Dynamic Reorganization of the Cardiomyocyte Transcriptome in Response to TNFα-induced Proinflammatory Signaling [ChIP-Seq]. Homo sapiens

Inflammation is associated with many cardiovascular pathologies, but the underlying mechanisms remain unclear. To explore this in more detail, we characterized the transcriptome of an immortalized adult human ventricular cardiomyocyte cell line (AC16) in response to tumor necrosis factor (TNFa). Using a combination of genomic approaches, including global nuclear run-on sequencing (GRO-seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-seq), we identified ~30,000 transcribed regions in AC16 cells, which includes a set of RNA polymerases I and III (Pol I and Pol III) transcribed regions revealed in the presence of α-amanitin. The set of transcribed regions produces both protein-coding and non-coding RNAs, many of which have not been annotated previously, including enhancer RNAs originating from NF-κB binding sites. In addition, we observed that AC16 cells rapidly and dynamically reorganize their transcriptomes in response to TNFa stimulation in an NF-κB-dependent manner, switching from a basal state to a proinflammatory state affecting a spectrum of cardiac-associated protein-coding and non-coding genes. Moreover, we observed distinct Pol II dynamics for up- and downregulated genes, with a rapid release of Pol II into productive elongation for TNFa-stimulated genes. Our studies shed new light on the regulation of the cardiomyocyte transcriptome in response to a proinflammatory signal and help to clarify the link between inflammation and cardiomyocyte function at the transcriptional level. Overall design: Using GRO-seq and ChIP-seq (p65 and RNA Pol II) over a time course of TNFα signaling in AC16 human cardiomyocytes.

炎症与多种心血管病理状态密切相关，但其潜在分子机制仍未明确。为更深入地探索该关联，我们针对肿瘤坏死因子α（TNFα）刺激下的永生化成人心室心肌细胞系（AC16）的转录组进行了系统表征。本研究结合全局核运行测序（GRO-seq）与染色质免疫共沉淀测序（ChIP-seq）等多种基因组学手段，在AC16细胞中鉴定出约30000个转录区域，其中包含在α-鹅膏蕈碱存在条件下显现的、由RNA聚合酶I和III（Pol I和Pol III）转录的区域。该类转录区域可编码蛋白RNA与非编码RNA，其中多数为此前未被注释的序列，包括源自核因子κB（NF-κB）结合位点的增强子RNA。此外，我们观察到AC16细胞可通过核因子κB（NF-κB）依赖的方式，对肿瘤坏死因子α（TNFα）刺激产生快速且动态的转录组重编程，从基础状态切换至促炎状态，进而影响一系列心脏相关的蛋白编码基因与非编码基因的表达。更为重要的是，我们发现上调与下调基因呈现出截然不同的RNA聚合酶II（Pol II）动态调控模式：肿瘤坏死因子α（TNFα）刺激的基因可快速释放RNA聚合酶II，使其进入有效延伸阶段。本研究为心肌细胞响应促炎信号的转录组调控机制提供了新视角，有助于从转录层面阐明炎症与心肌细胞功能之间的关联。整体实验设计：在人类AC16心肌细胞的肿瘤坏死因子α（TNFα）信号通路时间进程实验中，采用全局核运行测序（GRO-seq）与针对p65及RNA聚合酶II的染色质免疫共沉淀测序（ChIP-seq）开展检测。