A Positive Feedback Amplifier Circuit That Regulates Interferon (IFN)-Stimulated Gene Expression and Controls Type I and Type II IFN Responses

Abstract

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.

Keywords: JAK/signal transducer and activator of transcription signaling pathway; antiviral activity; interferon; interferon regulatory factor 1; interferon-stimulated gene factor 3; signal transducer and activator of transcriptions; transcriptional regulation.

Figure 1

IFN-activated ISG transcription mediated by ISGF3, GAF, and STAT2/IRF9 complexes. IFN-I is recognized by a heterodimeric receptor composed of IFNAR1 and IFNAR2 subunits. After IFN binding and receptor dimerization, juxtapositioning of JAK1 and TYK2 results in increased kinase activity via transphosphorylation and subsequent STAT protein recruitment. Receptor-bound STAT proteins are successively phosphorylated, dimerize, and translocate to nucleus, where ISG transcription is initiated after binding ISRE or GAS sites. Thus, in response to IFN-I three active complexes are formed that play a crucial role in transcriptional regulation. A STAT1/STAT2 heterodimer associated with IRF9, known as ISGF3, binds the ISRE motif present in >300 ISGs. Second, with the same mode of action, an alternative complex built of STAT2 homodimers and IRF9 (STAT2/IRF9). In addition, STAT1 homodimers (known as GAF), which specifically recognize the GAS sequence. On the other hand, IFN-II interacts with a different receptor built of two IFNGR1 and two IFNGR2 subunits connected with JAK1 and JAK2 kinases, which are capable of phosphorylating only STAT1 proteins, resulting in dimerization and formation of GAF. GAF translocates to the nucleus and targets GAS-containing genes, in a similar way as in response to IFN-I. Abbreviations: IFN, interferon; STAT, signal transducer and activator of transcription; IRF, interferon regulatory factor; JAK, Janus kinase; TYK, tyrosine kinase; ISGF3, interferon-stimulated gene factor 3; GAF, γ-activated factor; ISRE, interferon-stimulated response element; GAS, γ-activated sequence; ISG, interferon-stimulated gene; P, phosphate; IFNAR, IFNα receptor.

Figure 2

IFN-dependent and -independent ISG transcription mediated by complexes comprised unphosphorylated STATs. In addition to the classical IFN signaling pathway based on complexes of phosphorylated STAT proteins (early response—details Figure 1), evidence exists for the involvement of unphosphorylated versions of ISGF3 (U-ISGF3), GAF (U-STAT1), and STAT2/IRF9 (U-STAT2/IRF9), in maintaining basal expression of certain ISGs in unstimulated cells (IFN independent). In addition, these unphosphorylated complexes are instrumental in sustaining expression of ISGs in response to long-term IFN-I or IFN-II stimulation (IFN dependent), when the level of phosphorylated STAT proteins is decreasing, but de novo synthesized unphosphorylated STAT proteins accumulate and can be incorporated in unphosphorylated transcription factors (late response). Abbreviations: IFN, interferon; STAT, signal transducer and activator of transcription; IRF, interferon regulatory factor; JAK, Janus kinase; TYK, tyrosine kinase; ISGF3, interferon-stimulated gene factor 3; GAF, γ-activated factor; ISRE, interferon-stimulated response element; GAS, γ-activated sequence; ISG, interferon-stimulated gene; P, phosphate; U, unphosphorylated form.

Figure 3

IFN-dependent and -independent ISG transcription mediated by IRF1. According to the current state of knowledge, expression of IRF1 is driven by homodimers of STAT1 (GAF), as well as STAT1/STAT2 heterodimers, recognizing a single GAS sequence in the IRF1 promoter. Recent studies confirmed that basal expression of IRF1 can be detected in many cell types under basal conditions. Moreover, binding of IRF1 to ISRE-containing genes can play a role in maintaining constitutive expression of certain group of ISGs (left panel). After IFN-I or IFN-II stimulation GAF and GAF-like (STAT1/STAT2 heterodimers) complexes are rapidly formed and translocated to the nucleus to initiate IRF1 expression. Subsequently, IRF1 as an additional abundant transcription factor can collaborate with ISGF3 and GAF complexes stimulating ISRE-containing genes, thus appearing as an important link between IFN-I and IFN-II responses. Abbreviations: IFN, interferon; STAT, signal transducer and activator of transcription; IRF, interferon regulatory factor; JAK, Janus kinase; TYK, tyrosine kinase; ISGF3, interferon-stimulated gene factor 3; GAF, γ-activated factor; ISRE, interferon-stimulated response element; GAS, γ-activated sequence; ISG, interferon-stimulated gene; P, phosphate.

Figure 4

Representative modes of STAT1, STAT2, IRF9, and IRF1 binding to ISRE and GAS-containing ISGs in GM12878, K562, and 2fTGH cells treated with or without IFN-I or IFN-II. To further study genome-wide binding of STAT1, STAT2, IRF9, and IRF1, a ChIP-seq dataset from K562 cells treated with IFNα or IFNγ for 0.5 or 6 h and GM12878 cells used as a control were retrieved from the ENCODE database (GSE31477) and compared to our recently dataset from 2fTGH cells treated with or without IFNα for 2 and 24 h (unpublished data). Antibodies used in given experiments are indicated in the left panel and by color of the track—STAT1—green, STAT2—blue, IRF9—red, and IRF1—orange. Tracks derived from input sequencing are indicated in black. The bottom panel indicates ISRE or/and GAS sequences located under the peaks (see also Table 1). According to DNA motifs, ISGs can be divided into three groups: only ISRE-containing genes (examples are presented in red boxes—ISG15, MX1, OAS3, and IFIT3), only GAS-containing genes (examples in green boxes—IRF1, SOCS3, and ICAM1), and genes with both sequences (examples in yellow boxes—IFI35, DTX3L, TRIM69, AIM2, BST2, SOCS1, STAT1, STAT2, and IRF9). The blue frame marks components of ISGF3, GAF, and IRF1. Each group of genes displays differences in binding after IFN stimulation. ISRE-only genes show strong binding of components of ISGF3 (STAT1, STAT2, and IRF9) after IFNα, as well as IRF1 after IFNα and IFNγ, but no binding of STAT1 after IFNγ treatment. On the other hand, GAS-only genes demonstrate strong binding of STAT1 and STAT2 (components of GAF and GAF-like complexes) after IFNγ and IFNα treatment. The third group consists of ISRE + GAS composite-site containing ISGs, which display binding of all the components in response to both types of IFNs pointing to the possible cooperation and mechanism of co-binding of ISGF3, GAF and IRF1 for optimal gene expression. Abbreviations: IFN, interferon; STAT, signal transducer and activator of transcription; IRF, interferon regulatory factor; ISGF3, interferon-stimulated gene factor 3; GAF, γ-activated factor; ISRE, interferon-stimulated response element; GAS, γ-activated sequence; ISG, interferon-stimulated gene.

Figure 5

Cooperation of unphosphorylated and phosphorylated ISGF3 and GAF components and IRF1 in the regulation of basal and IFN-induced transcription. In unstimulated cells, most of the known ISGs are expressed at low but detectable levels. Among these genes, there are examples of components (STATs and IRFs) of transcription factors, which in the absence of stimulation can combine in unphosphorylated complexes (U-ISGF3, U-STAT1, U-STAT2/IRF9, and IRF1) and drive the constitutive expression of ISGs. After cell stimulation with IFN type I or II, receptors dimerize and enable receptor-bound kinases (JAK1 and TYK2 for IFN-I or JAK1 and JAK2 for IFN-II) transphosphorylation and STAT protein recruitment. Next step is phosphorylation of STATs, which can now dimerize and with or without a partner (IRF9) create complexes—ISGF3, ST2/IRF9, and GAF, then translocate to the nucleus and start a high and robust but transient expression (yellow dotted graph) of GAS and/or ISRE-containing ISGs. This leads to, inter alia, rapid accumulation of newly synthesized STAT1, STAT2, IRF9, and IRF1 proteins in the cytoplasm, which can create new transcription factors in unphosphorylated form (U-ISGF3, U-STAT1, and U-STAT2/IRF9), and while level of phospho-proteins is dropping (dotted line) support or take over the role of phosphorylated complexes in sustaining the expression of ISGs. Important role in the circuit plays IRF1, which can occupy ISRE-containing genes and co-binds with other factors to modulate IFN-induced response, as well as basal ISG expression. As shown in the graph, responses to both types of IFN rely on common elements and events of the JAK–STAT signaling pathway and result in partially shared outcome. Thus, IFN-I and IFN-II induce a common as well as a unique set of genes. These facts clearly point to functional and biological overlap between IFN-I and IFN-II action and additionally to existence of mechanisms allowing cells to manage and adjust both responses. Abbreviations: IFN, interferon; STAT, signal transducer and activator of transcription; IRF, interferon regulatory factor; JAK, Janus kinase; TYK, tyrosine kinase; ISGF3, interferon-stimulated gene factor 3; GAF, γ-activated factor; ISRE, interferon-stimulated response element; GAS, γ-activated sequence; ISG, interferon-stimulated gene; P, phosphate; U, unphosphorylated form.