Regulation of RNA polymerase II promoter-proximal pausing by DSIF

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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.