Interferon (IFN)-I and IFN-II both induce IFN-stimulated gene (ISG) expression through Janus kinase (JAK)-dependent phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT2. STAT1 homodimers, known as γ-activated factor (GAF), activate transcription in response to all types of IFNs by direct binding to IFN-II activation site (γ-activated sequence)-containing genes. Association of interferon regulatory factor (IRF) 9 with STAT1–STAT2 heterodimers [known as interferon-stimulated gene factor 3 (ISGF3)] or with STAT2 homodimers (STAT2/IRF9) in response to IFN-I, redirects these complexes to a distinct group of target genes harboring the interferon-stimulated response element (ISRE). Similarly, IRF1 regulates expression of ISGs in response to IFN-I and IFN-II by directly binding the ISRE or IRF-responsive element. In addition, evidence is accumulating for an IFN-independent and -dependent role of unphosphorylated STAT1 and STAT2, with or without IRF9, and IRF1 in basal as well as long-term ISG expression. This review provides insight into the existence of an intracellular amplifier circuit regulating ISG expression and controlling long-term cellular responsiveness to IFN-I and IFN-II. The exact timely steps that take place during IFN-activated feedback regulation and the control of ISG transcription and long-term cellular responsiveness to IFN-I and IFN-II is currently not clear. Based on existing literature and our novel data, we predict the existence of a multifaceted intracellular amplifier circuit that depends on unphosphorylated and phosphorylated ISGF3 and GAF complexes and IRF1. In a combinatorial and timely fashion, these complexes mediate prolonged ISG expression and control cellular responsiveness to IFN-I and IFN-II. This proposed intracellular amplifier circuit also provides a molecular explanation for the existing overlap between IFN-I and IFN-II activated ISG expression.

Interferon regulatory factors (IRFs) are a family of homologous proteins that regulate the transcription of interferons (IFNs) and IFN-induced gene expression. As such they are important modulating proteins in the Toll-like receptor (TLR) and IFN signaling pathways, which are vital elements of the innate immune system. IRFs have a multi-domain structure, with the N-terminal part acting as a DNA binding domain (DBD) that recognizes a DNA-binding motif similar to the IFN-stimulated response element (ISRE). The C-terminal part contains the IRF-association domain (IAD), with which they can self-associate, bind to IRF family members or interact with other transcription factors. This complex formation is crucial for DNA binding and the commencing of target-gene expression. IRFs bind DNA and exert their activating potential as homo or heterodimers with other IRFs. Moreover, they can form complexes (e.g., with Signal transducers and activators of transcription, STATs) and collaborate with other co-acting transcription factors such as Nuclear factor-κB (NF-κB) and PU.1. In time, more of these IRF co-activating mechanisms have been discovered, which may play a key role in the pathogenesis of many diseases, such as acute and chronic inflammation, autoimmune diseases, and cancer. Detailed knowledge of IRFs structure and activating mechanisms predisposes IRFs as potential targets for inhibition in therapeutic strategies connected to numerous immune system-originated diseases. Until now only indirect IRF modulation has been studied in terms of antiviral response regulation and cancer treatment, using mainly antisense oligonucleotides and siRNA knockdown strategies. However, none of these approaches so far entered clinical trials. Moreover, no direct IRF-inhibitory strategies have been reported. In this review, we summarize current knowledge of the different IRF-mediated transcriptional regulatory mechanisms and how they reflect the diverse functions of IRFs in homeostasis and in TLR and IFN signaling. Moreover, we present IRFs as promising inhibitory targets and propose a novel direct IRF-modulating strategy employing a pipeline approach that combines comparative in silico docking to the IRF-DBD with in vitro validation of IRF inhibition. We hypothesize that our methodology will enable the efficient identification of IRF-specific and pan-IRF inhibitors that can be used for the treatment of IRF-dependent disorders and malignancies.

In the canonical pathway of IFN-I-mediated signaling, phosphorylation of STAT1 and STAT2 leads to heterodimerization and interaction with IRF9. This complex, also known as IFN-stimulated gene factor 3 (ISGF3), then translocates into the nucleus and binds the IFN-I-stimulated response element (ISRE) leading to the activation of transcription of over 300 interferon stimulated genes (ISGs). In addition, STAT1 homodimers [known as γ-activated factor (GAF)] are formed and translocate to the nucleus, where they target genes containing the γ-activated sequence (GAS). The primary function of ISGF3 is to mediate a rapid and robust IFN-I activated response by regulating transient transcription of antiviral ISGs. This requires the quick assembly of ISGF3 from its pre-existing components STAT1, STAT2 and IRF9 and transport to the nucleus to bind ISRE-containing ISGs. The exact events that take place in formation, nuclear translocation and DNA-binding of active ISGF3 are still not clear. Over the years many studies have provided evidence for the existence of a multitude of alternative STAT2-containing (ISRE or GAS-binding) complexes involved in IFN-I signaling, emphasizing the importance of STAT2 in the regulation of specific IFN-I-induced transcriptional programs, independent of its involvement in the classical ISGF3 complex. This review describes the unique role of STAT2 in differential complex formation of unphosphorylated and phosphorylated ISGF3 components that direct constitutive and IFN-I-stimulated transcriptional responses. In addition, we highlight the existence of a STAT1-independent IFN-I signaling pathway, where STAT2/IRF9 can potentially substitute for the role of ISGF3 and offer a back-up response against viral infection.

Transcriptional responses to interferon (IFN) are mediated by tyrosine phosphorylation and nuclear translocation of transcription factors of the signal transducer and activator of transcription (Stat) family. The Stat1 protein is required for all transcriptional responses to IFN (both type I and type II). Responses to type I IFN (alpha and beta) also require Stat2 and the IFN regulatory factor family protein p48, which form a heterotrimeric transcription complex with Stat1 termed ISGF3. Stat1 homodimers formed in response to IFN-gamma treatment can also interact with p48 and function as transcriptional activators. We now show that Stat2 is capable of forming a stable homodimer that interacts with p48, can be recruited to DNA, and can activate transcription, raising a question of why Stat1 is required. Analysis of the transcriptional competence, affinity, and specificity of Stat2-p48 complexes compared with other Stat protein-containing transcription factor complexes suggests distinct roles for each component. Although Stat2 is a potent transactivator, it does not interact stably with DNA in complex with p48 alone. Adding Stat1 increases the affinity and alters the sequence selectivity of p48-DNA interactions by contacting a half-site of its palindromic recognition motif adjacent to a p48 interaction sequence. Thus, ISGF3 assembly involves p48 functioning as an adaptor protein to recruit Stat1 and Stat2 to an IFN-alpha-stimulated response element, Stat2 contributes a potent transactivation domain but is unable to directly contact DNA, while Stat1 stabilizes the heteromeric complex by contacting DNA directly.

VHL inactivation is the most established molecular characteristic of clear cell renal cell carcinoma (ccRCC), with only a few additional genes implicated in development of this kidney tumor. In recently published ccRCC gene expression meta-analysis study we identified a number of deregulated genes with limited information available concerning their biological role, represented by gene transcripts belonging to transmembrane proteins family (TMEMs). TMEMs are predicted to be components of cellular membranes, such as mitochondrial membranes, ER, lysosomes and Golgi apparatus. Interestingly, the function of majority of TMEMs remains unclear. Here, we analyzed expression of ten TMEM genes in the context of ccRCC progression and development, and characterized these proteins bioinformatically. The expression of ten TMEMs (RTP3, SLC35G2, TMEM30B, TMEM45A, TMEM45B, TMEM61, TMEM72, TMEM116, TMEM207 and TMEM213) was measured by qPCR. T-test, Pearson correlation, univariate and multivariate logistic and Cox regression were used in statistical analysis. The topology of studied proteins was predicted with Metaserver, together with PSORTII, Pfam and Localizome tools. We observed significant deregulation of expression of 10 analyzed TMEMs in ccRCC tumors. Cluster analysis of expression data suggested the down-regulation of all tested TMEMs to be a descriptor of the most advanced tumors. Logistic and Cox regression potentially linked TMEM expression to clinical parameters such as: metastasis, Fuhrman grade and overall survival. Topology predictions classified majority of analyzed TMEMs as type 3 and type 1 transmembrane proteins, with predicted localization mainly in ER. The massive down-regulation of expression of TMEM family members suggests their importance in the pathogenesis of ccRCC and the bioinformatic analysis of TMEM topology implies a significant involvement of ER proteins in ccRCC pathology.