Transcriptome characterization through comparative genome-wide analysis of nuclear RNA and RNAPII association in erythroid cells

Current evidence suggests that more than half of the mammalian genome is transcribed, yet how this vast transcriptome is regulated in vivo remains poorly understood. We demonstrate here an integrated, straightforward and widely applicable approach to characterize cell type-specific transcriptional programs and regulatory mechanisms by generating two genome-wide data sets. We used deep sequencing of nuclear RNA (nucRNA-Seq) to comprehensively describe the nuclear transcriptome in ex vivo murine erythroid cells. In parallel, we generated a profile of active RNA polymerase II (RNAPII) binding by chromatin-immunoprecipitation (ChIP-Seq), allowing us to explore the relationship between RNAPII occupancy and transcriptional output in erythroid cells on a genome-wide scale. Comparative analysis of both data sets enables us to not only measure primary transcriptional output and identify genes associated with more efficient polymerase usage, but also to identify putative regulatory elements such as enhancers and novel non-coding transcripts. Application of this method to different cell types allows for the characterization of important aspects of gene regulation in a cell type-specific manner. Our findings demonstrate the complex ways in which RNAPII is associated with the genome and how this affects transcription of target genes, highlighting the importance of approaching transcriptome characterization from multiple angles.

现有研究证据表明，超过半数的哺乳动物基因组会发生转录，但这一庞大转录组在活体中的调控机制仍有待阐明。本研究报道了一种整合化、简便且普适性强的研究策略，通过构建两套全基因组数据集以解析细胞类型特异性转录程序与调控机制。我们通过核RNA深度测序（nucRNA-Seq）全面描述了离体小鼠红系细胞的核转录组；同时，借助染色质免疫沉淀测序（ChIP-Seq）获取了活性RNA聚合酶II（RNAPII）的结合图谱，从而能够在全基因组范围内探究红系细胞中RNAPII占据度与转录产出之间的关联。对这两套数据集开展比较分析，不仅可以量化初级转录产出、鉴定出与聚合酶利用效率更高相关的基因，还能识别出增强子等潜在调控元件以及新型非编码转录本（non-coding transcripts）。将该方法应用于不同细胞类型，即可实现细胞类型特异性的基因调控关键特征解析。本研究结果揭示了RNAPII与基因组关联的复杂模式及其对靶基因转录的影响，凸显了从多维度开展转录组表征研究的重要意义。