CK2 is specifically associated with initiation-competent Pol Iβ. A. Kinase activity copurifies with Pol Iβ. Fractions from the Mono S (MS) column for Pol Iα and for Pol Iβ (a final step in the purification of Pol I complexes from HeLa nuclei [34]) were assayed in a nonspecific transcription assay and the activities expressed as a percentage of maximal transcription activity for each form of Pol I. The same fractions were assayed in a reconstituted rDNA promoter-specific transcription assay with SL1 (arrowhead; transcripts). The fractions were assayed for kinase activity in autophosphorylation reactions (arrow; ³³P). B. The Pol Iβ-associated kinase phosphorylates a single predominant protein. The Mono S fractions containing the peak transcription activities of Pol Iα and β were pooled and analyzed in autophosphorylation reactions. Phosphorylated polypeptides were visualized by phosphorimaging. C. The Pol Iβ-associated kinase is CK2. CK2 inhibitors heparin (0, 1, 5, or 10 μg; lanes 1 to 4), CK2 peptide RRREEETEEE (0, 1, 5, or 10 ng; lanes 1 to 4), DBC (0, 5, 10, 15, 20, and 25 μM), and TBB (0, 5, 10, 15, 20, and 25 μM) were analyzed for their effect on Pol Iβ-associated kinase activity in autophosphorylation reactions. For DBC and TBB, the data were quantitated and expressed as percentages of the phosphorylation activity detected in the absence of the inhibitors (set at 100%). The bars represent the standard error. D. CK2 subunit β is detectable in Pol Iβ. Pol Iα (lane 1) and Pol Iβ (lane 2) immunoblots were probed with antibodies specific to Pol I subunit A190, PAF53, or AC19 or to CK2 subunit β. Nonspecific band marked by asterisk. E. Kinase activity coimmunoprecipitates with RRN3 in Pol Iβ. Pol Iβ (3 μl) was immunoprecipitated with affinity-purified rabbit polyclonal RRN3-specific antibodies. RRN3-immunoprecipitated Pol Iβ (RRN3-IP; lane 2), a control IgG immunoprecipitation (IgG-IP; lane 3), or 2 μl of Pol Iβ (Input; lane 1) was assayed in an autophosphorylation assay (³³P) and with Western blotting (WB). Phosphorylated protein was visualized as in panel A.

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 50 μM 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 10 μl of M280 “empty” beads, subsequently washed in TM10/0.05. The immunoblots are representative of two independent experiments.

CK2 phosphorylation of SL1 can inhibit specific transcription by preventing SL1 binding at the promoter. A. CK2 can inhibit specific Pol I transcription during formation of the SL1 and Pol I-containing preinitiation complex. Pol Iβ, SL1, and rDNA promoter template (Fr4) were incubated with CK2 (0, 100, or 500 U; lanes 1, 2, and 3, respectively) in the presence of ATP for 15 min at room temperature, and then transcription was initiated by addition of NTPs. The reactions were incubated for 30 min at 30°C and transcripts analyzed by S1 nuclease protection assay and autoradiography (arrowhead). B. CK2 has no detectable effect on nonspecific RNA synthesis. Pol Iβ was preincubated with 0, 50, or 500 U of CK2 and ATP for 15 min at room temperature and then added to a nonspecific transcription assay. Nonspecific transcription (txn) detected from CK2-treated Pol Iβ is expressed as a percentage of transcription detected in the absence of CK2. C. Schematic representation of experiments to determine the effect of phosphorylation of SL1 by CK2 on Pol I transcription (D) and on rDNA-promoter binding (E). The experiments were repeated twice (in duplicate), and a representative is shown. To test the effect of CK2 added “before” SL1 binding to the rDNA promoter, CK2 (100 U) was incubated with or without DBC (50 μM) for 10 min at room temperature. SL1 was added, and incubation continued for 15 min at room temperature in the presence of ATP. After incubation the reactions were divided in two. For transcription analysis, IT-rDNA and Pol Iβ were added and transcription was initiated upon addition of NTPs. The transcription reactions were incubated for 30 min at 30°C and specific transcripts detected by S1 nuclease protection (see panel D). For analysis of rDNA promoter binding by SL1, IT-rDNA was mixed into the reactions, left on ice for 15 min, and then washed with TM10/0.05. IT-rDNA-bound proteins were eluted in SDS-sample buffer and immunoblotted (see panel E). To test the effect of CK2 added “after” SL1 binding to the rDNA promoter, CK2 (100 U) was incubated with or without DBC (50 μM) for 10 min at room temperature, SL1 prebound (for 15 min on ice) to IT-rDNA was added, and incubation was continued for another 15 min at room temperature in the presence of ATP. The beads were washed in TM10/0.05 buffer and divided in two, for transcription and immunoblot analysis, as described above. D. CK2 enzymatic activity inhibits Pol I transcription when added before, but not after, SL1 binding to DNA. Using the procedures outlined for panel C, the effects of CK2 on promoter-specific Pol I transcription, when added before (lane 4) or after (lane 7) SL1 was bound to IT-rDNA, were analyzed. Control reactions contained no CK2 (lane 1), CK2 preincubated with CK2 inhibitor DBC (lanes 3 and 6), or DBC alone (lanes 2 and 5). E. CK2 enzymatic activity decreases the ability of SL1 to bind DNA but does not cause SL1 to dissociate from DNA. Using the procedures outlined for panel C, the effects of CK2 on SL1 binding to IT-rDNA, when added before (lane 4) or after (lane 8) SL1 was bound to the IT-rDNA, were analyzed and compared to SL1 binding without CK2 (lanes 1 and 5), with CK2 preincubated with CK2 inhibitor DBC (lanes 3 and 7), or with DBC alone (lanes 2 and 6). Antibodies specific for TAF_I110, TAF_I63, or TBP were used in immunodetection.