Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation

Abstract Using affinity purification mass spectrometry (AP-MS) approaches, we have identified a novel role for CKII in the modification of the Polymerase Associated Factor Complex (PAF-C). Our data indicates that the FAcilitates Chromatin Transcription complex (FACT) interacts with CKII and may facilitate PAF Complex phosphorylation. Post-translational modification analysis of affinity isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C although CKII subunits were not detected as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate PAF-C in vitro whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with SILAC quantitation for PAF-C phosphorylation from wild−type and CKII temperature sensitive strains (cka1Δ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1Δ cka2-8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation - a modification dependent on FACT and PAF-C. Taken together, our results define a coordinated role of CKII and FACT in the regulation of RNAPII transcription through chromatin via phosphorylation of PAF-C.