Defining the cyclin K-SET1A regulatory axis through biophysical characterisation and proteomic mapping

Abstract

Cyclin K functions as the regulatory partner of cyclin-dependent protein kinase CDK12 and CDK13, where it plays essential roles in coordinating transcription elongation and termination through phosphorylation of the RNA polymerase II C-terminal domain. Beyond its core activity as a CTD kinase, cyclin K contributes to several co-transcriptional processes, including mRNA splicing and translation. It is also a key player in the DNA damage response (DDR): its expression is driven by p53, and it modulates the transcription of DNA repair genes. Cyclin K directly associates with the chromatin regulator SET1A, a histone methyltransferase that is a component of the COMPASS complex. Cyclin K also associates with the PAF1 complex through the CDC73 subunit. Although multiple proteins have been identified as cyclin K interactors, the precise binding interfaces on cyclin K remain unmapped. To address this gap, a crystallographic fragment screen was carried out using FragLites, a small set of fragments that both sensitively identify interaction hotspots on a protein surface and provide leads for subsequent chemical probe and drug discovery. Using the interaction hotspots identified on the cyclin K FragLite map, targeted mutations at putative protein- binding regions were engineered to pinpoint the SET1A interaction interface. Cyclin K variants carrying multi-residue substitutions at two distinct regions, Site 1 and Site 2, were expressed in HEK293T cells and examined through FLAG pull-down combined with mass spectrometry. Further characterisation demonstrated that these mutations define the interaction surfaces required not only for cyclin K binding to SET1A and the COMPASS complex, but also for the association of cyclin K with the PAF1 complex. Site 1 serves as a shared interaction platform for both COMPASS and PAF1C, whereas Site 2 appears to be selectively important for COMPASS association. These separation of function mutants provide a powerful framework for understanding the mechanistic specificity of cyclin K and open avenues for selectively modulating CDK12-cyclin K activity.