Regulatory Networks Involving STATs, IRFs, and NFκB in Inflammation

Abstract

Cells engaging in inflammation undergo drastic changes of their transcriptomes. In order to tailor these alterations in gene expression to the requirements of the inflammatory process, tight and coordinate regulation of gene expression by environmental cues, microbial or danger-associated molecules or cytokines, are mandatory. The transcriptional response is set off by signal-regulated transcription factors (SRTFs) at the receiving end of pathways originating at pattern recognition- and cytokine receptors. These interact with a genome that has been set for an appropriate response by prior activity of pioneer or lineage determining transcription factors (LDTFs). The same types of transcription factors are also critical determinants of the changes in chromatin landscapes and transcriptomes that specify potential consequences of inflammation: tissue repair, training, and tolerance. Here we focus on the role of three families of SRTFs in inflammation and its sequels: signal transducers and activators of transcription (STATs), interferon regulatory factors (IRFs), and nuclear factor κB (NFκB). We describe recent findings about their interactions and about their networking with LDTFs. Our aim is to provide a snapshot of a highly dynamic research area.

Keywords: IRF; NFκB; STAT; chromatin; epigenetic; inflammation; macrophage; transcription.

Figure 1

Structural attributes of STAT-, IRF-, and Rel family transcription factors. STATs. All mammalian STAT members share a common structural motif consisting of an N-terminal domain, which plays a role in dimerization (DD), followed by a coiled-coil domain (CC), that can be involved in interactions with other proteins, a DNA-binding domain (DBD), a linker domain (L), an SH2 domain for reciprocal phospho-tyrosine interaction and a transactivation domain (10, 11). Upon receptor engagement Janus kinases lead to the activation of the latent cytoplasmic STATs, via phosphorylation on single tyrosine residues (Y701 on STAT1 and Y690 on STAT2). The STAT1-STAT2 dimer associates with interferon regulatory factor 9 (IRF9) to form a transcriptionally active IFN-stimulated gene factor 3 (ISGF3). This complex controls gene expression by binding to interferon-stimulated response elements (ISRE) present in promoters of IFN stimulated gene (ISG). Additionally, STAT1 homodimers, translocate to the nucleus and stimulate ISG expression by binding to gamma interferon-activated sites (GAS) (17). IRFs. All IRFs harbor a conserved N-terminal DNA-binding domain (DBD), which forms a helix-turn-helix domain with a conserved tryptophan cluster that recognizes DNA sequences in interferon induced genes (18). An analysis of the crystal structure of the DBD of IRF1 bound to the Ifnb promoter revealed that 5′-GAAA-3′ is the consensus sequence recognized by the helix-turn-helix motif of IRF1 (19). This DNA motif is known as the IRF-element (IRF-E) (20). All IRFs harbor a C-terminal IRF association domain (IAD), which is responsible for homo- and heteromeric interactions with other family members or transcription factors (21, 22). IAD1 and IAD2 domains can be distinguished by structural criteria and are found, respectively, in IRF1 and IRF2 or all other IRFs. Rel (NFκB). One of the best studied NFκB dimers is the p50/p65 heterodimer, whose crystal structure has been solved (23). NFκB recognizes 9–11 bp (base pair) DNA-elements, which are often located within promoters and enhancers of NFκB target genes. The consensus sequence 5′-GGGRNWYYCC-3′, where R denotes a purine base, N means any base, W stands for adenine or thymine and Y represents a pyrimidine base, is recognized by the Rel-homology domain [RHD; (12)]. The C-terminal domain of RelA (p65) contains two strong and independent transactivation domains (TAD) providing full transcriptional activity (24). The p100 precursor protein is proteolytically processed to the NFκB subunit p50. The mature p50 protein contains the RHD followed by glycine-rich region, a region that is essential for directing the cleavage and proteolytic processing of a long IκB-like C-terminal part of the precursors (25). IκBα regulates rapid and transient induction of NFκB activity. The crystal structure of IκBα bound to the p65/p50 heterodimer revealed that one IκBα molecule binds to an NFκB dimer and masks the NLS of p65. IKKβ is necessary and sufficient for phosphorylation of IκBα, leading to IκBα ubiquitination, and further degradation by the proteasome.

Figure 2

Interplay between LDTFs and SRTFs. Pu.1 is an important macrophage lineage–determining factor (LDTF) and a major driving force behind setting up macrophage enhancers for further action by SRTFs. Enhancers are distinguished by high levels of H3K4me1 and are primed by LDTFs, which further displace nucleosomes. Stimulus responsive enhancers and promoters are bound by stimulus-regulated transcription factors (SRTFs), such as STAT, NFκB, and IRF transcription factors, to direct transcriptional responses in the course of inflammation. The binding of SRTFs to primed promoters and enhancers leads to further recruitment of co-activators that deposit the activation mark H3K27ac (5).

Figure 3

Transcription factors shaping macrophage polarity. M1 stimuli lipopolysaccharide (LPS) and interferon γ (IFNγ) trigger the activation of several transcription factors such as IRF1, IRF5, IRF8 STAT1, STAT2, and NFκB (64). M1 macrophages play key roles in inflammation as well as antibacterial responses. IL4 and IL13 induce M2 polarity in macrophages. M2 transcriptomes are determined by different transcription factors such as IRF4, STAT6, JMJD3, PPARγ, PPARδ, and C/EBPβ. M2 macrophages exert anti-inflammatory activities such as tissue repair. M1 macrophages are glycolytic whereas mitochondrial oxidative phosphorylation is required for M2 macrophage development (65).

Figure 4

STAT-NFκB cooperativity shapes the transcriptional response to Listeria monocytogenes. (A) NFκB association with the Nos2 promoter is the initial step and leads to recruitment of TFIIH which is further required for Pol II phosphorylation. Brd4 stabilizes the NFκB-TFIIH complex. In addition, association of HAT and the mediator kinase module (CDK8) strongly depend on NFκB. ISGF3, which recruits the core mediator, is essential for the formation of a pre-initiation complex (PIC) and further provides a critical prerequisite for TFIID and RNA polymerase II (Pol II) binding (–51). These references also describe the experimental procedures for separate analysis of STAT and NFκB signaling during L. monocytogenes infection. (B) Binding of NFκB RelA/p65 and STAT1 to promoters of co-regulated ISGs. The igv browser tracks show induced RelA/p65 binding in macrophages treated with IFNβ and heat-killed Listeria (hkl). STAT1 binding sites are shown from IFNβ-stimulated macrophages. A ChIP-seq data set described in reference 50 was used.

Figure 5

STAT1 and IRF1 synergistically drive expression of Gbp2. A large group of IFNγ-induced genes such as Gbp2 requires both STAT1 and IRF1 for transcriptional activation (52). STAT1 associates with the Gbp2 promoter and is responsible for the ordered recruitment of the coactivator/histone acetyl transferase CREB-binding protein (CBP) and histone hyperacetylation. CBP recruitment requires phosphorylation of the STAT1 TAD at S727. Irf1 is a STAT1 target gene and, following IRF1 synthesis, its association with the Gbp2 promoter follows that of STAT1, but in respective knockout cells the two transcription factors bind without requiring each other's presence. RNA polymerase II (Pol II) association with the Gbp2 promoter requires both STAT1 and IRF1, but only IRF1 is found in a complex with RNA polymerase II.