PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation.
PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation.
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DOI：
10.1371/journal.pgen.1005794
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发表时间：
2016-01
期刊：
影响因子：
4.5
通讯作者：
Dynlacht BD
中科院分区：
文献类型：
--
作者：
Yang Y;Li W;Hoque M;Hou L;Shen S;Tian B;Dynlacht BD
PAF复合物（Paf1C）已被证明调节染色质修饰，基因转录和RNA聚合酶II （PolII）延伸。在这里，我们使用染色质免疫沉淀和高通量测序提供了整个复合物在哺乳动物细胞中分布的第一个全基因组图谱。我们发现Paf1C不仅被招募到启动子和基因体，而且还被招募到3 '端切割/聚腺苷化（pA）位点下游的区域，这与酵母复合体形成鲜明对比。值得注意的是，我们利用RNAi结合转录本3 '端深度测序，发现了Paf1C与选择性切割和聚腺苷化（APA）调控以及上游反义转录之间的新型亚基特异性联系。此外，我们发现Paf1C亚基的缺失导致PolII在基因体上的积累，这与APA相吻合。特异性Paf1C亚基的缺失导致组蛋白H2B泛素化的整体缺失，尽管Paf1C缺失对其他组蛋白修饰的影响很小，包括先前与该复合物相关的赖氨酸4和36 （H3K4me3和H3K36me3）上组蛋白H3的三甲基化。我们的研究结果提供了与酵母的惊人差异，同时将Paf1C与PolII延伸和RNA加工联系起来，并表明Paf1C亚基可以通过抑制转录起始位点(TSS)-近端pA位点的PolII积累和调节3 ' utr中pA位点的选择来控制转录物长度。基因转录可以通过多种机制进行调节，例如组蛋白修饰导致染色质的结构变化，从而导致基因激活或抑制，或mRNA的3 '切割位点的调节，称为选择性切割和聚腺苷化（APA），从而产生不同长度的转录异构体。在这里，我们介绍了PAF复合物（Paf1C）在转录调控的两种机制中的全基因组亚单位特异性作用。使用小鼠肌肉细胞，我们展示了与酵母对比的结果，即Paf1C亚基的消耗不影响先前与该复合物相关的某些组蛋白修饰，并且该复合物表现出亚基特异性功能。我们还发现了Paf1C在APA中的新作用，其中三个亚基耗尽后发生全基因组转录缩短。然而，在某些亚基缺失后，APA会发生变化，这加强了我们关于亚基特异性的结论。此外，通过比较两个亚基的缺失，我们发现转录起始位点（TSS）附近的RNA聚合酶II （PolII）的积累与TSS-近端pA位点的激活特异性相关，在一个缺失中观察到，而在另一个缺失中观察到。
The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and RNA polymerase II (PolII) elongation. Here, we provide the first genome-wide profiles for the distribution of the entire complex in mammalian cells using chromatin immunoprecipitation and high throughput sequencing. 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, we identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription using RNAi coupled with deep sequencing of the 3’ ends of transcripts. Moreover, we found that depletion of Paf1C subunits resulted in the accumulation of PolII over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits led to global loss of histone H2B ubiquitylation, although there was little impact of Paf1C depletion on other histone modifications, including 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 indicate that Paf1C subunits could play roles in controlling transcript length through suppression of PolII accumulation at transcription start site (TSS)-proximal pA sites and regulating pA site choice in 3’UTRs. Gene transcription can be regulated through multiple mechanisms, such as histone modifications that create structural changes of the chromatin leading to gene activation or suppression, or regulation of the 3’ cleavage site of the mRNA, known as alternative cleavage and polyadenylation (APA), resulting in the generation of transcript isoforms with various lengths. Here we present genome-wide subunit-specific roles of the PAF complex (Paf1C) related to both mechanisms of transcriptional regulation. Using mouse muscle cells, we show contrasting results with yeast, namely, that depletion of Paf1C subunits does not affect certain histone modifications previously associated with this complex and that the complex exhibits subunit-specific functions. We also discovered a novel role of Paf1C in APA, wherein genome-wide transcript shortening occurs after depletion of three of the subunits. However, APA varies after depletion of certain subunits, reinforcing our conclusions regarding subunit specificity. Furthermore, by comparing depletions of two subunits, we show that the accumulation of RNA polymerase II (PolII) near the transcription start site (TSS) is specifically associated with the activation of TSS-proximal pA sites observed in one depletion but not the other.