Background:
This project focuses on the role of transcriptional cyclin-dependent kinases (CDKs) in regulating DNA replication and cancer proliferation. Transcriptional CDKs play a crucial role in regulating RNA polymerase II (RNAPII)-mediated transcription, which is essential for cell-fate and -type-specific gene expression. Unlike cell cycle CDKs, which are involved in cell cycle transitions, transcriptional CDKs (tCDKs) such as CDK7-9 and CDK11-13 regulate transcription initiation, pause release, and elongation by phosphorylating the carboxy-terminal domain (CTD) of RNAPII. CDK12 and CDK13, in complex with their cyclin partner CycK, phosphorylate serine-2 of the RNAPII CTD, promoting transcriptional elongation. These kinases are critical for sustaining the transcriptional programs that support cellular proliferation. CDK12 and CDK13 play critical roles in sustaining cancer cell transcription, and their inhibition can lead to significant disruptions in both transcription and DNA replication. We have shown that pharmacological inhibition of CDK12 and CDK13 leads to the disruption of these transcriptional processes in cancer cells, causing a near-total shutdown of nascent transcription and DNA replication. Within 6 hours of CDK12/CDK13 inhibition, we observed a significant downregulation of RNAPII phosphorylation, leading to a sharp decrease in nascent mRNA synthesis. To track the kinetics of this effect, we performed SLAM-seq at two, four-, and six-hours post-inhibition. To explore whether these changes result from change in chromatin accessibility, we also conducted ATAC-seq at the same time points. We are currently mapping RNAPII localization and replication timing for these time points, and these data will be available for analysis during this thesis project.
Aims:
The overarching aim of this master project is to understand how CDK12/13 inhibition affects genome-wide transcriptional and DNA replication processes. This work seeks to uncover potential vulnerabilities in cancer cells that could be exploited for therapeutic purposes, offering an exciting opportunity to contribute to cancer biology.
Further, our aim is to identify and analyze key pathways and gene sets involved in transcriptional addiction and replication stress. This project will utilize bioinformatic approaches to analyze how CDK12/13 inhibition affects transcription and replication dynamics in cells which require CDK12/CDK13 function, providing insights into potential therapeutic strategies for cancer treatment.
Project and tasks:
This is a bioinformatics-based master’s project and will focus on analyzing transcriptional and DNA replication data from cells treated with CDK12/13 inhibitors. Using cancer cells which are highly sensitive to CDK12/13 inhibition, we will investigate the link between transcriptional perturbations and DNA replication. The candidate will analyze changes in nascent mRNA levels, RNAPII occupancy, chromatin accessibility, and replication timing across the genome. The goal is to understand how transcriptional dysregulation affects DNA replication in pancreatic cancer cells and potentially uncover new targets for cancer therapy.
The specific tasks of the project will be analyzing pre-existing datasets:
- SLAM-seq analyses to study changes in nascent and steady-state mRNA levels. SLAM-seq will help identify which mRNAs are affected early, along with any enriched functional categories (1-3 months), and track the kinetics of mRNA synthesis changes, potentially extending to transcript decay (1-6 months).
- ATAC-seq analyses to assess changes in chromatin accessibility. ATAC-seq analyses will provide insights into changes in chromatin accessibility (4-6 months) and its correlation with mRNA synthesis (4-9 months).
- RNAPII ChIP-seq and Ori-seq analysis. These data will assess RNAPII occupancy and replication origins, and this data will be integrated with SLAM-seq and ATAC-seq (7-15 months).
With these data, the student will have the opportunity to identify change in RNA Pol II localization during cell cycle phases, perform replication timing analysis to identify regions of the genome that are replicated early or late, and perform integrative analyses to identify the relationship between change in transcriptional output and replication timing. Together, these analyses will identify and place the mechanism through which CDK12/CDK13 regulate transcription and genome maintenance.
Supervision:
We are seeking a motivated master student for this project, which will be jointly supervised by Dr. Deo Prakash Pandey from the Department of Molecular Medicine at Rikshospitalet (see Pandey group: https://www.ous-research.no/pandey) and Dr. Thomas Fleischer from the Institute for Cancer Research at Radiumhospitalet (see Fleischer group: https://www.ous-research.no/fleischer/). The internal supervisor at IBV will be either Prof. Rein Aasland or Dr. Jonas Paulsen. Progress will be closely monitored through regular one-on-one meetings with both supervisors, along with monthly joint meetings. The student will have the opportunity to participate in group meetings in both labs and present their work at the section or department level.