Pcf11 Is a Termination Factor in Drosophila that Dismantles the Elongation Complex by Bridging the CTD of RNA Polymerase II to the Nascent Transcript  Zhiqiang Zhang, David S. Gilmour  Molecular Cell  Volume 21, Issue 1, Pages 65-74 (January 2006) DOI: 10.1016/j.molcel.2005.11.002 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Immunofluorescence Analysis of dPcf11 on Polytene Chromosomes (A and B) Polytene chromosomes from nonheat-shocked larvae stained with antibody against Pol II and dPcf11, respectively. Loci were mapped based on the DNA staining pattern (Figure S1A). Pol II was detected with the ARNA-3 antibody, which binds the largest subunit outside the CTD (Kramer et al., 1980). (C and D) Polytene chromosomes from heat-shocked larvae stained with antibody against Pol II and dPcf11. (E) Western blot analysis of whole-cell lysates from salivary glands and S2 cells with antibody raised against dPcf11. The band at ∼62 kDa is close to the predicted mobility of Pcf11. (F) High magnification view of the 87A locus in a stretched polytene chromosome from a heat-shocked larva. The schematic shows the two divergently transcribed copies of hsp70 that generate the heat-shock puff at 87A. The bottom two panels show individual staining for Pol II and dPcf11. Color images of (A)–(D) and (F) are provided in Figure S1. Molecular Cell 2006 21, 65-74DOI: (10.1016/j.molcel.2005.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Analysis of dPcf11 Function In Vivo Using ChIP and RNAi (A) Western blot analysis for dPcf11 and a control protein, CBP80, in LacZ RNA-treated (lane 1), dPcf11 RNAi-treated (lane 2), and untreated (lane 3) Drosophila cells. CBP80 is the largest subunit of the cap binding protein (Izaurralde et al., 1994) and serves as a loading control. (B) One data set for a ChIP analysis of dPcf11 and Pol II on the hsp70 gene in heat-shocked cells. The bands correspond to radiolabeled DNA PCR amplified from immunoprecipitates of dPcf11 (lanes 5–12 and 17–20) and Pol II (lanes 25–32 and 37–40) from crosslinked cells previously treated with lacZ RNAi (lanes 5–8 and 25–28), dPcf11 RNAi (lanes 9–12 and 29–32), or no RNAi (lanes 17–20 and 37–40). Samples in lanes 13–16 and 33–36 provide a measure of background and represent material immunoprecipitated from untreated cells with dPcf11 preimmune antiserum. Samples in lanes 1–4 and 21–24 represent 2% of the material subjected to immunoprecipitation (input). The band marked with an asterisk is from a region of the genome lacking any known transcription units. The bands delimited by the two vertical bars are PCR products from hsp70 spanning +10 to +181, +1201 to +1364, +2003 to +2166, and +2433 to +2601: each region corresponds respectively to the first through the fourth lane in each group of four lanes. (C and D) Quantification of ChIP data from two independent experiments for hsp70. The polyadenylation signal for hsp70 is at +2326. The error bars indicate the range of values for the two experiments. Molecular Cell 2006 21, 65-74DOI: (10.1016/j.molcel.2005.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 dPcf11 Dismantles Pol IIA, but Not Pol IIB ECs (A) Depiction of the EC used in these studies. (B) Native gel analysis of Pol II ECs (IIA EC and IIB EC). ECs were formed and then treated with buffer (lane 1), various derivatives of dPcf11 (lanes 2–5), or the CTD antibody (lane 6). Finally, the integrity of the ECs were evaluated by native gel electrophoresis. The numbers associated with each derivative of dPcf11 identify the amino acid region constituting the protein. dPcf11 1–574 is the full-length protein. (C) Native gel analysis of Pol II ECs treated with dCstF50 (lane 2) or dPcf11 1–283 (lane 3). (D) Transcripts released from immobilized Pol II ECs after incubation with derivatives of dPcf11. A mixture of Pol IIA and Pol IIB ECs (lanes 1–10) or Pol IIB ECs (lanes 11–16) alone were formed on immobilized DNA templates. Immobilized ECs were incubated for 15 min with various derivatives of dPcf11 and then separated into bound (“B”) and released (“R”) fractions. Radiolabeled transcripts were isolated and analyzed on a denaturing gel. (E) Measurement of Pol II released from immobilized Pol IIA/IIB ECs. Immobilized Pol IIA/IIB ECs were incubated with buffer, dCstF50, or dPcf11 1–238 for 15 min and then separated into B and R fractions. The largest subunit of Pol II was detected in each fraction by Western blotting with the ARNA-3 antibody, which binds outside the CTD (Kramer et al., 1980). IIa is the largest subunit derived from Pol IIA, and IIb is the largest subunit derived from Pol IIB. Lanes 1 and 2 show Pol IIB and the mixture of Pol IIA and Pol IIB. Pol IIB was prepared as previously described (Zhang et al., 2004). Molecular Cell 2006 21, 65-74DOI: (10.1016/j.molcel.2005.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 dPcf11 and a CTD Antibody Can Bridge the CTD to RNA (A) GST-CTD pull-down assays to detect interaction between dPcf11 and the CTD. GST (lane 1) or GST-CTD (lanes 2–5) were immobilized on glutathione Sepharose and then incubated with various derivatives of dPcf11. Bound protein was eluted and analyzed by Western blotting with antibody against the histidine tag on dPcf11. (B) Coomassie blue-stained gel of the dPcf11 preparations used in (A). (C) UV crosslinking assay to detect protein-RNA interactions. One microgram of each of the proteins listed at the top of each lane was incubated with radiolabeled RNA, followed by UV irradiation. RNA was degraded with RNase, and radioactively tagged proteins were detected after SDS-PAGE. mPcf11 1–238 has a three amino acid mutation that inactivates its dismantling activity (data not shown). The weak signal in lane 3 that comigrates with GST-CTD was not reproducible. (D) Analysis of CTD-RNA bridging activity. Glutathione Sepharose was first incubated with buffer (lane 1), GST (lanes 2 and 9), GST-CTD (lanes 3, 5, 6, and 8), dPcf11 1–238 (lane 4), or CTD antibody (lane 7) for 30 min. After several washes, the beads were incubated with buffer (lanes 1–4, and lane 7), dPcf11 1–238 (lane 5), mPcf11 1–238 (lane 6), or CTD antibody (lane 8 and 9). Beads were again washed and then incubated with radiolabeled RNA. After this last incubation, beads were extensively washed to remove unbound RNA. Radiolabeled RNA was recovered from the beads and analyzed on a denaturing polyacrylamide gel. Lane 10 (Input) shows 50% of the total RNA added to each sample. (E) UV crosslinking analysis to detect CTD antibody-RNA interactions. Two dPcf11 derivatives or the CTD antibody were incubated with radiolabeled RNA and subjected to crosslinking analysis. Lanes 1–3 show the radioactively tagged proteins. Lanes 4–6 show Coomassie blue staining of proteins in the gel. Molecular Cell 2006 21, 65-74DOI: (10.1016/j.molcel.2005.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 dPcf11 Associates Preferentially with a Part of the CTD Containing the Only Two Heptad Motifs that Match the Consensus (A) The amino acid sequence of the CTD of Drosophila Pol II. The sequence is displayed to emphasize the heptads matching the consensus YSPTSPS. Asterisks demarcate the only two heptads that precisely match the consensus heptad. Each of the four copies of the sequence PSYSPTSP shown in bold matches the part of the CTD that contacts yPcf11 in a crystal (Meinhart and Cramer, 2004). The vertical lines demarcate regions of the CTD that have been fused to GST. (B) GST-CTD pull-down assays to detect interaction between dPcf11 and subregions of the CTD. Derivatives of the CTD fused to GST (lanes 1–4) or GST alone (lane 5) were immobilized on glutathione Sepharose. CTD1 spans the entire CTD, whereas CTD2, CTD3, and CTD4 are demarcated in (A). Each immobilized GST derivative was incubated with dPcf11 1–238. Bound Pcf11 was eluted and analyzed by Western blotting with antibody against the histidine tag on dPcf11 1–238 (top). The bottom panel shows the Coomassie-stained GST derivatives that had been immobilized and subsequently run on SDS-PAGE. Molecular Cell 2006 21, 65-74DOI: (10.1016/j.molcel.2005.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 dPcf11 Inhibits Transcription by Pol IIA at Low, but Not High, Nucleotide Concentrations Pol II was incubated with the tailed template in the presence or absence of dPcf11 derivatives for 5 min prior to the addition of ATP, CTP, and radioactive UTP. Transcription was allowed to proceed for 20 min, and the resulting transcripts were analyzed on denaturing gels. (A) Transcripts produced by the Pol IIA/IIB mixture or Pol IIB at high and low nucleotide concentrations. High nucleotide conditions: 500 μM ATP, 500 μM CTP, and 25 μM α-32P UTP. Low nucleotide conditions: 25 μM ATP, 25 μM CTP, and 10 μM α-32P UTP. (B) Quantification of transcript levels. The graph shows the amount of transcript formed in the presence of each dPcf11 derivative divided by the amount of transcript produced in the absence of dPcf11. Error bars represent standard deviations from the mean for three independent experiments. Molecular Cell 2006 21, 65-74DOI: (10.1016/j.molcel.2005.11.002) Copyright © 2006 Elsevier Inc. Terms and Conditions