Casein Kinase 2 Associates with Initiation-Competent RNA Polymerase I and Has Multiple Roles in Ribosomal DNA Transcription

ChIP analysis indicates that CK2α is present at the rDNA promoter and to some extent throughout the rDNA repeat in cells. Chromatin immunoprecipitation (from HEK293 cells) with antibodies specific to CK2α (A), specific to TAF_I110 of SL1 (B), or specific to the A135 subunit of Pol I (C) or the corresponding sheep or rabbit IgG controls, followed by quantitative real-time PCR with primers specific for the promoter region (P1 and P2), transcribed regions Tr1 (18S gene), or Tr2 (28S gene) and the intergenic spacer (IGS). The data, expressed as percentages of input chromatin, are from two independent experiments.

TopoIIα, rather than the largest Pol I subunit, A190, is the substrate for Pol Iβ-associated CK2 in Pol Iβ. A. Pol Iβ (lanes 1, 4, and 7), Pol Iα (lanes 2, 5, and 9), and TopoIIα (lanes 3, 6, and 8) were incubated in the presence of [γ-³³P]ATP for 15 min at 30°C. Pol Iα and TopoIIα reactions were supplemented with 25 U of recombinant CK2. Proteins were separated by Tris-acetate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Invitrogen) and immunoblotted using A190-specific antibodies. After immunodetection (ECL panel), phosphorylated proteins were detected by autoradiography under conditions where a residual ECL signal from immunodetection of A190 (asterisk) was detectable (ECL+³³P panel) or was undetectable (³³P panel). B. TopoIIα is present in Pol Iβ and not in Pol Iα. Pol Iα (lane 1) and Pol Iβ (lane 2) complexes were immunoblotted with antibodies specific for the largest (A190), the second-largest (A135), or the PAF53 core Pol I subunit or with TopoIIα- or RRN3-specific antibody. C. Pol Iβ-associated kinase phosphorylates the same substrate as exogenous CK2 in Pol Iβ, whereas Pol Iα contains neither CK2 enzyme activity nor a substrate for CK2. Pol Iα was incubated, in the absence (lanes 1 and 3) or presence (lane 2) of CK2 and in the absence (lanes 1 and 2) or presence of TopoIIα (lane 3) with [γ-³³P]ATP for 30 min at 30°C. Pol Iβ was incubated, in the absence (lane 4) or presence (lane 5) of CK2 with [γ-³³P]ATP for 30 min at 30°C. De novo phosphorylated proteins were visualized by phosphorimaging.

CK2-specific inhibitor DBC inhibits multiple rounds of specific Pol I transcription but has no effect on elongation following random initiation events. A. CK2 phospho-acceptor peptide inhibits specific Pol I transcription. A 2.5-μl sample of highly purified Pol Iβ, in a 10-μl reaction mixture, was incubated with 0, 5, 10, or 50 ng of CK2 phospho-acceptor peptide (RRREEETEEE; New England Biolabs) for 15 min on ice. Two hundred nanograms of template DNA (prHu3) and 1 μl of highly purified SL1 were added to each reaction mixture. Transcription was initiated with the addition of NTPs. Transcript synthesis was analyzed in an S1 nuclease protection assay. The autoradiograph shows the transcript levels. B. CK2 phospho-acceptor peptide inhibits nonspecific transcription by Pol I, independent of its effect on CK2. A 2.5-μl sample of highly purified Pol Iα (which does not contain CK2) in a 10-μl reaction mixture was incubated with 0, 5, 10, or 50 ng of CK2 phospho-acceptor peptide (black) or control peptide (gray) for 15 min on ice. Nonspecific transcription was initiated by the addition of a transcription mixture containing [α-³²P]CTP, NTPs, and calf thymus DNA. Radioactivity incorporated in the acid-insoluble fraction was Cerenkov counted and expressed as a percentage of that without peptide, which was set at 100%. Experimental errors are indicated. C. DBC has no effect on nonspecific Pol I transcription in nuclear extract. HeLa nuclear extract was incubated with DMSO alone or 100 μM DBC (in DMSO) for 15 min at room temperature. Nonspecific transcription reactions were initiated and analyzed over time as in panel A, and synthesis was expressed in cpm and plotted against time (for two independent experiments). D. DBC inhibits multiple rounds of specific Pol I transcription. HeLa nuclear extract was incubated with immobilized rDNA promoter template (Fr4) (39) for 15 min on ice. The templates were washed in TM10/0.05, and then 0 or 100 μM DBC was added to the preformed PICs on these promoter templates. Incubation was continued for another 15 min at room temperature. Transcription was initiated with NTPs, and at each time point, transcription was quantitated by phosphorimaging, expressed in arbitrary units (AU) and plotted against time (for two independent experiments).

Pol Iβ-associated kinase phosphorylates UBF and SL1 subunit TAF_I110. A. Pol Iβ-associated kinase phosphorylates UBF. Pol Iβ (lanes 1 to 5) or recombinant CK2 (lane 6) was incubated with (lanes 1, 4, 5, and 6) or without (lanes 2 and 3) recombinant UBF for 15 min on ice. rDNA promoter-containing fragment (Fr4) was also present in the reactions of lanes 3 and 5. Incubation was then continued with [γ-³³P]ATP for 30 min at 30°C. Proteins were immunoblotted and probed with antibodies specific for TopoIIα, UBF, or Pol I subunit A127 or PAF53 (lane 1, W), and in parallel de novo phosphorylated proteins were detected by autoradiography (lanes 2 to 6; ³³P). B. Pol Iβ-associated kinase phosphorylates TAF_I110 in SL1. Pol Iβ was incubated with TBP-antibody (monoclonal 3G3, a kind gift from L. Tora) immunoaffinity-purified SL1 in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of rDNA promoter template for 15 min on ice. Incubation was then continued with [γ-³³P]ATP for 30 min at 30°C. Proteins were immunoblotted and probed with antibodies specific for TAF_I110, TAF_I63, or TBP (lanes 1 and 2; W), and de novo phosphorylated proteins were detected by autoradiography (lanes 3 and 4; ³³P).

CK2 phosphorylation activates UBF, increasing UBF-dependent activated but not basal transcription by Pol I, and stabilizes UBF at the rDNA promoter in an SL1-dependent manner. A. CK2 inhibitor DBC does not affect SL1- and Pol I-dependent basal transcription. Pol Iβ was preincubated in the absence of DBC (DMSO) or in the presence of 50μ M or 100 μM of DBC for 15 min at room temperature as outlined. rDNA promoter template (Fr4) and SL1 were added to each reaction, and incubation was continued for another 15 min on ice. Specific transcription (specific txn) was initiated at 30°C upon addition of NTPs, and samples were taken at the time points indicated. At each time point, transcription was quantitated by phosphorimaging and expressed as a percentage of the highest level of transcription, which was set at 100%. B. CK2 inhibitor DBC affects UBF-dependent activation of Pol I transcription. As in panel A, except that recombinant UBF was added to determine the effect of DBC on UBF-dependent activation of Pol I transcription (at the same time as SL1). C. Schematic representation of the purification of CK2-phosphorylated Flag-tagged UBF (CK2-P-UBF; see Materials and Methods). A 1.5-μl sample of either (Flag-)UBF (lane 1) or CK2-P-(Flag-)UBF (lane 2) and 2.5 μl (400 ng) of highly purified Flag-tagged UBF, which was the input for the phosphorylation reaction (lane 3), were resolved on a 4 to 20% bis-Tris Novex gel (Invitrogen). The gel was stained with Sypro-Ruby (Invitrogen). Lane 5 contained the “Mark 12” protein ladder (Invitrogen). D. CK2 phosphorylation of UBF increases UBF activity. Pol Iβ and SL1 were incubated with rDNA promoter template (Fr4) in the absence of UBF (basal transcription; lanes 1 and 4) or in the presence of (Flag-tagged) UBF (5 and 20 ng; lanes 2 and 3) or CK2-phosphorylated (Flag-tagged) UBF (5 and 20 ng; lanes 5 and 6) (see panel C). Incubation was for 15 min on ice, and transcription was initiated upon addition of NTPs. Transcript synthesis after 30 min was quantitated by phosphorimaging from two independent experiments (in duplicate). n-fold stimulation is indicated (2.1 ± 0.3 and 2.8 ± 0.4). E. CK2 phosphorylation of UBF reduces the rate of dissociation of UBF from an SL1-rDNA promoter fragment but not from the promoter fragment alone. Recombinant UBF (300 ng) was incubated with 100 U of recombinant CK2 and 0.5 mM ATP in the absence of DBC (in DMSO) or in the presence of 100 μM of DBC for 20 min at room temperature. One hundred fifty nanograms of CK2-phosphorylated UBF (CK2-P-UBF; lanes 1 to 6 and 14 to 19) or nonphosphorylated UBF (UBF; lanes 8 to 13 and 21 to 26) was incubated for 20 min at 0°C with 70 μl of IT-rDNA or IT-rDNA to which SL1 had been prebound for 20 min at 0°C (IT-rDNA + SL1; excess SL1 removed by TM10/0.05 wash). Templates were subsequently washed with TM10/0.05, and sheared ctDNA was added (at time zero; final concentration, 0.5 mg/ml). Equal aliquots were removed at 0, 5, 10, 20, 30, and 45 min, and the recovered templates were washed with TM10/0.05 to remove factors no longer associated with the IT-rDNA. Template-associated UBF was analyzed by immunoblotting following elution with 5 M urea. Lanes 7 and 20 represent controls in which 21 ng of CK2-P-UBF was incubated with 10μ l of M280 “empty” beads, subsequently washed in TM10/0.05. The immunoblots are representative of two independent experiments.