alvocidib and triptolide

Maintenance of transcription programs is challenged during mitosis when chromatin becomes condensed and transcription is silenced. How do the daughter cells re-establish the original transcription program? Here, we report that the TATA-binding protein (TBP), a key component of the core transcriptional machinery, remains bound globally to active promoters in mouse embryonic stem cells during mitosis. Using live-cell single-molecule imaging, we observed that TBP mitotic binding is highly stable, with an average residence time of minutes, in stark contrast to typical TFs with residence times of seconds. To test the functional effect of mitotic TBP binding, we used a drug-inducible degron system and found that TBP promotes the association of RNA Polymerase II with mitotic chromosomes, and facilitates transcriptional reactivation following mitosis. These results suggest that the core transcriptional machinery promotes efficient transcription maintenance globally.

Topics: Animals; Cell Line; Chromosomes; Diterpenes; Epoxy Compounds; Flavonoids; Mice; Mitosis; Molecular Imaging; Mouse Embryonic Stem Cells; Phenanthrenes; Piperidines; Promoter Regions, Genetic; Protein Binding; RNA Polymerase II; Single-Cell Analysis; TATA-Box Binding Protein; Transcriptional Activation

Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ∼3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes.DOI: http://dx.doi.org/10.7554/eLife.02407.001.

Topics: Animals; Cells, Cultured; Chromatin; Chromatin Immunoprecipitation; Diterpenes; Embryonic Stem Cells; Epoxy Compounds; Exons; Flavonoids; Gene Library; Genetic Association Studies; Genetic Variation; Humans; Mice; Phenanthrenes; Piperidines; Promoter Regions, Genetic; RNA Polymerase II; RNA, Messenger; Sequence Alignment; Sequence Analysis, DNA; Transcription, Genetic