CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes.

Anil Paul Chirackal Manavalan,Dalibor Blazek,Michael Kluge,Koen Bartholomeeusen,Jan Oppelt,Prashant Khirsariya,Lumir Krejci,Kveta Pilarova,Caroline C Friedel,Kamil Paruch,Michal Rajecky

doi:10.15252/embr.201847592

Abstract

CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog‐sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA‐seq and ChIP‐seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3′ends of predominantly long, poly(A)‐signal‐rich genes. CDK12 inhibition does not globally reduce levels of RNAPII‐Ser2 phosphorylation. However, individual CDK12‐dependent genes show a shift of P‐Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.

Highlights

Transcription of protein-coding genes is mediated by RNA polymerase II (RNAPII) and represents an important regulatory step of many cellular processes
Immunoprecipitation (IP) of CDK12 from the WT and AS CDK12 HCT116 cells followed by Western blotting showed that equal amounts of cyclin K (CCNK) associated with CDK12, and that comparable levels of CDK12 were expressed in both cell lines, confirming the functionality of the AS variant (Fig EV1C)
Our genome-wide studies of total and modified RNAPII suggest that CDK12 kinase does not globally control P-Ser2 levels on transcription units; it is crucial for RNAPII processivity on a subset of long and poly(A)signal-rich genes, those involved in DNA replication and DNA damage response

Summary

Introduction

Transcription of protein-coding genes is mediated by RNA polymerase II (RNAPII) and represents an important regulatory step of many cellular processes. Various kinases have been implicated in CTD phosphorylation [7,8,9,10], and the kinase CDK12 is thought to phosphorylate predominantly Ser2 [11,12,13,14,15,16,17,18] These findings were based on the use of phospho-CTD specific antibodies combined with various experimental approaches including in vitro kinase assays, long-term siRNAmediated depletion of CDK12 from cells or application of the CDK12 inhibitor THZ531. Each of these experiments has caveats with respect to the physiological relevance. The specific impact of a short-term CDK12-selective inhibition on CTD phosphorylation and genome-wide transcription in cells remains an important question to be addressed

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