ABSTRACT: In macrophages, homeostatic and immune signals induce distinct sets of transcriptional responses, defining the cellular identity and function. The activity of lineage specific and signal induced transcription factors are regulated by chromatin accessibility and other epigenetic modulators. Glucocorticoids are potent anti-inflammatory drugs. Acting through the glucocorticoid receptor (GR), glucocorticoids directly repress inflammatory responses at transcriptional and epigenetic levels in macrophages. In this study, we identified bromodomain containing 9 (BRD9), a component of SWI/SNF chromatin remodeling complex, as a novel modulator for glucocorticoids responses in macrophages. Inhibition, degradation, or genetic depletion of BRD9 in bone marrow derived macrophages (BMDM), significantly compromised their responses to inflammatory stimuli, such as liposaccharides (LPS), and interferons. A large portion of BRD9-regulated genes are also known to be regulated by dexamethasone, a synthetic glucocorticoid. Importantly, pharmacologic inhibition of BRD9 is able to further potentiate the anti-inflammatory responses of dexamethasone, by further repressing the GR downstream targets. Mechanistically, BRD9 co-localized with a subset of GR binding sites. Depletion of BRD9 enhanced GR occupancy at a subset of its targets. Enhanced occupancy of GR at these sites is associated with further repression of inflammation-related genes. Collectively, these findings establish BRD9 as a key modulator of macrophage inflammatory responses, revealing the therapeutic potential of BRD9 inhibitors as modulators for glucocorticoids action.
Project description:In macrophages, homeostatic and immune signals induce distinct sets of transcriptional responses, defining the cellular identity and function. The activity of lineage specific and signal induced transcription factors are regulated by chromatin accessibility and other epigenetic modulators. Glucocorticoids are potent anti-inflammatory drugs. Acting through the glucocorticoid receptor (GR), glucocorticoids directly repress inflammatory responses at transcriptional and epigenetic levels in macrophages. In this study, we identified bromodomain containing 9 (BRD9), a component of SWI/SNF chromatin remodeling complex, as a novel modulator for glucocorticoids responses in macrophages. Inhibition, degradation, or genetic depletion of BRD9 in bone marrow derived macrophages (BMDM), significantly compromised their responses to inflammatory stimuli, such as liposaccharides (LPS), and interferons. A large portion of BRD9-regulated genes are also known to be regulated by dexamethasone, a synthetic glucocorticoid. Importantly, pharmacologic inhibition of BRD9 is able to further potentiate the anti-inflammatory responses of dexamethasone, by further repressing the GR downstream targets. Mechanistically, BRD9 co-localized with a subset of GR binding sites. Depletion of BRD9 enhanced GR occupancy at a subset of its targets. Enhanced occupancy of GR at these sites is associated with further repression of inflammation-related genes. Collectively, these findings establish BRD9 as a key modulator of macrophage inflammatory responses, revealing the therapeutic potential of BRD9 inhibitors as modulators for glucocorticoids action.
Project description:In macrophages, homeostatic and immune signals induce distinct sets of transcriptional responses, defining the cellular identity and function. The activity of lineage specific and signal induced transcription factors are regulated by chromatin accessibility and other epigenetic modulators. Glucocorticoids are potent anti-inflammatory drugs. Acting through the glucocorticoid receptor (GR), glucocorticoids directly repress inflammatory responses at transcriptional and epigenetic levels in macrophages. In this study, we identified bromodomain containing 9 (BRD9), a component of SWI/SNF chromatin remodeling complex, as a novel modulator for glucocorticoids responses in macrophages. Inhibition, degradation, or genetic depletion of BRD9 in bone marrow derived macrophages (BMDM), significantly compromised their responses to inflammatory stimuli, such as liposaccharides (LPS), and interferons. A large portion of BRD9-regulated genes are also known to be regulated by dexamethasone, a synthetic glucocorticoid. Importantly, pharmacologic inhibition of BRD9 is able to further potentiate the anti-inflammatory responses of dexamethasone, by further repressing the GR downstream targets. Mechanistically, BRD9 co-localized with a subset of GR binding sites. Depletion of BRD9 enhanced GR occupancy at a subset of its targets. Enhanced occupancy of GR at these sites is associated with further repression of inflammation-related genes. Collectively, these findings establish BRD9 as a key modulator of macrophage inflammatory responses, revealing the therapeutic potential of BRD9 inhibitors as modulators for glucocorticoids action.
Project description:Glucocorticoids are widely used to treat inflammatory disorders. Prolonged use results in side effects including osteoporosis, diabetes and obesity. The selective glucocorticoid receptor (GR) modulator Compound A (CpdA) exhibits an inflammation-suppressive effect, largely in absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated using an unbiased proteomics approach. We found that the autophagy receptor p62 but not GR mediates the anti-inflammatory action of CpdA in macrophages. CpdA drives the upregulation of p62 by recruiting the NRF2 transcription factor to its promoter. Contrarily, the classic GR ligand dexamethasone recruits GR to p62 and other NRF2 controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA are able to induce autophagy, albeit in a cell-type and time-dependent manner. Suppression of LPS-induced IL-6 and MCP1 genes in bone marrow-derived macrophages by CpdA is hampered upon p62 silencing, confirming that p62 is essential for the anti-inflammatory capacity of CpdA. Together, these results demonstrate how off-target mechanisms of selective GR ligands may establish a more efficient anti-inflammatory therapy
Project description:Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts in solid cancers. However, their use is associated with severe side effects and therapy resistance. Novel glucocorticoid receptor (GR) ligands with a patient-validated reduced side effect profile have not yet reached the clinic. GR is a member of the nuclear receptor family of transcription factors and heavily relies on interactions with coregulator proteins for its transcriptional activity. To elucidate the role of the GR interactome in the altered transcriptional activity of GR following treatment with agonists, antagonists or lead selective GR agonists and modulators (SEGRAMs), we generated comprehensive interactome maps by high-confidence proximity proteomics in lung epithelial carcinoma cells. We found that the GR antagonist RU486 and the SEGRAM Dagrocorat both reduced GR interaction with CREB-binding protein (CBP)/p300 and the mediator complex compared to the full agonist Dexamethasone. Our data offer new insights into the role differential coregulator protein recruitment in shaping specific GR-mediated transcriptional responses.
Project description:Macrophages are amongst the major targets of glucocorticoids (GC) as therapeutic anti-inflammatory agents. Here we show that GC treatment of mouse and human macrophages initiates a cascade of induced gene expression including many anti-inflammatory genes. Inducible binding of the glucocorticoid receptor (GR) was detected at candidate enhancers in the vicinity of induced genes in both species and this was strongly associated with canonical GR binding motifs. However, the sets of inducible genes, the candidate enhancers, and the GR motifs within them, were highly-divergent between the two species. 3 biological replicates of a 6 point time series for the transcriptional response to dexamethasone 100nM
Project description:Macrophages are amongst the major targets of glucocorticoids (GC) as therapeutic anti-inflammatory agents. Here we show that GC treatment of mouse and human macrophages initiates a cascade of induced gene expression including many anti-inflammatory genes. Inducible binding of the glucocorticoid receptor (GR) was detected at candidate enhancers in the vicinity of induced genes in both species and this was strongly associated with canonical GR binding motifs. However, the sets of inducible genes, the candidate enhancers, and the GR motifs within them, were highly-divergent between the two species.. The data cast further doubt upon the predictive value of mouse models of inflammatory disease. Four biological replicates of a 6 point 24h time series transcriptional response of human monocyte derived macrophages to dexamethasone 100nM.
Project description:Glucocorticoids are widely prescribed anti-inflammatory drugs with tissue-specific effects. Beneficial anti-inflammatory effects are caused in cells of the immune system whereas metabolic adverse effects of glucocorticoid therapy are seen in metabolic tissues such as liver. The glucocorticoid receptor (GR), a nuclear receptor targeted by glucocorticoids, bind its DNA response elements upon ligand exposure in a tissue-specific manner. Tissue-specific GR binding patterns depend on the access of its binding sites, which determines the tissue-specific glucocorticoid response. Here, we investigated this response by nascent RNAseq in murine bone marrow-derived macrophages (BMDMs) after stimulation with lipopolysaccharide (LPS) and dexamethasone (Dex). After labelling newly synthesized transcripts with 4-thiouracile (4sU), we identified macrophage-specific non-coding transcripts expressed at intergenic GR binding sites. Those transcripts are regulated by the GR and correlate with its anti-inflammatory function in macrophages. Those findings add another layer to the mechanisms underpinning GR's tissue-specific gene regulation and represent potential drug targets in anti-inflammatory therapy and/or management of adverse effects in glucocorticoid therapy.
Project description:Macrophages are amongst the major targets of glucocorticoids (GC) as therapeutic anti-inflammatory agents. Here we show that GC treatment of mouse and human macrophages initiates a cascade of induced gene expression including many anti-inflammatory genes. Inducible binding of the glucocorticoid receptor (GR) was detected at candidate enhancers in the vicinity of induced genes in both species and this was strongly associated with canonical GR binding motifs. However, the sets of inducible genes, the candidate enhancers, and the GR motifs within them, were highly-divergent between the two species.. The data cast further doubt upon the predictive value of mouse models of inflammatory disease. human monocyte derived macrophages were treated with dexamethasone for 2h, fixed and ChIP performed. Material from 4 volunteers was pooled for the IP.
Project description:Glucocorticoids are widely used to treat inflammatory disorders; however, prolonged use of glucocorticoids results in side effects including osteoporosis, diabetes and obesity. Compound A (CpdA), identified as a selective NR3C1/glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1) modulator, exhibits an inflammation-suppressive effect, largely in the absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated in bone marrow-derived macrophages using an unbiased proteomics approach. We found that the autophagy receptor SQSTM1 but not NR3C1 mediates the anti-inflammatory action of CpdA. CpdA drives SQSTM1 upregulation by recruiting the NFE2L2 transcription factor to its promoter. In contrast, the classic NR3C1 ligand dexamethasone recruits NR3C1 to the Sqstm1 promoter and other NFE2L2-controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA induce autophagy, with marked different autophagy characteristics and morphology. Suppression of LPS-induced Il6 and Ccl2 genes by CpdA in macrophages is hampered upon Sqstm1 silencing, confirming that SQSTM1 is essential for the anti-inflammatory capacity of CpdA, at least in this cell type. Together, these results demonstrate how off-target mechanisms of selective NR3C1 ligands may contribute to a more efficient anti-inflammatory therapy.
Project description:Glucocorticoids are primary stress hormones and their synthetic derivatives are widely used clinically. The therapeutic efficacy of these steroids is limited by severe side effects and glucocorticoid resistance. Multiple glucocorticoid receptor (GR) isoforms are produced by alternative translation initiation; however, the role individual isoforms play in controlling tissue-specific responses to glucocorticoids in vivo is unknown. We have generated knockin mice that exclusively express the most active receptor isoform, GR-C3. The GR-C3 knockin mice die at birth due to respiratory distress but can be completely rescued by antenatal glucocorticoid administration. To evaluate the GR-C3 transcriptome, we prepared mouse embryonic fibroblasts (MEFs) from E12.5 wild-type (WT) and GR-C3 knockin embryos and treated the cells with vehicle or the synthetic glucocorticoid Dexamethasone (Dex) for 6 hours. The GR-C3 isoform was found to have a markedly different gene-regulatory profile than GR in WT MEFs.