Volume 45, Issue 1, Pages 51-63 (January 2012) Transcriptional Regulation by Pol II(G) Involving Mediator and Competitive Interactions of Gdown1 and TFIIF with Pol II  Miki Jishage, Sohail Malik, Ulrich Wagner, Beatrix Uberheide, Yasushi Ishihama, Xiaopeng Hu, Brian T. Chait, Averell Gnatt, Bing Ren, Robert G. Roeder  Molecular Cell  Volume 45, Issue 1, Pages 51-63 (January 2012) DOI: 10.1016/j.molcel.2011.12.014 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Gdown1 Competes with TFIIF for Binding to Pol II In Vitro (A) p53-dependent and basal transcription with Pol II and Pol II(G). Transcription was assayed in a completely defined system containing all GTFs, PC4 and plasmids containing the adenovirus major late (AdML) core promoter and either p53-binding sites (GADD45) (An et al., 2004) or no activator binding sites (MLΔ53). p53 and Mediator were added as indicated. (B) Inhibitory effect of recombinant Gdown1 on basal transcription. In vitro transcription reactions were reconstituted with 10 ng of TBP, 10 ng of TFIIB, 25 ng of TFIIF, and 50 ng of Pol II. The indicated amounts of rGdown1 were added to reaction mixtures. Reaction mixtures also contained 50 ng of pMLΔ53. (C) Partial recovery of basal transcription by addition of excess TFIIF. In vitro transcription reaction mixtures were as described in (B). Reaction mixtures contained 5- or 10-fold excesses of rTFIIB or rTFIIF and 20 ng of rGdown1 as indicated. (D) Inhibition of PIC assembly by Gdown1. An end-labeled Ad ML oligonucleotide (−40 to +20) probe was incubated with the indicated combinations of factors: TFIIB (10 ng, lanes 2–5), TFIIF (50 ng, lanes 2–5), and bovine Pol II (50 ng). All reactions contained PC4 (65 ng) and TBP (10 ng). Reactions were incubated at 30°C for 40 min and purified RNA products resolved by native PAGE. (E) Pol II(G)-promoter complex formation in the absence of TFIIF. EMSA was performed as described in (D). Indicated combinations of factors were incubated with the probe. All the reactions contained TFIIB (10 ng), PC4 (65 ng), and TBP (10 ng). One microgram of a purified IgG (lane 6) or Gdown1 antibody (lane 7) was added to the reactions. A single asterisk denotes the upper band and two asterisks denote the middle band. See also Figure S1. Molecular Cell 2012 45, 51-63DOI: (10.1016/j.molcel.2011.12.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Analysis of Pol II Complexes (A) Purification of Gdown1 complex. Nuclear extracts from HeLa cells (lane 1) or from cell lines that stably express FLAG-tagged Gdown1 (lane 2) or FLAG-tagged RPB9 (lane 3) were directly purified on M2 agarose in buffer C containing 0.18 M KCl and 0.1% NP40 and bound proteins were eluted and analyzed by SDS-PAGE with silver staining. (B) Mass spectrometric analysis of the Gdown1 complex. Proteins identified by MALDI mass spectrometry are indicated. (C and D) Gel filtration (Superose 6) analyses of FLAG-Gdown1 (C) and FLAG-RPB9 (D) complexes. Fractions were analyzed by immunoblot. (E) Pol II(G) does not interact with TFIIF. Fractions 24 and 25 in (C) were immunoprecipitated with rabbit IgG or anti-Gdown1 antibodies. The antibody-bound or unbound fractions were analyzed by immunoblot with indicated antibodies. Molecular Cell 2012 45, 51-63DOI: (10.1016/j.molcel.2011.12.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Gdown1 Interacts with RPB5 and RPB1 (A) Far-western blot assay. Purified Pol II was resolved by SDS-PAGE and blotted to a membrane that then was incubated with GST (lane 1) or GST-Gdown1 (lane 2) protein as a probe. Membrane bound proteins were detected with HRP-labeled anti-GST antibody. The membrane that was probed with GST-Gdown1 was stripped and immunoblotted with an anti- RPB5 antibody (lane 3). (B) GST pull-down assay. Selected subunits of Pol II were expressed as 35S-labeled proteins in a rabbit reticulocyte coupled transcription and translation system and incubated with immobilized GST or GST-Gdown1. Bound proteins were analyzed by SDS-PAGE and autoradiography. (C) Crosslinking assay. Immobilized GST or GST-Gdown1 was incubated with purified Pol II. Proteins bound to the beads were incubated with/without DSP for the indicated times. After washing with 8 M urea buffer, the crosslinks were reversed and bound proteins were analyzed by immunoblot. (D) GST pull-down assay. Immobilized GST or GST-Gdown1 was incubated with 35S-labeled RPB5-jaw domain and bound proteins were analyzed by SDS-PAGE and autoradiography. (E) Dissociation of Gdown1 from Pol II by excess TFIIF. Pol II(G) was purified as described in the Experimental Procedures. A 5-, 10-, or 25-fold excess of rTFIIF was added to the Pol II(G) immobilized beads in buffer C containing 0.3 M KCl and 0.1% NP40. After washing the beads with buffer C containing 0.1 M KCl and 0.1% NP40, the proteins that remained on the beads were analyzed by immunoblot. Molecular Cell 2012 45, 51-63DOI: (10.1016/j.molcel.2011.12.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Pol II(G) Is Recruited to the Promoter in an Activator- and Mediator-Dependent Fashion (A) Comparison of the levels of Pol II(G) in HeLa or HeLa f:Gdown1 nuclear extracts. Pol II in HeLa (“C”) or HeLa f:Gdown1 (“Gdn”) nuclear extracts was immunoprecipitated with mouse IgG (lanes 5 and 6) or 8WG16 (lanes 3 and 4) antibodies. Immunoprecipitates were analyzed by immunoblot. (B) Schematic of the immobilized template used. (C) Pol II(G) is recruited to the promoter in an activator- and Mediator-dependent fashion. M280-streptavidin Dynabeads carrying a biotinylated DNA fragment were incubated with HeLa f:Gdown1 or HeLa nuclear extract with p53 (lanes 3 and 6) or without p53 (lanes 2 and 5). After 1.5 hr incubation, the beads were washed, and bound proteins were analyzed by immunoblot. (D) Mediator-dependent Pol II(G) recruitment to DNA template. Mediator was immunodepleted from HeLa f:Gdown1 nuclear extract as described in the Experimental Procedures. Factor recruitment was monitored as described in (C). See also Figure S2. Molecular Cell 2012 45, 51-63DOI: (10.1016/j.molcel.2011.12.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Pol II(G) Is Enriched at Promoter Regions of a Subset of Genes ChIP-seq data for Gdown1, Pol II, and IgG with antibodies that recognize Gdown1 (black), the RPB1 subunit (blue), and IgG (orange) are shown for four genes, plotted as relative read density versus base pair units. The start site and direction of transcription are shown by arrows, with boxes depicting exons and lines representing introns. FUS (A), RPL19 (B), PRKCI (D), and ANKRD13a (E) are shown. Averaged read densities of Pol II (blue) and Pol II(G) (black) with class II (C) or class I (F) genes identified by ChIP-seq are shown. See also Table S1. Molecular Cell 2012 45, 51-63DOI: (10.1016/j.molcel.2011.12.014) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Effects of Gdown1 Knockdown on Gene Expression (A) Gene expression of Pol II(G) target genes in Gdown1 knockdown cells. Error bars represent the standard deviation from five independent experiments. Inset: Gdown1 knockdown in IMR90 cells. Inset: Whole-cell lysates were prepared from control or knockdown cells and analyzed by immunoblot. (B–F) Doxorubicin effects on expression of the Pol II(G) target genes (B–D) or apoptotic genes (E and F). Error bars represent the standard deviation from three independent experiments. (G and H) ChIP-seq data for PIK3R3 (G) and GADD45A (H). (I) ChIP analysis on the promoter regions of PIK3R3 and GADD45A genes. ChIP analysis was performed with the indicated antibodies. Error bars represent the standard deviation from three independent experiments. See also Figures S3 and S4. Molecular Cell 2012 45, 51-63DOI: (10.1016/j.molcel.2011.12.014) Copyright © 2012 Elsevier Inc. Terms and Conditions