Multi-omics studies of RNA polymerase II transcription regulation by cyclin-dependent kinases
by Taras Velychko
Date of Examination:2022-12-01
Date of issue:2024-10-30
Advisor:Prof. Dr. Patrick Cramer
Referee:Prof. Dr. Patrick Cramer
Referee:Prof. Dr. Henning Urlaub
Persistent Address:
http://dx.doi.org/10.53846/goediss-10831
Files in this item
Name:PhD_Thesis_Velychko_2024-10-15.pdf
Size:3.74Mb
Format:PDF
The following license files are associated with this item:
Abstract
English
Phosphorylation of the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) plays a central role in the regulation of gene transcription in eukaryotes. In yeast and mammals, the CTD is highly conserved and consists of 26 and 52 repeats of the consensus heptapeptide sequence (Y1S2P3T4S5P6S7), respectively. Changes in Pol II CTD phosphorylation patterns regulate the transcription cycle via recruitment of transcription and RNA processing factors. Transcriptional cyclin-dependent kinases (CDKs) are serine-threonine protein kinases that are responsible for Pol II CTD phosphorylation during the transcription cycle. Over the years, numerous studies have investigated the roles of CDKs in transcription, yet the use of various inhibition strategies, inhibition durations, and methods to monitor transcriptional changes have resulted in inconsistent findings so that the direct function of transcriptional CDKs remains to be fully established. To shed light on the matter, here we combine state-of-the-art technologies by performing rapid and selective inhibition of CDK8, CDK7, and CDK12 kinases in human cells with time-resolved detection of genome-wide changes in RNA synthesis and Pol II occupancy. Inhibition of CDK8, a part of the kinase module of Mediator, does not affect Pol II transcription globally and results in downregulation of only a subset of genes. However, acute depletion of Mediator leads to a pronounced transcription disruption of cell-type-specific genes, which feature exceptionally high Pol II initiation rates. Housekeeping genes are less affected by Mediator depletion due to CDK9-dependent compensation that increases the release of paused Pol II molecules. CDK7 kinase inhibition leads to an immediate global downregulation of transcript synthesis due to decreased Pol II initiation, whereas elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although Pol II, initiation factors, and Mediator accumulate at promoters, a fraction of Pol II complexes escapes and proceeds into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. This indicates that loss of Pol II interactions with initiation factors is not required for Pol II release from promoters. Further downstream, Pol II CTD phosphorylation levels increase, initiation factors and Mediator get released, allowing for recruitment of elongation factors and increase in Pol II elongation velocity. Inhibition of CDK12 kinase activity leads to a global transcription elongation defect due to the loss of elongation factors from newly elongated Pol II molecules. Collectively, these results show that despite the pronounced stimulation of cell-type-specific gene transcription by Mediator, CDK8 kinase module is mostly inactive in steady-state; CDK7 kinase activity promotes the release of initiation factors and Mediator from Pol II, facilitating Pol II escape from the promoter; CDK9 kinase activity can compensate for the inefficient initiation in response to Mediator loss; and CDK12 is a general activator of Pol II transcription elongation.
Keywords: CDK7; CDK8; CDK9; CDK12; Mediator; Gene regulation; CTD; Analog sensitive; RNA polymerase II; Transcription; Pre-initiation complex; Promoter escape
