Genome-wide localization of exosome components to active promoters and chromatin insulators in Drosophila

Chromatin insulators are functionally conserved DNA-protein complexes that are situated throughout the genome and organize independent transcriptional domains.  Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the mechanisms by which RNA affects insulator activity are not yet understood.  Here we identify the exosome, the highly conserved major cellular 3’ to 5’ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila.  High resolution genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32, and CTCF insulator proteins.  Colocalization occurs mainly at promoters but also well-characterized boundary elements, such as scs, scs’, Mcp, and Fab-8.  Surprisingly, exosome associates primarily with promoters but not gene bodies, arguing against simple cotranscriptional recruitment to RNA substrates.  We find that exosome is recruited to chromatin in a transcription dependent manner, preferentially to highly transcribed genes.  Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters.  Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is specifically required for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment.  Taken together, our results reveal a novel relationship between exosome and chromatin insulators throughout the genome. ChIP-seq of exosome components. RNA-seq after control and exosome subunit knockdown in Drosophila cell lines.

染色质绝缘子（chromatin insulator）是一类功能保守的DNA-蛋白质复合物，广泛分布于基因组中，可构建独立的转录结构域。既往研究表明RNA是染色质绝缘子活性的重要辅因子，然而RNA调控绝缘子活性的具体分子机制尚未明确。本研究鉴定出外切体（exosome）——高度保守的主要细胞内3’→5’ RNA降解机器——是果蝇中依赖CP190的染色质绝缘子复合物的物理互作因子。本研究通过染色质免疫共沉淀测序（ChIP-seq），在两种不同的果蝇胚胎细胞系中对外切体开展高分辨率全基因组图谱分析，发现其与CP190、BEAF-32及CTCF绝缘子蛋白存在广泛且特异性的共定位信号。这类共定位主要发生在启动子区域，同时也富集于已被充分表征的边界元件，如scs、scs’、Mcp及Fab-8。令人意外的是，外切体主要结合启动子区域，而非基因体（gene body），这与简单的共转录招募至RNA底物的假说相悖。研究发现外切体以转录依赖的方式被招募至染色质，且优先结合高转录活性的基因。与绝缘子蛋白类似，外切体在双向转录启动子区域也呈现显著富集。在绝缘子蛋白敲低的细胞系中开展外切体定向染色质免疫共沉淀实验，结果显示CTCF仅在Mcp和Fab-8位点对外切体的染色质结合是必需的，而在其他位点则并非如此，这表明外切体的染色质招募还存在其他替代机制。综上，本研究结果揭示了全基因组范围内外切体与染色质绝缘子之间的新型关联。本研究包含两类测序实验：外切体组分的染色质免疫共沉淀测序，以及果蝇细胞系中外切体亚基敲低和对照处理后的RNA测序（RNA-seq）。