Inherited Diseases - Transcriptional Regulation – Dalibor Blažek

• Regulation of eukaryotic transcription
• Role of transcription cycle-related Cdks in maintenance of genome stability
• Transcription cycle-related Cdks in human disease

• Proteomics, Genomics, Chemical Biology
• Control of DNA damage response and genome stability via regulation of expression of DNA damage response genes

RNA polymerase II (RNAPII) directs transcription of protein coding genes and this process consists of several stages including preinitiation complex formation, productive elongation and termination. This transcription cycle is tightly linked to co-transcriptional maturation of nascent transcripts including pre-mRNA splicing and polyadenylation. RNAPII contains an unstructured C-terminal domain (CTD) with repeats of evolutionarily conserved heptapeptide YSPTSPS, where individual serines get phosphorylated. Several cyclin-dependent kinases (Cdks) regulate the phosphorylation status of the CTD and subsequent binding of transcription and pre-mRNA processing factors. Thus, the patterns of phosphorylation of the CTD direct actions of RNAPII during transcriptional cycle and co-transcriptional processing of nascent transcripts. Moreover, CTD was also functionally linked to DNA damage response and maintenance of genome stability via regulation of transcription, mRNA processing and recombination. Thus, CTD and its posttranslational modifications, associated proteins and modifying enzymes are emerging as new players in cellular response to DNA damage. Our recent work led to the identification of the Cyclin K/Cdk12 complex that phosphorylates serine 2 in the CTD of RNAPII and directs expression of several crucial DNA damage response genes including BRCA1, ATR or FANCI. In my lab we apply a combination of biochemical, proteomics and genome-wide techniques to determine the molecular mechanism that regulates the expression of Cdk12-dependent genes with a focus on DNA damage response genes. The ultimate goal of our research is to uncover how the CycK/Cdk12 complex and the CTD of RNAPII contribute to the maintenance of genome stability, and how disruption of their functions lead to the onset of a malignant state.

Research overview

Regulation of Gene Expression by Cyclin-dependent Kinases 11 and 12 (CDK11 and CDK12)

RNA Polymerase II (RNAPII) directs the transcription of protein-coding genes in a process consisting of initiation, elongation and termination. It contains an unstructured C-terminal domain (CTD) with 52 repeats of evolutionarily conserved heptapeptide YSPTSPS, where individual serines (Ser) (Ser2, Ser5, Ser7), threonine 4 (Thr4) and tyrosine 1 (Tyr1) are phosphorylated. The CTD is necessary for the regulation of the transcription cycle and for coupling transcription with co-transcriptional processing. P-Ser2 is a marker of active elongation and plays a stimulatory role in co-transcriptional mRNA processing via the recruitment of splicing, cleavage and polyadenylation factors.

Several kinases, including well-studied CDK7 and CDK9, phosphorylate the CTD of RNAPII and other transcription factors to regulate individual steps of transcription and co-transcriptional processing. Our work has indicated important roles of other less-studied CDKs, including CDK11 and CDK12, in the regulation of transcription elongation and co-transcriptional mRNA processing. Notably, all the known P-Ser2 CTD and elongation-associated kinases are dysregulated in human diseases, including cancer, and their inhibition is considered to be a very promising strategy for fighting various diseases.

Ongoing work in my lab will further explore the roles of CDK11 and CDK12 in the regulation of RNAPII-directed gene expression, particularly in transcription elongation and mRNA-processing. We use genomic, proteomic and biochemical approaches to achieve these goals.

Regulation of transcriptional elongation and genome stability by CDK12

CDK12 phosphorylates the CTD of RNAPII and other substrates to regulate the elongation of transcription. We found that CDK12 maintains genome stability via regulating the transcription elongation of key DNA damage response genes including BRCA1, ATR, ATM, FANCD2 and FANCI (Blazek et al, G&D, 2011). CDK12 kinase activity is also needed for the optimal transcription of core DNA replication genes and thus for G1/S cell cycle progression (Chirackal Manavalan et al., EMBO reports, 2019). We propose that by regulating gene-specific expression, CDK12 has pleiotropic effects on genome stability in cell lines and various tumors (Pilarova et al, NAR Cancer, 2020). Consistent with this, comprehensive genomic analyses of patient samples revealed CDK12 to be disregulated in various types of cancer. We showed that CDK12 mutations in high-grade serous ovarian carcinoma prevent the formation or activation of the CDK12/CYCK complex, rendering the kinase inactive (Ekumi et al, NAR, 2015).

Ongoing research is focusing on the molecular mechanism of CDK12-dependent gene expression including the identification of novel substrates. We are also interested in exploiting CDK12 vulnerabilities in cancer treatment.

Roles of CDK11 in gene-specific expression

CDK11 is a little-studied, but essential kinase, and is believed to play a role in RNA polymerase II (RNAPII)-directed transcription and co-transcriptional mRNA-processing. However, so far its specific roles in gene expression have been unknown. Importantly, numerous recent studies have demonstrated many cancers to be dependent on CDK11 activity.

We have found that CDK11 is required for the transcription of replication-dependent histone genes. The kinase binds FLASH protein, and with the contribution of replication-dependent histone mRNAs, recruits CDK11 to the middle of the histone genes, where it is needed for Ser2 phosphorylation and their optimal elongation and 3´ end processing (Gajduskova et al, Nature Structural & Molecular Biology, 2020).

Ongoing research in my lab is focusing on uncovering the molecular mechanisms and cellular functions of this enigmatic kinase. We are also interested in exploiting CDK11 vulnerabilities in human diseases.

Summary of Main Areas of Research and Approaches

• Roles of CDK11 and CDK12 in the regulation of gene expression
• Roles of CDK12 in the maintenance of genome stability
• Roles of CDK12 in the regulation of cell cycle progression
• Phosphorylation of the C-terminal domain of RNA polymerase II and other substrates by CDK11 and CDK12
• The deregulation of CDK11 and CDK12 in the onset and development of cancer
• Proteomics, Genomics, Chemical Biology