Bromodomain protein 9 (BRD9) regulates interferon-stimulated genes during macrophage activation via cooperation with BET protein BRD4

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.

巨噬细胞经微生物配体刺激后，可诱导一系列炎症应答基因的表达。在内毒素脂质A（Lipid A）刺激下，细胞谱系特异性转录因子与刺激应答型转录因子协同作用，触发初级应答基因再到次级应答基因的基因激活级联反应。表观遗传状态是调控这两类基因激活的动力学特性、特异性与激活机制的重要因素。具体而言，SWI/SNF染色质重塑复合物（SWI/SNF chromatin remodeling complex）是次级应答基因激活所必需的，而通常染色质呈开放状态的初级应答基因则无需该复合物。

本研究发现，近期发现的SWI/SNF复合物变体——非经典BAF复合物（non-canonical BAF complex, ncBAF），可调控干扰素（IFN）应答通路中的次级应答基因。使用BRD9溴结构域抑制剂（BRD9 bromodomain inhibitors, BRD9i）或降解剂（degrader, dBRD9）对非经典BAF复合物的亚基含溴结构域蛋白9（bromodomain-containing protein 9, BRD9）进行抑制，可降低内毒素脂质A刺激后干扰素刺激基因（interferon-stimulated genes, ISGs）的表达水平。BRD9依赖性基因与内毒素刺激后经BET蛋白抑制剂JQ1差异调控的部分基因存在高度重叠。

我们发现，在未受刺激的巨噬细胞中，BET蛋白BRD4可与BRD9发生共结合；经刺激后，二者可与IFNAR信号通路下游激活的ISGF3复合物组分信号转导与转录激活因子1（STAT1）、信号转导与转录激活因子2（STAT2）及干扰素调节因子9（IRF9）一同被招募至干扰素刺激基因的启动子区域。在使用BRD9i或dBRD9处理后，STAT1、STAT2及IRF9的结合水平出现下降，部分情况下BRD4的结合也会随之降低。

上述结果阐明了BRD9与非经典BAF复合物在干扰素刺激基因激活中的特异性作用，并证实BRD9抑制剂与降解剂可抑制内毒素及干扰素依赖的基因表达。

整体实验设计：
1. RNA测序（RNA sequencing, RNA-seq）：分别在0h、1h、4h脂质A刺激下，对经BET抑制剂（BRDi）处理的骨髓源性巨噬细胞（bone-marrow derived macrophages, BMDMs）进行检测；
2. 染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）：分别在0h或4h脂质A刺激下，对RAW 264.7巨噬细胞系中的BRD4、BRD9、BRD2进行检测；
3. ChIP-seq：分别在0h或4h脂质A刺激下，对BRD9进行检测；
4. ChIP-seq：分别在0h或4h脂质A刺激且经BRDi处理的条件下，对IRF9、STAT1及STAT2进行检测；
5. ChIP-seq：在经BRD9i处理且4h脂质A刺激的骨髓源性巨噬细胞中，对BRD4进行检测；
6. RNA-seq：分别在0h或4h干扰素-α刺激下，对经BRDi处理的骨髓源性巨噬细胞进行检测；
7. ChIP-seq：经JQ1处理并在0h或4h脂质A刺激下，对BRD4进行检测；
8. ChIP-seq：分别经溶剂对照、BI9564、dBRD9、IBRD9、JQ1处理并在0h或4h脂质A刺激下，对BRD9进行检测；
9. ChIP-seq：分别经溶剂对照或BI9564处理并在4h脂质A刺激下，对IRF9、STAT1及STAT2进行检测。