Dynamic Reorganization of the Cardiomyocyte Transcriptome in Response to TNFα-induced Proinflammatory Signaling [GRO-Seq]

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. Using GRO-seq and ChIP-seq (p65 and RNA Pol II) over a time course of TNFα signaling in AC16 human cardiomyocytes.

炎症与诸多心血管病理状态密切相关，但其潜在分子机制仍未明确。为更深入地探究该关联，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）的转录组开展了系统表征。我们联合使用全局核连缀测序（GRO-seq）、染色质免疫共沉淀测序（ChIP-seq）等基因组学研究手段，在AC16细胞中鉴定出约3万个转录区域，其中包含在α-鹅膏蕈碱处理条件下被检测到的RNA聚合酶I和III（Pol I和Pol III）转录区域。该转录区域集可产生蛋白编码RNA与非编码RNA，其中多数此前未被注释，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，快速且动态地重编程其转录组以响应TNFα刺激，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与非编码基因的表达。进一步研究发现，上调与下调基因具有截然不同的RNA聚合酶II（Pol II）动力学特征：对于TNFα刺激的基因，RNA聚合酶II可快速释放并进入有效延伸阶段。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。本研究在AC16人心肌细胞的TNFα信号传导时间进程中，采用了针对p65与RNA聚合酶II的全局核连缀测序及染色质免疫共沉淀测序技术。