Transcription kinetics in pluripotent cells : RNA turnover, transcription velocity, and epigenomic regulation

Abstract: Transcriptional regulation is one of the primary steps in gene expression control. It is now appreciated that a large fraction of coding genome is transcribed in concert of other functional RNAs. A quantitative method for transient transcriptome sequencing (TT-seq) allows profiling of entire transcriptional activities, de novo transcription unit (TU) annotation, and estimation of transcription kinetics from initiation to termination. In Paper I, we showed the establishment of TT-seq method in mouse embryonic stem cells (mESCs) to understand transcriptome plasticity for both coding and non-coding RNAs. With external references in form of a spike-in RNA mix, we were able to estimated RNA synthesis and turnover rates, which consolidated the attenuation under inhibitor-induced pluripotent states (naïve 2i and paused mTORi). We also extended the estimation of transcription velocity to each annotated TU, by integration of RNA polymerase II (Pol II) quantitative profiles from MINUTE-ChIP (quantitative multiplexed ChIP). After explaining transcription velocity with chromatin features, we also evaluated its genome-wide contribution to termination distance. In Paper II, we mapped endogenous genomic G-quadraplex structures (G4) with CUT&Tag in HEK293T and mESCs. We verified the high signal-to-ratio G4 peaks to reflect the DNA motifs of both canonical and trans-strand putative quadraplex sequences (PQS), which enriched on both gene and active enhancer TSSs (transcription start sites). After stabilizing G4 with the small molecule PDS, we observed a genome-wide reduction of RNA synthesis (by TT-seq). The co-occupancy of G4 and R-loop was further verified at transcribed promoters and enhancers. However, promoter G4s could consistently form after transcription inhibition, which suggests an intricate cause-consequence relationship between G4 and transcription activity. In Paper III, we evaluated the regulatory role of repressive histone modifications, H2AK119 ubiquitination and H3K27 tri-methylation. We introduced a rapid H2Aub depletion by BAP1 pulse expression with the amber-suppression system, and observed a wide Polycomb target genes de-repression, especially in the bivalent chromatin state (H3K4me3 + H3K27me3). Further, we observed that H2Aub-mediated repression strength was associated with H3K27me3 occupancy. However, double depletion of H3K27me3 by Ezh2 inhibition with ectopic BAP1 failed to enlarge Polycomb genes de-repression. We also measured transcriptional responses with TT-seq and observed that H2Aub depletion immediately triggered transcription activation before the redistribution of Polycomb proteins and their associated nucleosomes decompaction. Together, our results indicate that H2Aub directly mediates Polycomb integrity and nucleosome barrier that limits early transcription checkpoints.