Using affinity purification MS approaches, we have identified a novel role for casein kinase II (CKII) in the modification of the polymerase associated factor complex (PAF-C). Our data indicate that the facilitates chromatin transcription complex (FACT) interacts with CKII and may facilitate PAF complex phosphorylation. Posttranslational 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 stable isotope labeling in cells (SILAC) quantitation for PAF-C phosphorylation from wild-type and CKII temperature-sensitive strains (cka1⌬ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1⌬ 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 RNA polymerase II transcription through chromatin via phosphorylation of PAF-C.

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The main goal of this project is to identify how cells respond and adapt to genetic changes that alter the process of RNA synthesis. Using baker's yeast as the study system, experimental approaches provide undergraduate students with early exposure to cutting‐edge research technologies that are broadly applicable in both academia and industry. The project introduces students at all levels of training to the importance of quantitative methods to interrogate biology. The specific focus of the research is to understand the consequences of genetic perturbations that knock out or reduce expression of non‐essential elongation factors on RNA polymerase II‐catalyzed RNA synthesis (transcription). Preliminary data suggest that such genetic perturbations lead to significant adaptations within the RNA polymerase II protein‐protein interaction networks. These adaptations may include (but are not limited to) disruption of interactions with RNA polymerase II, compensation leading to novel protein recruitment, and activation of rescue pathways to dispose of arrested RNA polymerase II. The laboratory portion of an undergraduate course in molecular biology was redesigned to generate elongation factor knock‐out yeast strains with a focus on key molecular biology concepts and techniques including: genomic DNA isolation, oligonucleotide design, PCR, genotyping, protein isolation, and western blotting. In addition to these key concepts, we introduced students to the theory and practice of proteomics analysis through mass spectrometry including discussions of bioinformatics analysis of proteomics data, calculation of false discovery rates, and affinity purification mass spectrometry approaches. Thus far in this study, 9 elongation factor knock out strains have been made and verified by DNA sequencing and are in the process of being analyzed by quantitative mass spectrometry. Data from pre‐ and post‐instructional tests will be presented which demonstrate increased student understanding of mass spectrometry. Additionally, students participated in an 8 week summer internship held at the IU School of Medicine where they extended the project from the classroom lab to performing quantitative mass spectrometry analysis of their elongation factor knock‐outs.Support or Funding InformationDePauw University Student Faculty Research Fund, Showalter Fund (IU), NSF Award Number:1515748 PI Amber Mosley, PhD