CK2 Phosphorylation of Bdp1 Executes Cell Cycle-Specific RNA Polymerase III Transcription Repression  Ping Hu, Kalpana Samudre, Si Wu, Yuling Sun, Nouria Hernandez  Molecular Cell  Volume 16, Issue 1, Pages 81-92 (October 2004) DOI: 10.1016/j.molcel.2004.09.008

Figure 1 CK2 Phosphorylation of Bdp1 Inhibits U6 Transcription In Vitro (A) Bdp1, but not TBP or Brf2, is phosphorylated by CK2 in vitro. 5 μg of E. coli-expressed TBP and Brf2 and 8 μg of E. coli-expressed Bdp1 were incubated with 5 units of CK2α and [γ-32P]ATP, fractionated by two-dimensional gel electrophoresis, and visualized by autoradiography. The two upper panels are 5 day exposures, and the lowest panel is an 8 hr exposure. (B) CK2 phosphorylation of Bdp1 inhibits U6 transcription in vitro. SNAPc and the factors labeled “I” above the lanes (either TBP, Brf2, or Bdp1) were incubated with 60 μM of LY294002 followed by addition of 5 units of CK2α, 0.65 mM ATP, 1.25 mM phosphocreatine, and 0.25 ng/μl creatine phosphokinase. Pol III and the factors labeled “CK2” above the lanes were incubated first with the enzyme mix and then with LY294002. U6, correctly initiated RNA; IC, internal control for RNA recovery. (C) CK2α is associated with Bdp1. Immunoprecipitation from HeLa cell extracts with anti-Bdp1 (lanes 1 and 2) or preimmune (lane 3) antibodies. The membrane was probed with the anti-CK2α antibody 245 (Yu et al., 1991). Molecular Cell 2004 16, 81-92DOI: (10.1016/j.molcel.2004.09.008)

Figure 2 The Conserved Region Downstream of the SANT Domain Is Required for CK2 Phosphorylation (A) Bdp1-truncated proteins. Black boxes, SANT domain; hatched boxes, other conserved regions; black arrows, repeats in the C-terminal part of H. sapiens (Hs) Bdp1; numbers, aa positions; percentages, aa identical in S. cerevisiae (Sc) and Hs Bdp1. (B) Expression of truncated forms of Bdp1 in E. coli. The proteins were purified by consecutive nickel and anti-Flag affinity chromatography, fractionated by SDS-PAGE, and stained with Coomassie blue. (C) Only truncated forms of Bdp1 containing the conserved region downstream of the SANT domain are efficiently phosphorylated by CK2. 1 μg of each truncated Bdp1 protein was incubated with CK2 holoenzyme and [γ-32P]ATP, fractionated by SDS-PAGE, and visualized by autoradiography. Asterisks, band obtained even when the CK2 holoenzyme is incubated alone (lane 1); arrowheads, CK2-phosphorylated Bdp1 proteins. Molecular Cell 2004 16, 81-92DOI: (10.1016/j.molcel.2004.09.008)

Figure 3 The Bdp1 Conserved Region Is Necessary and Sufficient for Pol III-Directed Transcription (A) The conserved Bdp1 region is necessary and sufficient for VAI transcription in a Bdp1-depleted extract. HeLa cell extracts were incubated with either preimmune (lanes 1 and 13) or anti-Bdp1 antibody (lanes 2–12 and 14–21) beads. The depleted extracts were then complemented with increasing amounts of Bdp1 (lanes 3–6), or 5 (lane 7), 10 (lane 8), 20 (lane 9), 50 (lanes 10 and 15), 100 (lanes 11 and 16), and 400 ng (lane 12) of Bdp11-355, or 50 (lane 17) and 100 ng (lane 18) of Bdp11-470, or 50 (lane 19), 100 (lane 20), and 400 ng (lane 21) of Bdp1167-470, and tested for VAI transcription. The brackets under lanes 17–20 indicate samples with comparable amounts of added Bdp1-truncated proteins, and the numbers below are a quantitation of the VAI signal in lanes 17, 18, 19, and 20 in arbitrary units. (B) The Bdp1 conserved region is necessary and sufficient for U6 transcription in a minimal transcription system. The pol III complex and SNAPc were treated as in Figure 1B, and TBP and Brf2 purified from E. coli were left untreated. Full-length Bdp1, Bdp11-355, Bdp11-470, and Bdp1167-470 were either treated first with LY294002 and then CK2 (lanes 1–6 and 12–16) or treated first with CK2 and then LY294002 (lanes 7–11 and 17–21, labeled “CK2” above the lanes). The amounts of the Bdp1 truncated proteins used were 200 (lane 3) and 400 ng (lanes 4 and 9) of Bdp11-355; 100 (lanes 5, 10, 12, and 17) and 200 ng (lanes 6, 11, 13, and 18) of Bdp11-470; and 100 (lanes 14 and 19), 200 (lanes 15 and 20) and 400 ng (lanes 16 and 21) of Bdp1167-470. The brackets under lanes 12–15 are as in (A). Molecular Cell 2004 16, 81-92DOI: (10.1016/j.molcel.2004.09.008)

Figure 4 Mutation of the CK2 Phosphorylation Sites in Bdp11-470 Abolishes CK2 Transcription Regulation (A) Structure of Bdp1-470S/T→A. (B) Expression of Bdp1-470S/T→A in E. coli. Bdp1-470S/T→A was purified through consecutive nickel and anti-Flag affinity chromatography, fractionated by SDS-PAGE, and stained with Coomassie blue. (C) Bdp1-470S/T→A is not phosphorylated by CK2. 5 μg of Bdp11-470 or Bdp1-470S/T→A was incubated with CK2 holoenzyme and [γ-32P]ATP, fractionated by SDS-PAGE, and visualized by autoradiography. Asterisks and arrowheads highlight bands appearing upon incubation of the CK2 holoenzyme alone (lane 1). (D) Mutation of the CK2 phosphorylation sites in Bdp11-470 abolishes CK2 transcription regulation. Pol III, TBP, Brf2, and SNAPc were treated as in Figure 3B. Bdp11-470 or Bdp1-470S/T→A was either treated first with LY294002 and then CK2 (lanes 1, 2, 5, and 6) or treated first with CK2 and then LY294002 (lanes 3, 4, 7, and 8, labeled “CK2” above the lanes). The amounts of the Bdp1 proteins used were 100 (lanes 1 and 3) and 200 ng (lanes 2 and 4) of Bdp11-470; and 100 (lanes 5 and 7) and 200 ng (lanes 6 and 8) of Bdp1-470S/T→A. Molecular Cell 2004 16, 81-92DOI: (10.1016/j.molcel.2004.09.008)

Figure 5 Bdp1 Is Phosphorylated In Vivo during Mitosis, and Extracts from Mitotic Cells, but Not from Mitotic Cells Treated with a CK2 Inhibitor, Are Inactive for Pol III Transcription (A) Bdp1 is phosphorylated in vivo during mitosis. Synchronized HeLa cells were lysed under denaturing conditions in the presence of phosphatase inhibitors. The resulting extracts were directly analyzed by immunoblotting with anti-β-actin and anti-RPC6 antibodies (two lower panels) and used for denaturing immunoprecipitations with an anti-Bdp1 antibody. The immunoprecipitates were analyzed by immmunoblot with an anti-phospho-S/T and an anti-Bdp1 antibody (upper two panels). (B) Bdp1 purified from HeLa cells can be phosphorylated by CK2. Bdp1 was purified from HeLa cell extracts by nickel affinity chromatography and phosphorylated by CK2 in vitro. The in vitro-phosphorylated Bdp1 was then bound to anti-Bdp1 beads and either treated (lane 2) or not treated (lane 1) with calf intestine phosphatase followed by elution in Laemmli buffer. The eluted materials as well as Bdp1 immunoprecipitated as in (A) from mitotic cells (lane 3) or from mitotic cells treated with LY294002 (lane 4) were immunoblotted with an anti-phospho-S/T antibody. (C) Pol III transcription in extracts from synchronized cells. Extracts were prepared from synchronized cells, normalized for protein concentration, and tested for transcription. In lane 4, the cells were treated with LY294002. Molecular Cell 2004 16, 81-92DOI: (10.1016/j.molcel.2004.09.008)

Figure 6 Inhibition of CK2 and Addition of Bdp1 Rescue Transcription in Mitotic Extracts, Whereas Inhibition of CK2 Inhibits Transcription in S Phase Extracts (A) Mitotic extracts are activated by inhibition of CK2 and addition of Bdp1. S phase (lane 1) and mitotic (lanes 2–10) extracts were used. 100 (lanes 3, 5, and 9) and 400 ng (lanes 4, 6, and 10) of Bdp11-470 alone (lanes 3 and 4), or with 150 μM PKA peptide (LRRASLG) (lanes 5 and 6), or with 180 μM CK2 peptide (RRREEETEEE) (lanes 9 and 10) were added to the mitotic cell extract. In lanes 7 and 8, 180 and 360 (VAI panel) or 60 and 180 μM (U6 panel) CK2 peptide were added, respectively. (B) Bdp1 specifically activates pol III transcription in mitotic cell extracts where CK2 is inhibited. A mitotic extract was complemented either with just buffer (lane 1), or 60 μM CK2 peptide (lanes 2–12) and either 100 (lane 3) and 400 ng (lane 4) of Bdp11-470 or 30 (lane 5) and 60 ng (lane 6) of TBP, or 80 (lane 7) and 160 ng (lane 8) of Brf1 for VAI (upper panel), or 50 (lane 7) and 100 ng (lane 8) of Brf2 for U6 (lower panel), or 500 (lane 9) and 800 ng (lane 10) of pol III, or 400 (lane 11) and 800 ng (lane 12) of SNAPc for U6 (lower panel). (C) Inhibition of CK2 has opposite effects in extracts from M and S phase cells. An S phase cell extract was complemented either with just buffer (lane 1), 60 (lane 2) and 180 μM (lane 3) of CK2 substrate peptide, or 60 (lane 4) and 150 μM (lane 5) of PKA substrate peptide. Molecular Cell 2004 16, 81-92DOI: (10.1016/j.molcel.2004.09.008)

Figure 7 Bdp1 Dissociates from DNA during Mitosis (A) Bdp1 dissociates from the U6 promoter during mitosis. The y axis indicates the ratios of the signals obtained in material immunoprecipitated from either asynchronous cells, mitotic cells, or mitotic cells treated with LY294002 to the signals obtained with mitotic cells. The error bars are based on six independent qPCR reactions with the same template and primers. (B) Fractionation scheme to obtain cytoplasmic, nuclear soluble, and chromatin fractions. (C) Bdp1 dissociates from chromatin at mitosis as visualized by biochemical fractionation. The S2 (cytoplasmic), S3 (nuclear soluble), and P3 (chromatin bound) fractions from either asynchronous cells (lanes 1–3), mitotic cells (lanes 4–6), or mitotic cells treated with 20 μM LY294002 (lanes 7–9) were immunoblotted with anti-Bdp1, Brf2, SNAP45, TBP, MEK2, and Orc2p antibodies, as indicated. Molecular Cell 2004 16, 81-92DOI: (10.1016/j.molcel.2004.09.008)

Figure 8 Bdp1 Dissociates from Chromatin at Mitosis as Visualized by Immunofluorescence (A) HeLa cells incubated with or without the CK2 inhibitors LY294002 (20 μM) and TBB (50 μM in the experiment shown; similar results were obtained with 25 μM) were stained either with DAPI or immunostained with an anti-α-tubulin antibody (Sigma), an anti-Bdp1 antibody (CS842), or an anti-condensin antibody (Kimura et al., 2001), as indicated. The secondary antibodies were Alexa Fluor 488-conjugated goat anti-mouse IgG (anti-α-tubulin) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (anti-Bdp1 and anti-condensin) (Molecular Probes). Arrowheads, M phase cells; arrows, interphase cells. (B) The Bdp1 immunostaining is blocked by preincubation of the antibody with the peptide against which it was raised. HeLa cells were stained with DAPI or immunostained as in (A) with an anti-α-tubulin antibody or an anti-Bdp1 antibody either in the absence (−) or presence (+) of the peptide against which the anti-Bdp1 antibody was raised (HB″-3). (C) Quantitation of mitotic cells showing Bdp1 on and off the chromatin in the absence and presence of CK2 inhibitor. A total of 120 mitotic cells were counted in each case. Molecular Cell 2004 16, 81-92DOI: (10.1016/j.molcel.2004.09.008)