Mechanism of Transcriptional Repression of E2F by the Retinoblastoma Tumor Suppressor Protein

Molecular Cell, Vol. 3, 195–205, February, 1999, Copyright 1999 by Cell Press

Mechanism of Transcriptional Repression of E2F by the Retinoblastoma Tumor Suppressor Protein John F. Ross,* Xuan Liu,† and Brian David Dynlacht*‡ * Harvard University Department of Molecular and Cellular Biology Cambridge, Massachusetts 02138 † University of California Department of Biochemistry Riverside, California 92521

Summary The retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor, critical for normal cell cycle progression. We have undertaken studies using a highly purified reconstituted in vitro transcription system to demonstrate how pRB can repress transcriptional activation mediated by the E2F transcription factor. Remarkably, E2F activation became resistant to pRB-mediated repression after the establishment of a partial (TFIIA/TFIID) preinitiation complex (PIC). DNase I footprinting studies suggest that E2F recruits TFIID to the promoter in a step that also requires TFIIA and confirm that recruitment of the PIC by E2F is blocked by pRB. These studies suggest a detailed mechanism by which E2F activates and pRB represses transcription without the requirement of histone-modifying enzymes. Introduction The retinoblastoma protein (pRB) is a tumor suppressor involved in the transcriptional regulation of genes required for the G1-to-S phase transition of the cell cycle (reviewed in Dyson, 1998). pRB interacts with a multitude of proteins, and it is thought to execute its cell cycle regulatory function primarily through interactions with transcriptional activator proteins. pRB regulates the activity of its targets in both a negative and positive manner (reviewed in Sanchez and Dynlacht, 1996; Mulligan and Jacks, 1998). Mutations in the RB gene are associated with approximately 30% of all human tumors, highlighting its importance as a regulator of cell proliferation (reviewed in Weinberg, 1995). Because of the diversity of proteins with which it appears to interact, its variable activity, and its importance as a tumor suppressor protein, it is essential to understand how pRB carries out its functions at the molecular level. Most studies of pRB function have focused on its interactions with the E2F transcription factor. E2F designates a family of heterodimers composed of any one of six E2F subunits (E2Fs 1–6), in combination with either of two DP polypeptides, DP-1 or DP-2. With the exception of heterodimers containing E2F-6, E2F is a transcriptional activator thought to play a critical role in S phase entry (reviewed in Dyson, 1998). Exemplifying its role as a regulator of cell growth, at least one of the E2F ‡ To whom correspondence should be addressed (e-mail: dynlacht@ biosun.harvard.edu).

family members, E2F-1, appears to exhibit properties of both an oncogene and a tumor suppressor: E2F-1 knockout mice exhibit a broad range of tumors, prompting its classification as a tumor suppressor (Field et al., 1996; Yamasaki et al., 1996), yet overexpression of E2F-1 in transgenic mice promotes tumorigenesis (Pierce et al., 1998). In addition, in transformation assays E2F-1 is oncogenic (Singh et al., 1994; Xu et al., 1995). It is thought that E2F-1 may serve as a tumor suppressor at least in part by virtue of its ability to interact with pRB, which converts the activator into a repressor (Dyson, 1998; Yamasaki et al., 1998). Many E2F-responsive genes are activated in mid to late G1 phase of the cell cycle and play a crucial role in cell proliferation. Thus, several genes involved in DNA replication, such as DNA polymerase a, proliferating cell nuclear antigen (PCNA), Orc 1, Cdc6, and the Mcm proteins, are induced in the presence of exogenous E2F (DeGregori et al., 1995; Leone et al., 1998). E2F-binding sites are also found in the promoters of genes encoding several known cell cycle regulators, such as B-myb, cdc2, cyclin A, and cyclin E. The cyclins, together with their catalytic partners, the cyclin-dependent kinases (cdks), are crucial elements in the control of the cell cycle, and progression through the cell cycle is characterized by the activity of specific cyclin/cdk complexes (reviewed in Sherr, 1996). Upon binding pRB, E2F activation is abolished in early G1 in vivo and in vitro (Helin et al., 1993a; Hiebert, 1993; Dynlacht et al., 1994). Later in G1, the pRB–E2F interaction is abrogated by the action of kinases that phosphorylate pRB, effectively activating E2F (Dyson, 1998). pRB remains hyperphosphorylated during S phase and through the late stages of mitosis (Buchkovich et al., 1989; Chen et al., 1989; DeCaprio et al., 1989; Ludlow et al., 1990). It is the underphosphorylated form of pRB that associates in vivo with E2F as well as all other cellular and viral proteins (reviewed in Weinberg, 1995; Dyson, 1998). pRB has been shown to repress transcription by all three eukaryotic RNA polymerases (reviewed in Dynlacht, 1997), and several models for pRB-mediated transcriptional repression have been suggested. For RNA polymerase II (pol II) regulation, the simplest model suggests that pRB blocks activation by certain sequencespecific transcription factors by inhibiting the interaction between their activation domains and undefined components of the basal transcription initiation machinery. Alternatively, pRB may function less passively, by directly interacting with the basal transcription initiation machinery itself. In support of the latter hypothesis, it has been shown that chimeric pRB-DNA-binding domain fusion proteins can directly repress transcription subsequent to promoter binding (Adnane et al., 1995; Bremner et al., 1995; Sellers et al., 1995; Weintraub et al., 1995). Although additional in vitro studies suggested that pRB may directly contact the TAFII250 subunit of the basal transcription factor TFIID, the functional consequences of such an interaction were not determined (Shao et al., 1995). Moreover, other in vitro binding experiments suggested that pRB does not directly interact

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with native TFIID (Weintraub et al., 1995). Such contradictory data regarding the interaction of pRB with components of the general transcription machinery highlight the need for an investigation of functional interactions of pRB with the basal transcription machinery once it is recruited to a promoter. Recently, a mechanism involving chromatin has been proposed to explain pRB-mediated transcriptional repression of pol II. These data suggested that pRB may function through the recruitment of a histone deacetylase, HDAC1, which is thought to repress transcription by promoting nucleosome formation (Brehm et al., 1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998). Interestingly, it has been noted that certain promoters were repressed in response to HDAC1 recruitment, while others were insensitive to HDAC1-mediated inhibition (Luo et al., 1998). This suggests that pRB-mediated repression could occur through distinct, promoter-specific mechanisms. Indeed, it has been shown previously that pRB is able to repress transcription in a reconstituted in vitro transcription system lacking histones (Dynlacht et al., 1994). Thus, although pRB is known to repress E2F in vivo and in vitro, the molecular mechanisms by which this occurs remain elusive. To better understand the function of pRB at the molecular level, an in vitro transcription assay using highly purified basal transcription factors was developed. This in vitro transcription system was exploited to identify the minimal requirements for E2F activation and pRB repression. Further, promoter assembly and order-of-addition experiments were performed in parallel with DNase I footprint assays to identify which, if any, of the basal factors were targets of pRB regulation. We found that pRB functions exclusively during transcriptional initiation at a very early stage of preinitiation complex formation. These findings suggest that pRB may repress transcription by targeting a key step in E2F activation, namely, the recruitment of the basal transcription factors, TFIIA and TFIID.

Results pRB-Mediated Repression Reconstituted In Vitro To understand the mechanisms of transcriptional repression by pRB, we studied its activity in a partially purified system. Thus, each reaction contained recombinant TFIIB, partially purified factors (TFIIA, TFIID, TFIIE, TFIIF, and TFIIH derived from HeLa cells), and affinitypurified RNA polymerase II (pol II). Initial experiments were carried out using the partially purified system since it was possible that an unknown cofactor(s) may be required for E2F activation and/or pRB inhibitory activity. This system supported robust activation by the E2F4/DP-1 heterodimer from a previously characterized E2F-responsive promoter (Figure 1, lanes 1–4) (Helin et al., 1993a; Dynlacht et al., 1994). This transcription factor was used in all subsequent experiments as well, and for simplicity, we shall refer to this heterodimer as E2F. Importantly, we established that E2F activity was completely and specifically repressed by pRB, while neither basal transcription nor activation by an unrelated factor,

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.

Gal4-VP16, was diminished by pRB (lanes 5–9 and data not shown). Additional evidence that this system was able to faithfully recapitulate the natural function of these proteins is indicated by experiments that included E1A in the transcription reactions. The addition of E1A, a viral oncoprotein known to reverse the inhibitory function of pRB in vivo, was able to eliminate repression by pRB (lanes 10–13).

Minimum Factor Requirements for E2F and pRB Activity To identify the minimal factor requirements for E2F and pRB activity, the partially purified basal factor fractions described above were replaced with recombinant and affinity-purified ones. Thus, these reactions contained recombinant TFIIA, TFIIB, TFIIE, and TFIIF, and native, immunoaffinity-purified TFIID, TFIIH, and pol II (Figure 2A). The method of isolation and purity of these factors is comparable to that of the most highly purified in vitro transcription system currently in use (LeRoy et al., 1998). The high level of purity of this system is demonstrated by Figure 2B, which shows that basal and E2F-activated transcription were completely dependent on each of the general transcription factors. However, there did not appear to be a complete dependence on TFIIE, since omission of this factor did not abolish transcription. There is precedence for TFIIE-independent transcription of certain promoters (Parvin et al., 1992; Holstege et al., 1995), although we have not excluded the possibility that one of our fractions contains trace amounts of this factor. Further, we cannot rigorously rule out the possibility that a cofactor may be required in addition to the basal factors for E2F and pRB activity. However, given the fact that this highly purified system supported robust E2F activation and complete pRB repression to virtually the same extent as that seen with the partially purified factors (Figure 2C, compare to Figure 1, lanes 5–9), the

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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.

existence of a putative cofactor absolutely required for E2F activation and/or pRB repression appears unlikely. pRB Functions during the Early Stages of the Preinitiation Complex Formation It is well established that the preinitiation complex (PIC) assembles in an ordered, stepwise fashion in which TFIID binding to the TATA box nucleates the assembly of TFIIA, recruiting other basal factors in turn (Van Dyke et al., 1988; Buratowski et al., 1989). Because many transcriptional activators have been shown to function by recruitment of specific components of the PIC to the promoter, we sought to determine if this was so for E2F, and if pRB acted at a specific step during the assembly of the PIC as well. We therefore performed order-ofaddition experiments in which pRB was added to the transcription reaction at different stages during the assembly of basal factors on the promoter. Remarkably, we found that E2F activation was largely resistant to repression when pRB was introduced after the assembly of the complete preinitiation complex (Figure 3A, lanes 17–20). As we analyzed the earlier stages of the PIC assembly, we found that even the relatively small complex containing E2F, TFIIA, and TFIID was strikingly resistant to pRB repression (lanes 5–8), as were each of the larger complexes (lanes 9–20). There was a reduction in overall levels of transcription, as well as relative activation, when the first incubation included TFIID without TFIIA (lanes 1–4) as expected (Chi and Carey, 1996), and this complex was significantly less resistant to pRB than even the next larger complex, which included TFIIA and TFIID. These data suggest that the E2F-4/DP-1 complex activates transcription through interactions with TFIID and/or TFIIA and that pRB could potentially inhibit this activity.

We next addressed the possibility that pRB was able to repress subsequent initiation events during the course of our standard transcription reaction (45 min), causing the partial repression observed when pRB was added after PIC assembly. Further, it was possible that besides functioning at the level of recruitment and assembly of the PIC, E2F and pRB could also function later, during transcriptional elongation and reinitiation. A role for sequence-specific activators in reinitiation has been described (Kraus and Kadonaga, 1998). We therefore carried out the order-of-addition experiments under conditions that allowed only a single round of transcription (Figure 3B; see Experimental Procedures). Despite the expected overall reduction of transcription levels when initiation was restricted to a single round, relative levels of activation by E2F, as well as repression by pRB, were virtually indistinguishable from those obtained under conditions allowing multiple rounds of initiation (compare panels A and B). That we were observing only the product labeled in the initial round is demonstrated by the absence of a signal in the control lanes, in which the radioactive label was introduced after the 2 min “pulse” period (lanes 13 and 14). These data suggest that both E2F and pRB function during the early stages of the PIC assembly. The transcription reactions shown in Figures 3A and 3B also included the USA (upstream activator-dependent stimulatory activity) fraction. The USA fraction is known to contain both positive and negative cofactors, including PC-1-4 and NC-1 (Ge and Roeder, 1994). Inclusion of this fraction resulted in an additional 1.5-fold increase in the level of activation (but not basal transcription) relative to reactions lacking USA (data not shown). Results similar to the ones shown were also obtained without the USA fraction (not shown).

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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 308C. 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.

To further characterize the components required for pRB inhibitory activity, we replaced TFIID with only the TATA box–binding protein (TBP) subunit, thereby excluding TBP-associated factors (TAFs) from the reaction. Although we observed robust basal transcription activity under these conditions, the system was no longer responsive to either E2F or pRB (panel C). These

data suggest that TAFs are necessary both for E2F activation as well as pRB repression, since pRB was nevertheless recruited to the promoter by E2F, and under these conditions E2F and TBP bind the promoter simultaneously (see below, Figure 5A). Although interpretation of these results is complicated by the fact that omitting TAFs abrogates E2F activation, the data suggest that

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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 110, relative to the transcription start site, is indicated. Large arrows denote hypersensitive sites.

active repression of at least some promoters by pRB requires both recruitment to the promoter and a target present in TFIID. E2F and TFIIA Together Promote Stable Binding of TFIID to the Promoter To further study the functional interactions between E2F, TFIIA, and TFIID on a promoter, in vitro DNase I footprinting was carried out using a promoter identical to the one used in the in vitro transcription reactions. Initially, we verified that purified E2F-4/DP-1 and TBP bound specifically to the four tandem E2F-binding sites and to the TATA box, respectively (Figure 4A). The former footprint was efficiently competed by unlabeled double-stranded oligonucleotides containing the consensus E2F site, but not by the corresponding oligonucleotides containing a mutated E2F site (lanes 1–4). Similarly, protection over the TATA box was observed with recombinant TBP, and protection of this site was competed by wild-type, but not mutant, TATA box oligonucleotides (lanes 5–11).

To analyze the individual contributions of E2F, TFIIA, and TFIID to preinitiation complex assembly at the promoter, each was titrated into footprinting reactions while keeping the quantities of the remaining factors fixed (Figure 4B). Interestingly, incubation with TFIID alone or TFIIA and TFIID in the absence of E2F resulted in weak protection over the TATA box and a putative initiator element (Inr) (lane 10 and data not shown). Addition of TFIIA alone had no effect on E2F binding (lane 6), and coincubation of E2F with TFIID resulted in a pattern that was essentially a combination of either one alone (lane 2 and data not shown). However, when all three proteins were present simultaneously, several dramatic changes were evident. First, there was a distinct, extended footprint characteristic of the native TFIID complex (Figure 4B, lanes 5, 9, and 13) (Zhou et al., 1992). The footprint induced by TFIID binding was clearly distinguishable from the smaller TATA box footprint produced with TBP alone (Figure 4A). This included strong protection over the TATA box as well as the putative Inr region, and weaker protection downstream of the Inr, corresponding

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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 mg) or the GST-E1A fusion protein (1.0 mg) 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 308C 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.

to a position between 19 and 111. In addition, multiple, strong hypersensitive sites were generated at the borders of each footprint. These hypersensitive cleavage sites were absolutely dependent on the presence of E2F, TFIIA, and TFIID, since omission of any of these factors precluded their appearance (Figure 4B). The fact that hypersensitive sites and more complete protection of the TATA box and Inr were obtained only when all three proteins were present simultaneously supports the hypothesis that E2F, TFIIA, and TFIID bind cooperatively to form a unique, and possibly more stable, complex than any subset of factors. Moreover, the cooperative assembly of a stable complex containing all three proteins is consistent with the transcription data presented above in which this ternary complex was resistant to the effects of pRB (Figure 3). pRB Disrupts the Cooperative Footprint of E2F/TFIIA/TFIID Having observed a specific footprint with the E2F/TFIIA/ TFIID complex, we next tested the effects of pRB on the stability of this complex. pRB had no effect on the

DNase I protection pattern produced by E2F alone or a combination of either E2F and TFIIA or E2F and TFIID (Figure 5A, lanes 2–5, and data not shown). This suggests that E2F binding to DNA is not altered qualitatively by interactions between pRB and E2F. Importantly, however, when pRB was included in reactions containing all three proteins, there was a striking change in the footprint pattern relative to reactions lacking pRB (compare lanes 7–10). Here, the strong protection over the TATA box as well as the Inr element was dramatically diminished. In addition, each of the hypersensitive sites unique to the E2F/TFIIA/TFIID complex was completely eliminated (lanes 7–10). Indeed, the resulting weak protection of the TATA box and Inr, and the elimination of the hypersensitive sites resulted in a footprint pattern reminiscent of the one obtained with E2F and TFIID alone (compare Figure 4B, lane 2, and Figure 5A, lane 10), suggesting that TFIIA may have been displaced from the promoter. Alternatively, the presence of pRB may change the conformation of the TFIIA/IID complex to a less active, more weakly bound state. The transcription data presented above suggested

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the importance of TAFs for E2F activation and pRB repression (Figure 3C). Therefore, to test the importance of native TFIID in the context of pRB-induced alterations, experiments were carried out in which we replaced this basal factor with TBP. The footprint pattern obtained with the combination of E2F, TFIIA, and TBP appeared to be largely a composite of those obtained with E2F and TBP added separately, with the exception of the establishment of a single hypersensitive site between the two protected regions (Figure 5A, lanes 11 and 12; compare with Figure 4A, lanes 2 and 5). Both TBP and E2F are known to significantly bend DNA (Kim and Burley, 1994; Cress and Nevins, 1996). Whether the induced hypersensitivity arose from a cumulative effect of having two DNA bending proteins binding adjacently, thereby increasing the susceptibility to the DNase I cleavage, or whether it was due to direct interactions between these proteins is unknown. While incubation of TBP with E2F and TFIIA produced a strong footprint over the TATA box, there was no protection of downstream elements or hypersensitive sites in that region, in contrast to that obtained with TFIID. These results are consistent with previous observations that the downstream footprint pattern of native TFIID is due to the presence of one or more of the TAFs, including TAFII250, TAFII150, or dTAFsII40/60 (Verrijzer et al., 1995; Burke and Kadonaga, 1997). Importantly, when we included pRB in reactions containing TBP, E2F, and TFIIA, there were no alterations of the DNase I protection pattern (lanes 12–15). This is entirely consistent with our demonstrated requirement for TAFs in pRB-mediated transcriptional repression (Figure 3C). Although the recombinant pRB appeared homogeneous in its purity (Figure 2A), we considered the possibility that an undetectable, contaminating activity could be present and responsible for the alterations in the E2F/TFIIA/TFIID footprint pattern induced upon addition of pRB. To test this notion and to attempt to recapitulate one mechanism of pRB deregulation that can occur in the cell upon oncogenic transformation, E1A was included in the footprint reactions. Here, the GST-E1A fusion protein completely reversed the effects of pRB (Figure 5B, lane 8), while GST alone had no effect (lane 7). GST-E1A had no effect on the footprint pattern when it was included in reactions with E2F/TFIIA/TFIID (lane 5) or alone (lane 3). These results demonstrate that pRB is functioning specifically and are in agreement with the ability of E1A to inactivate pRB and with the transcription results presented in Figure 1. To extend the above transcription studies regarding the effect of pRB on the assembly of E2F/TFIIA/TFIID on a promoter, an order-of-addition footprinting experiment was carried out. This was of particular interest in light of our results suggesting the resistance of the ternary complex to transcriptional repression by pRB (Figure 3). pRB was added either during or after the assembly of these factors on the promoter. In agreement with transcription experiments, the preformed ternary complex was largely resistant to the effects of pRB (Figure 5C, lanes 2–4). These data further support the model that if pRB is present during the formation of the preinitiation complex, it will destabilize it, preventing the enhanced TFIID binding, as measured by DNase I footprinting, and

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.

if the complex forms in the absence of pRB, it is resistant to the subsequent addition of pRB. Discussion While a considerable amount of data have accumulated over the last few years regarding the mechanism of transcriptional regulation by pRB, a detailed mechanistic description for how this important tumor suppressor functions is lacking. We have undertaken a study of the mechanism of pRB repression in a well-defined and simplified system. This study takes advantage of a reconstituted in vitro transcription assay that circumvents some of the complications linked to cell-based (transfection) assays. The data presented here show that pRB repression can occur in a system containing only recombinant and affinity-purified basal factors. One important initial result was the observation that pRB could repress E2F activation in a chromatin-free system. The observation that repression was dependent on the presence of E2F and that it was effectively reversed by the viral E1A protein indicates that our in vitro system recapitulates several important aspects of pRB regulation in vivo. The observation that pRB can repress E2F activation in the absence of chromatin is important in light of several recent reports suggesting that pRB recruits a histone deacetylase, HDAC1, that presumably remodels chromatin, resulting in transcriptional repression (Brehm et al., 1998; Luo et al., 1998; MagnaghiJaulin et al., 1998). It had been further suggested that

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pRB may function through an alternate mechanism in a manner that is dependent on promoter context but that does not require HDAC recruitment (Luo et al., 1998). It is clear from the data presented here that pRB can function very efficiently in the absence of chromatin, and future experiments will determine whether we can discriminate in vitro between pRB-repressible promoters that require chromatin assembly and those that do not. We were able to pinpoint pRB function to the early stages of preinitiation complex formation and thereby rule out an obligate role for pRB in preventing reinitiation and elongation of transcription. A complex containing E2F, TFIIA, and TFIID was largely resistant to the effects of pRB as determined by transcription and DNase I footprinting assays. The fact that the E2F/TFIID/TFIIA complex was highly resistant to pRB indicates that these factors can cooperate to form a stable complex that may exclude pRB. Further evidence of cooperative interactions between E2F and these basal factors was obtained with DNase I footprinting experiments. Taken together, these experiments are consistent with others demonstrating that activator-dependent recruitment of TFIIA and TFIID forms a stable complex (Lieberman and Berk, 1994; Kobayashi et al., 1995; Shykind et al., 1997). A model summarizing these results is shown in Figure 6. Our data suggest that recruitment of the TFIIA/TFIID complex by E2F may confer resistance to the effects of pRB due to masking of the pRB–E2F interface, since the activation and pRB-binding domains are juxtaposed in E2F (Figure 6A). Thus, if the E2F activation domain contacts a target(s) in TFIID, simultaneous E2F activation and binding of pRB might be prevented. Based on experiments in which we have substituted TBP for native TFIID, we infer that E2F interacts with a TAF(s) in the TFIID complex. Further, it is possible that E2F might simultaneously interact with TFIIA. While we cannot formally rule out the possibilty that pRB may still bind to E2F under conditions in which an active PIC is formed, a subcomplex formed during early preinitiation complex assembly is resistant to the repressive effects of pRB. On the other hand, binding of a pRB–E2F repressor complex to the promoter could prevent TFIIA/IID from forming an active complex, either by directly interfering with their association, or by causing them to assemble in a weakly binding, inactive conformation (Figure 6B). Thus, in vitro footprint analysis revealed that when pRB was included in reactions with E2F, TFIIA, and TFIID, the unique footprint pattern of this ternary complex disappeared, resulting in apparent weak binding by TFIID. It is especially interesting to note that the DNase I footprint of this complex in the presence of pRB looked very similar to the footprint pattern of E2F and TFIID in the absence of TFIIA (compare Figures 4 and 5). Since pRB clearly did not disrupt E2F binding to the promoter, and TFIID retained its ability to bind (weakly) to the TATA box, the simplest explanation is that the association of TFIIA was disrupted, resulting in weakened TFIID binding. This may be true whether or not pRB interacts directly with TFIID. If a target for pRB does exist in the TFIID complex, it is possible that pRB binding and TFIIA binding to TFIID could be mutually exclusive. It will also be important to determine which TAFs, if any, are prevented from contacting downstream promoter elements

in the presence of pRB. Furthermore, it will be very interesting to examine other E2F-responsive promoters to investigate whether there are natural promoters that do not require TFIIA or TAFs, and if so, whether these are repressed by pRB. Transcription experiments in which TFIID was replaced with TBP indicated that the TAFs were necessary for E2F activation. This is consistent with the notion that TAFs are important coactivators of transcription and are required by many activators in vitro (Verrijzer and Tjian, 1996). Additional experiments will be required to determine which subset of TAFs is required for E2F activation. That TAFs are required for pRB activity is less clear, since TBP is unable to support E2F activity. However, multiple studies have shown that pRB can actively repress transcription (Adnane et al., 1995; Bremner et al., 1995; Sellers et al., 1995; Weintraub et al., 1995). These studies were carried out by fusing pRB to a heterologous DNA-binding domain, thereby directly recruiting it to a promoter carrying the requisite binding site. In these studies, pRB was capable of repressing transcription potentiated by proximal activators. However, these studies presented contradictory conclusions regarding the ability of pRB to repress basal transcription. If pRB were able to repress transcription without the requirement for trans-activation, then repression should have been observed in the presence of TBP, since pRB was nevertheless recruited to the promoter through E2F. This clearly was not the case, and two possible explanations can account for this observation. First, pRB might function exclusively to block interactions between transactivators and the PIC. Second, in contrast with a passive role for pRB, pRB might actively interact with a component of the PIC, such as TFIID, to change the conformation or stability of this complex on the promoter. While we cannot formally exclude either possibility, the apparent ability of pRB to alter the assembly of TFIID, but not TBP, on the promoter and the ability of pRB to interact with recombinant TAFII250 in vitro (Shao et al., 1995) suggest a more active role for pRB in transcriptional repression. It is interesting to note that another mammalian transcriptional repressor protein studied in detail, RBP, functions by targeting the TFIID/IIA complex (Olave et al., 1998). In that elegant study, RBP was shown to bind directly to the promoter in a sequence-specific manner, where it repressed transcription by directly interacting with TFIIA and precluding association between TFIIA and one member of the TFIID complex, TAFII110 (Olave et al., 1998). pRB has been shown to repress transcription by RNA polymerases I (pol I) and III (pol III) as well (reviewed in Dynlacht, 1997, and references therein). Interestingly, pRB repression of pol III, but not pol I, can occur before or after a preinitiation complex is assembled (Larminie et al., 1997; Voit et al., 1997). It is intriguing to speculate that the mechanism of inhibition of pol II transcription could more closely resemble that of pol I, and future experiments will determine the similarity of mechanisms used by each. Experimental Procedures Purification of Transcription Factors TFIIA was prepared in two different ways. Native TFIIA was purified from HeLa cell nuclear extracts essentially as described (Maldonado

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et al., 1990), with the following exceptions. The DEAE-Sepharose column was eluted with a linear gradient of KCl (0.1–0.6 M) over 5 column volumes. The resulting fractions were dialyzed against BC100 (20 mM HEPES-KOH [pH 7.9], 0.1 mM EDTA, 20% glycerol, 0.1 M KCl, 1.0 mM DTT, and 0.2 mM PMSF) and assayed for transcriptional activity, as well as for the activity of other potentially contaminating factors. The fraction containing the highest level of TFIIA activity with the least evidence of other contaminating factors was used in the subsequent transcription assays. Highly purified recombinant TFIIA containing the fused a/b subunits and the g subunit was prepared essentially as described (Ozer et al., 1994). Recombinant HA-tagged TFIIB was expressed in bacteria, and an extract was prepared essentially as described (Maldonado et al., 1996). Lysate (4 mg) was applied to a 1 ml FPLC Mono S column (Pharmacia) and eluted with a linear gradient of KCl (0.1–1.0 M) over 30 column volumes. TFIIB eluted with greater than 99% purity at a salt concentration near 0.25 M. The fractions containing TFIIB were dialyzed against BC100 and stored at 2808C. The HeLa cell fraction containing TFIID was prepared essentially as described (Maldonado et al., 1996), up to and including the DEAESepharose column, which was eluted with a linear gradient of KCl. Highly purified epitope-tagged TFIID was prepared by affinity chromatography as described (Zhou et al., 1992) or by affinity chromatography using an anti-TAF130 antibody (a generous gift of R. Tjian) with identical results. Purified His6-tagged TBP was kindly provided by J. Parvin. A HeLa cell fraction containing TFIIE, TFIIF, and TFIIH activity was purified essentially as described (Maldonado et al., 1996), with the following changes. Dialyzed protein from the DEAE-Sepharose column was applied to a 1 ml HPLC Biosep-DEAE-P column (Phenomenex) and was eluted with a linear gradient of KCl (0.1–0.6 M) over 20 column volumes. The resulting fractions were dialyzed against BC100 and assayed for transcriptional activity. The peak activity eluted with approximately 0.2 M KCl. Recombinant TFIIE was prepared essentially as described (Peterson et al., 1991). Recombinant TFIIF was expressed by coinfecting High Five insect cells with recombinant baculoviruses encoding RAP30 and RAP74 (Tan et al., 1994). Cells were harvested 72 hr after infection, and TFIIF was purified essentially as described (Holstege et al., 1995), up to and including the phosphocellulose column. Affinity-purified holo-TFIIH was prepared by affinity chromatography using the partially purified DEAE-Sepharose fraction containing TFIIE, TFIIF, and TFIIH activity (described above) as starting material. The affinity purification was carried out as described (LeRoy et al., 1998). Highly purified RNA polymerase II was obtained by immunoaffinity chromatography essentially as described (Maldonado et al., 1996), with the following changes. The eluate from the DE52 column was dialyzed against TEG/0.2 buffer (50 mM Tris–HCl [pH 7.9], 0.1 mM EDTA, 20% glycerol, 0.2 M (NH4)2SO4, 1 mM DTT, and 0.2 mM PMSF). Binding and washing of the affinity column was carried out with TEG/0.2 buffer. Pol II was eluted from the affinity column for 10 min at 308C three times with two column volumes of elution buffer (50 mM Tris–HCl [pH 7.9] at 48C, 0.1 mM EDTA, 0.75 M (NH4)2SO4, 40% ethylene glycol, 0.5 mg/ml CTD tetraheptapeptide). The CTD tetraheptapeptide was as described (Thompson et al., 1990). The three elutions were pooled and dialyzed against BC100. The USA fraction was prepared using the DEAE-Sepharose flowthrough fraction from the TFIID purification as input for a heparin-Sepharose column. The USA fraction was purified over the heparin-Sepharose column essentially as described (Meisterernst et al., 1991). Tubulin-tagged E2F-4 and GST-tagged DP-1 were purified and allowed to dimerize as described (Dynlacht et al., 1997). His6-tagged pRB was purified using an E7 peptide column as described (Dynlacht et al., 1994), except that it was washed with buffer containing 1.0 M KCl prior to elution. The E1A protein consisted of a GSTtagged fragment containing residues 1–139 (Fattaey et al., 1993) and was expressed and purified in a manner similar to GST-DP-1. Plasmid Construction The G-less cassette transcription template, p(E2F)4BG-, was prepared as follows. A restriction fragment containing four tandem E2F sites upstream of the E1B TATA box was excised from p(E2F)4BCAT

(Helin et al., 1993b) using the restriction enzymes XhoI and KpnI. The ends were blunted using T-4 polymerase, and the fragment was ligated into the blunted SacI site of p(C2AT)19 (Sawadogo and Roeder, 1985). In Vitro Transcription Assays Transcription reactions were carried out using either the highly purified factors or a combination of partially purified and highly purified factors as indicated. The basal transcription factors of the highly purified system included recombinant TFIIA, TFIIB, TFIIE, and TFIIF, and immunoaffinity-purified TFIID, TFIIH, and pol II. These factors were used in the experiments shown in Figure 2. The general transcription factors of the partially purified system included recombinant TFIIB, affinity-purified pol II, and the HeLa cell fractions containing TFIIA, TFIID, and TFIIE/F/H. These factors were used in the experiments shown in Figures 1 and 3. Typically, transcription reactions were carried out as follows. The proteins were combined, and the reaction volume was brought to 12.5 ml using BC100 that contained 0.2 mg/ml highly purified BSA (Boehringer-Mannheim). Reactions also included 12.5 ng of supercoiled p(E2F)4BG- template DNA and were allowed to incubate for 30 min at 308C. The following reagents were then added to the indicated final concentrations: 25 mM HEPES [pH 7.6], 4.0 mM MgCl2, 1.2% PEG8000, 0.16 U/ml RNase inhibitor (Promega), 0.1 mM ATP, 0.1 mM UTP, 2.5 mM CTP, 50 mM 39-OMe-GTP (Pharmacia), and 0.5 mCi/ml a-32P-CTP (800 Ci/mmol). Water was added to bring the final volume to 25 ml. The reactions were allowed to proceed for 45 min at 308C and were subsequently treated as described (Kadonaga, 1990), with the exclusion of the primer extension step. For the order-of-addition experiments shown in Figure 3, the proteins were combined in the order indicated in the figure. In addition, the USA fraction was included in the first incubation of these reactions. This resulted in an approximate 1.5-fold increase in the overall level of activation. Similar results were obtained without the USA fraction, but with lower levels of activation, relative to basal activity. The single-round transcription experiment shown in Figure 3B was carried out using a modification of a previously published pulsechase method (Olave et al., 1998). The reactions were set up under standard conditions, except that unlabeled CTP was omitted. The reactions were pulsed for 2 min in the presence of ATP and UTP (0.1 mM final), and 0.5 mCi/ml a-32P-CTP (800 Ci/mmol), after which time the reactions were chased for 5 min by the addition of unlabeled CTP to 1.0 mM. The products were then treated as described above. For the control reactions (lanes 13 and 14) to show that products synthesized after the first 2 min were not visualized, the unlabeled CTP was added first, followed by the 32P-CTP after 2 min. In Vitro DNase I Footprint Assays The footprint probe was prepared essentially as described (Marshak et al., 1996). In brief, p(E2F)4BG- was digested with EarI, labeled with g-32P-ATP, then digested with EcoRI. This resulted in a 208 bp fragment spanning the promoter region, labeled at the downstream end of the promoter. The in vitro DNase I footprint assays were carried out using recombinant TFIIA and affinity-purified TFIID as indicated. The proteins were allowed to incubate with the probe for 1 hr at 308C prior to DNase I digestion. DNase I digestion and all subsequent steps were carried out as described (Marshak et al., 1996). For the pRB order-of-addition experiment shown in Figure 5C, after 30 min of incubation without pRB, pRB was added, and an additional 30 min incubation was carried out prior to DNase I digestion. Because of the difficulty in quantitating such a large multiprotein complex such as TFIID, the amount of this factor is indicated in transcription units. One transcription unit is defined as the minimum amount that results in the optimal level of E2F activation using the standard transcription conditions described above. Acknowledgments We thank J. and R. Conaway for TFIIF baculoviruses; J. Parvin for purified his6-TBP; D. Reinberg for expression vectors for TFIIA subunits, and HA-tagged TFIIB; R. Tjian and R. Burgess for antibodies; and I. Sanchez for helpful discussions. We are grateful to the

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American Cancer Society for their initial support of this work (Research Project Grant RPG-98-074-01-GMC) and to the NIH/NCI for their current support (1R01 CA77245-01). B. D. D. is also most grateful to E. and K. Langone for their generous donation of a Damon Runyon Scholar Award (DRS-01), part of which funded the very initial stages of this work. B. D. D. is a Pew Scholar in the Biomedical Sciences. B. D. D. is grateful to R. Tjian for his continued support and encouragement. Received September 28, 1998; revised December 11, 1998. References Adnane, J., Shao, Z., and Robbins, P.D. (1995). The retinoblastoma susceptibility gene product represses transcription when directly bound to the promoter. J. Biol. Chem. 270, 8837–8843. Brehm, A., Miska, E.A., McCance, D.J., Reid, J.L., Bannister, A.J., and Kouzarides, T. (1998). Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature 391, 597–601. Bremner, R., Cohen, B.L., Sopta, M., Hamel, P.A., Ingles, C.J., Gallie, B.L., and Phillips, R.A. (1995). Direct transcriptional repression by pRB and its reversal by specific cyclins. Mol. Cell. Biol. 15, 3256– 3265. Buchkovich, K., Duffy, L.A., and Harlow, E. (1989). The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell 58, 1097–1105. Buratowski, S., Hahn, S., Guarente, L., and Sharp, P.A. (1989). Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56, 549–561. Burke, T.W., and Kadonaga, J.T. (1997). The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila. Genes Dev. 11, 3020–3031. Chen, P.-L., Scully, P., Shew, J.-Y., Wang, J.Y.J., and Lee, W.-H. (1989). Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell 58, 1193– 1198. Chi, T., and Carey, M. (1996). Assembly of the isomerized TFIIATFIID-TATA ternary complex is necessary and sufficient for gene activation. Genes Dev. 10, 2540–2550. Cress, W.D., and Nevins, J.R. (1996). A role for a bent DNA structure in E2F-mediated transcription activation. Mol. Cell. Biol. 16, 2119– 2127. DeCaprio, J.A., Ludlow, J.W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica-Worms, H., Huang, C.-M., and Livingston, D.M. (1989). The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell 58, 1085–1095. DeGregori, J., Kowalik, T., and Nevins, J.R. (1995). Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol. Cell. Biol. 15, 4215–4224. Dynlacht, B.D. (1997). Regulation of transcription by proteins that control the cell cycle. Nature 389, 149–152. Dynlacht, B.D., Flores, O., Lees, J.A., and Harlow, E. (1994). Differential regulation of E2F trans-activation by cyclin-cdk2 complexes. Genes Dev. 8, 1772–1786. Dynlacht, B.D., Moberg, K., Lees, J.A., Harlow, E., and Zhu, L. (1997). Specific regulation of E2F family members by cyclin-dependent kinases. Mol. Cell. Biol. 17, 3867–3875. Dyson, N. (1998). The regulation of E2F by pRB-family proteins. Genes Dev. 12, 2245–2262. Fattaey, A.R., Harlow, E., and Helin, K. (1993). Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes. Mol. Cell. Biol. 13, 7267–7277. Field, S.J., Tsai, F.-Y., Kuo, F., Zubiaga, A.M., Kaelin, W.G., Jr., Livingston, D.M., Orkin, S.H., and Greenberg, M.E. (1996). E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell 85, 549–561. Ge, H., and Roeder, R.G. (1994). Purification, cloning, and characterization of a human coactivator, PC4, that mediates transcriptional activation of class II genes. Cell 78, 513–523.

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