Preparing the First Responders: Building the Inflammatory Transcriptome from the Ground Up

Abstract

In cells of the immune system, inflammatory stimuli trigger highly coordinated cascades of gene activation that are precisely calibrated to the nature and strength of the stimulus. Herein, we describe the forces that control inflammatory gene transcription and highlight that many critical determinants of responsiveness are established prior to challenge. We discuss key steps in the transcription cycle that are regulated during gene activation and the importance of the underlying enhancer landscape. Further, we illustrate how the diversity in regulatory strategies employed at inflammatory genes provides novel opportunities for therapeutic intervention.

Introduction

Immune cells have evolved to respond in a balanced fashion to a myriad of environmental challenges and stimuli (Medzhitov, 2008). Moreover, the response is highly tailored to the stimulus and specific for each individual cell type (Smale, 2012b). For example, cells of the innate immune system, such as neutrophils, macrophages and dendritic cells, serve as the body’s first line of defense against infection and other insults. When challenged, these cells express a number of genes that combat infection both directly (e.g., anti-microbial peptides, reactive oxygen species) and indirectly (e.g., chemokines and cytokines) through attracting and activating cells of the adaptive immune system.

Given their critical position at the interface between organisms and their environment, inflammatory responses must be precisely controlled: inadequate responses impose a danger of systemic infection, whereas excessive inflammation can lead to tissue damage. Furthermore, chronic inflammation contributes to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis and has been implicated in type II diabetes, atherosclerosis and cancer (Karin et al., 2006). Thus, there is an ever pressing need to develop therapies that curb excessive inflammation without blunting the natural defenses from dangerous pathogens. Reaching this goal, however, requires a detailed understanding of the molecular pathways and mechanisms governing inflammatory gene expression programs. By identifying the key rate-limiting steps in expression of disease-specific proinflammatory genes or gene classes, we hope to expose their “Achilles heels” to be exploited in the design of more selective therapies.

Recent years have revolutionized our understanding of immune recognition of diverse stimuli and the signaling cascades activated by different effector molecules. Immune cells typically sense challenge through pattern recognition receptors such as Toll-like receptors (TLRs) located at the cell surface or on the endosomal membranes. These receptors bind structurally conserved microbial products termed ‘pathogen-associated molecular patterns’, e.g., components of the bacterial cell wall, lipoproteins, single- or double-stranded nucleic acids. The liganded receptor activates specific signal transduction pathways that, through sequential phosphorylation of adapter proteins, ultimately converge upon transcription factors of the NF-κB/Rel, AP-1, IRF and STAT families to induce their nuclear translocation and/or DNA binding (Takeuchi and Akira, 2010). For example, first described over 25 years ago, the NF-κB family of transcription factors plays a dominant role in the inflammatory response. In unstimulated cells, the majority of NF-κB is sequestered in the cytoplasm by the Inhibitor (IκB) proteins. Immune stimulation leads to IκB phosphorylation and ubiquitin-mediated proteasomal degradation (Hayden and Ghosh, 2012) thereby releasing free NF-κB into the nucleus where it binds DNA and activates transcription.

The expression of inflammatory genes reflects the integration of many steps, including transcription of messenger RNA, RNA processing, RNA turnover and protein translation (Caput et al., 1986; Sariban et al., 1988). However, a number of recent studies have highlighted the paramount importance of transcription regulation in globally defining the inflammatory response (Bhatt et al., 2012; Escoubet-Lozach et al., 2011; Rabani et al., 2011). Indeed, within the minutes and hours following immune challenge, affected cells elaborate a sophisticated transcriptional program, during which hundreds of genes undergo highly-choreographed, successive waves of activation and attenuation (Amit et al., 2009). This temporal regulation allows for coordinated induction of genes encoding functionally related cytokines, chemokines and signaling molecules that cooperate to influence later stages of the inflammatory response. For instance, a number of genes activated immediately upon immune stimulation encode transcription factors that contribute to the subsequent induction of additional genes (Fowler et al., 2011). In addition to this diversity in timing of gene induction following immune challenge, the magnitude of activation varies greatly between genes, with transcript accumulation increasing from several-fold up to several thousand-fold. Despite the complexity of these expression profiles, each cell type responds to a particular stimulus with rather predictable cascades of gene activity, consistent with the idea that these patterns are fine-tuned to achieve an appropriate functional outcome. As such, gene expression in activated macrophages - cells highly responsive to pro- and anti-inflammatory signals - has served as a paradigm for dissecting environmentally-responsive transcriptional control in an experimentally tractable system.

This review focuses on recent advances in our understanding of transcriptional regulation of the inflammatory response. Although there is still much to learn about the forces that drive the transcriptional program in activated innate immune cells, accumulating evidence supports key roles for the following components: i) the characteristics of inflammatory gene promoters prior to immune challenge, including occupancy by the transcription machinery, chromatin architecture and basal activity; ii) the repertoire of transcription factors present in a given cell type and activated by a particular stimulus; iii) the binding pattern of these transcription factors near promoters and at cis-regulatory elements across the genome. We present each of these interrelated topics, beginning with the manner in which the localization and activity of RNA polymerase II (Pol II) is regulated before and during immune challenge. We describe the signal-dependent activation of transcription factors and the features that determine where and when these factors bind DNA, both at promoters and distal regulatory regions proposed to function as enhancers of transcription. Finally, we illustrate how the emerging diversity in mechanisms underlying gene regulation is suggesting different routes for blocking pathogenic proinflammatory gene activation or conferring repression – a standing goal in managing inflammatory and autoimmune conditions.

Be prepared: Pol II sets the stage for gene activation

Recent global studies of gene expression in stimulated macrophages have emphasized the central role of transcription in shaping the inflammatory response (Bhatt et al., 2012; Escoubet-Lozach et al., 2011; Rabani et al., 2011). Moreover, analyses of nascent RNA profiles have revealed a diversity of regulatory strategies governing gene induction, including modulation of Pol II recruitment to promoters as well as release of the early elongation complex into productive transcription elongation. These studies have also uncovered a surprising level of Pol II activity at cis-regulatory elements, suggesting that transcription of enhancer regions also impacts inflammatory gene activation.

Transcription regulation at gene promoters: recruiting and releasing Pol II

Metazoan gene transcription can be regulated at several distinct steps in the transcription cycle (Adelman and Lis, 2012). The promoter region surrounding the transcription start site must first be rendered accessible through the removal of repressive nucleosomes (Figure 1A–B). Pol II can then be recruited, along with the general transcription factors that enable the initiation of RNA synthesis (Figure 1C). After synthesis of a short, 25–60 nucleotide-long RNA, Pol II pauses, awaiting a signal for productive elongation into the coding region (Figure 1D). Pausing of the early elongation complex involves the pause-inducing factors DRB sensitivity-inducing factor (DSIF) and Negative Elongation Factor (NELF) (reviewed in (Yamaguchi et al., 2012). In particular, the NELF complex is critical for the stable pausing of polymerase within the promoter-proximal region (Henriques et al., 2013; Li et al., 2013). Accordingly, release of paused Pol II requires the dissociation of NELF from the elongation complex, through the kinase activity of the Positive Transcription Elongation Factor-b (P-TEFb), which phosphorylates DSIF and/or NELF, as well as the regulatory C-terminal domain (CTD) of the Pol II largest subunit (Figure 1E; (Peterlin and Price, 2006). Notably, Pol II CTD phosphorylation by P-TEFb creates a binding platform for a plethora of RNA processing and chromatin modifying factors that facilitate processive elongation and maturation of the transcript (Figure 1F). In this way, release of promoter-paused Pol II by P-TEFb is coupled with the creation of a productive elongation complex that can transcribe many kilobases before perceiving a signal to terminate transcription and release the nascent RNA.

Figure 1. Key points of regulation in the transcription cycle.

(A) Repressed promoter: A gene promoter is shown with the transcription start site (depicted by an arrow) occluded by a nucleosome.

(B) Chromatin Rremodeling: A chromatin remodeler recruited by a transcription factor (TF) can remove nucleosomes from around the promoter, rendering the region accessible for recruitment of the transcription machinery.

(C) Pre-initiation complex formation: this involves the recruitment of a set of general transcription factors (GTFs), the multi-subunit Mediator complex and Pol II. The regulatory C-terminal domain (CTD) of Pol II is unphosphorylated in this state.

(D) Pausing during early elongation: Pol II initiates RNA synthesis and pauses during early transcription elongation, remaining associated with the short nascent RNA (blue). The pause-inducing factors DSIF and NELF are shown.

(E) Pause release: recruitment of the kinase P-TEFb releases Pol II into the gene through phosphorylation (green P) of pause-inducing factors to dissociate NELF from the elongation complex and transform DSIF into a positive elongation factor that associates with Pol II throughout the gene. The Pol II CTD is also a target of P-TEFb activity.

(F) Productive elongation and recruitment of new Pol II: an ensemble of elongation, RNA processing and chromatin modifying factors (ovals) associate with the phosphorylated Pol II CTD to facilitate productive RNA synthesis and nucleosome disassembly. Additional Pol II is recruited, allowing for multiple rounds of RNA production.

Our growing appreciation that pause release is a crucial regulatory step defining the levels of RNA expression has stimulated great interest in the events that determine P-TEFb localization and activity. These studies have revealed a myriad of mechanisms for bringing P-TEFb to promoters, including the association of P-TEFb with signal-dependent transcription factors such as NF-κB (Barboric et al., 2001). P-TEFb can also be recruited by the Mediator complex (Takahashi et al., 2011) and Bromodomain and Extra-Terminal (BET) proteins that bind acetylated histones (discussed in detail below; Prinjha and Tarakhovsky, 2013; Jang et al., 2005; Yang et al., 2005). Further, many factors that enhance P-TEFb recruitment (e.g., NF-κB, Mediator) can also stimulate additional Pol II loading and transcription initiation, which is necessary to achieve high-level gene activation. Thus, the ensemble of transcription factors and co-factors that facilitate Pol II and P-TEFb recruitment can tailor both the level and timing of activity from gene to gene.

Given the sophistication of the inflammatory gene expression program, any of the steps above could be rate-limiting for a given gene. A number of well-studied inflammatory genes require chromatin remodeling to remove nucleosomes prior to Pol II recruitment and transcription initiation (Ramirez-Carrozzi et al., 2006; Weinmann et al., 2001). Formation of pre-initiation complexes is facilitated by the signal-dependent recruitment of coactivators (Figure 2A, green arrow) such as the large, multi-subunit Mediator complex (van Essen et al., 2009) or the histone acetyltransferase p300/CBP (Zhong et al., 1998). Many genes however, including critical inflammatory genes like TNFα, exhibit accessible promoter regions, histone acetylation and Pol II occupancy in unstimulated macrophages (Figure 2B; Bhatt et al., 2012; Escoubet-Lozach et al., 2011; Ramirez-Carrozzi et al., 2009). Activation of these genes involves the stimulus-induced recruitment of P-TEFb and release of paused Pol II into productive elongation (Figure 2B, green arrow). A central implication of these findings is that such genes could be rendered competent or ‘primed’ for activation prior to immune challenge.

Figure 2. Signal-dependent transcription factors can stimulate diverse steps in the transcription cycle, providing multiple targets for inhibition.

(A) Activation (green arrow): Signal-dependent TFs can stimulate Pre-initiation complex formation through recruitment of coactivators such as Mediator that stimulate association of Pol II with the promoter, or of p300 that acetylates (Ac) adjacent histones to generate a binding site for BET proteins.

Repression (Red T): I-BETs can block the acetylation-dependent association of BET proteins, leading to transcription repression. Signal-dependent Pol II recruitment can be blocked through the activity of hormone-activated (H) Glucocorticoid Receptor when associated with GRIP1 (GR:GRIP1).

(B) Inflammatory genes that exhibit paused Pol II and acetylated histones prior to immune challenge are typically CpG-island promoters.

Activation (green arrow): Induction of paused genes is triggered by signal-dependent recruitment of P-TEFb and pause release.

Repression (Red T): GR:GRIP1 blocks P-TEFb recruitment and NELF release, thereby enforcing Pol II pausing.

Pol II transcription within intergenic regions: more than just noise

A striking observation arising from genomic studies of Pol II activity was the level of transcription occurring at intergenic loci, kilobases away from the nearest known protein-coding transcript (Carninci et al., 2005; Cheng et al., 2005). Suggestive of functional importance, these sites of Pol II activity were often within nucleosome-depleted regions that contained binding motifs for several transcription factors (De Santa et al., 2010; Kapranov et al., 2007; Kim et al., 2010; Koch and Andrau, 2011). Further, transcription at many such regions was induced by activating stimuli, as was the acquisition of histone modifications typical of enhancers (De Santa et al., 2010; Hah et al., 2013; Kim et al., 2010; Koch and Andrau, 2011). Thus, the non-coding RNA species generated at these sites were termed enhancer RNAs or eRNAs (Kim et al., 2010).

Enhancers are defined as sequences that can increase transcription of a target promoter at a considerable distance, from several kilobases to several hundred kilobases, in an orientation-independent manner (Bulger and Groudine, 2011). Enhancers are broadly characterized by a depletion of stable nucleosomes that causes DNA hypersensitivity to nucleases (e.g., DNAse I, MNase). Further, enhancer regions exhibit certain post-translational histone modifications (Heintzman et al., 2007; Visel et al., 2009), such as mono-methylation of histone H3 lysine 4 (H3-K4me1) and acetylation of histone H3 lysine 27 (H3-K27Ac). Although the mechanisms underlying enhancer function are still poorly defined, current models suggest that enhancers facilitate recruitment of coactivators and/or the transcription machinery to the target promoter, perhaps through the formation of DNA loops that bring promoter and enhancer into physical proximity (reviewed in Calo and Wysocka, 2013; Spitz and Furlong, 2012).

Distant control of transcription by associated enhancers had been demonstrated at several cytokine genes (e.g., IL4, IL12b; Agarwal et al., 2000; Zhou et al., 2007), raising the possibility that transcription activity at enhancers may play a role in immune regulation. Indeed, activation of macrophages increased Pol II occupancy and non-coding eRNA production at enhancer-like sites upstream of activated genes (De Santa et al., 2010). Moreover, at a few genes studied in detail, the kinetics of nascent mRNA and eRNA synthesis following immune challenge were very similar, suggesting a mechanistic link between their production (De Santa et al., 2010). The purpose of non-coding transcription at enhancer-like regions is still enigmatic, and the mechanisms of eRNA action are difficult to study because these transcripts are of low-abundance, nuclear, and very short-lived (reviewed in Orom and Shiekhattar, 2013). Nonetheless, recent work suggests that eRNAs facilitate transcription activation of neighboring genes by altering chromatin organization or enabling promoter-enhancer looping (Lam et al., 2013; Melo et al., 2013). Interestingly, several compelling studies argue for the transcription process, rather than eRNAs per se, as being critical (Hah et al., 2013; Kaikkonen et al., 2013). In one such study, transcription of cis-regulatory regions during development appeared to enable the deposition of H3-K4me1, thereby marking these regions as potential enhancers for use in fully differentiated macrophages (Kaikkonen et al., 2013).

The interplay between Pol II and chromatin

Chromatin structure around inflammatory gene promoters and their distal enhancers critically contributes to cell type-specific induction. Importantly, chromatin accessibility informs not only the ‘geography’ of transcription factor binding but also the dynamics of gene activation itself. For example, if a transcription factor has to bind a given site to activate gene expression, the kinetics of activation may reflect whether or not prior chromatin remodeling is required. Indeed, NF-κB binds some genomic loci extremely rapidly after immune stimulation, but shows delayed association dynamics with other regions, consistent with a need for local chromatin remodeling to enable some interactions (Saccani et al., 2001).

As described above, the generation of a nucleosome-deprived region and the presence of Pol II appear to go hand in hand, suggesting a functional connection. In support of this, studies in Drosophila have revealed that stable pausing of Pol II facilitates transcription activity by preventing nucleosome assembly over promoters (Gilchrist et al., 2010). Another feature of mammalian promoters that influences their basal nucleosome occupancy is the presence of extended regions of high CG dinucleotide content known as CpG-islands. The DNA within CpG-islands is thought to be structurally constrained and unfavorable for bending around histone proteins, leading CpG-island promoters to be intrinsically depleted of stable nucleosomes (Fenouil et al., 2012; Ramirez-Carrozzi et al., 2009). Such promoters are frequently decorated with paused Pol II and histone modifications characteristic of active chromatin such as histone H3 and H4 acetylation and H3-K4me3 (Core et al., 2008). Consequently, CpG-island promoters could be envisioned to be poised for more rapid activation (Ramirez-Carrozzi et al., 2009). Indeed, promoters of many rapidly-activated inflammatory genes are within CpG-islands, nucleosome-deprived and occupied by paused Pol II in unstimulated macrophages (Adelman et al., 2009; Ramirez-Carrozzi et al., 2009; Ramirez-Carrozzi et al., 2006). However, 70% of mammalian promoters are estimated to fall within CpG-islands, including a majority of genes activated both ‘early’ and ‘late’ in the inflammatory response (Bhatt et al., 2012). Thus, promoter accessibility cannot be the sole key to the differential kinetics of gene activation. A recent analysis of the relationship between activation kinetics and CpG content found no significant enrichment of CpG-island promoters among the most rapidly induced inflammatory genes versus those with slower induction profiles (Bhatt et al., 2012). This finding is in agreement with recent work in other systems showing that the presence of paused Pol II and open chromatin architecture are not necessary for rapid gene activation (Danko et al., 2013; Gilchrist et al., 2012; Lin et al., 2011).

Notably, the constitutively open chromatin and engagement of the transcription machinery at many inflammatory genes could establish the appropriate level of basal RNA synthesis. Indeed, most inflammatory genes are transcribed at detectable levels prior to stimulation, with activation turning them ‘UP’ rather than ‘ON’ from an inactive state (Bhatt et al., 2012; Escoubet-Lozach et al., 2011). In this way, signal-dependent factors largely play upon an established landscape of gene activity, amplifying gene expression. Further, the broad DNA accessibility surrounding CpG-island promoters has been proposed to facilitate binding by a range of signal-dependent transcription factors (Bhatt et al., 2012), as exemplified by immediate early genes such as c-fos, c-jun and Egr-1 that are induced by diverse stimuli (Fowler et al., 2011). In contrast, genes characterized by repressive promoter chromatin prior to stimulation have been suggested to undergo activation in a more cell type- or signal-specific manner (Bhatt et al., 2012). Genes with this kind of stringent control of expression include a number of activating cytokines (e.g., IL12b, IL6) that may be important to keep fully repressed in resting inflammatory cells (Bhatt et al., 2012). A role of chromatin in influencing the activation state of inflammatory genes is further supported by studies of ‘endotoxin tolerance’ – a process whereby prior exposure of macrophages to endotoxin or other inflammatory triggers causes a number of genes to become refractory to repeated stimulation. In particular, the attenuated recruitment of chromatin remodelers to such tolerized genes and persistent occupancy by corepressor complexes have been linked to the reduced transcriptional response (Chen and Ivashkiv, 2010; Foster et al., 2007; Yan et al., 2012). In summary, the dynamic and sophisticated relationship between gene responsiveness, Pol II occupancy and chromatin architecture around gene promoters and enhancers is an exciting area for further exploration.

Transcription factors: a division of labor

Developments in genomic techniques have greatly expanded our knowledge of where inflammatory signal-dependent factors bind during immune challenge, revealing that a major determinant in this process lies in the chromatin organization and accessibility of potential binding sites. Many critical transcription factors such as NF-κB are unable to bind their cognate motifs when these are occluded by nucleosomes (Lone et al., 2013) and accordingly, a majority of the rapid, signal-dependent NF-κB binding events occur within regions that were depleted of nucleosomes in unstimulated cells (Saccani et al., 2001). In the past few years, we have gained exciting insights into how and when such open chromatin is generated through the activity of pioneer transcription factors involved in cell differentiation and lineage commitment (Ghisletti et al., 2010; Heinz et al., 2010). The implication of these findings is that specific patterns of inflammatory gene expression result from two distinct inputs: one from signal-independent factors that establish the appropriate chromatin landscape over the course of development, and a second from the signal-dependent factors that are activated by challenge.

Inflammatory signal-independent transcription factors: priming the response

Pioneer transcription factors are able to bind their DNA motifs even when embedded in condensed chromatin, and to promote local chromatin remodeling, creating accessible regions (Gualdi et al., 1996). These factors are typically expressed early in development in a lineage-restricted manner and persist throughout cell differentiation, ensuring local opening of chromatin over developmental time. The association of pioneer factors with gene promoters and cis-regulatory regions establishes a cell type-specific chromatin architecture that can later be targeted by stimulus-induced transcription factors in fully differentiated tissues (Figure 3A). The predominant pioneer factor in several immune cell types, including B-cells, macrophages and dendritic cells is PU.1 (also called Spi1). PU.1 is a member of the ETS family of transcription factors that recognizes a sequence motif that is somewhat distinct from that bound by other ETS proteins (Wei et al., 2010). Importantly, PU.1 does not work alone: its binding sites in macrophages are often close to motifs for other transcription factors critical for development such as C/EBP and AP-1 (Figure 3B), suggesting that a key set of transcription factors work together to define and poise enhancer elements (Heinz et al., 2010).

Figure 3. Interplay between transcription factors and Pol II at enhancers.

(A) Chromatin opening is initiated during development by the pioneer factor PU.1 binding to its recognition motif within repressive chromatin.

(B) PU.1-binding is accompanied by a reduction in stable nucleosome occupancy over the region, which either occurs indirectly through competition with nucleosomes, or directly through association of PU.1 with nucleosome remodelers. Binding is often stabilized by additional transcription factors such as C/EBP (shown) or AP-1.

(C) The opening of chromatin is proposed to allow for Pol II recruitment and a low level of eRNA transcription, which could attract chromatin modifying enzymes (ovals) that establish histone marks such as H3-K4me1 (Me), consistent with the formation of a ‘poised’ enhancer.

(D) The combination of open chromatin, appropriate histone modifications and, in many cases, Pol II generates an optimal target for binding of signal-dependent transcription factors and their associated coactivators (e.g., p300) during immune challenge. This leads to local histone acetylation (Ac) and often the increased production of eRNAs (blue), which correlate with ‘active’ enhancers.

Although how PU.1 binding leads to chromatin remodeling is not yet clear, its presence coincides with depletion of nucleosomes over the binding site, and the acquisition of H3-K4me1 on flanking histones, perhaps due to a low level of transcription by Pol II (Figure 3C; Ghisletti et al., 2010; Heinz et al., 2010; Kaikkonen et al., 2013). Notably, the H3-K4me1 modification is thought to represent an early mark in the development of an enhancer that facilitates the binding of chromatin modifying factors involved in acetylation of nearby histones during enhancer activation (Figure 3D; Calo and Wysocka, 2013; Jeong et al., 2011). Indeed, consistent with a role of PU.1 in defining putative enhancer regions, mapping of sites bound by the histone acetyltransferase p300 during immune challenge revealed that p300 generally associated with regions pre-marked by H3-K4me1 and enriched in binding sites for PU.1 (Ghisletti et al., 2010; Heinz et al., 2010).

Thus, the prevailing picture is one wherein the competence for immune responsiveness is programmed at an early stage of development, through the evolution of appropriate chromatin structure and transcription profiles. However, many questions remain. One such question revolves around the sheer number of PU.1-bound regions, which approaches 45,000 in unstimulated murine macrophages, including nearly 9,000 in the vicinity of gene promoters (Ghisletti et al., 2010). Given that very few of these PU.1-bound genes are in fact activated by signal-dependent factors upon immune challenge (Ostuni et al., 2013), there must be more to specificity than the presence of accessible transcription factor binding sites. A related question has to do with the fraction of cis-regulatory regions bound by PU.1 that are functional enhancers: a strikingly small number of such putative regulatory sites have been validated or causally linked to gene expression. In this regard, the development of genome editing technologies (e.g., CRISPR/Cas9 or TALEN) that allow for specific deletions or mutations of endogenous enhancers will greatly expand our ability to both validate and investigate enhancer function.

Inflammatory signal-dependent transcription factors: acting according to plan

The primary signal-dependent transcription factors in innate immune cells are represented by the NF-κB, AP-1, IRF and STAT families, with each factor playing a distinct role during gene activation, often targeting a particular stage of the response or a discrete subset of genes. For most signal-dependent transcription factors, the determinants of in vivo DNA binding remain enigmatic despite well-defined consensus recognition motifs. For example, activated NF-κB does not occupy the majority of high-affinity motifs present in the genome, displaying a remarkable level of cell type selectivity for distinct sites (Smale, 2012a). Accordingly, selective DNA binding has been proposed to be specified by the repertoire of accessible genomic sites available for binding and cooperation between the ensemble of transcription factors that are present in a given cell type and can be activated by a specific stimulus (e.g., TLR4 ligation).

Going beyond cooperative DNA binding, is the notion of temporal regulation of transcription factor activity, which influences the dynamic nature of the inflammatory gene expression program. A number of genes activated early in the inflammatory response themselves encode transcription factors or signaling proteins that together sculpt later stages of the response. Thus, the cascades of gene expression are in part shaped by an evolving arsenal of transcription factors available within the cell, each with their own target gene specificities. It is therefore important, when considering the differential kinetics of gene expression, to bear in mind that the activation of many late-response genes requires production and deployment of additional transcription factors (Ramirez-Carrozzi et al., 2006). Notably, because activation of these genes requires new protein synthesis, they can be distinguished using translation inhibitors such as cyclohexamide (Ramirez-Carrozzi et al., 2006; Yamamoto and Alberts, 1976). This enables broad classification of inflammatory genes as ‘primary’, which can be induced in the absence of protein synthesis, and ‘secondary’, whose key transcriptional activator must be translated first to enable expression. As anticipated, secondary response genes are often induced during the later cascades of gene activation; however, the activation kinetics of ‘primary’ response genes is also non-uniform. Several landmark studies following the dynamics of the macrophage transcriptome during and post stimulation reveal a remarkable diversity in the timing of activation among primary response genes (Adelman et al., 2009; Bhatt et al., 2012; Ramirez-Carrozzi et al., 2009; Ramirez-Carrozzi et al., 2006). Thus, the speed of gene induction is not a simple reflection of transcription factor availability.

Another fundamental layer of functional cooperativity between transcription factors lies in their ability to target distinct steps in the transcription cycle. For example, NF-κB was shown many years ago to stimulate recruitment of both Pol II and P-TEFb to gene promoters (Barboric et al., 2001), providing an explanation for the potent activation function of this transcription factor at many inflammatory genes. However, as described above, NF-κB binding does not induce local chromatin remodeling nor can NF-κB interact with its cognate sites within nucleosomes, such that stimulation of nucleosome-occupied genes requires additional regulators (Weinmann et al., 2001). Thus, NF-κB seems perfectly suited for generating high-level induction of CpG-island genes that reside within open chromatin, but needs assistance to activate nucleosome-repressed promoters. Consistent with this, NF-κB is not sufficient to induce a subset of nucleosome-repressed primary response genes: these require signal-dependent transcription factors such as IRF3 that can efficiently recruit ATP-dependent remodelers (Ramirez-Carrozzi et al., 2009). Further, a number of immune targets in unstimulated macrophages were found to be occupied by corepressor complexes, which could be released in a stimulus-dependent fashion by NF-κB and AP-1 (Huang et al., 2009b). Thus, to fully appreciate the coordination and cooperation between signal-dependent transcription factors, we must further probe the manner in which they stimulate transcription.

Feedback: staying flexible in the face of challenge

Inflammatory cells must respond dynamically to their environments, and recent findings suggest that the cis-regulatory landscape established during development can be ‘re-wired’ by immune exposure (Kaikkonen et al., 2013; Ostuni et al., 2013; Qiao et al., 2013). These studies noted a number of regions, termed latent enhancers, which were unmarked in resting cells but gained PU.1 binding, Pol II transcription and histone modifications typical of active enhancers following immune challenge. Interestingly, while signal-dependent DNA accessibility, transcription and H3-K27Ac at these sites were relatively short lived, the H3-K4me1 mark persisted for several days after challenge. Given the proposed role of H3-K4me1 in priming sites for enhancer activity, the maintenance of this mark raises the possibility that inflammatory genes in this novel transcriptional state could respond differently to a secondary stimulus. Remarkably, several genes adjacent to these latent enhancers were more rapidly induced by secondary challenge, suggesting that the newly established regions are functionally relevant (Ostuni et al., 2013). These studies, combined with those of endotoxin tolerance described above, suggest that the generation of novel regulatory sites or closure of existing sites in mature innate immune cells contributes to establishing a cellular memory of past stimuli, setting the stage for future transcriptional responses to challenge. Understanding the critical epigenetic determinants of such memory functions as well as the longevity of these effects upon immune stimulation or in the face of inflammatory gene repression will be fascinating to define in future work.

Exploiting differences in mechanisms to influence specific gene sets

Inflammation is often viewed as a double-edged sword: essential for host defenses against pathogens yet, dangerous to the host and even fatal when deregulated. Thus, extensive efforts have been aimed at designing compounds that can interrupt inflammatory cascades or mimic the function of their endogenous inhibitors. Historically, the upstream steps in TLR and cytokine signaling were used as therapeutic targets in many autoimmune and inflammatory diseases. Indeed, antibodies and decoy receptors to cytokines (e.g., IL1b, TNFα and IL6) have been on the market for over a decade (Maini and Taylor, 2000). The utility of macromolecules as drugs, however, is limited by their extremely high cost, complex delivery systems and difficulties in titrating dosage, as well as the inherent risks of immunosuppression upon extended treatment. Thus, there is great interest in development of highly-specific small molecule-based alternatives. Deciphering the rate-limiting steps in activation of specific proinflammatory genes and gene classes may enable us to selectively target the factors that drive their expression.

Blocking activation through epigenetic “readers”

Although transcription regulators were once considered too general to be ‘druggable’, pioneering studies in cancer therapy have revealed the benefits of targeting epigenetic regulators of histone modifications (Prinjha and Tarakhovsky, 2013). Indeed, histone deacetylase inhibitors have become an integral part of chemotherapy for T-cell lymphoma, and the list of indications is expanding (Slingerland et al., 2013). An emerging target that has received much attention is the “readers” of histone acetylation, including the BET proteins that bind acetyl-lysine residues. BET proteins facilitate the recruitment of P-TEFb (Hargreaves et al., 2009; Jang et al., 2005; Yang et al., 2005), Mediator, and several chromatin modifying enzymes to target genes (Prinjha and Tarakhovsky, 2013). Among the four BET family members, Brd2 and Brd4 have been implicated in inflammatory gene activation. Indeed, small molecules that block BET protein binding to acetylated histone tails (I-BETs) have conferred protection in mouse models of endotoxin shock and bacterially induced sepsis (Nicodeme et al., 2010). I-BETs displayed substantial selectivity for a group of late-response genes (Nicodeme et al., 2010), consistent with the idea that such genes possess a distinct promoter chromatin structure that requires histone acetylation and signal-induced BET binding (Figure 2A). Notably, histones are not the only substrates for the BET proteins: specific acetylation of NF-κB promotes the recruitment of Brd4 to several inflammatory genes, which potentiates NF-κB activity (Huang et al., 2009a). Given this broad range of potential activities, it is intriguing that only a subset of late-response genes is sensitive to I-BETs; thus, the mechanisms of selectivity in BET recruitment and function are attractive areas for further investigation.

Active repression by Glucocorticoid receptor and Co

A sound alternative to blocking proinflammatory regulators is to mobilize endogenous systems that counter inflammation. Indeed, mammals possess an arsenal of transcription factors that actively repress inflammatory genes by engaging activation complexes at promoters and/or enhancers and disabling their function. Among such regulators, the glucocorticoid (GC) receptor (GR) has been a key therapeutic target in autoimmune and inflammatory diseases for over 60 years (Hench et al., 1949; Ward et al., 1951), however, the lack of selectivity and side effects of traditional GCs demand a better understanding of GR biology (Rhen and Cidlowski, 2005). A prototypic ligand-dependent transcription factor, GR is activated by endogenous or synthetic GCs and regulates hundreds of genes, which in macrophages dramatically re-shapes the inflammatory transcriptome. In addition to binding specific DNA sites directly, GR can tether to other DNA-bound regulators, most prominently, AP-1 and NF-κB, and rapidly and directly repress their target proinflammatory genes (Figure 2) (Yamamoto et al., 1998). Notably, AP-1 and NF-κB remain bound at their GC-repressed targets and typical nuclear receptor corepressors NCoR and SMRT do not appear to engage in these repression complexes. Instead, a regulator of the NCoA family termed the GR-Interacting Protein (GRIP)1 (NCoA2/TIF2/SRC2) (Hong et al., 1997; Voegel et al., 1998) was recently shown to impart repression when recruited by activated GR (Chinenov et al., 2012). Indeed, GRIP1 deletion in macrophages attenuated glucocorticoid repression of numerous proinflammatory genes and, consistently, sensitized mice to LPS-induced endotoxin shock (Chinenov et al., 2012).

Interestingly, GR:GRIP1 complexes repress genes that are activated primarily via Pol II recruitment and transcription initiation (Figure 2A), as well as those controlled by the signal-dependent release of the paused Pol II into productive elongation (Figure 2B) by targeting distinct molecular steps: GR:GRIP1 can block recruitment of both Pol II and P-TEFb (Gupte et al., 2013). Additionally, GRIP1 interacts with numerous histone modifiers as well as components of ATP-dependent chromatin remodeling machinery (reviewed in Xu et al., 2009); however, the roles of these enzymes in GR repression await further investigation. Notably, GR appears to prevent the recruitment of P-TEFb to all GC-sensitive inflammatory genes investigated thus far (Gupte et al., 2013) suggesting that GCs can impose a broad block to productive elongation. Further support for this idea comes from studies of the IL8 gene, which is activated predominantly by Pol II recruitment but can be repressed by GR during early elongation and remain occupied by paused Pol II (Luecke and Yamamoto, 2005; Nissen and Yamamoto, 2000). Therefore, as we define the rate-limiting steps of the transcription cycle for activation of specific genes, it will be important to consider that these may be distinct from the GC-sensitive step. Thus, as disease-specific cytokine signatures continue to emerge, understanding the key determinants that specify the behavior of GR:GRIP1 complexes at different gene classes should help direct synthetic chemistry efforts toward new-generation GR ligands which ‘customize’ GR complexes to such regulatory hallmarks thereby improving anti-inflammatory therapies.

Conclusions

Recent findings have transformed our views on inducible gene expression in higher organisms. It is now appreciated that the promoters of many inflammatory genes are poised in unstimulated cells, allowing for engagement of the transcription machinery and basal gene expression that can be readily amplified by signal-dependent transcription factors (Bhatt et al., 2012; Escoubet-Lozach et al., 2011). Consequently, in order to understand and manipulate the response to inflammatory stimuli we need a better understanding of how the pre-challenge ‘ready’ state of the genome is established in differentiated immune cells, and how it is modified by incoming signals. Furthermore, we now recognize the pivotal importance of both Pol II recruitment and transcription elongation at gene promoters as well as distal cis-regulatory regions. In particular, the discovery that inducible gene expression is often regulated through the establishment and release of promoter-proximally paused Pol II (Adelman and Lis, 2012) raises a multitude of questions about how the process of pause release is controlled. The varied mechanisms described for P-TEFb recruitment and pause release suggest that this process might be disrupted at specific gene subsets to block activation. Indeed, our growing appreciation of the diversity of molecular strategies underlying inflammatory gene regulation provides a unique opportunity to selectively target particular genes or gene classes therapeutically. Thus, there is much excitement that our expanding understanding of gene regulation will yield novel means to modulate aberrant gene activation without dampening the healthy immune response.