Involvement of Transcription Termination Factor 2 in Mitotic Repression of Transcription Elongation  Yan Jiang, Mingyi Liu, Charlotte A. Spencer, David H. Price  Molecular Cell  Volume 14, Issue 3, Pages 375-386 (May 2004) DOI: 10.1016/S1097-2765(04)00234-5

Figure 1 Expression of TTF2 Is Cell Cycle Regulated (A) HeLa S3 cells were immunostained with affinity-purified TTF2 antibody (red), and DNA was visualized by DAPI staining (blue) during (a) interphase, (b) early prophase, (c) late prophase, (d) metaphase, (e) anaphase, and (f) telophase. (B) Immunoblot of HeLa nuclear extract (HNE), whole-cell extract from cycling cells, or cells in G1, S, G2, or M and increasing amounts of recombinant human TTF2 (rTTF2) using anti-TTF2 antibodies. The highest level of rTTF2 is 8 ng, and each lane contains twice as much rTTF2 as the one on its left. Two exposures of the blot are shown. The loading control shows nonspecific detection of blotted proteins. (C) Northern blot of total RNA from synchronized (or cycling) HeLa S3 cells using TTF2 coding region as probe. TTF2 mRNA was approximately 3.5–4 kb. Molecular Cell 2004 14, 375-386DOI: (10.1016/S1097-2765(04)00234-5)

Figure 2 TTF2 Releases Both Pol II and Nascent Transcripts Independent of the Phosphorylation State of the Polymerase (A) Isolated EECs (on an immobilized template) were phosphorylated with P-TEFb and γ 32 P-ATP as described in the Experimental Procedures and then reisolated. The phosphorylated EECs (EEC-P) were then incubated with the indicated amounts of TTF2 and ATP for 5 min. The reactions were separated into bead (B) and supernatant (S) fractions, and proteins were analyzed by SDS-PAGE (9% gel) and autoradiography. (B) EECs were phosphorylated with P-TEFb (EEC-P) as described in the text and Experimental Procedures or left in a hypophosphorylated state (EEC) and then subjected to standard termination assays. Transcripts in the bead (B) and supernatant (S) fractions were analyzed on a 15% denaturing RNA gel. For (A) and (B) 1× TTF2 is ∼5 ng recombinant TTF2. Molecular Cell 2004 14, 375-386DOI: (10.1016/S1097-2765(04)00234-5)

Figure 3 TTF2 Is the Only Termination Factor Associated with Early Elongation Complexes (A) Depletion of TTF2 from HeLa nuclear extract (HNE) and immunoblot were carried out as described in the Experimental Procedures section. From left to right, 0, 1 μl undepleted HNE; 1, 2 μl HNE after first depletion; 2, 1.45 μl HNE after second depletion; 3, 1.85 μl HNE after third depletion. The amount loaded was normalized to total protein as determined by a Bio-Rad assay. 1, 2, 4, 8, 16, and 32 ng recombinant TTF2 (rTTF2) was loaded as standard. (B) Low salt-washed early elongation complexes (EECs) were prepared using either HNE or TTF2-depleted HNE (ΔHNE). EECs were incubated with buffer, TTF2, and/or ATP for 5 min. Fractions (Fxn) containing the total nascent transcripts in EECs (T) or supernatant fractions containing only released transcripts (S) are shown. 1× TTF2 is 12 ng (the amount of native TTF2 in 1 μl HNE). 3× TTF2 for lanes 4–6 and 13–15 is a total of 36 ng. Molecular Cell 2004 14, 375-386DOI: (10.1016/S1097-2765(04)00234-5)

Figure 4 TTF2 Terminates Productive Elongation Complexes (A) PECs and EECs were produced as described in the Experimental Procedures. DRB addition to the pulse (P) or the chase (C) is indicated. Reactions were stopped immediately after the 2 min pulse (lanes 1 and 4) or after a 3 min chase (lanes 2, 3, and 5). Transcripts were analyzed in a 6% denaturing RNA gel. A specific DRB-sensitive 40 nt transcript is labeled with an asterisk. tRNAs labeled in the nuclear extract by CCA addition are indicated. (B) Termination assay as in Figure 3 except that PECs or EECs were washed with high salt and Sarkosyl before being incubated with ATP or ATP and TTF2. (C) Termination assay as in (B). The left six lanes were the same as those in (B). The TTF2-resistant PECs in the lane marked B* were washed, chased for 1.5 min, reisolated (chased B*), and then subjected to the termination assay. In (B) and (C) transcripts in the bead (B) and supernatant (S) fractions were analyzed on a 15% denaturing RNA gel. Molecular Cell 2004 14, 375-386DOI: (10.1016/S1097-2765(04)00234-5)

Figure 5 Increasing Levels of TTF2 Causes Termination at Most Positions and Eliminates Run-Off Transcripts (A) EECs were produced and chased for the indicated amounts of time and washed with high salt and Sarkosyl. Standard termination assays were performed with the first of each set of three lanes being the total (T) transcripts produced and the second and third lanes being the transcripts released into the supernatant (S) after addition of ATP or ATP and TTF2 as indicated. The asterisk indicates the transcript identified in Figure 4 that was not efficiently terminated. (B) Standard pulse-chase assays using either HeLa nuclear extract (HNE) or TTF2-depleted HNE (ΔHNE). TTF2 was added to the extracts during the preincubation. Pairs of reactions were either pulsed or pulsed and then chased for 10 min. Six hundred and sixty nucleotide run-off transcripts, RO. A longer exposure of the indicated region of the gel is also shown. 1× TTF2 is 12 ng. Molecular Cell 2004 14, 375-386DOI: (10.1016/S1097-2765(04)00234-5)

Figure 6 Treatment with TTF2-siRNA Reduces TTF2 Protein (A) Immunoblot of whole-cell extract from HeLa cells transfected with negative control siRNA or TTF2-siRNA and harvested at the indicated times after transfection. The blot was bisected at about 50 kDa, and the upper portion was probed with anti-TTF2 antiserum and the bottom portion with anti-actin monoclonal antibody. (B) Relative amounts of TTF2 protein. The signals from the immunoblot shown in (A) and another identical blot were quantitated, and the TTF2 signal was normalized to actin as described in the Experimental Procedures. Molecular Cell 2004 14, 375-386DOI: (10.1016/S1097-2765(04)00234-5)

Figure 7 Immunofluorescence Analysis of TTF2-siRNA-Treated HeLa Cells (A) Effect of TTF2-siRNA on Pol II localization during mitosis. HeLa cells were treated with negative siRNA or TTF2-siRNA for 48 hr before being stained with DAPI for DNA (blue), affinity-purified anti-TTF2 antibodies for TTF2 (red), and H5 monoclonal antibody for Ser2 phosphorylated Pol II or ARNA3 antibody that recognizes non-CTD, large subunit sequences (green) as described in the Experimental Procedures. (B) Quantitation of the number of multinucleate cells. The percentage of cells with aberrant nuclear content is plotted at 0, 24, 48, and 72 hr after TTF2-siRNA or negative siRNA. (C) Chromosome segregation defects caused by TTF2-siRNA. Cells transfected with TTF2-siRNA were stained with α-tubulin (green) and DAPI (red). Molecular Cell 2004 14, 375-386DOI: (10.1016/S1097-2765(04)00234-5)