Transcriptional elongation is tightly controlled by cyclin-dependent kinases CDK12 and CDK13, which coordinate co-transcriptional histone H2B ubiquitination through the heterodimeric E3 ligase complex RNF20/40. (1) The WW-containing adaptor protein WAC serves as a critical molecular bridge linking RNA polymerase II phosphorylation to targeted ubiquitin deposition at actively transcribed loci. While defects in this pathway are implicated in neurodevelopmental disorders and cancer, the direct molecular mechanisms remain unclear. (2)
To investigate CDK12/13-specific regulation of WAC, we generated analog-sensitive kinase alleles and degron-tagged cell lines enabling rapid and reversible control of kinase activity. Global phosphoproteomics revealed CDK13-specific phosphorylation sites in WAC that are not targeted by CDK12, indicating functionally distinct regulatory mechanisms. Immunoprecipitation-mass spectrometry identified novel WAC interactions with transcription elongation complexes and nucleosome remodeling machinery. Acute WAC degradation followed by RNA-seq analysis demonstrated that lowly-expressed genes are more sensitive to WAC loss than highly-expressed genes, revealing expression level-dependent requirements for WAC-mediated ubiquitination. While highly expressed genes showed modest upregulation and lowly expressed genes showed modest downregulation upon WAC depletion, the overall transcriptional effects were minimal, suggesting gene-specific regulatory roles.
These findings establish WAC as a phosphorylation-dependent coordinator linking CDK13 activity to co-transcriptional chromatin modifications. Future work will encompass biochemical reconstitution of WAC-containing complexes, structural characterization of phospho-dependent interactions, rescue experiments with phospho-mutant WAC variants, and development of ubiquitin-tagging methodologies.
This work provides crucial mechanistic insights into transcriptional kinase regulation of chromatin modifications and identifies potential therapeutic targets for diseases involving defective transcription-coupled ubiquitination.