Dynamic regulation of promoter-proximal RNA polymerase II pausing

Abstract

Transcriptional pausing is a widely used regulatory mechanism to control precise gene expression from bacteria to humans. In metazoans, transcription initiation requires the recruitment of general transcription factors and RNA polymerase II (Pol II) to gene promoters. After it initiates transcription and synthesizes a short RNA, Pol II pauses at the promoter-proximal region, standing by for further cues to enter the productive elongation stage. Promoter-proximal Pol II pausing is one of the rate-limiting steps during transcription, functioning as an early transcriptional elongation check point, to ensure the correct modification of transcription machinery and the addition of the 7-methylguanosine cap to nascent transcripts. Pol II pausing at promoter-proximal region is stabilized by the negative elongation factor (NELF) and the 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). In response to developmental signals or environmental stress, the release of paused Pol II can be triggered by the positive transcription elongation factor b (P-TEFb), which is composed of the kinase CDK9 and its regulatory subunit CCNT1/2. The kinase activity of P-TEFb towards Pol II carboxy-terminal domain (CTD), NELF and DSIF is critical for P-TEFb to execute its function in pause release, and is thus tightly controlled. To date, it has been identified that P-TEFb mainly exists in three different complexes: the super elongation complex (SEC), BRD4-P-TEFb, and the inactive 7SK snRNP-P-TEFb complex. SEC, one of the most active P-TEFb containing complexes, is required for rapid gene expression in response to stress or developmental signals. The active and inactive forms of P-TEFb complexes co-exist in vivo , reaching equilibrium to meet the transcriptional needs of cells. The transition of Pol II from pause to productive elongation stage is achieved by dynamic exchange between the pausing factors and the positive elongation factors at the proximal promoter region. Liquid-liquid phase separation (LLPS) in living cells is a physicochemical process that confines biochemical reactions within membrane-less compartments. Low complexity intrinsically disordered regions (IDRs) within proteins can initiate and promote phase separation and formation of liquid droplets, which in turn facilitate the multivalent weak interactions among these IDR-containing proteins. Recent studies using super-resolution live cell imaging technology have revealed that RNA Pol II can form dynamic foci in cells through LLPS and transitions between different phases, which are likely mediated by phosphorylated CTD. Moreover, factors involved in various transcription steps can rapidly and dynamically concentrate and exchange at the hot regulatory genomic loci. For example, SEC components are able to compartmentalize and concentrate P-TEFb from its inactive partners. Upon serum stimulation, SEC assembles promptly into phase-separated droplets at the immediate early genes (IEG), triggering rapid transcription induction. Mutations in transcription regulators can lead to developmental defects and many human diseases, including cancers. For instance, abnormal stabilization of SEC components via MLL fusion plays a contributing role in leukemogenesis. Fusion of the SEC core component ENL with MLL can greatly enhance phase separation of SEC, through which the leukemic genes might be mis-activated. In addition, there is growing evidence that disease related mutations in transcriptional regulators can cause malfunction through aberrant condensation, providing potential targets for designing therapeutic strategies through phase separation interventions.