Volume 45, Issue 4, Pages 541-552 (February 2012) Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1  Jennifer A. Fairley, Louise E. Mitchell, Tracy Berg, Niall S. Kenneth, Conrad von Schubert, Herman H.W. Silljé, René H. Medema, Erich A. Nigg, Robert J. White  Molecular Cell  Volume 45, Issue 4, Pages 541-552 (February 2012) DOI: 10.1016/j.molcel.2011.11.030 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Endogenous Plk1 Associates with Pol III-Transcribed Genes (A) ChIP for endogenous Plk1, TAFI48, and TFIIA at tRNA, 5S and U1 genes in asynchronous HeLa cells. (B) Mean ratios ± standard deviations of ChIP signals (IP/input) for the indicated proteins at 5S genes in asynchronous and nocodozole-arrested mitotic HeLa cells. n = 3. (C) ChIP to compare relative levels of crosslinking to 5S genes of endogenous Plk1, TAFI48, and TFIIIC in cycling HeLa cells separated by elutriation into G1, S, and G2/M phase-enriched populations. Alongside representative examples of ChIPs are shown flow-cytometric analyses of the relative DNA content of each cell population; the y axis indicates relative cell number, and the x axis indicates relative DNA content. (D) Western blots comparing levels of the indicated proteins in the cell fractions used in Figure 1C. (E) Mean ChIP signals (IP/input) ± standard deviations as determined by qPCR of endogenous Plk1 and TAFI48 at 5S genes and a control gene desert locus in the HeLa cell populations used in Figures 1C and 1D. n = 3. See also Figure S1. Molecular Cell 2012 45, 541-552DOI: (10.1016/j.molcel.2011.11.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Plk1 Binds and Phosphorylates Brf1 (A) Mean ChIP signals (IP/input) ± standard deviations as determined by qPCR of endogenous Plk1 and TAFI48 in asynchronous HeLa cells at tRNA genes that are occupied by pol III transcription apparatus (active) or unoccupied (silent). n = 2. (B) Western blot of Brf1, Plk1, and actin in Brf1.CHO cells in which Brf1 is uninduced (U) or induced (I) for 48 hr. (C) ChIP showing crosslinking of TFIIA, Brf1, TFIIIC, and Plk1 to 5S genes in Brf1.CHO cells in which Brf1 is uninduced (U) or induced (I) for 48 hr. Two precent and 0.4% inputs are shown for both U and I. Quantitation of each signal, as determined by densitometry, is indicated. (D) Endogenous Brf1 coimmunoprecipitates with endogenous Plk1 from asynchronous HeLa cells. Western blot for Plk1 in 10% input and material immunoprecipitated with beads alone (mock), preimmune serum, antisera against two different regions of Brf1, and Plk1 antibody as positive control. (E) Pull-down assay with GST or GST-Plk1 and in vitro-translated Brf1 and TBP. (F) Recombinant Plk1 is pulled-down specifically by a GST fusion with residues 393-513 of human Brf1. The top panel shows a western blot for bound Plk1. The bottom panel shows Coomassie staining to reveal levels of GST proteins. Molecular Cell 2012 45, 541-552DOI: (10.1016/j.molcel.2011.11.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 S450 of Brf1 Is Phosphorylated by Plk1 and Influences Binding to TFIIIC (A) Western blot of Brf1 S450 phosphorylation, total Brf1, and actin in asynchronous HeLa cells treated for 2 hr with vehicle (–) or BI2536 Plk1 inhibitor (+). (B) Autoradiographs of peptide spots after incubation with γ-[32P]-ATP ± recombinant Plk1 (top three rows) or Cdk1 (bottom three rows). Rows 1 and 4 use peptides corresponding to Brf1 residues 444–455 (left lane) or an S450A substitution mutant (right lane). Rows 2 and 5 use peptides corresponding to Brf1 residues 264–275 (left lane) or a T270A substitution mutant (right lane). Row 3 has peptides corresponding to residues 807–818 of the established Plk1 substrate pericentrin (left lane) or an S813A substitution mutant (right lane). Row 6 has peptides corresponding to residues 1037–1048 of the established Cdk1 substrate BubR1 (left lane) or an S1043A substitution mutant (right lane). The wild-type peptide sequences are indicated alongside, beneath a minimal consensus sequence for Plk1 phosphoacceptors, where X indicates any residue and ϕ indicates a hydrophobic residue. A more detailed consensus is L(ϕ)-E/N/D(Q)-X-S/T-L(ϕ). Kinases were omitted in the –Plk1 and –Cdk1 panels. (C) RT-PCR analysis of tRNA, 5S rRNA and TFIIB mRNA expression in asynchronous HeLa cells transfected with empty vector or vector encoding HA-Brf1 wild-type (WT), HA-Brf1.S450A, or HA-Brf1.S450D, as indicated. (D) Mean tRNA expression ± standard deviations normalized to TFIIB mRNA in the experiment shown in Figure 3C and two additional, independent experiments. (E) Western blots of total HA-Brf1 (top), HA-Brf1 coimmunoprecipitated with TFIIIC (middle), and total actin (bottom) from asynchronous HeLa cells transfected with empty vector or vector encoding HA-Brf1 wild-type (WT), S450A, or S450D, as indicated. (F) ChIP showing crosslinking of HA-Brf1 and TFIIA (control) to tRNA and 5S genes in asynchronous HeLa cells transfected with vector encoding WT or S450A HA-Brf1. Quantitation of 5S signals, as determined by densitometry, is indicated. See also Figure S2. Molecular Cell 2012 45, 541-552DOI: (10.1016/j.molcel.2011.11.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Plk1 Stimulates tRNA and 5S rRNA Expression in Interphase but Represses It During Mitosis (A) RT-PCR of tRNA, 5S rRNA and TFIIB mRNA expression in asynchronous HeLa cells transfected with empty vector or vector encoding Plk1. (B) RT-PCR of tRNA, 5S rRNA and TFIIB mRNA expression in asynchronous HeLa cells treated for 2 hr with vehicle (–) or BI2536 Plk1 inhibitor (+). (C) Transcription of a tRNA gene in 15 μg of asynchronous HeLa cell extract with addition of buffer (–), 62.5 ng (+), or 125ng (++) of wild-type (WT) or kinase-dead (KD) recombinant Plk1. (D) Transcription of a tRNA gene in 15 μg S or M phase HeLa cell extract with addition of buffer (–) or 125 ng of recombinant Plk1 (+). (E) Mean expression of 5S rRNA ± standard deviations after normalization to TFIIB mRNA in S or M phase-arrested HeLa cells treated for 2 hr with vehicle (–) or BI2536 Plk1 inhibitor (+). Value for S phase without inhibitor is designated 1.0. (F) RT-PCR of the indicated transcripts in S or M phase-arrested HeLa cells transfected with control or Plk1 siRNA. (G) Westerns of Plk1, S10-phosphorylated histone H3, T15-phosphorylated Cdk1 and actin in S or M phase HeLa cells transfected with control or Plk1 siRNA. See also Figure S3. Molecular Cell 2012 45, 541-552DOI: (10.1016/j.molcel.2011.11.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 T270 of Brf1 Is Phosphorylated When Plk1 Becomes Activated (A) Westerns of Brf1 phosphorylated at T270 and S450, total Plk1, Plk1 phosphorylated at T210, histone H3 phosphorylated at serine 10 and TFIIB loading control in HeLa cells fractionated by elutriation. Beneath are shown flow-cytometric profiles of the relative DNA content of each fraction; the y axis indicates relative cell number, and the x axis indicates relative DNA content. (B) Westerns of Brf1, TFIIB, and phosphorylated T270 of Brf1 in HeLa cells arrested in S or M phase and treated for 2 hr with vehicle (–) or BI2536 Plk1 inhibitor (+). The band marked ∗ corresponds to phosphorylated Brf1. (C) Western of Plk1, actin, Brf1, and phosphorylated T270 of Brf1 in M phase-arrested HeLa cells transfected with control nontargeting siRNA or two siRNAs against distinct Plk1 sequences. (D) Western blots of proteins (top five panels) and RT-PCRs of RNAs (bottom two panels) showing expression of the indicated products in asynchronous U2OS cells harvested without induction (–) or after 8 hr induction of wild-type or T210D mutant Plk1, as indicated. Phosphospecific antibodies to S450 and T270 of Brf1 were used in the top two panels. (E) Flow-cytometric profiles of the relative DNA content of the cells used in Figure 5D; the y axis indicates relative cell number, and the x axis indicates relative DNA content. See also Figure S4. Molecular Cell 2012 45, 541-552DOI: (10.1016/j.molcel.2011.11.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 T270 of Brf1 Mediates Repression by Plk1 of Pol III Binding and Transcription (A) RT-PCR of tRNA, 5S rRNA and TFIIB mRNA and westerns of HA-Brf1 and actin in asynchronous HeLa cells transfected with empty vector or vector encoding HA-Brf1 wild-type, T270A, or T270D mutants. (B) Relative mean tRNA expression ± standard deviations after normalization to TFIIB mRNA in Figure 6A and two additional independent experiments. The effect of the T270D substitution is highly significant (p = 0.0003). (C) Western of total HA-Brf1 (middle), actin (bottom), and HA-Brf1 coimmunoprecipitated with pol III (top) from asynchronous HeLa cells transfected with empty vector or vector encoding HA-Brf1 wild-type (WT), T270A, or T270D, as indicated. (D) RT-PCR of tRNA, 5S rRNA, and ARPP P0 mRNA and western blots of HA-Brf1, Plk1, and actin in asynchronous U2OS cells transfected with empty vector or vector encoding wild-type or T270A HA-Brf1 and harvested without induction (–) or after 8 hr induction (+) of T210D mutant Plk1, as indicated. (E) Mean relative tRNA expression ± standard deviations normalized to ARPP P0 mRNA in the experiment shown in Figure 6D and two additional, independent experiments. (F) Model for cell cycle-dependent regulation of pol III transcription by Plk1. Plk1-mediated phosphorylation of Brf1 on S450 during interphase promotes interaction of Brf1 with TFIIIC and transcription of tRNA and 5S genes. In mitosis, T210-phosphorylated Plk1 instigates Brf1 phosphorylation on T270, inhibiting interaction with pol III and repressing transcription. See also Figure S5. Molecular Cell 2012 45, 541-552DOI: (10.1016/j.molcel.2011.11.030) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 T270A Mutant Brf1 Induces Chromosomal Abnormalities (A) Mean frequencies of micronuclei ± standard deviations in cells transfected with vectors expressing wild-type or T270A Brf1. (B) Characteristic appearance of a T270A-transfected cell with a micronucleus, indicated with arrow. Alongside is a transfected cell without a micronucleus. Cells are stained for the HA tag on transfected Brf1 (green) and DNA (blue). (C–E) Extreme examples of abnormalities seen in cells transfected with T270A Brf1. Molecular Cell 2012 45, 541-552DOI: (10.1016/j.molcel.2011.11.030) Copyright © 2012 Elsevier Inc. Terms and Conditions