Bioinformatic analysis of multi-omics data elucidates transcriptional cyclin dependent kinase mediated transcriptional regulation

Abstract

Transcriptional regulation is a highly dynamic biological process which is governed by a complex network of proteins for precise expression of a gene. One of the most important regulators of gene expression are cyclin dependent kinases (CDKs), which transduces signal by phosphorylation of its substrate. CDKs are broadly classified into two groups – cell cycle CDKs and transcriptional CDKs(tCDKs). Due to limited structural and functional knowledge of tCDKs, till date it remains to be a fascinating field of research. One of the early functions discovered for tCDKs is the phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II), but the knowledge remains poorly understood in vivo till date. This phosphorylation cycle of Pol II CTD is crucial to co-ordinate between different transcriptional processes namely initiation, pause release, elongation, as well as termination of transcription. Additionally, tCDKs can also interact with other proteins in transcription cycle for orchestrating regulation intertwined with chromatin accessibility and epigenetic mechanisms. Since the discovery of tCDKs, scientists have been trying to unravel the functional mystery of the individual kinases in transcriptional cycle regulation. tCDKs are structurally and functionally conserved, therefore dissecting the role of individual kinases is a challenging task. This thesis work presents a collaborative effort to piece together the transcriptional kinase puzzle by defining the primary role of individual tCDKs in transcription cycle. This will eventually lead to uncover the detailed molecular mechanism of tCDKs and their impact on transcriptional regulation. Research focused on tCDKs for functional interpretation are mostly done with chemical inhibitors e.g., THZ1 which has off target effects as well as longer inhibition duration introduces secondary effects. To circumvent these limitations, the studies presented in the thesis uses an analog sensitive kinase approach as a powerful tool to study the function of individual kinases with specific inhibition in vivo. Restricting the inhibition duration to a shorter time span allows to investigate the primary function of the individual kinases. Precisely, we investigated the function of three different kinases, CDK7, CDK12 in human cell line and CDK8 in yeast. To obtain novel insights into the underlying mechanism, we combined complementary functional genomic methods, for example, Transient Transcriptome Sequencing (TT-seq) and mammalian Native Elongating Transcript Sequencing (mNET-seq) with proteomics and structural studies which gives us promising results to determine the exact functionality of the individual CDKs. This broadens our knowledge in the field of tCDK mediated transcriptional regulation and its involvement in all of the relevant biological processes. More careful analysis needs to be done to interpret how kinases interact under physiological conditions, but the work presented in this thesis significantly contributed to the understanding of individual CDKs function in vivo. In recent times, tCDKs have been implicated in cancer and their inhibition in cancer therapy and presence as biomarkers are being explored extensively. The knowledge of individual tCDK function will help us to manipulate the activity of these kinases to substantially increase the therapeutic potential.