Genome-scale chromatin interaction dynamic measurements for key components of the RNA Pol II general transcription machinery [ChIP-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已有大量研究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）在启动子区域组装形成预起始复合物（preinitiation complex, PIC），该组装过程需辅因子与位点特异性转录因子（site-specific transcription factors, TFs）的活性辅助。 然而，目前学界对体内预起始复合物的组装通路及其动力学机制的认知仍相对不足，而明确这些机制对于从分子层面解析体内RNA合成速率的建立方式，以及辅因子与转录因子如何调控该速率，均具有至关重要的意义。 本研究采用竞争染色质免疫沉淀（competition ChIP）技术，对酿酒酵母中5种通用转录因子——TBP、TFIIA、TFIIB、TFIIE及TFIIF的全基因组范围停留时间进行了定量估算。研究发现，尽管多数通用转录因子与染色质的相互作用寿命较短（<1分钟），但仍存在大量寿命为数分钟量级的相互作用。具有共同功能的基因集合，其通用转录因子的动力学行为模式也较为相似。其中，TFIIE作为一类在预起始复合物组装后期才加入的通用转录因子，其停留时间与RNA合成速率呈显著相关。 本研究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、基因调控与转录之间的分子机制关系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提示，共享的转录因子集合可通过调控预起始复合物组装动力学，以确保基因表达水平的适宜性。 相关脚本与处理后文件可从以下网址获取：https://github.com/AubleLab/PIC_competition_ChIP_scripts