Paf1C plays novel subunit-specific roles in alternative cleavage and polyadenylation

The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and PolII elongation. Here, we provide the first genome-wide analysis of chromatin occupancy by the entire PAF complex in mammalian cells. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3' ends, a profile that sharply contrasted with the yeast complex. Remarkably, our studies identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription. Moreover, we found that depletion of Paf1C subunits also resulted in the accumulation of RNA polymerase II (PolII) over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits leads to global loss of histone H2B ubiquitylation, but surprisingly, there is little impact of Paf1C depletion on other histone modifications, including the tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and suggest that Paf1C could play a role in protecting transcripts from premature cleavage by preventing PolII accumulation at TSS-proximal pA sites. Overall design: ChIP-seq, RNA-seq and 3''READS of Paf1C factors in mouse C2C12 myoblast cells

PAF复合物（Paf1C）已被证实可调控染色质修饰、基因转录以及RNA聚合酶II（PolII）延伸。本研究首次针对哺乳动物细胞内完整PAF复合物的染色质结合谱开展全基因组分析。研究发现，Paf1C不仅被招募至启动子区域与基因本体区域，还分布于基因3'端剪切/多聚腺苷酸化（pA）位点下游区域，这一分布特征与酵母中的Paf1C形成鲜明对比。值得注意的是，本研究揭示了Paf1C与可变剪切及多聚腺苷酸化（APA）、上游反义转录调控之间全新的亚基特异性关联。此外，我们发现敲减Paf1C亚基同样会导致RNA聚合酶II（PolII）在基因本体区域的积累，该现象与APA事件相伴随。敲减特定Paf1C亚基会引发全局组蛋白H2B泛素化水平缺失，但令人意外的是，Paf1C敲减对其他组蛋白修饰几乎无影响，包括此前被认为与该复合物相关的组蛋白H3赖氨酸4和36三甲基化（H3K4me3与H3K36me3）。本研究结果与酵母中的相关研究存在显著差异，同时统一了将Paf1C与PolII延伸及RNA加工相关联的观测结论，并提示Paf1C可通过阻止PolII在转录起始位点（TSS）近端pA位点的积累，从而保护转录本免于过早剪切。实验整体设计：针对小鼠C2C12成肌细胞中的Paf1C因子开展染色质免疫共沉淀测序（ChIP-seq）、RNA测序（RNA-seq）及3''READS测序。