Abstract
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 (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 RNA polymerase II transcription through chromatin via phosphorylation of PAF-C.
Highlights
Transcription elongation by RNA polymerase II (RNAPII)4 is a coordinated process that is regulated to ensure the proper expression of protein-coding genes
In support of this idea, we show that casein kinase II (CKII) copurified with Spt16-TAP readily phosphorylates polymerase-associated factor complex (PAF-C) in vitro, whereas no detectable kinase activity copurified with PAF-C through Ctr9-FLAG
We chose to perform four biological replicate purifications of Spt16-TAP under low-salt (LS, 100 mM NaCl) conditions to facilitate the capture of transient interacting proteins because facilitates chromatin transcription (FACT) was the lowest-level RNAPII interaction partner known from previous studies identified in Rpb3-TAP
Summary
Yeast Strains and Growth Conditions—All yeast strains are listed in supplemental Table S1. For stable isotope labeling in cells (SILAC) experiments, precultures of Ctr9-FLAG WT and cka1⌬ cka were grown in yeast nitrogen base 2% glucose without arginine or lysine overnight. FLAG tag affinity purifications were performed as described above with the following changes. The elutions were performed by incubation of the resin with 250 l of a 1 mg/ml solution of 3ϫ FLAG peptide resuspended in lysis buffer. Four-step MudPIT consisted of 8-l injections of 50, 100, 200, and 300 mM ammonium acetate, respectively, followed by a 20-min wash with buffer A (5% acetonitrile and 0.1% formic acid) and a 90-min organic gradient of 5– 80% buffer B (80% acetonitrile and 0.1% formic acid) to facilitate peptide elution from the reverse-phase resin. HRP-conjugated anti-rabbit or anti-mouse secondary (1:10,000) antibody was used, and the signal was detected using ECL Prime or ECL (Amersham Biosciences)
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