Mechanisms of Epigenomic and Functional Convergence Between Glucocorticoid- and IL4-Driven Macrophage Programming.

Macrophages adopt distinct phenotypes in response to environmental cues, with type-2 cytokine interleukin-4 promoting a tissue-repair homeostatic state (M2IL4). Glucocorticoids, widely used anti-inflammatory therapeutics, reportedly impart a similar phenotype (M2GC), but how such disparate pathways may functionally converge is unknown. We show using integrative functional genomics that M2IL4 and M2GC transcriptomes share a striking overlap mirrored by a shift in chromatin landscape in both common and signal-specific gene subsets. This core homeostatic program is enacted by transcriptional effectors KLF4 and the GC receptor, whose genome-wide occupancy and actions are integrated in a stimulus-specific manner by the nuclear receptor cofactor GRIP1. Indeed, many of the M2IL4:M2GC-shared transcriptomic changes were GRIP1-dependent. Consistently, GRIP1 loss attenuated phagocytic activity of both populations in vitro and macrophage tissue-repair properties in the murine colitis model in vivo. These findings provide a mechanistic framework for homeostatic macrophage programming by distinct signals, to better inform anti-inflammatory drug design.

Genomic glucocorticoid action in embryonic mouse neural stem cells.

Prenatal exposure to synthetic glucocorticoids (sGCs) reprograms brain development and predisposes the developing fetus towards potential adverse neurodevelopmental outcomes. Using a mouse model of sGC administration, previous studies show that these changes are accompanied by sexually dimorphic alterations in the transcriptome of neural stem and progenitor cells (NSPCs) derived from the embryonic telencephalon. Because cell type-specific gene expression profiles tightly regulate cell fate decisions and are controlled by a flexible landscape of chromatin domains upon which transcription factors and enhancer elements act, we multiplexed data from four genome-wide assays: RNA-seq, ATAC-seq (assay for transposase accessible chromatin followed by genome wide sequencing), dual cross-linking ChIP-seq (chromatin immunoprecipitation followed by genome wide sequencing), and microarray gene expression to identify novel relationships between gene regulation, chromatin structure, and genomic glucocorticoid receptor (GR) action in NSPCs. These data reveal that GR binds preferentially to predetermined regions of accessible chromatin to influence gene programming and cell fate decisions. In addition, we identify SOX2 as a transcription factor that impacts the genomic response of select GR target genes to sGCs (i.e., dexamethasone) in NSPCs.

Transcription cofactor GRIP1 differentially affects myeloid cell-driven neuroinflammation and response to IFN-ß therapy.

Macrophages (MФ) and microglia (MG) are critical in the pathogenesis of multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). Glucocorticoids (GCs) and interferon ß (IFN-ß) are frontline treatments for MS, and disrupting each pathway in mice aggravates EAE. Glucocorticoid receptor-interacting protein 1 (GRIP1) facilitates both GR and type I IFN transcriptional actions; hence, we evaluated the role of GRIP1 in neuroinflammation. Surprisingly, myeloid cell-specific loss of GRIP1 dramatically reduced EAE severity, immune cell infiltration of the CNS, and MG activation and demyelination specifically during the neuroinflammatory phase of the disease, yet also blunted therapeutic properties of IFN-ß. MФ/MG transcriptome analyses at the bulk and single-cell levels revealed that GRIP1 deletion attenuated nuclear receptor, inflammatory and, interestingly, type I IFN pathways and promoted the persistence of a homeostatic MG signature. Together, these results uncover the multifaceted function of type I IFN in MS/EAE pathogenesis and therapy, and an unexpectedly permissive role of myeloid cell GRIP1 in neuroinflammation.

Negative elongation factor complex enables macrophage inflammatory responses by controlling anti-inflammatory gene expression.

Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report that widespread promoter-proximal RNA polymerase II pausing in resting macrophages is marked by co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.

Redox Sensing by PecS from the Plant Pathogen Pectobacterium atrosepticum and Its Effect on Gene Expression and the Conformation of PecS-Bound Promoter DNA.

The plant pathogen Pectobacterium atrosepticum encounters a stressful environment when it colonizes the plant apoplast. Chief among the stressors are the reactive oxygen species (ROS) that are produced by the host as a first line of defense. Bacterial transcription factors in turn use these signals as cues to upregulate expression of virulence-associated genes. We have previously shown that the transcription factor PecS from P. atrosepticum binds the promoters that drive expression of pecS and pecM, which encodes an efflux pump, to repress gene expression. We show here that addition of oxidant relieves repression in vivo and in vitro. While reduced PecS distorts promoter DNA on binding, oxidized PecS does not, as evidenced by DNaseI footprinting. PecS oxidation is reversible, as shown by an oxidant-dependent quenching of the intrinsic tryptophan fluorescence that is completely reversed upon addition of a reducing agent. Cysteine 45 positioned at the PecS dimer interface is the redox sensor. Reduced PecS-C45A causes less DNA distortion on binding compared to wild-type PecS; addition of an oxidant has no effect on binding, and PecS-C45A cannot repress gene expression. Our data suggest that reduced PecS distorts its cognate DNA on binding, perhaps inducing a conformation in which promoter elements are suboptimally aligned for RNA polymerase binding, resulting in transcriptional repression. In contrast, oxidized PecS binds promoter DNA such that RNA polymerase may successfully compete with PecS for binding, allowing gene expression. This mode of regulation would facilitate induction of the PecS regulon when the bacteria encounter host-derived ROS in the plant apoplast.

Gene-specific mechanisms direct glucocorticoid-receptor-driven repression of inflammatory response genes in macrophages.

The glucocorticoid receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis in mouse macrophages reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB-binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class; however, it effects repression by targeting distinct temporal events and components of transcriptional machinery.

The Stringent Response Induced by Phosphate Limitation Promotes Purine Salvage in Agrobacterium fabrum.

Agrobacterium fabrum induces tumor growth in susceptible plant species. The upregulation of virulence genes that occurs when the bacterium senses plant-derived compounds is enhanced by acidic pH and limiting inorganic phosphate. Nutrient starvation may also trigger the stringent response, and purine salvage is among the pathways expected to be favored under such conditions. We show here that phosphate limitation induces the stringent response, as evidenced by production of (p)ppGpp, and that the xdhCSML operon encoding the purine salvage enzyme xanthine dehydrogenase is upregulated ∼15-fold. The xdhCSML operon is under control of the TetR family transcription factor XdhR; direct binding of ppGpp to XdhR attenuates DNA binding, and the enhanced xdhCSML expression correlates with increased cellular levels of (p)ppGpp. Xanthine dehydrogenase may also divert purines away from salvage pathways to form urate, the ligand for the transcription factor PecS, which in the plant pathogen Dickeya dadantii is a key regulator of virulence gene expression. However, urate levels remain low under conditions that produce increased levels of xdhCSML expression, and neither acidic pH nor limiting phosphate results in induction of genes under control of PecS. Instead, expression of such genes is induced only by externally supplemented urate. Taken together, our data indicate that purine salvage is favored during the stringent response induced by phosphate starvation, suggesting that control of this pathway may constitute a novel approach to modulating virulence. Because bacterial purine catabolism appears to be unaffected, as evidenced by the absence of urate accumulation, we further propose that the PecS regulon is induced by only host-derived urate.

MarR family transcription factors: dynamic variations on a common scaffold.

Members of the multiple antibiotic resistance regulator (MarR) family of transcription factors are critical for bacterial cells to respond to chemical signals and to convert such signals into changes in gene activity. Obligate dimers belonging to the winged helix-turn-helix protein family, they are critical for regulation of a variety of functions, including degradation of organic compounds and control of virulence gene expression. The conventional regulatory paradigm is based on a genomic locus in which the gene encoding the MarR protein is divergently oriented from a gene under its control; MarR binding to the intergenic region controls expression of both genes by changing the interaction of RNA polymerase with gene promoters. MarR protein oxidation or binding of a small molecule ligand adversely affects DNA binding, resulting in altered expression of the divergent genes. The generality of this simple paradigm, including the regulation of Escherichia coli MarR by direct binding of antibiotics, has been challenged by reports published in recent years. In addition, structural and biochemical analyses of ligand binding to numerous MarR homologs are converging to identify a shared ligand-binding "hot-spot". This review highlights recent research advances that point to shared features, yet at the same time highlights the remarkable flexibility with which members of this protein family implement responses to inducing signals. A more comprehensive understanding of protein function will pave the way towards the development of both antibacterial agents and biosensors that are based on MarR family proteins.

pH-Dependent DNA Distortion and Repression of Gene Expression by Pectobacterium atrosepticum PecS.

Transcriptional activity is exquisitely sensitive to changes in promoter DNA topology. Transcription factors may therefore control gene activity by modulating the relative positioning of -10 and -35 promoter elements. The plant pathogen Pectobacterium atrosepticum, which causes soft rot in potatoes, must alter gene expression patterns to ensure growth in planta. In the related soft-rot enterobacterium Dickeya dadantii, PecS functions as a master regulator of virulence gene expression. Here, we report that P. atrosepticum PecS controls gene activity by altering promoter DNA topology in response to pH. While PecS binds the pecS promoter with high affinity regardless of pH, it induces significant DNA distortion only at neutral pH, the pH at which the pecS promoter is repressed in vivo. At pH ∼8, DNA distortions are attenuated, and PecS no longer represses the pecS promoter. A specific histidine (H142) located in a crevice between the dimerization- and DNA-binding regions is required for pH-dependent changes in DNA distortion and repression of gene activity, and mutation of this histidine renders the mutant protein incapable of repressing the pecS promoter. We propose that protonated PecS induces a DNA conformation at neutral pH in which -10 and -35 promoter elements are suboptimally positioned for RNA polymerase binding; on deprotonation of PecS, binding is no longer associated with significant changes in DNA conformation, allowing gene expression. We suggest that this mode of gene regulation leads to differential expression of the PecS regulon in response to alkalinization of the plant apoplast.

A moonlighting function of Mycobacterium smegmatis Ku in zinc homeostasis?

Ku protein participates in DNA double-strand break repair via the nonhomologous end-joining pathway. The three-dimensional structure of eukaryotic Ku reveals a central core consisting of a ß-barrel domain and pillar and bridge regions that combine to form a ring-like structure that encircles DNA. Homologs of Ku are encoded by a subset of bacterial species, and they are predicted to conserve this core domain. In addition, the bridge region of Ku from some bacteria is predicted from homology modeling and sequence analyses to contain a conventional HxxC and CxxC (where x is any residue) zinc-binding motif. These potential zinc-binding sites have either deteriorated or been entirely lost in Ku from other organisms. Using an in vitro metal binding assay, we show that Mycobacterium smegmatis Ku binds two zinc ions. Zinc binding modestly stabilizes the Ku protein (by ∼3°C) and prevents cysteine oxidation, but it has little effect on DNA binding. In vivo, zinc induces significant upregulation of the gene encoding Ku (∼sixfold) as well as a divergently oriented gene encoding a predicted zinc-dependent MarR family transcription factor. Notably, overexpression of Ku confers zinc tolerance on Escherichia coli. We speculate that zinc-binding sites in Ku proteins from M. smegmatis and other mycobacterial species have been evolutionarily retained to provide protection against zinc toxicity without compromising the function of Ku in DNA double-strand break repair.