Regulation of RNA polymerase II promoter-proximal pausing by DSIF

高等真核生物的RNA聚合酶II（Pol II）通常在大多数基因的转录起始位点的下游，在所谓的启动子近端区域中暂停。主要基于使用基于细胞的测定的间接观察，似乎Pol II暂停是由因子DSIF和NELF诱导的。这些因子的动态磷酸化触发Pol II释放进入生产性伸长。虽然暂停是转录的关键调控步骤，但DSIF如何同时作为负延伸因子和正延伸因子发挥作用仍不清楚。为了进一步阐明DSIF的机制作用，我将评估其潜在的合作与NELF使用体外转录测定，使用纯化的因子（无提取物）。基于体外数据，我将设计基于细胞的测定来进一步测试所提出的机制。这将依赖于PRO-seq和使用degron或Trim-Away方法的快速蛋白质去除。尽管已经努力了解暂停背景下的DSIF磷酸化，但详细的理解仍然难以捉摸。因此，我将其次包括P-TEFb，一个负责DSIF激酶，来分析这个问题。将在基于细胞的研究中进一步测试使用体外系统鉴定的对Pol II起始、暂停或暂停释放的任何P-TEFb或DSIF磷酸化位点特异性作用。除了机械的理解，基于细胞的方法将提供有价值的见解的动力学和范围的启动子近端暂停。在未来的工作中，同样的系统将允许分析DSIF在生产延伸中的作用。

RNA Polymerase II (Pol II) of higher eukaryotes commonly pauses downstream of the transcription start site of most genes, in the so-called promoter-proximal region. Based primarily on indirect observations using cell-based assays, it appears that Pol II pausing is induced by the factors DSIF and NELF. Dynamic phosphorylations of these factors trigger the release of Pol II into productive elongation. Although pausing is a key regulatory step of transcription, it remains unclear how DSIF functions as both a negative and positive elongation factor. To further elucidate the mechanistic role of DSIF, I will assess its potential cooperation with NELF using an in vitro transcription assay that uses purified factors (no extracts). Based upon the in vitro data, I will design cell-based assays to further test the proposed mechanism. This will rely on PRO-seq and rapid protein depletion with degron or Trim-Away methods.Although efforts have been made to understand DSIF phosphorylation in the context of pausing, a detailed understanding remains elusive. Hence, I will secondly include P-TEFb, a responsible DSIF kinase, to analyze this question. Any P-TEFb or DSIF phospho-site specific effects on Pol II initiation, pausing or pause release that are identified using the in vitro system will be tested further in cell-based studies. In addition to mechanistic understanding, cell-based approaches will provide valuable insights into the dynamics and scope of promoter-proximal pausing. In future efforts, the same systems will allow analysis of the role of DSIF in productive elongation.