Yurii Chinenov

The GRIP1:IRF3 interaction as a target for glucocorticoid receptor-mediated immunosuppression.

Glucocorticoids dramatically inhibit cytokine and chemokine production. They act through the glucocorticoid receptor (GR), a ligand‐dependent transcription factor that binds to and represses activities of other DNA‐bound regulators, activator protein 1 and nuclear factor κB, utilizing a p160 GRIP1 as a corepressor. A yeast two‐hybrid screen with the GRIP1 corepression domain (RD) yielded interferon (IFN) regulatory factor (IRF)3—a downstream effector of Toll‐like receptors (TLR) 3/4 and an essential activator of several IFN and chemokine genes. We defined the GRIP1:IRF3 interface and showed that endogenous GRIP1 and IRF3 interact in mammalian cells. Interestingly, GR and IRF3 competed for GRIP1 binding; GR activation or GRIP1 knockdown in macrophages blocked whereas GRIP1 overexpression rescued IRF3‐dependent gene expression. GR interference persisted in MyD88‐ and IFNA receptor‐deficient mice, suggesting a specific disruption of TLR3–IRF3 pathway, not of autocrine IFN signaling. Finally, IRF3‐stimulated response elements were necessary and sufficient for TLR3‐dependent induction and glucocorticoid inhibition. Thus, GRIP1 plays a cofactor role in innate immunity. Competition with GR for GRIP1 antagonizes IRF3‐mediated transcription, identifying the GRIP1:IRF3 interaction as a novel target for glucocorticoid immunosuppression.

Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling

The kinetics and magnitude of cytokine gene expression are tightly regulated to elicit a balanced response to pathogens and result from integrated changes in transcription and mRNA stability. Yet, how a single microbial stimulus induces peak transcription of some genes (TNFα) within minutes whereas others (IP-10) require hours remains unclear. Here, we dissect activation of several lipopolysaccharide (LPS)-inducible genes in macrophages, an essential cell type mediating inflammatory response in mammals. We show that a key difference between the genes is the step of the transcription cycle at which they are regulated. Specifically, at TNFα, RNA Polymerase II initiates transcription in resting macrophages, but stalls near the promoter until LPS triggers rapid and transient release of the negative elongation factor (NELF) complex and productive elongation. In contrast, no NELF or polymerase is detectible near the IP-10 promoter before induction, and LPS-dependent polymerase recruitment is rate limiting for transcription. We further demonstrate that this strategy is shared by other immune mediators and is independent of the inducer and signaling pathway responsible for gene activation. Finally, as a striking example of evolutionary conservation, the Drosophila homolog of the TNFα gene, eiger, displayed all of the hallmarks of NELF-dependent polymerase stalling. We propose that polymerase stalling ensures the coordinated, timely activation the inflammatory gene expression program from Drosophila to mammals.

Glucocorticoids and the innate immune system: Crosstalk with the Toll-like receptor signaling network

Toll-like receptors (TLRs) are responsible for the recognition of a variety of microbial pathogens and the initial induction of immune and inflammatory responses. These responses are normally restricted by the adrenally produced glucocorticoid hormones which provide a feedback mechanism to curb unabated inflammation. Glucocorticoids act through a ligand-dependent transcription factor-the glucocorticoid receptor (GR), which engages in a complex network of protein:protein and protein:DNA interactions ultimately activating or repressing target gene transcription. Not surprisingly, multiple mechanisms account for the glucocorticoid interference with TLR signaling including enhanced expression of the natural inhibitors of TLR pathways, direct repression of TLR-activated transcriptional regulators and cross-utilization of cofactors essential for both GR and TLR signaling. Here we discuss recent and unexpected examples of crosstalk between the two transcriptional networks and the emerging role of GR in the regulation of innate immunity.

The type I interferon signaling pathway is a target for glucocorticoid inhibition.

ABSTRACT Type I interferon (IFN) is essential for host defenses against viruses; however, dysregulated IFN signaling is causally linked to autoimmunity, particularly systemic lupus erythematosus. Autoimmune disease treatments rely on glucocorticoids (GCs), which act via the GC receptor (GR) to repress proinflammatory cytokine gene transcription. Conversely, cytokine signaling through cognate Jak/STAT pathways is reportedly unaffected or even stimulated by GR. Unexpectedly, we found that GR dramatically inhibited IFN-stimulated gene (ISG) expression in macrophages. The target of inhibition, the heterotrimeric STAT1-STAT2-IRF9 (ISGF3) transcription complex, utilized the GR cofactor GRIP1/TIF2 as a coactivator. Consequently, GRIP1 knockdown, genetic ablation, or depletion by GC-activated GR attenuated ISGF3 promoter occupancy, preinitiation complex assembly, and ISG expression. Furthermore, this regulatory loop was restricted to cell types such as macrophages expressing the GRIP1 protein at extremely low levels, and pharmacological disruption of the GR-GRIP1 interaction or transient introduction of GRIP1 restored RNA polymerase recruitment to target ISGs and the subsequent IFN response. Thus, type I IFN is a cytokine uniquely controlled by GR at the levels of not only production but also signaling through antagonism with the ISGF3 effector function, revealing a novel facet of the immunosuppressive properties of GCs.

Role of transcriptional coregulator GRIP1 in the anti-inflammatory actions of glucocorticoids

Inhibition of cytokine gene expression by the hormone-activated glucocorticoid receptor (GR) is the key component of the anti-inflammatory actions of glucocorticoids, yet the underlying molecular mechanisms remain obscure. Here we report that glucocorticoid repression of cytokine genes in primary macrophages is mediated by GR-interacting protein (GRIP)1, a transcriptional coregulator of the p160 family, which is recruited to the p65-occupied genomic NFκB-binding sites in conjunction with liganded GR. We created a mouse strain enabling a conditional hematopoietic cell-restricted deletion of GRIP1 in adult animals. In this model, GRIP1 depletion in macrophages attenuated in a dose-dependent manner repression of NFκB target genes by GR irrespective of the upstream Toll-like receptor pathway responsible for their activation. Furthermore, genome-wide transcriptome analysis revealed a broad derepression of lipopolysaccharide (LPS)-induced glucocorticoid-sensitive targets in GRIP1-depleted macrophages without affecting their activation by LPS. Consistently, conditional GRIP1-deficient mice were sensitized, relative to the wild type, to a systemic inflammatory challenge developing characteristic signs of LPS-induced shock. Thus, by serving as a GR corepressor, GRIP1 facilitates the anti-inflammatory effects of glucocorticoids in vivo.

Nuclear receptors in inflammation control: repression by GR and beyond.

Inflammation is a protective response of organisms to pathogens, irritation or injury. Primary inflammatory sensors activate an array of signaling pathways that ultimately converge upon a few transcription factors such as AP1, NFκB and STATs that in turn stimulate expression of inflammatory genes to ultimately eradicate infection and repair the damage. A disturbed balance between activation and inhibition of inflammatory pathways can set the stage for chronic inflammation which is increasingly recognized as a key pathogenic component of autoimmune, metabolic, cardiovascular and neurodegenerative disorders. Nuclear receptors (NRs) are a large family of transcription factors many of which are known for their potent anti-inflammatory actions. Activated by small lipophilic ligands, NRs interact with a wide range of transcription factors, cofactors and chromatin-modifying enzymes, assembling numerous cell- and tissue-specific DNA-protein transcriptional regulatory complexes with diverse activities. Here we discuss established and emerging roles and mechanisms by which NRs and, in particular, the glucocorticoid receptor (GR) repress genes encoding cytokines, chemokines and other pro-inflammatory mediators.

Glucocorticoid receptor represses proinflammatory genes at distinct steps of the transcription cycle

Widespread anti-inflammatory actions of glucocorticoid hormones are mediated by the glucocorticoid receptor (GR), a ligand-dependent transcription factor of the nuclear receptor superfamily. In conjunction with its corepressor GR-interacting protein-1 (GRIP1), GR tethers to the DNA-bound activator protein-1 and NF-κB and represses transcription of their target proinflammatory cytokine genes. However, these target genes fall into distinct classes depending on the step of the transcription cycle that is rate-limiting for their activation: Some are controlled through RNA polymerase II (PolII) recruitment and initiation, whereas others undergo signal-induced release of paused elongation complexes into productive RNA synthesis. Whether these genes are differentially regulated by GR is unknown. Here we report that, at the initiation-controlled inflammatory genes in primary macrophages, GR inhibited LPS-induced PolII occupancy. In contrast, at the elongation-controlled genes, GR did not affect PolII recruitment or transcription initiation but promoted, in a GRIP1-dependent manner, the accumulation of the pause-inducing negative elongation factor. Consistently, GR-dependent repression of elongation-controlled genes was abolished specifically in negative elongation factor-deficient macrophages. Thus, GR:GRIP1 use distinct mechanisms to repress inflammatory genes at different stages of the transcription cycle.

Glucocorticoid receptor coordinates transcription factor-dominated regulatory network in macrophages

BackgroundInflammation triggered by infection or injury is tightly controlled by glucocorticoid hormones which signal via a dedicated transcription factor, the Glucocorticoid Receptor (GR), to regulate hundreds of genes. However, the hierarchy of transcriptional responses to GR activation and the molecular basis of their oftentimes non-linear dynamics are not understood.ResultsWe investigated early glucocorticoid-driven transcriptional events in macrophages, a cell type highly responsive to both pro- and anti-inflammatory stimuli. Using whole transcriptome analyses in resting and acutely lipopolysaccharide (LPS)-stimulated macrophages, we show that early GR target genes form dense networks with the majority of control nodes represented by transcription factors. The expression dynamics of several glucocorticoid-responsive genes are consistent with feed forward loops (FFL) and coincide with rapid GR recruitment. Notably, GR binding sites in genes encoding members of the KLF transcription factor family colocalize with KLF binding sites. Moreover, our gene expression, transcription factor binding and computational data are consistent with the existence of the GR-KLF9-KLF2 incoherent FFL. Analysis of LPS-downregulated genes revealed striking enrichment in multimerized Zn-fingers- and KRAB domain-containing proteins known to bind nucleic acids and repress transcription by propagating heterochromatin. This raises an intriguing possibility that an increase in chromatin accessibility in inflammatory macrophages results from broad downregulation of negative chromatin remodelers.ConclusionsPro- and anti-inflammatory stimuli alter the expression of a vast array of transcription factors and chromatin remodelers. By regulating multiple transcription factors, which propagate the initial hormonal signal, GR acts as a coordinating hub in anti-inflammatory responses. As several KLFs promote the anti-inflammatory program in macrophages, we propose that GR and KLFs functionally cooperate to curb inflammation.

Glucocorticoid Signaling: An Update from a Genomic Perspective

Glucocorticoid hormones (GC) regulate essential physiological functions including energy homeostasis, embryonic and postembryonic development, and the stress response. From the biomedical perspective, GC have garnered a tremendous amount of attention as highly potent anti-inflammatory and immunosuppressive medications indispensable in the clinic. GC signal through the GC receptor (GR), a ligand-dependent transcription factor whose structure, DNA binding, and the molecular partners that it employs to regulate transcription have been under intense investigation for decades. In particular, next-generation sequencing-based approaches have revolutionized the field by introducing a unified platform for a simultaneous genome-wide analysis of cellular activities at the level of RNA production, binding of transcription factors to DNA and RNA, and chromatin landscape and topology. Here we describe fundamental concepts of GC/GR function as established through traditional molecular and in vivo approaches and focus on the novel insights of GC biology that have emerged over the last 10 years from the rapidly expanding arsenal of system-wide genomic methodologies.

GRIP1-associated SET-domain methyltransferase in glucocorticoid receptor target gene expression

Transcriptional regulators such as the glucocorticoid receptor (GR) recruit multiple cofactors to activate or repress transcription. Although most cofactors are intrinsically bifunctional, little is known about the molecular mechanisms dictating the specific polarity of regulation. Furthermore, chromatin modifications thought to be confined to silent loci appear in actively transcribed genes suggesting that similar enzymatic activities may mediate constitutive and transient chromatin states. GRIP1, a GR ligand-dependent coregulator of the p160 family can potentiate or inhibit transcription but the molecular contexts and mechanisms that enable GRIP1 corepressor activity are poorly understood. In a yeast 2-hybrid screen with GRIP1 repression domain (RD)-containing fragment, we repeatedly isolated the C-terminal region of a SET domain-containing protein subsequently identified as histone H4 lysine 20 trimethyltransferase, Suv4-20h1. We cloned a full-length Suv4-20h1 and dissected its interaction with GRIP1 in yeast, in vitro, and in mammalian cells. Strict nuclear localization and high salt concentration required for Suv4-20h1 extraction were consistent with its tight association with chromatin. Overexpression of Suv4-20h1 in human U2OS and A549 cells expressing integrated and endogenous GR, respectively, antagonized ligand-dependent induction of a subset of GR target genes, whereas Suv4-20h1 siRNA-mediated depletion had a reciprocal effect. Inhibition of GR transactivation required both the GRIP1 interacting region of Suv4-20h1 and its catalytic activity. Thus, Suv4-20h1 known exclusively as a factor involved in constitutive heterochromatin maintenance, actively participates in hormone-dependent transcriptional regulation affecting GR target gene expression in a promoter- and cell type-specific manner.