Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
Macrophages induce a number of inflammatory response genes in response to stimulation with microbial ligands. In response to endotoxin Lipid A, lineage-defining and stimulation-responsive transcription factors cooperate to induce a gene activation cascade of primary followed by secondary response genes. Epigenetic state is an important regulator of the kinetics, specificity, and mechanism of gene activation of these two classes. In particular, the SWI/SNF chromatin remodeling complex is required for the activation secondary response genes, but not primary response genes, which generally exhibit open chromatin. Here we show that a recently discovered variant of the SWI/SNF complex, the non-canonical BAF complex (ncBAF), regulates secondary response genes in the interferon (IFN) response pathway. Inhibition of bromodomain-containing protein 9 (BRD9), a subunit of the ncBAF complex, with BRD9 bromodomain inhibitors (BRD9i) or a degrader (dBRD9), led to reduction in a number of interferon-stimulated genes (ISGs) following stimulation with endotoxin lipid A. BRD9-dependent genes overlapped highly with a subset of genes differentially regulated by BET protein inhibition with JQ1 following endotoxin stimulation. We find that the BET protein BRD4 is co-bound with BRD9 in unstimulated macrophages and co-recruited upon stimulation to ISG promoters along with STAT1, STAT2, and IRF9, components of the ISGF3 complex activated downstream of IFNAR stimulation. In the presence of BRD9i or dBRD9, STAT1, STAT2, and IRF9 binding is reduced, in some cases with reduced binding of BRD4. These results demonstrate a specific role for BRD9 and the ncBAF complex in ISG activation and identify an activity for BRD9 inhibitors and degraders in dampening endotoxin- and IFN-dependent gene expression.
Overall design
RNA-seq of BRDi-treated bone-marrow derived macrophages (BMDMs) in 0h, 1h, or 4h Lipid A stimulation; ChIP-seq of BRD4, BRD9, BRD2 in RAW 264.7 macrophage cell line in 0h or 4h Lipid A stimulation; ChIPseq of BRD9 in 0h or 4h Lipid A stimulation; ChIPseq of IRF9, STAT1, and STAT2 in BRDi treatment in 0h or 4h Lipid A; ChIPseq of BRD4 in BMDMs in BRD9i in 4h Lipid A. RNA-seq of BRDi-treated bone-marrow derived macrophages (BMDMs) in 0h or 4h Interferon-alpha stimulation; ChIP-seq of BRD4 in 0h or 4h Lipid A stimulation with treatment with JQ1; ChIPseq of BRD9 in 0h or 4h Lipid A stimulation with treatment with vehicle, BI9564, dBRD9, IBRD9, JQ1; ChIPseq of IRF9, STAT1, and STAT2 4h Lipid A with vehicle or BI9564.
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