95% confidence internal (CI): 0.964-0.996; rs2280381: CC vs. TT, P = 0.005, OR = 0.150, 95% CI: 0.033-0.676). Conditional logistic regression analysis showed that the association of rs2280381 remained significant (P adjusted = 0.028) after adjustment for rs11644034, but not vice versa (P adjusted = 0.361).When stratifying patients with SLE according to clinical subtypes, SNP rs2280381 was found to be associated with low complement in patients with SLE. However, SNP rs11644034 was not found to be associated with SLE clinical subgroups. Analysis of the haplotypes revealed that haplotype G-T and G-C were also significantly associated with SLE (P = 0.002 and P = 0.012, respectively). Our study indicated that the IRF8 gene polymorphisms might be associated with susceptibility to SLE and with disease-related clinical manifestations in Chinese Han population.

Implication of IRF4 Aberrant Gene Expression in the Acute Leukemias of Childhood

The most frequent targets of genetic alterations in human leukemias are transcription factor genes with essential functions in normal blood cell development. The Interferon Regulatory Factor 4 (IRF4) gene encodes a transcription factor important for key developmental stages of hematopoiesis, with known oncogenic implications in multiple myeloma, adult leukemias and lymphomas. Very few studies have reported an association of IRF4 with childhood malignancy, whereas high transcript levels have been observed in the more mature immunophenotype of ALL. Our aim was to investigate the expression levels of IRF4 in the diagnostic samples of pediatric leukemias and compare them to those of healthy controls, in order to determine aberrant gene expression and whether it extends to leukemic subtypes other than the relatively mature ALL subpopulation. Quantitative real-time RT-PCR methodology was used to investigate IRF4 expression in 58 children with acute leukemias, 4 leukemic cell lines and 20 healthy children. We show that aberrant IRF4 gene expression is implicated in a variety of leukemic subtypes; higher transcript levels appear in the more immature B-common ALL subtype and in T-cell than in B-cell leukemias, with the highest expression levels appearing in the AML group. Interestingly, we show that childhood leukemia, irrespective of subtype or cell maturation stage, is characterised by a minimum of approximately twice the amount of IRF4 gene expression encountered in healthy children. A statistically significant correlation also appeared to exist between high IRF4 expression and relapse. Our results show that ectopic expression of IRF4 follows the reverse expression pattern of what is encountered in normal B-cell development and that there might be a dose-dependency of childhood leukemia for aberrantly expressed IRF4, a characteristic that could be explored therapeutically. It is also suggested that high IRF4 expression might be used as an additional prognostic marker of relapse at diagnosis.

Interferon Regulatory Factor 1 Polymorphisms Previously Associated with Reduced HIV Susceptibility Have No Effect on HIV Disease Progression

IntroductionInterferon regulatory factor 1 (IRF1) is induced by HIV early in the infection process and serves two functions: transactivation of the HIV-1 genome and thus replication, and eliciting antiviral innate immune responses. We previously described three IRF1 polymorphisms that correlate with reduced IRF1 expression and reduced HIV susceptibility.ObjectiveTo determine whether IRF1 polymorphisms previously associated with reduced HIV susceptibility play a role in HIV pathogenesis and disease progression in HIV-infected ART-naïve individuals.MethodsIRF1 genotyping for polymorphisms (619, MS and 6516) was performed by PCR in 847 HIV positive participants from a sex worker cohort in Nairobi, Kenya. Rates of CD4+ T cell decline and viral loads (VL) were analyzed using linear mixed models.ResultsThree polymorphisms in the IRF1, located at 619, microsatellite region and 6516 of the gene, previously associated with decreased susceptibility to HIV infection show no effect on disease progression, either measured by HIV-1 RNA levels or the slopes of CD4 decline before treatment initiation.ConclusionWhereas these three polymorphisms in the IRF1 gene protect against HIV-1 acquisition, they appear to exert no discernable effects once infection is established.

Poly I:C induces a protective antiviral immune response in the Pacific oyster (Crassostrea gigas) against subsequent challenge with Ostreid herpesvirus (OsHV-1 μvar)

In-vivo studies were carried out to investigate the protective effect of a synthetic viral analogue (poly I:C) against Ostreid herpes virus (OsHV-1 μvar). Pacific oysters (Crassostrea gigas) were immune-primed by intramuscular injection of 240 μg of poly I:C or sterile seawater at 1 day prior to infection with OsHV-1 μvar. Poly I:C injection induced an antiviral state in C. gigas as the percentage of viral-infected oysters at 48 h post infection was significantly lower in the poly I:C treatment (11%) compared to seawater controls (100%). In an additional experiment, we demonstrated that the protective role of poly I:C is reproducible and elicits a specific antiviral response as immune-priming with heat-killed Vibrio splendidus provided no protection against subsequent viral infection. In both experiments, genes homologous to a toll-like receptor (TLR), MyD88, interferon regulatory factor (IRF) and protein kinase R (PKR) were up-regulated in oysters immune-primed with poly I:C compared to seawater controls (p < 0.05). The MyD88, IRF and PKR genes were also significantly up-regulated in response to OsHV-1 μvar infection (p < 0.05), which is suggestive that they are implicated in the antiviral response of C. gigas. Our results demonstrate that C. gigas can recognise double-strand RNA to initiate an innate immune response that inhibits viral infection. The observed response has striking similarities to the hallmarks of the type-1 interferon response of vertebrates.

HnRNP A1/A2 and SF2/ASF Regulate Alternative Splicing of Interferon Regulatory Factor-3 and Affect Immunomodulatory Functions in Human Non-Small Cell Lung Cancer Cells

Heterogeneous nuclear ribonucleoparticule A1/A2 (hnRNP A1/A2) and splicing factor 2/alternative splicing factor (SF2/ASF) are pivotal for precursor messenger RNA (pre-mRNA) splicing. Interferon regulatory factor-3 (IRF-3) plays critical roles in host defense against viral and microbial infection. Truncated IRF-3 proteins resulting from alternative splicing have been identified and characterized as functional antagonists to full-length IRF-3. In this study, we examined the molecular mechanism for splicing regulation of IRF-3 pre-mRNA and first reported the regulatory effect of hnRNP A1/A2 and SF2/ASF on IRF-3 splicing and activation. RNA interference-mediated depletion of hnRNP A1/A2 or SF2/ASF in human non-small cell lung cancer (NSCLC) cells increased exclusion of exons 2 and 3 of IRF-3 gene and reduced expression levels of IRF-3 protein and IRF-3 downstream effector molecules interferon-beta and CXCL10/IP-10. In addition, direct binding of hnRNP A1 and SF2/ASF to specific binding motifs in IRF-3 intron 1 was confirmed by RNA electrophoretic mobility shift assay. Subsequent minigene splicing assay showed that IRF-3 minigenes with mutated hnRNPA 1/A2 or SF2/ASF binding motifs increased exclusion of exons 2 and 3. Moreover, knockdown of hnRNP A1/A2 or SF2/ASF in NSCLC cells reinforced phytohemagglutinin-induced tumor necrosis factor-alpha release by peripheral blood mononuclear cells (PBMC) but suppressed that of interleukin-10 in NSCLC/PBMC co-cultures. Taken together, our results suggest that specific knockdown for hnRNP A1/A2 or SF2/ASF increase exclusion of exons 2 and 3 of IRF-3 pre-mRNA and influence immunomodulatory functions of human NSCLC cells.

Interferon regulatory factor 3 alters glioma inflammatory and invasive properties

Glioblastoma multiforme (GBM) is the most common, highly malignant primary tumor of the brain with poor prognosis. Even with the improved therapy regimen including temozolomide, the average survival rate is less than 2years. Additional approaches to therapy targeting multiple aspects of glioma progression are in need. In the present work, we have tested the possibility that upregulation of the transcription factor interferon regulatory factor 3 (IRF3) can inhibit glioma invasiveness, proliferation and production of pro-inflammatory and pro-angiogenic factors in cultures of malignant glioma cell lines (U271, U87 and SNB-19). IRF3 is an essential transcription factor involved in TLR3/4-mediated signaling and generation of type I interferons. Although IRF3 has been suggested as a potential tumor suppressor gene, its role in glioma remains uninvestigated. In this study, we find that human glioma immune activation is potently elicited by a cytokine combination, IL-1/IFNγ (or poly IC), but not by bacterial lipopolysaccharide (LPS), similar to primary human astrocytes. GBM biopsy specimens show little detectable IRF3 immunoreactivity, and in vitro adenovirus-mediated IRF3 gene transfer in glioma cells modulates IL-1/IFNγ-induced cytokine and chemokine genes, resulting in upregulation of IFNβ and IP-10 (IRF3-stimulated genes) and downregulation of proinflammatory and angiogenic genes including IL-8, TNFα and VEGF (IRF3-represssed genes). Cytokines (IL-1β and TNFα) also induce the expression of miR-155 and miR-155*, the microRNAs crucial in immunity and inflammation-induced oncogenesis and this is dose-dependently suppressed by IRF3. Importantly, IRF3 also inhibits glioma proliferation, migration and invasion. Together, these data suggest that IRF3 can suppress glioma progression. Agents that promote IRF3 activation and expression (such as IRF3 gene transfer) could be explored as potential future therapy.

Association between SNPs in interferon regulatory factor 2 (IRF-2) gene and resistance to Aeromonas hydrophila in freshwater mussel Hyriopsis cumingii

Interferon regulatory factor 2 (IRF-2) is a multi-functional transcription factor in the IRF family exhibiting both transcriptional activating and repressing activities. In this study, an IRF-2 gene (HcIRF-2) from Hyriopsis cumingii was identified and characterized. The cDNA sequence consisted of 2688 bp, encoding a 329 amino acid-protein. The amino acid sequence had a highly conserved N-terminal DBD structure, containing characteristic repeats of six tryptophan residues. The 5′-flanking region contained several transcription regulation elements such as AP1, CdxA, HSF, NIT2 and HNF-3b. Nine SNPs were obtained through direct sequencing of HcIRF-2 from resistant and susceptible stock. Only +2365T/C SNP was significantly associated with resistance/susceptibility of H. cumingii to Aeromonas hydrophila both in genotype (P = 0.021) and allele (P = 0.006) analysis. The SNPs +2248T/C and +2365T/C were in high linkage disequilibrium, and haplotype analysis revealed that haplotype TT frequency in the resistant group was significantly higher than in the susceptible group. The mortality in +2248CC genotype individuals was significantly higher than in CT and TT genotype individuals. These results indicated that haplotype TT and genotype +2248CT and +2248GT individuals were resistant to A. hydrophila, which could make them potential markers in selective breeding of H. cumingii.

Cross talk between cancer and immune cells: exploring complex dynamics in a microfluidic environment

The reconstitution of a complex microenvironment on microfluidic chips is one of the cornerstones to demonstrate the improved flexibility of these devices with respect to macroscale in vitro approaches. In this work, we realised an on-chip model to investigate the interactions between cancer and immune system. To this end, we exploited mice deficient (Knock Out, KO) for interferon regulatory factor 8 (IRF-8), a transcription factor essential for the induction of competent immune responses, to investigate how IRF-8 gene expression contributes to regulate immune and melanoma cells crosstalk. In vivo, IRF-8 KO mice are highly permissive to B16 melanoma growth due to failure of immune cells to properly exert immunosurveillance. B16 cells and immune cells isolated from the spleen of wild type (WT) and IRF-8 KO mice were co-cultured for one week in a PDMS platform and monitored by fluorescence microscopy and time-lapse recordings. We observed that WT spleen cells migrated through microchannels connecting the culturing chambers towards B16 cells and tightly interacted with tumor cells, forming clusters of activation. In contrast, IRF-8 KO immune cells poorly interacted with melanoma cells. In parallel, B16 cells were more attracted towards microchannels, acquiring a more invasive behaviour in the presence of IRF-8 KO spleen cells, with respect to WT cells. Our results strongly confirm the in vivo observations and highlight the value of on-chip co-culture systems as a useful in vitro tool to elucidate the reciprocal interactions between cancer cells and host immune system, with relevant impact in the development of more effective anti-tumor therapeutic strategies.

Interferon-γ Upregulates Expression of IFP35 Gene in HeLa Cells via Interferon Regulatory Factor-1

BackgroundInterferon-induced 35-kDa protein (IFP35) plays important roles in antiviral defense and the progression of some skin cancer diseases. It can be induced by interferon-γ (IFN-γ) in multiple human cells. However, the mechanisms by which IFN-γ contributes to IFP35 induction remain to be elucidated.Methods/Principal FindingsWe identified the transcription start sites of IFP35 by 5′ rapid amplification of cDNA ends (RACE) and cloned the promoter of IFP35. Sequence analysis and luciferase assays revealed two GC boxes and an IFN-stimulated response element (ISRE) in the 5′ upstream region of the transcription start sites, which were important for the basal transcription of IFP35 gene. Furthermore, we found that interferon regulatory factor 1 (IRF-1) and IRF-2 could bind to IFP35 promoter and upregulate endogenous IFP35 protein level. Depletion of endogenous IRF-1 by interfering RNA reduced the constitutive and IFN-γ-dependent expression of IFP35, whereas depletion of IRF-2 had little effect on IFN-γ-inducible IFP35 expression. Moreover, IRF-1 was recruited to the ISRE site in IFP35 promoter in IFN-γ treated HeLa cells, as demonstrated by electrophoretic mobility shift and chromatin immunoprecipitation assays.Conclusions/SignificanceThese findings provide the first evidence that IRF-1 and IRF-2 are involved in constitutive IFP35 expression in HeLa cells, while IRF-1 also activates IFP35 expression in an IFN-γ-inducible manner. Our data therefore identified a new IRF-1 and IRF-2 target gene, which may expand our current understanding of the versatile functions of IRF-1 and IRF-2.

Expression of interferon-γ, interferon-α and related genes in individuals with Down syndrome and periodontitis

BackgroundRecently, attenuation of anti-inflammatory and increase of pro-inflammatory mediators was demonstrated in individuals with Down syndrome (DS) in comparison with euploid patients during periodontal disease (PD), suggesting a shift to a more aggressive inflammation in DS. AimTo determine the influence of DS in the modulation of interferons (IFNs) signaling pathway in PD. Materials and methodsClinical periodontal assessment was performed and gingival tissue samples obtained from a total of 51 subjects, including 19 DS individuals with PD, 20 euploid individuals with PD and 12 euploid individuals without PD. Expression levels of interferon-gamma (IFNG) and interferon-alpha (IFNA), and their receptors IFNGR1, IFNGR2, IFNAR1 and IFNAR2, the signaling intermediates Janus kinase 1 (JAK1), signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1) were determined using real time quantitative polymerase chain reaction (qPCR). ResultsClinical signs of periodontal disease were markedly more severe in DS and euploid patients with PD in comparison to euploid and periodontally healthy patients. There was no difference on mRNA levels of IFNA, IFNG, INFGR2, IFNAR1 and IFNAR2 between DS and euploid individuals, even though some of these genes are located on chromosome 21. STAT1 and IRF1 mRNA levels were significantly lower in DS patients in comparison with euploid individuals with PD. In euploid individuals, PD was associated with an increased expression of IFNGR1, IFNGR2, IFNAR1, STAT1 and IRF1. ConclusionsReduced expression of STAT1 and IRF1 genes indicate an impaired activation of IFNs signaling in individuals with DS and PD. Expression of IFNA, IFNG and IFN receptors was not altered in DS patients, indicating that indirect mechanisms are involved in the reduced activation of IFN signaling.

Biological activity of lenalidomide in myelodysplastic syndromes with del5q: results of gene expression profiling from a multicenter phase II study

In vitro studies suggest that haploinsufficiency is involved in the pathogenesis of myelodysplastic syndromes (MDS). In patients with del5q cytogenetic abnormality, RPS-14 and microRNAs (miRNAs) play a major role. In a multicenter phase II single-arm trial with lenalidomide in anemic primary del5q MDS patients with low- or int-1 risk IPSS, biological changes from baseline were investigated. Gene expression profiling of selected genes was performed (TaqMan® Low Density Array Fluidic card, Applied Biosystems PRISM® 7900HT) and normalized against the expression of the 18S housekeeping gene from a pool of healthy subjects. Thirty-two patients were evaluated at baseline and after 3 and 6months of treatment. RPS-14, miR-145, and miR-146 were downregulated at baseline and significantly increased during treatment. Nuclear factor kappa B, IL-6, interferon regulatory factor-1, IFNγ-R2, IL-2, and many genes in the apoptotic pathways (TNF, IL-1B, and IL-10) were upregulated at baseline and significantly downregulated during lenalidomide treatment, while forkhead box P3, FAS, IFNγ, IL-12A, and IL-12B were downregulated at baseline and progressively upregulated during treatment. The crucial role of aberrant immunological pathways and haploinsufficiency in the pathogenesis of del5q MDS is confirmed in the present patient setting. Our results indicate that lenalidomide may act through defined immunological pathways in this condition.

A FOXO3–IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses

Antiviral responses must be tightly regulated to rapidly defend against infection while minimizing inflammatory damage. Type 1 interferons (IFN-I) are crucial mediators of antiviral responses1 and their transcription is regulated by a variety of transcription factors2; principal amongst these is the family of interferon regulatory factors (IRFs)3. The IRF gene regulatory networks are complex and contain multiple feedback loops. The tools of systems biology are well suited to elucidate the complex interactions that give rise to precise coordination of the interferon response. Here we have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. Genome-wide location analysis combined with gene deletion studies identified the Irf7 gene as a critical target of FOXO3. FOXO3 was identified as a negative regulator of Irf7 transcription and we have further demonstrated that FOXO3, IRF7 and IFN-I form a coherent feed-forward regulatory circuit. Our data suggest that the FOXO3-IRF7 regulatory circuit represents a novel mechanism for establishing the requisite set points in the interferon pathway that balances the beneficial effects and deleterious sequelae of the antiviral response.

Molecular cloning, expression, mapping of interferon regulatory factor 2 (IRF2) gene and its association with immune traits in pigs

Interferon regulatory factor 2 (IRF2) gene is a member of IRF-family and shown to play functionally diverse roles in the regulation of the immune system. In this report, the porcine IRF2 cDNA was cloned and its 14,000bp genomic DNA structure was also identified. The putative IRF2 protein included 352 amino acids. Alignment analysis of the predicted porcine IRF2 amino acid sequences with their homologies of other species showed high identity (over 93%). Tissues expression of IRF2 mRNA was observed by RT-PCR, the results revealed IRF2 expressed widely in all analyzed tissues except skeletal muscle. Using the radiation hybrid panel, the porcine IRF2 gene was mapped to chromosome 15 and closely linked to microsatellite S0149 (LOD=6.17, 26 cR). A SNP (GU295944:c.1383 G>C) in exon 6 of porcine IRF2 gene was demonstrated by sequencing and PCR-RFLP analysis. The further association analysis indicated that the SNP was significantly associated with level of IL10 (at day 20), IFN-γ (at day 35) and ratio of IFN-γ/IL10 (at day 35) in serum (P<0.05). The results suggested that IRF2 could be served as a promising candidate gene for pig breeding of disease resistance.

Molecular cloning and expression analysis of interferon regulatory factor 8 (IRF8) in turbot, Scophthalmus maximus

Interferon regulatory factor 8 (IRF8) in mammals is known to be involved in antiviral response. In this study, the gene of IRF8 was cloned from the turbot (Scophthalmus maximus) fish and its expression in response to polyinosinic:polycytidylic acid (poly I:C) and turbot reddish body irrdovirus (TRBIV) challenges was studied. Turbot (Sm)IRF8 gene is 4363bp long, comprises nine exons and eight introns and encodes a putative 420 amino acid (aa) protein. The predicted protein sequence possesses a DNA binding domain (DBD), an IRF association domain (IAD) and a nuclear localization signal (NLS). Constitutive expression of SmIRF8 was detectable in all tested organs, with higher levels observed in the spleen, kidney and head kidney. SmIRF8 transcript levels were up-regulated by both poly I:C and TRBIV treatments in the spleen, head kidney, gills and muscle in an early phase of a 7-day time course and the poly I:C was a quicker inducer. In both challenge cases, the highest and earliest inductions were detected in the spleen, while the induction in the muscle was quite faint. These results provide insights into the role of SmIRF8 in antiviral response.

Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells

The expression of the major histocompatibility complex (MHC) classical class I genes is important for the adaptive immune response to target virus-infected cells and cancer cells. The up-regulation of the MHC is achieved by hormonal/cytokine signals including IFN-γ-inducible elements. The swine leukocyte antigen (SLA), the MHC class I region of pigs, consists of the duplicated classical class I genes, SLA-1, SLA-2 and SLA-3, but the molecular mechanisms involved in their up-regulation after T cell stimulation have not been fully elucidated. In order to better understand some of the putative regulatory mechanisms of SLA class I gene expression in activated T cells, we examined the coordinated expression of the SLA classical class I, IFN-γ and interferon regulatory factor-1 (IRF-1) genes in the peripheral blood mononuclear cells (PBMCs) of SLA homozygous Clawn miniature swine stimulated for 72h with either IFN-γ or an enterotoxin produced by Staphylococcus aureus. This enterotoxin, toxic shock syndrome-1 (TSST-1), is known to act as a superantigen (sAG) to activate the T cells in various vertebrate species. We showed by using mAbs and flow cytometry that the CD4+CD25+ cell number of swine PBMCs was also increased by TSST-1 and to a lesser degree by IFN-γ. Time course analyses of the expression of the IFN-γ, IRF-1 and the three classical class I genes, SLA-1, SLA-2, and SLA-3, in PBMCs by quantitative real-time PCR revealed a transitory response to TSST-1 or IFN-γ stimulation. The IFN-γ mRNA levels in the PBMCs were continuously up-regulated over the first 48h by TSST-1 or IFN-γ. In contrast, SLA class I expression moderately increased at 24h and then decreased to a baseline level or less at 72h of IFN-γ or TSST-1 stimulation. The three classical SLA class I genes showed similar expression kinetics, although SLA-3 mRNA level was consistently lower than those of SLA-1 and -2. The expression of IRF-1, a modulator of SLA expression, showed similar kinetics to those of the three classical SLA class I genes. The expression profiles detected by flow cytometry of the SLA molecules on the cell surface of PBMCs were maintained at a consistently high level during cell stimulation with either TSST-1 or IFN-γ, which was distinct from the kinetics of mRNA expression. These results showed that miniature swine SLA class I mRNA expression was effectively and equally up-regulated among the three loci and coordinately with IRF-1 gene expression after stimulation of T cell activation by sAG or IFN-γ.

Transcription factors Sp1 and Sp3 regulate basal transcription of the human IRF-3 gene

Interferon regulatory factor 3 (IRF-3) plays a crucial role in initiation and development of the IFN antiviral response. The expression level of human IRF-3 is thought to be closely related to antiviral state of cells. However, the mechanisms of the transcription regulation of IRF-3 have remained largely unknown. We previously reported that transcription factor E2F1 negatively regulates the basal transcriptional activity of IRF-3. Here we demonstrate that transcription factors Sp1 and Sp3 up-regulate the basal transcriptional activity of IRF-3 and increase IRF-3 expression at mRNA level. By transient transfection analysis we revealed that mutation of Sp1/NRF-1 binding site resulted in a profound reduction of IRF-3 promoter activity. Overexpression of Sp1 and Sp3, but not NRF-1, transactivated the IRF-3 promoter activity in reporter gene assays while knocking-down of endogenous Sp1 and Sp3 by a shRNA strategy markedly inhibited IRF-3 promoter activity. Chromatin immunoprecipitation (ChIP) assays showed that Sp1 and Sp3 interact with the IRF-3 promoter invivo. These results suggest that basal expression level of IRF-3 is regulated by transcription factors Sp1 and Sp3.

Characterization of a spliced variant of human IRF-3 promoter and its regulation by the transcription factor Sp1

Interferon regulatory factor 3 (IRF-3), an essential transcriptional regulator of the interferon genes, plays an important role in host defense against viral and microbial infection as well as in cell growth regulation. Promoter plays a crucial role in gene transcription. We have reported the characterization of the wide type of human IRF-3 promoter, but the characterization of the spliced variant of human IRF-3 Int2V1 promoter has not been systematically analyzed. To observe the spliced variant of human IRF-3 promoter, we have cloned the human IRF-3 gene promoter region containing 300 nucleotides upstream the transcription start site (TSS). Transient transfection of 5' deleted promoter-reporter constructs and luciferase assay illustrated the region -159/-100 relative to the TSS is sufficient for full promoter activity. This region contains GATA1 and specific protein-1 (Sp1) transcription factor binding sites. Interestingly, mutation of this Sp1 site reduced the promoter activity by 50%. However, overexpression of Sp1 increased the transcription activity by 2.4-fold. These results indicated that the spliced variant of human IRF-3 gene core promoter was located within the region -159/-100 relative to the TSS. Sp1 transcription factor upregulates the spliced variant of human IRF-3 gene promoter.

Knockdown of menin affects pre-mRNA processing and promoter fidelity at the interferon-gamma inducible IRF1 gene

BackgroundThe tumor suppressor menin (MEN1) is mutated in the inherited disease multiple endocrine neoplasia type I, and has several documented cellular roles, including the activation and repression of transcription effected by several transcription factors. As an activator, MEN1 is a component of the Set1-like mixed lineage leukemia (MLL) MLL1/MLL2 methyltransferase complex that methylates histone H3 lysine 4 (H3K4). MEN1 is localized to the signal transducer and activator of transcription 1 (STAT1)-dependent gene, interferon regulatory factor 1 (IRF1), and is further recruited when IRF1 transcription is triggered by interferon-γ signaling.ResultsRNAi-mediated knockdown of MEN1 alters the H3K4 dimethylation and H3 acetylation profiles, and the localization of histone deacetylase 3, at IRF1. While MEN1 knockdown does not impact the rate of transcription, IRF1 heteronuclear transcripts become enriched in MEN1-depleted cells. The processed mRNA and translated protein product are concomitantly reduced, and the antiviral state is attenuated. Additionally, the transcription start site at the IRF1 promoter is disrupted in the MEN1-depleted cells. The H3K4 demethylase, lysine specific demethylase 1, is also associated with IRF1, and its inhibition alters H3K4 methylation and disrupts the transcription start site as well.ConclusionsTaken together, the data indicate that MEN1 contributes to STAT1-activated gene expression in a novel manner that includes defining the transcription start site and RNA processing.

Overexpression of interferon regulatory factor 1 enhances chemosensitivity to 5-fluorouracil in gastric cancer cells

To investigate the effects of interferon regulatory factor 1 (IRF-1) gene overexpression on chemotherapeutic sensitivity of gastric cancer cells. An AGS cell system with tetracycline-inducible IRF-1 expression (AGS/IRF-1) was established. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of the IRF-1 gene. Chemosensitivity to 5-fluorouracil (5-FU) was assessed by cell proliferation assay and cell apoptosis. IRF-1 mRNA and protein level were significantly increased in AGS/IRF-1 cells induced with tetracycline. Compared with control cells, the growth inhibition rate of cells with IRF-1 overexpression was significantly increased when treated with 5-FU (P<0.01). Treatment with 5 μmol/l 5-FU resulted in 12.6% apoptotic cells, whereas such treatment after overexpression of IRF-1 resulted in 39.4% apoptotic cells. Moreover, more poly(adenosine diphosphate-ribose) polymerase (PARP) cleavage was seen in cells with IRF-1 overexpression. Overexpression of IRF-1 enhanced the chemosensitivity of gastric cancer cells to 5-FU through induction of apoptosis.

γ-IFN May Act As a Positive Regulator to Hematogenesis Through PI3K/Akt and JAK2/STAT5 Pathways

Abstract 4785It is known to us all that γ-IFN is a growth inhibitory to hematogenesis and plays a critical role in the pathophysiology of aplastic anemia(AA). However, γ-IFN actually is a bilateral regulatory factor. In certain experimental situations, it can facilitate the proliferation and growth of hematopoitic stem cells. Interferon regulatory factor-1 (IRF-1) and IRF-2 are the key regulators of IFN system and IRF-1 has been shown to participate in development of CD8+ T cells, Th1 differentiation, and NK cell development. Thus, IRF-1 may has crucial functions against hematopoietic stem and/or progenitors cells. To investigate the function of IRF-1and the molecular mechanism of proliferative effect of γ-IFN, IRF-1-siRNA was designed to knocked out IRF-1 gene in 32D cells, mouse multipotent progenitor cells, and the apoptosis was assayed by flow cytometry 48 hr after incubation with γ-IFN in three different concentrations. It showed that after IRF-1 gene was knocked out, γ-IFN reversed its action on hematogenesis and apoptosis,it potentiated the proliferation and growth of 32D cells. Interestingly, at this situations, the inhibitory effect of γ-IFN on Akt activation was abolished completely and reversed to enhanced Akt phosphorylation significantly. The expression of pStat5 also changed when γ-IFN only exert proliferative effect.Nevertheless,The negative effect of γ-IFN on Erk1/2 activation was not blocked after IRF-1 gene was knocked out. Taken together, γ-IFN can enhance or antagonize cell proliferation depending on the function of IRF-1 and may act as a positive regulator to hematogenesis through PI3K/Akt and JAK2/STAT5 signaling pathways. Our findings may contribute to understanding the decreased number of stem cells characteristic of aplastic anemia and will provide novel therapeutic strategies for this disease. Disclosures:No relevant conflicts of interest to declare.