CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation.

Michael Tellier,Ivan Ferrer-Vicens,Taras Velychko,Shona Murphy,Livia Caizzi,Justyna Zaborowska,Björn Schwalb,Daniel Blears,Patrick Cramer,Takayuki Nojima,Eusra Mohammad

doi:10.1093/nar/gkaa514

Abstract

Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

Highlights

The phosphorylation status of the C-terminal domain (CTD) of RNA polymerase II plays a major role in expression of non-coding and protein-coding genes [1]
Pol II distribution on expressed transcription units in the parental cells is not affected by short-term NM treatment as measured by polymerase II (pol II) chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) of KPNB1 and Chromatin immunoprecipitation (ChIP)-sequencing (ChIP-seq) (Supplementary Figure S2A and B)
As transcription of histone genes is relatively unaffected by CDK12as inhibition, we have investigated whether pol II carboxyl-terminal domain (CTD) Ser2P and Ser5P levels are affected by CDK12as inhibition

Summary

Introduction

The phosphorylation status of the C-terminal domain (CTD) of RNA polymerase II (pol II) plays a major role in expression of non-coding and protein-coding genes [1]. The CTD comprises 52 repeats of the consensus heptapeptide Tyr1/Ser2/Pro3/Thr4/Ser5/Pro6/Ser and is reversibly phosphorylated by several kinases during transcription [1]. CDK12 is the metazoan ortholog of the Saccharomyces cerevisiae CTD Ser kinase, Ctk1 [2,3]. Knockdown of CDK12 results in loss of CTD Ser phosphorylation in Drosophila, Caenorhabditis elegans and human cells [3,4,5,6,7,8,9,10]. Treatment with the CDK12/CDK13 inhibitor THZ531 in Jurkat cells affects pol II CTD Ser phosphorylation in a concentration-dependent manner [7]. Analog-sensitive (as) kinase technology has been used to demonstrate rapid loss of Ser phosphorylation after inhibiting the Schizosaccharomyces pombe CDK12 homologue Lsk1 [11]. CDK12 mainly phosphorylates Ser in vitro [15,16]

Methods

Results

Conclusion