Mechanism of Transcriptional Repression of E2F by the Retinoblastoma Tumor Suppressor Protein  John F Ross, Xuan Liu, Brian David Dynlacht  Molecular Cell  Volume 3, Issue 2, Pages 195-205 (February 1999) DOI: 10.1016/S1097-2765(00)80310-X

Figure 1 E2F Activation and pRB Repression of Transcription Reconstituted In Vitro In vitro transcription reactions were carried as described in Experimental Procedures. Increasing amounts (4, 10, and 20 ng, respectively) of the E2F-4/DP-1 heterodimer were included in the reactions shown in lanes 2–4. Subsequent reactions that included E2F are indicated in the figure, and each contained 20 ng. E1A (250 ng per reaction) and pRB were included in the indicated reactions. The amount of pRB used is indicated above lanes 7–9, and all other reactions with pRB contained 200 ng. For each panel, basal transcription was arbitrarily set to 1 (lanes 1, 5, and 10), and transcription levels relative to basal conditions are shown. Transcription levels were quantitated using a phosphorimager. Molecular Cell 1999 3, 195-205DOI: (10.1016/S1097-2765(00)80310-X)

Figure 2 Minimal Requirements for E2F and pRB Activity (A) Silver-stained polyacrylamide gels of the purified factors used in the transcription reactions. The asterisk indicates BSA added to the protein preparations after purification. The lanes labeled “M” contain molecular weight markers, 100 ng per band. (B) In vitro transcription reactions were carried out using highly purified transcription factors shown in (A). Transcription was carried out with the omission of each of the basal factors as indicated. Lanes 15 and 16 contained the full complement of basal factors. E2F was included in the reactions indicated. (C) Both E2F activation and pRB repression are observed using the purified factors. E2F and pRB were included in the reactions as indicated. Molecular Cell 1999 3, 195-205DOI: (10.1016/S1097-2765(00)80310-X)

Figure 3 pRB Functions at the Early Stages of PIC Formation (A) In vitro transcription was carried out as described in Experimental Procedures. Each of the indicated transcription factors was allowed to assemble on the promoter in the presence of the USA fraction for 30 min at 30°C. E2F and pRB were included as indicated. After the initial incubation, pRB was added to the indicated reactions, together with each of the other remaining GTFs, and transcription was allowed to proceed. A diagram of the transcription protocol is shown. (B) Single-round transcription reactions. Preinitiation subcomplexes were formed in a manner similar to that described in (A), and transcription was carried out using the single-round procedure described in Experimental Procedures. A diagram of the transcription protocol is shown. The control lanes (13 and 14) included the unlabeled CTP for the first 2 min, followed the by the addition of the 32P-CTP prior to the 5 min “chase” period. (C) Native TFIID is required for E2F and pRB activity. The products of transcription reactions in which TBP replaced TFIID are shown. E2F and pRB were included in the reactions as indicated. In each panel, basal transcription for each set of reactions was arbitrarily set to 1 ([A], lanes 1, 5, 9, 13, and 17; [B], lanes 1, 5, and 9; [C], lane 1), and the levels of transcriptional activity in the corresponding reactions are displayed relative to basal transcription. The products were quantitated as described in Figure 1. Molecular Cell 1999 3, 195-205DOI: (10.1016/S1097-2765(00)80310-X)

Figure 5 pRB Disrupts the Extended Footprint Observed with the Combination of E2F, TFIIA, and TFIID In vitro DNase I footprint reactions were carried out as described in Experimental Procedures. (A) Increasing amounts of pRB (4, 20, and 100 ng) were included in footprint reactions containing E2F alone (10 ng), or in combination with E2F, TFIIA (50 ng), and TFIID (5 transcription units) or TBP (20 ng), as indicated in the figure. (B) E1A reverses the effect of pRB. GST (1.0 μg) or the GST-E1A fusion protein (1.0 μg) were included in the footprint reactions as indicated. The other factors (the same amounts as indicated in [A] and 100 ng of pRB) were included in the footprint reactions as indicated. (C) A preassembled complex containing E2F, TFIIA, and TFIID is resistant to pRB. E2F, TFIIA, and TFIID were incubated with the probe for 30 min at 30°C either in the presence (lane 3) or absence (lanes 2 and 4) of pRB. pRB was then added to the reaction in lane 4, and all reactions were incubated for an additional 30 min prior to carrying out DNase I digestion. The amounts of each protein used were the same as indicated in (B). The relevant promoter elements (shaded boxes) and hypersensitive sites (arrows) are indicated to the left of each panel. Reactions that contained no protein are indicated above the figure by a minus sign. Molecular Cell 1999 3, 195-205DOI: (10.1016/S1097-2765(00)80310-X)

Figure 4 E2F, TFIIA, and TFIID Cooperate to Form a Stable TFIID Footprint on the Promoter In vitro DNase I footprint reactions were carried out as described in Experimental Procedures. (A) Approximately 20 ng of E2F with or without unlabeled competitor oligonucleotides (200 ng) containing a wild-type or mutant E2F-binding site was included in the reactions as indicated. The position of the E2F-binding sites is indicated by a shaded rectangle to the left of the figure. Approximately 10 ng of TBP was included in the reactions represented by lanes 5–10. Unlabeled double-stranded competitor oligonucleotide containing a wild-type or mutated TATA box was included in the reactions as indicated. The amount, in nanograms, of oligonucleotide used is indicated. The position of the TATA box is indicated by a shaded rectangle on the right. Reactions that contained no protein, lanes 1 and 11, are indicated by a minus sign. (B) TFIIA (40, 80, and 200 ng), TFIID (2, 4, and 8 transcription units), and E2F (10, 20, and 40 ng) were titrated individually into the footprint reactions while holding the other two constant, each at its highest amount, as indicated in the figure. The relevant promoter elements, indicated to the left of the panel, were determined by comparisons with Maxam-Gilbert sequencing ladders loaded in parallel. The nucleotide corresponding to +10, relative to the transcription start site, is indicated. Large arrows denote hypersensitive sites. Molecular Cell 1999 3, 195-205DOI: (10.1016/S1097-2765(00)80310-X)

Figure 6 A Model for How pRB Could Repress Transcription (A) E2F, TFIIA, and TFIID cooperate to form a complex and activate transcription. This complex is resistant to pRB when pRB is introduced subsequent to the assembly of the partial preinitiation complex. (B) When pRB is present during the assembly process, the extended TFIID footprint that results from the cooperativity of E2F with TFIIA and TFIID is no longer observed, and transcription is repressed. This could occur through conformational differences within the TFIIA/IID complex, or could possibly be due to the exclusion of TFIIA. That pRB may function through direct interactions with components of TFIID is possible but is as yet unknown. Molecular Cell 1999 3, 195-205DOI: (10.1016/S1097-2765(00)80310-X)