Characterizing CDK12 and CDK13 Activity in Transcription and mRNA Biogenesis in Mouse Embryonic Stem Cells

Abstract

The RNA Polymerase II (RNAPII) C‐terminal domain (CTD) heptapeptide repeat (YSPTSPS) is dynamically phosphorylated during transcription. Serine 2 phosphorylation of the RNAPII CTD (Ser2P) occurs upon the transition to productively elongating RNAPII and extends through the gene body. Ser2P recruits mRNA processing machinery, histone modifying enzymes, chromatin remodelers, and transcription elongation factors directly to the transcription machinery consequently coupling transcription and mRNA processing. Recently, two paralogous kinases in mammals, CDK12 and CDK13, were identified as members of the family of cyclin‐dependent kinases responsible for Ser2P. Additionally, CDK12 has been identified as a tumor suppressor in several tumor types. However, the function of each kinase in regulating transcription and mRNA biogenesis remains unclear.Here, we report the establishment of a genetic system to interrogate CDK12 and CDK13 activity. CDK12 or CDK13 knockouts were generated in mouse embryonic stem cells using CRISPR/Cas9 genome editing. The rescue of knockout cells with an inducible CDK12 or CDK13 epitope‐tagged expression system permits high‐confidence genomic characterization. Currently RNA sequencing (RNA‐seq) and Chromatin Immunoprecipitation Sequencing (ChIP‐seq) with antibodies specific for phosphorylated and total RNAPII, CDK12, and CDK13 are being performed in knockout and rescued cells. The derivation of this cell system and progress in CDK12 and CDK13 characterization will be presented.NIH RO1‐CA133404 to P.A. Sharp, NIH Pre‐Doctoral Training Grant T32GM007287 to MIT Biology Dept., and MIT School of Science Fellowship in Cancer Research Award to S. Dubbury supported this work.