Genome-scale chromatin interaction dynamic measurements for key components of the RNA Pol II general transcription machinery [RNA-seq]

A great deal of work has revealed in structural detail the components of the machinery responsible for mRNA gene transcription initiation. These include the general transcription factors (GTFs) which assemble at promoters along with RNA Polymerase II (Pol II) to form a preinitiation complex (PIC) aided by the activities of cofactors and site-specific transcription factors (TFs). However, less well understood are the in vivo PIC assembly pathways and their kinetics, an understanding of which is vital for determining on a mechanistic level how rates of in vivo RNA synthesis are established and how cofactors and TFs impact them. We used competition ChIP to obtain genome-scale estimates of the residence times for five GTFs: TBP, TFIIA, TFIIB, TFIIE and TFIIF in budding yeast. While many GTF-chromatin interactions were short-lived (< 1 min), there were numerous interactions with residence times in the several minutes range. Sets of genes with shared function also shared similar patterns of GTF kinetic behavior. TFIIE, a GTF that enters the PIC late in the assembly process, had residence times correlated with RNA synthesis rates. The datasets and results reported here provide kinetic information for most of the Pol II-driven genes in this organism and therefore offer a rich resource for exploring the mechanistic relationships between PIC assembly, gene regulation, and transcription. The relationships between gene function and GTF dynamics suggest that shared sets of TFs tune PIC assembly kinetics to ensure appropriate levels of expression. Scripts and processed fileas are available from: https://github.com/AubleLab/PIC_competition_ChIP_scripts大量研究已从结构层面揭示了负责mRNA基因转录起始的分子机器组分。其中包括通用转录因子（general transcription factors, GTFs），它们可与RNA聚合酶II（RNA Polymerase II, Pol II）一同在启动子区域组装，在辅因子与位点特异性转录因子（site-specific transcription factors, TFs）的活性辅助下形成预起始复合物（preinitiation complex, PIC）。然而，目前人们对体内预起始复合物的组装通路及其动力学机制的了解仍较为有限，而这一认知对于从分子机制层面确定体内RNA合成速率的建立方式，以及辅因子与转录因子如何影响该速率至关重要。本研究采用竞争染色质免疫沉淀（competition ChIP）技术，获取了酿酒酵母中五种通用转录因子——TBP、TFIIA、TFIIB、TFIIE与TFIIF的全基因组尺度停留时间估算值。尽管多数通用转录因子与染色质的相互作用寿命较短（小于1分钟），但仍存在大量停留时间为数分钟量级的相互作用。具有相似功能的基因集合，其通用转录因子的动力学行为模式也较为相似。在组装过程后期进入预起始复合物的通用转录因子TFIIE，其停留时间与RNA合成速率呈相关性。本文报道的数据集与研究结果，为该物种中绝大多数由RNA聚合酶II驱动的基因提供了动力学信息，因此为探索预起始复合物组装、基因调控与转录过程之间的机制关联提供了宝贵的研究资源。基因功能与通用转录因子动力学之间的关联表明，共享转录因子集合可通过调控预起始复合物的组装动力学，以确保基因表达水平处于合适范围。相关代码脚本与处理后的数据文件可从以下网址获取：https://github.com/AubleLab/PIC_competition_ChIP_scripts