Maintenance of gene expression homeostasis by systems-level feedback loops in response to changes in RNA polymerase II dosage [RNAPII_levels_DRB-TTseq]

Transcription is regulated by sequence-specific transcription factors and enzymes allowing access to genes in chromatin. However, recent data indicate that the abundance of RNA polymerase II (RNAPII) may itself under certain circumstances represent an additional, crucial determinant of transcription regulation. Here, we titrated the cellular dosage of human RPB1, the largest RNAPII subunit, using the dTAG system to more generally assess the importance of RNAPII levels. Unexpectedly, cells are extremely sensitive to RPB1 dosage, with a mere 30% reduction sufficient to perturb cell proliferation, cell cycle progression, and global transcription. Using a combination of sequencing and proteomic approaches, we uncover the existence of multiple feedback loops between transcriptional initiation, promotor-proximal pause release, transcript elongation, splicing, and mRNA half-life, which facilitate the maintenance of gene expression homeostasis. Together, our data suggests that gene expression is highly sensitive to RPB1 dosage and that the cellular levels of RNAPII must be tightly regulated. Overall design: To investigate the effect of RPB1 abundance on transcript elongation rates, we modulated RPB1 levels by treating RPB1 degron cells with 4 concentrations of dTAGv-1. After 12.5 hours, transcription was synchronized at the promoter proximal pause site through the addition of DRB. Afterwards, RNA polymerases were released for different times by washing out DRB, and nascent RNA was metabolically labeled with a short pulse of the uridine analog 4-thiouridine (4sU). Transient transcriptome chem-sequencing (TTchem-seq) was then used to fragment, affinity purify and sequence 4sU-labelled RNA for high-resolution profiles of nascent transcription. Finally, the transcription wave peak was measured at different time points to estimate elongation speed.