Systems-level feedback loops maintain gene expression homeostasis following RNA polymerase II dosage perturbation

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) itself may under certain circumstances represent an additional, crucial determinant of transcription regulation. Here, we used the dTAG system to titrate the cellular dosage of human RPB1, the largest RNAPII subunit, 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. Importantly, alterations in RPB1 abundance trigger hierarchical gene expression changes that are highly organized rather than stochastic. 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 together establish RNAPII abundance as a crucial systems-level regulator of transcriptional homeostasis.