JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.

溴结构域与额外末端结构域（BET）蛋白是治疗癌症与免疫疾病的极具潜力的药物靶点。然而相较于BRD2，目前针对BET抑制效应的研究更多聚焦于含溴结构域蛋白4（BRD4），且BET蛋白与组蛋白H4高乙酰化染色质的全基因组关联机制仍未阐明。本研究针对人非小细胞肺癌（NSCLC）细胞系H23开展了基于转录起始位点（TSS）分辨率的染色质免疫共沉淀测序（ChIP-seq）与转录组图谱整合分析。研究发现，在绝大多数活性增强子与启动子区域中，组蛋白H4的K5与K8位点双乙酰化（H4K5acK8ac）与H3K27乙酰化（H3K27ac）及BRD2共定位，且BRD2与H4K5acK8ac的结合亲和力强于其与H3K27ac的结合。尽管经JQ1介导的BET抑制可完全消除BRD2与染色质的结合，但仅能观察到H4K5acK8ac水平的局部变化，这表明BRD2的募集不会影响全局组蛋白H4的高乙酰化水平。该结果支持如下模型：通过组蛋白H4高乙酰化募集BET蛋白的过程，相较于BET蛋白结合的乙酰转移酶介导的组蛋白H4乙酰化，占据主导地位。此外，本研究发现，在经JQ1处理后，包括MYC及其下游靶基因在内的大量BRD2结合基因的转录水平显著上调。本研究通过BRD2富集位点分析与转录活性分析，筛选出了可能以BRD2依赖或非依赖方式参与JQ1应答的候选转录因子。