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Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein

Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein The EMBO Journal Vol.16 No.8 pp.2061–2071, 1997 Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein 1 1 We have demonstrated recently a novel function for RB Christopher G.C.Larminie , Carol A.Cairns , 2 3 in controlling RNA polymerase (pol III) transcription Renu Mital , Klaus Martin , 3 3 (White et al., 1996). Overexpression of RB represses pol Tony Kouzarides , Stephen P.Jackson and 1,3,4 III templates in transfected cells, whereas control pol II Robert J.White promoters are unaffected. Furthermore, purified recombin- Institute of Biomedical and Life Sciences, Division of Biochemistry ant RB inhibits pol III transcription when added to and Molecular Biology, Davidson Building, University of Glasgow, a system reconstituted with fractionated factors. These Glasgow, G12 8QQ, UK, Cold Spring Harbor Laboratory, experiments demonstrate that RB can repress pol III when Cold Spring Harbor, NY 11724, USA and Wellcome/CRC Institute, it is overexpressed. To determine whether endogenous Tennis Court Road, Cambridge CB2 1QR and Departments of Zoology and Pathology, University of Cambridge, Cambridge, UK levels of RB can control pol III under physiological circumstances, we investigated the expression of class III Corresponding author genes in cells that have lost RB function (White et al., 1996). When two human osteosarcoma lines were com- The tumour suppressor protein RB restricts cellular pared, RB-deficient SAOS2 cells were found to have a growth. This may involve inhibiting the synthesis of more active pol III transcription apparatus than RB- tRNA and 5S rRNA by RNA polymerase (pol) III. We positive U2OS cells. In addition, primary fibroblasts from have shown previously that RB can repress pol III RB-knockout mice were shown to have much higher pol transcription when overexpressed either in vitro or III activity than primary fibroblasts from wild-type mice. in vivo. We also demonstrated that pol III activity is No general increase in pol II activity was seen when elevated substantially in primary fibroblasts from SAOS2 cells were compared with U2OS cells or when RB-deficient mice. Here we address the molecular –/– / Rb fibroblasts were compared with Rb fibroblasts. mechanism of this regulation. RB is shown to repress These experiments established that endogenous RB plays all types of pol III promoter. It can do this even if an important role in suppressing pol III transcription. added after transcription complex assembly. Func- The minimal region of RB that is required for growth tional assays demonstrate that RB targets specifically suppression comprises amino acid residues 379–928 (Qin the general pol III factor TFIIIB. A physical interaction et al., 1992). The same sequences are sufficient to inhibit between TFIIIB and RB is indicated by fractionation, pol III transcription (White et al., 1996). Various deletions pull-down and immunoprecipitation data. We show that remove segments from within this region abolish the that TFIIIB activity is elevated in primary fibroblasts ability of RB to regulate pol III (White et al., 1996). from RB-deficient mice. TFIIIB is a multisubunit Several naturally occurring mutations that prevent RB complex that includes the TATA-binding protein (TBP) from functioning as a tumour suppressor also inhibit its and a TFIIB-related factor called BRF. We show that ability to repress pol III (White et al., 1996). This raises RB itself contains regions of homology to both TBP the possibility that regulation of pol III contributes to and BRF and propose a model in which RB disrupts the tumour suppressive activity of RB (Nasmyth, 1996; TFIIIB by mimicking these two components. Keywords: RB/RNA polymerase III/TBP/TFIIIB/ White, 1997). transcription The viral oncoproteins SV40 large T antigen and adenovirus E1A can bind to RB and neutralize its function (Whyte, 1995). Both T antigen and E1A can also activate pol III transcription (Loeken et al., 1988; Patel and Jones, Introduction 1990; White et al., 1996). One way in which they may achieve this is by overcoming the repression of pol III The retinoblastoma protein (RB) is a 105 kDa nuclear transcription by RB (White et al., 1996). The ability of phosphoprotein that is encoded by an important tumour these oncoproteins to activate pol III by relieving the suppressor gene (reviewed by Weinberg, 1995; Whyte, physiological constraint that is normally provided by RB 1995). In normal cells, RB is involved in constraining may contribute to their transforming capability. growth and proliferation; in its absence, the ability of cells Transcription by pol III involves at least two general to shut down these functions is compromised (Weinberg, factors, named TFIIIB and TFIIIC (reviewed by Willis, 1995; Whyte, 1995). RB is mutated in a variety of tumours, 1993; White, 1994; Geiduschek and Kassavetis, 1995). including retinoblastomas, small-cell lung carcinomas, TFIIIB is a multisubunit complex that contains the TATA- sarcomas and bladder carcinomas (Weinberg, 1995). In many other human malignancies, the function of RB is binding protein TBP (Hernandez, 1993; Rigby, 1993). lost due to the disruption of upstream control pathways One of the TBP-associated factors (TAFs) in TFIIIB is (Pines, 1995; Weinberg, 1995). It is therefore of consider- structurally and functionally related to the pol II general able importance to understand fully the ways in which factor TFIIB (Buratowski and Zhou, 1992; Colbert and RB is able to influence cellular activity. Hahn, 1992; Lopez-de-Leon et al., 1992; Khoo et al., © Oxford University Press 2061 C.G.C.Larminie et al. 1994; Wang and Roeder, 1995; Mital et al., 1996). This to be sufficient to allow VA to recruit TFIIIB into a TAF has been variously named TDS4, PCF4, BRF and stable complex and thereby exclude expression of the TFIIIB90, but we shall refer to it as BRF. Although second gene. yeast TFIIIB has been reconstituted from recombinant Having established that these conditions allow the stable components (Kassavetis et al., 1995; Roberts et al., 1996; assembly of TFIIIC and TFIIIB onto the VA promoter, Ruth et al., 1996), the mammalian factor is much less we then tested whether RB is able to disrupt the preformed well characterized (reviewed by Hernandez, 1993; Rigby, complex (Figure 1C). The pol III factors were mixed with 1993). This partly reflects a tendency for TFIIIB to either glutathione S-transferase (GST) or a GST fusion dissociate during purification (Lobo et al., 1992; Taggart protein containing residues 379–928 of RB. The recombin- et al., 1992; Chiang et al., 1993; Teichmann and Seifart, ant proteins were added either 15 min prior to the addition 1995). Several groups have identified polypeptides as of VA DNA (Figure 1C, lanes 1 and 2), simultaneously candidate TAFs for human TFIIIB (Lobo et al., 1992; with the addition of VA (lanes 3 and 4) or 15 min after Taggart et al., 1992; Chiang et al., 1993; Teichmann and the factors were mixed with VA DNA (lanes 5 and 6). Seifart, 1995; Wang and Roeder, 1995; Mital et al., 1996), Nucleotides were then added to allow transcription. RB but there is little consensus and BRF is the only subunit was found to repress transcription to a similar extent which has been cloned and had its function established whether it was added before, during or after initiation categorically (Wang and Roeder, 1995; Mital et al., 1996). complex assembly. Most pol III templates lack a TATA box and so are not recognized directly by TFIIIB; in these cases, TFIIIB is RB represses all types of pol III promoter recruited via protein–protein interactions with promoter- Three distinct types of promoter structure are utilized by bound TFIIIC (reviewed by Willis, 1993; White, 1994; pol III, and this diversity reflects clear differences in Geiduschek and Kassavetis, 1995). transcription factor requirements: type 1 promoters have In the current work, we have investigated the mechan- internal A and C blocks and are unique to 5S rRNA istic basis of pol III regulation by RB. We demonstrate genes; type 2 promoters have internal A and B blocks and that RB is able to disrupt the activity of a preassembled are utilized by most pol III templates, including tRNA pol III transcription complex. We also show that it can and VA genes; type 3 promoters are located entirely inhibit expression from all types of pol III promoter. upstream of the transcription unit, include a TATA box, Functional studies implicate TFIIIB as a specific target and are found in U6 and 7SK genes (reviewed by Willis, for repression by RB. In support of this, we find that a 1993; White 1994; Geiduschek and Kassavetis, 1995). We population of RB molecules consistently co-fractionates tested the ability of RB to regulate transcription directed with cellular TFIIIB. Furthermore, immunoprecipitation by each of these promoter types. Increasing quantities of and pull-down experiments demonstrate an association RB(379–928) were added to reactions containing 5S (type between RB and TFIIIB. RB itself contains a region of 1), VA (type 2) or U6 (type 3) templates (Figure 2). As homology to TBP that is followed by a region of homology controls, we added an equal amount of GST (Figure 2B) to BRF. We present a model in which RB inactivates or a GST fusion protein containing residues 612–711 of TFIIIB by mimicking TBP and BRF in order to disrupt RB (Figures 2A and C). We have demonstrated previously TFIIIB. that GST–RB(612–711) is unable to regulate VA transcrip- tion (White et al., 1996). With each template, transcription was strongly repressed by the GST–RB(379–928) protein, Results whereas an equal amount of the control protein had little RB can disrupt the activity of a preformed pol III or no effect. We conclude that RB is effective in repressing transcription complex transcription from all categories of pol III promoter. Fully formed pol III transcription complexes are extremely stable and support multiple rounds of transcription without RB inactivates TFIIIB specifically dissociating from the template (Lassar et al., 1983; Since RB can regulate each of these promoter types, it reviewed by White, 1994). We tested whether RB is able seemed likely that it acts upon a general component of to repress transcription from a preinitiation complex that the pol III transcription apparatus that is utilized by all had already assembled on the adenovirus VA gene (Figure of these templates. We therefore investigated its ability to 1). Initially, we used template commitment assays to inactivate the pol III enzyme and the general pol III factors establish that, under the conditions of our experiments, TFIIIB and TFIIIC. If RB represses transcription by a stable transcription complex had assembled on VA inhibiting a specific component, then it should be possible following a 15 min incubation. Figure 1A demonstrates to restore expression in the presence of RB by adding more that 15 min is sufficient for VA to recruit factors and of that component. We carried out add-back experiments to thereby exclude the expression of a second template that test whether this is the case. was added subsequently (lanes 4 and 5). However, pre- VA transcription was reconstituted using partially puri- emptive complex formation did not require the presence fied factors (Figure 3A). Recombinant RB repressed of TFIIIB (lanes 5 and 7). We therefore carried out expression in this system, as previously (Figure 3A, lanes additional assays to confirm that TFIIIB is recruited to 1 and 2). Inclusion of additional pol III or TFIIIC fractions the complex within the 15 min period. In these experiments did not restore transcription (lanes 3 and 4). In contrast, (Figure 1B), VA was again preincubated with a full set a fraction containing partially purified TFIIIB produced a of factors but was then challenged by a second template substantial reversal in the level of inhibition (lane 5). that had been preincubated separately with TFIIIC in the Figure 3B demonstrates that repression by RB can be absence of TFIIIB. The 15 min preincubation was found reversed completely in a dose-dependent manner using 2062 RB represses TFIIIB Fig. 1. RB inactivates pol III transcription whether added before, during or after initiation complex assembly. (A) Template 1 (500 ng), as indicated, was preincubated for 15 min at 30°C with either a full set of fractionated factors (all lanes except 5 and 7) or with 2 μl of CHep-1.0 alone (lanes 5 and 7). Template 2 (500 ng) was then added, together with 1 μl each of 0.38M-TFIIIB and 0.48M-TFIIIB in the case of lanes 5 and 7. Nucleotides were added immediately to initiate transcription. The VA template was pVA and the tRNA template was Mcet1. The short transcript that runs just below the tRNA in lanes 2–5 is derived from pVA .(B) Template 1 (500 ng), as indicated, was preincubated for 15 min at 30°C with a full set of fractionated factors. Template 2 (500 ng) was preincubated separately with 2 μl of CHep-1.0. The reactions were then mixed and nucleotides were added immediately to initiate transcription. The VA template was pVA and the B2 template was pAG38. (C) Fractionated factors were preincubated for 15 min at 30°C before the addition of pVA (250 ng); after a further 15 min at 30°C, nucleotides were added and transcription was allowed to proceed. Reactions were supplemented with 250 ng of GST or GST–RB(379–928), as indicated, which were added at the indicated times. Fig. 2. RB represses transcription of 5S, VA and U6 genes. (A) Transcription of pHu5S3.1 (250 ng) using nuclear extract (10 μg) preincubated (15 min at 30°C) with no addition (lanes 1 and 5) or with 100, 200 or 300 ng of GST–RB(379–928) (lanes 2, 3 and 4, respectively) or 100, 200 or 300 ng of GST–RB(612–711) (lanes 6, 7 and 8, respectively). (B) Transcription of pVA (250 ng) using fractionated factors preincubated (15 min at 30°C) with no addition (lanes 1 and 5) or with 75, 150 or 300 ng of GST–RB(379–928) (lanes 2, 3 and 4, respectively) or 75, 150 or 300 ng of GST (lanes 6, 7 and 8, respectively). (C) Transcription of pU6/Hae/RA.2 (500 ng) using nuclear extract (10 μg) preincubated (15 min at 30°C) with no addition (lanes 1 and 5) or with 100, 200 or 300 ng of GST–RB(379–928) (lanes 2, 3 and 4, respectively) or 100, 200 or 300 ng of GST–RB(612–711) (lanes 6, 7 and 8, respectively). the TFIIIB fraction (lanes 1–5). In contrast, the same Thus, TFIIIB activity is in excess in the absence of fraction has little or no stimulatory effect when added in recombinant RB, but becomes limiting when RB is added. the absence of recombinant RB (lanes 6–9). This is This demonstrates that RB is able to bring about a specific because TFIIIC is limiting and TFIIIB is in relative excess decrease in TFIIIB activity such that it becomes limiting in the reconstituted system, as shown by the ability of the for transcription. The fact that a TFIIIB fraction alone TFIIIC fraction to increase transcription (lanes 10–12). is sufficient to overcome repression indicates that the 2063 C.G.C.Larminie et al. Fig. 4. Repression by RB can be relieved using affinity-purified TFIIIB TAFs but not using TBP. Transcription of pVA (250 ng) using fractionated factors preincubated (15 min, 30°C) with 250 ng of GST (lane 1) or GST–RB (lanes 2–9), and 1, 3 or 6 μl of TBP (lanes 3–5, respectively), 3 or 6 μl of B-TAF fraction (lanes 6 and 7, respectively) or 3 or 6 μl of control fraction (lanes 8 and 9, respectively). the level of inhibition. In contrast, the affinity-purified B-TAF fraction produced a significant, dose-dependent reversal of repression. That this response is due to the presence of TBP-associated factors in the B-TAF fraction is demonstrated by the inability of the control fraction to relieve inhibition. This experiment suggests that a TAF component of TFIIIB is the specific target for repression Fig. 3. (A) TFIIIB relieves repression by recombinant RB whereas TFIIIC and pol III do not. Transcription of pVA (250 ng) using of pol III transcription by RB. fractionated factors preincubated (15 min, 30°C) with 250 ng of GST (lane 1) or GST–RB(379–928) (lanes 2–5) and 3 μl of A25(1.0) TFIIIB interacts with immobilized recombinant RB fraction containing pol III (lane 3), CHep-1.0 fraction containing We carried out pull-down experiments to test for a physical TFIIIC (lane 4) or A25(0.15) fraction containing TFIIIB (lane 5). (B) TFIIIB is limiting for transcription in the presence of recombinant interaction between RB and pol III factors (Figure 5). RB, whereas in the absence of recombinant RB TFIIIC is limiting and Crude phosphocellulose (PC) fractions were incubated TFIIIB is in excess. Transcription of pVA (250 ng) using fractionated on a vibrating platform with glutathione–agarose beads factors preincubated (15 min, 30°C) with 250 ng of GST (lanes 1 and carrying immobilized GST or GST–RB, and the unbound 6–12) or GST–RB(379–928) (lanes 2–5) and 1 μl (lanes 3 and 7), 2 μl material was then assayed for transcription factor activity. (lanes 4 and 8) or 4 μl (lanes 5 and 9) of A25(0.15) fraction containing TFIIIB or 1 μl (lane 10), 2 μl (lane 11) or 4 μl (lane 12) of When PC-C fractions were treated in this way, we detected CHep-1.0 fraction containing TFIIIC. no depletion of TFIIIC activity (Figure 5A). In contrast, TFIIIB activity was partially depleted from PC-B fractions activities of TFIIIC and pol III are not compromised by using GST–RB (Figure 5B). Depletion under these condi- RB. We conclude that RB inhibits TFIIIB specifically. tions was relatively inefficient and we were unable to improve it by extending the incubation period because A TBP-associated component of TFIIIB is prolonged treatment in this way resulted in a general inactivated by RB loss of transcriptional activity. Nevertheless, these results In order to determine which part of TFIIIB is the target suggest that the RB portion of GST–RB interacts with for repression by RB, we carried out add-back experiments TFIIIB. We confirmed this by Western blotting the material using TBP alone or the TAF component of the complex that remained bound to the beads after extensive washing. (Figure 4). TFIIIB TAFs (B-TAFs) were affinity-purified Figure 5C shows that both TBP and BRF were detected by chromatography on a column containing immobilized in association with the GST–RB beads, but were not TBP, as described previously (White and Jackson, 1992; bound to GST alone. These data provide evidence for a White et al., 1995a). We also prepared a control fraction physical association between RB and TFIIIB. by the same procedure but using a column without TBP. Whereas the B-TAF fraction can reconstitute pol III A population of RB molecules co-fractionates with transcription in the presence of TBP, TFIIIC and pol III, TFIIIB the control fraction is inactive (White et al., 1995a). The results above indicate a physical and functional Recombinant RB was used to repress VA transcription interaction between TFIIIB and RB when the latter is in a system reconstituted with fractionated factors (Figure overexpressed as a recombinant protein. A more important 4). Addition of recombinant TBP made no difference to issue is whether these factors interact with each other 2064 RB represses TFIIIB Fig. 5. Immobilized RB binds TFIIIB but not TFIIIC. (A) Transcription of pVA (250 ng) using 2 μl of PC-B (all lanes) and 2 μl of PC-C (lane 1), 2 μl of LDB (lane 2), 4 μl of supernatant from PC-C that had been preincubated with GST beads (lane 3) or 4 μl of supernatant from PC-C that had been preincubated with GST–RB beads (lane 4). (B) Transcription of pVA (250 ng) using 2 μl of CHep-1.0 (all lanes) and 2 μl of PC-B (lane 1), 2 μl of LDB (lane 2), 4 μl of supernatant from PC-B that had been preincubated with GST beads (lane 3) or 4 μl of supernatant from PC-B that had been preincubated with GST–RB beads (lane 4). (C) Proteins from PC-B that remained bound to GST–RB (lanes 1 and 4) or GST (lanes 2 and 5) beads after extensive washing were resolved on an SDS–7.8% polyacrylamide gel and then analysed by Western immunoblotting with antibodies against TBP (lanes 1–3) or BRF (lanes 4–6). Lane 3 contains 10 ng of recombinant TBP. Lane 6 contains 15 μl of PC-B. when they are present at physiological concentrations. To Endogenous RB associates with TFIIIB address this, we assayed for the presence of RB in TFIIIB We carried out immunoprecipitation assays as an fractions by immunoblotting fractionated factors and additional test of whether cellular RB associates with probing with an anti-RB antibody. Consistent co- TFIIIB (Figure 7). Fractions containing TFIIIB were purification would be suggestive of a stable interaction immunoprecipitated using a monoclonal antibody that between these two factors. specifically recognizes the hypophosphorylated (active) Figure 6A demonstrates that RB is readily detectable form of RB. After extensive washing in 200 mM KCl and in TFIIIB fractions that have been purified through 0.1% NP-40, the precipitated material was immunoblotted phosphocellulose and DEAE–Sephadex (lane 3) or phos- and probed with antibodies against TBP and BRF. We phocellulose and a glycerol gradient (lane 4). In contrast, found that both these TFIIIB subunits were co-precipitated little or no RB was detected in fractions containing TFIIIC with RB (Figure 7A, lanes 4 and 8). Neither TBP nor (lane 1), pol III (lane 2), or the TBP-containing complex BRF was immunoprecipitated using a control antibody SL1 (lane 5). These data support the contention that RB that recognizes Sp1 (Figure 7B, lanes 5 and 10). As an interacts specifically with TFIIIB. additional test of specificity, we carried out immuno- To provide an additional test for an association between precipitations with the anti-RB antibody in the presence RB and TFIIIB, we examined whether these factors co- of blocking agents. A short GST–RB polypeptide con- fractionate during gradient chromatography on heparin– taining the region of RB that is recognized by this antibody Sepharose. A phosphocellulose fraction containing RB was able to block the co-precipitation of TBP and BRF and TFIIIB was applied to a heparin–Sepharose column (Figure 7B, lanes 3 and 8). In contrast, an equal amount and the bound proteins were eluted with a linear salt of GST protein did not prevent the immunoprecipitation gradient. Individual fractions were assayed for their content of these subunits (Figure 7B, lanes 2 and 7). These of TFIIIB (Figure 6B, upper panel) and RB (Figure 6B, experiments provide direct evidence that endogenous RB lower panel). Neither TFIIIB nor RB was detectable before associates with TFIIIB at physiological concentrations. fraction 49; both factors began to elute in fraction 50, peaked in fractions 51 and 52, and then tailed off gradually Extracts of cells from RB-knockout mice have in the next few fractions. Thus, RB co-fractionates closely elevated TFIIIB activity with TFIIIB on a heparin–Sepharose salt gradient. We have shown previously that endogenous RB represses These data demonstrate that a population of RB mole- pol III transcription in primary fibroblasts from embryonic cules co-purifies with TFIIIB on phosphocellulose, DEAE– mice (White et al., 1996). If this effect is achieved through Sephadex and heparin–Sepharose columns, and on glycerol the repression of TFIIIB, then we would expect to find gradients. We have also found RB in TFIIIB fractions that an increase in TFIIIB activity in these cells following were prepared using Q-Sepharose, Mono Q and Macro- disruption of the Rb gene. To test this, we prepared Prep CM resins (data not shown). Such consistent co- extracts from primary cells that had been isolated from fractionation is suggestive of a stable interaction. In either wild-type or RB-knockout mice (Jacks et al., 1992). contrast, little or no RB was detected in fractions con- The RB-negative extracts supported higher rates of pol taining pol III, TFIIIC or the TBP-containing complex III transcription than the RB-positive extracts (Figure 8A). SL1 (Figure 6A). Together, these results suggest that there As a control for factor recovery, we compared the levels is a specific physical and functional interaction between of TFIIIC in these extracts and detected little or no endogenous RB and TFIIIB. difference (Figure 8B). In contrast, when TFIIIB activity 2065 C.G.C.Larminie et al. TFIIIB is required for the expression of all class III genes, its targeting by RB provides an explanation for the general increase in pol III transcription that is observed in RB- knockout mice (White et al., 1996). RB can repress transcription from a preformed pol III initiation complex The ability of RB to regulate transcription is believed to be restricted to a defined time window during the cell cycle. Thus, RB is active during early G phase, is inactivated at the G /S transition, and then remains inactive until the end of mitosis (reviewed by Weinberg, 1995; Whyte, 1995). Since DNA replication erases initiation complexes from class III genes (Wolffe and Brown, 1986), complexes must reform during S and G phases, when RB is believed to be inactive. For RB to repress TFIIIB during the subsequent G phase, it may need to regulate transcription complexes that had assembled during the previous cycle. We have confirmed that RB is indeed capable of inactivating a preassembled pol III preinitiation complex. Alternatively, it is possible that the pol III preinitiation complex is disrupted during mitosis; in this case RB would inhibit complexes that reform during the early part of G . Fig. 6. A population of RB molecules co-fractionates with TFIIIB. (A) RB is readily detectable in fractions containing TFIIIB but not in RB represses pol III transcription by inactivating fractions containing pol III, TFIIIC or SL1. Fractionated factors (15 μl) as indicated were resolved on an SDS–7.8% polyacrylamide TFIIIB gel and then analysed by Western immunoblotting with anti-RB We have presented several independent lines of evidence antibody G3-245. The TFIIIC fraction in lane 1 was affinity-purified that implicate TFIIIB as the component of the pol III and has a protein concentration of 93 ng/μl. The pol III fraction in transcription apparatus that is targeted by RB. (i) Addition lane 2 was A25(1.0) and has a protein concentration of 15 ng/μl. The of recombinant RB to reconstituted transcription reactions TFIIIB fraction in lane 3 was prepared by chromatography on phosphocellulose followed by glycerol gradient sedimentation; it has a causes a specific decrease in the activity of TFIIIB protein concentration of 200 ng/μl. The TFIIIB fraction in lane 4 was such that this factor becomes limiting for transcription. A25(0.15) and has a protein concentration of 80 ng/μl. The SL1 Repression by RB is overcome by the addition of more fraction in lane 5 was SS700 and has a protein concentration of TFIIIB, but not by TFIIIC or pol III. (ii) Recombinant RB 260 ng/μl. (B) RB co-fractionates with TFIIIB during gradient chromatography on heparin–Sepharose. The upper panel shows TFIIIB that has been immobilized on beads depletes fractions of activity of individual fractions and the lower panel shows the RB TFIIIB, but not TFIIIC activity. The BRF and TBP content of the same fractions. In each case lane 1 contains buffer, lane subunits of TFIIIB were shown to bind to the immobilized 2 contains input material (PC-B), lane 3 contains flow-through, and RB. (iii) A population of RB molecules co-purifies with lanes 4–18 contain fractions 46–60, respectively. TFIIIB activity was TFIIIB during a variety of distinct fractionation pro- assayed using 4 μl of the indicated fraction, 2 μl of PC-C and 500 ng of pVA ; after 15 min incubation at 30°C, nucleotides were added to I cedures. In contrast, little or no RB is detected in fractions assay transcription. RB content was assayed using 14 μlofthe containing TFIIIC, pol III or SL1. Although we cannot indicated fractions which were resolved on an SDS–7.8% exclude the possibility that the co-purification of TFIIIB polyacrylamide gel and then analysed by Western immunoblotting with and RB is fortuitous, we consider that this is unlikely anti-RB antibody G3-245. given the consistency with which it is observed under a variety of distinct chromatographic conditions. We have also fractionated TFIIIB from SAOS2 cells, which contain was measured by complementation assays, it was found inactive mutant RB. We find that TFIIIB can be readily to be significantly elevated in the RB-negative extracts separated from SAOS2 RB (data not shown), which (Figure 8C). We conclude that TFIIIB is a physiological supports the idea that co-fractionation of TFIIIB and RB target for repression by RB in mouse embryonic fibroblasts. reflects a functional interaction. In addition, we have examined the fractionation properties of RB that has been expressed in Sf9 cells. This recombinant RB displayed Discussion chromatographic behaviour that was distinct from that of We have shown previously that endogenous RB plays an TFIIIB (data not shown). It is therefore unlikely that the important role in suppressing the level of pol III transcrip- co-fractionation of TFIIIB with a population of cellular tion in primary murine fibroblasts (White et al., 1996). In RB molecules is due to these factors possessing identical this study, we have addressed the molecular mechanism chromatographic properties. The most plausible explana- of this regulation. We have demonstrated that RB is able tion of the co-purification data is that a proportion of to disrupt the function of a preformed pol III transcription endogenous TFIIIB exists in association with RB. (iv) complex. We have also shown that it can regulate the TFIIIB subunits (TBP and BRF) co-immunoprecipitate activity of each type of pol III promoter. This appears to with RB. This effect is specific, since it is not seen using be achieved by the specific inactivation of TFIIIB. Since control antibodies and it can be blocked using an excess 2066 RB represses TFIIIB Fig. 7. TBP and BRF co-immunoprecipitate with RB. (A) Proteins from PC-B that were immunoprecipitated using anti-TBP antibody bound to protein A–Sepharose (lanes 2 and 6), protein A–Sepharose alone (lanes 3 and 7) or anti-RB antibody bound to protein A–Sepharose (lanes 4 and 8) were resolved on an SDS–7.8% polyacrylamide gel and then analysed by Western immunoblotting with antibodies against TBP (lanes 1–4) or BRF (lanes 5–8). Lane 1 contains 10 ng of recombinant TBP. Lane 5 contains 15 μl of PC-B. (B) Proteins from PC-B that were immunoprecipitated using anti-RB antibody (lanes 2, 3, 7 and 8), anti-TBP antibody (lanes 4 and 9) or anti-Sp1 antibody (lanes 5 and 10) were resolved on an SDS–7.8% polyacrylamide gel and then analysed by Western immunoblotting with antibodies against TBP (lanes 1–5) or BRF (lanes 6–10). The reactions in lanes 2 and 7 contained 1.5 μg of GST. The reactions in lanes 3 and 8 contained 1.5 μg of GST–RB(379–611). Lane 1 contains 10 ng of recombinant TBP. Lane 6 contains 15 μl of PC-B. Fig. 8. Primary fibroblasts from RB-knockout mice have elevated TFIIIB activity. (A) Transcription of pVA (250 ng) using whole-cell extract / –/– (24 μg) prepared from RB (lanes 1 and 3) or RB (lanes 2 and 4) primary fibroblasts. (B) Band shift assay using 4 ng of radiolabelled B-block –/– / oligonucleotide probe, 500 ng of poly(dI-dC), no protein (lane 1), 12.7 μgofRB extract (lane 2) or 12.7 μgofRB extract (lanes 3–5) and 200 ng of B-block (lane 4) or MSV (lane 5) oligonucleotide competitor. (C) Transcription of pVA (500 ng) using 4 μl of PC-C and 5 ng of recombinant / –/– TBP (all lanes) and 25 μg of heat-treated RB extract (lane 1), 25 μg of heat-treated RB extract (lane 2), no addition (lane 3) or 3 μl of heat-treated Mono Q-purified TFIIIB (lane 4). of the epitope. In addition to detecting TBP and BRF in shown). (v) TFIIIB activity is elevated in extracts immunoprecipitates from an anti-RB antibody, we have prepared from RB-knockout fibroblasts. This provides also co-precipitated RB using antibodies against TBP and genetic evidence for a functional interaction between BRF (data not shown). These data provide direct evidence TFIIIB and RB. We consider that these independent for an interaction between RB and TFIIIB. We were lines of evidence together provide a compelling case for unable to detect this interaction using a TFIIIB fraction believing that RB regulates pol III transcription by target- from SAOS2 cells, in which RB is inactive (data not ing the general factor TFIIIB. 2067 C.G.C.Larminie et al. Fig. 9. Sequence homology between RB, TBP and BRF. The regions of RB that are related to TBP and BRF are indicated and the alignments are shown underneath. Sequence identities are indicated with black boxes and sequence similarities (using highly stringent criteria) are indicated with grey boxes. Adenovirus infection RB activity is high (in those experiments it was added in Although E1A can overcome the repressive effects of RB, excess) (White et al., 1996). In contrast to the results in there is little or no increase in TFIIIB activity when HeLa HeLa cells, we would predict that TFIIIB activity will cells are infected by adenovirus (Hoeffler and Roeder, increase significantly during adenovirus infection of cells 1985; Yoshinaga et al., 1986). Instead, the activation of such as fibroblasts that have high RB activity. pol III transcription that accompanies adenoviral infection is mediated by changes in TFIIIC (Hoeffler and Roeder, RB may bind TFIIIB by mimicking TBP and BRF 1985; Yoshinaga et al., 1986; Sinn et al., 1995). We Our results indicate that TFIIIB is subject to repression believe that the explanation for this apparent paradox lies by RB, but do not determine which of its subunits is in the fact that RB activity is very low in HeLa cells. targeted. The fact that repression is overcome using Although accurate quantitation is extremely difficult, our affinity-purified TFIIIB TAFs but not TBP alone suggests best estimate is that 5–10% of TFIIIB is associated with that RB targets one or more of the pol III TAFs. However, RB in extracts made from uninfected asynchronous HeLa the possibility remains that an unidentified intermediate cells (data not shown). Even if E1A displaces all of this component is required for the association between RB RB during adenovirus infection, the overall effect on total and TFIIIB. Proof of direct binding will require interaction TFIIIB activity is unlikely to be detectable. In order to studies with purified and separated subunits of TFIIIB. stimulate pol III transcription significantly, adenovirus Such analyses are hampered at present by the uncertainty must employ an additional mechanism. The activation of concerning the precise composition of human TFIIIB. TFIIIC is the decisive step in stimulating pol III transcrip- Much of the function of RB is carried out by a region tion during infection of HeLa cells (Hoeffler and Roeder, called the ‘pocket’ that stretches between residues 379 1985; Yoshinaga et al., 1986). and 792 (Weinberg, 1995; Whyte, 1995). The pocket is The relatively small proportion of TFIIIB that associates composed of two essential regions, A and B, that are with RB in HeLa cells is consistent with the high pol III separated by a spacer, the sequence of which is not activity of this line. It is likely that HPV E7 protein that required for function (Weinberg, 1995; Whyte, 1995). is expressed in these cells may help overcome repression Disruption of either the A domain or the B domain can by RB, although we have yet to confirm this for pol III. prevent RB from repressing pol III (White et al., 1996). In other cell types, RB appears to play a much more It has been observed previously that the A domain of the significant role in repressing pol III. This is particularly RB pocket bears 21% identity and 38% similarity to a obvious in mouse embryo fibroblasts, where deletion of 151 amino acid region from the C-terminus of TBP RB causes a 5-fold increase in pol III transcription (White (Hagemeier et al., 1993). The C-terminal domain of TBP et al., 1996). We have shown previously that E1A can that encompasses this homology is sufficient to support overcome repression by RB under conditions in which pol III transcription (White and Jackson, 1992). We found 2068 RB represses TFIIIB activated at the G /S transition, it seems likely that the increase in pol III transcription that accompanies progression into S phase may reflect, at least in part, the release of TFIIIB from inhibition by RB. We are currently investigating this possibility. The existing data suggest that a common mechanism for achieving cell cycle control of transcription by pols II and III may involve the regulation of TAF activity by RB. Fig. 10. Model proposing that RB may bind and inactivate TFIIIB by Materials and methods mimicking TBP and BRF. Plasmids The plasmids used in this work have all been described previously that an extended 230 amino acid region of BRF (residues (White et al., 1989, 1995a). 96–326) displays 15% identity and 33% similarity to the B domain and C-terminal region (residues 665–910) of Protein fractions RB (Figure 9). This homology was assessed using very Nuclear extracts were prepared from proliferating HeLa cells by the stringent criteria for similarity (R/K, E/D, S/T, F/Y and method of Dignam et al. (1983). The extracts were then chromato- L/I/V/M). Thus, RB bears homology to two of the subunits graphed on phosphocellulose according to the method of Segall et al. (1980). Direct assay confirmed that PC-B fractions (0.1–0.35 M KCl of the TFIIIB complex that it is able to regulate. step) contained TFIIIB and pol III, PC-C fractions (0.35–0.6 M KCl Because the level of homology between RB, TBP and step) contained TFIIIC and pol III, and PC-D fractions (0.6–1.0 M KCl BRF is relatively low, one cannot be certain that it is step) contained SL1. significant. However, we are struck by the fact that adjacent The 0.38M-TFIIIB and 0.48M-TFIIIB fractions were prepared from PC-B by Mono Q gradient chromatography, as described by Lobo et al. regions of RB bear a resemblance to two polypeptides that (1992). Fractions were dialysed into LDB buffer (20 mM HEPES–KOH, interact within TFIIIB. This apparent resemblance could pH 7.9, 17% glycerol, 100 mM KCl, 12 mM MgCl , 0.1 mM EDTA, provide the key to the mechanism of repression. The 2 mM DTT). The 0.38M-TFIIIB fraction eluted at ~340 mM KCl and adjacent homologous regions may allow RB to present a had a protein concentration of 0.45 mg/ml. The 0.48M-TFIIIB fraction eluted at ~450 mM KCl and had a protein concentration of 0.11 mg/ml. molecular surface that resembles TBP alongside BRF. Western immunoblotting analysis revealed that TBP and BRF are present This could enable it to associate with another TAF subunit in the 0.38M-TFIIIB but not in the 0.48M-TFIIIB (Mital et al., 1996). through a similar set of interactions to those that occur in The A25(0.15) and A25(1.0) fractions were prepared by chromatography intact TFIIIB. We therefore propose a model in which RB of PC-B fractions on DEAE–Sephadex, as previously (White et al., is able to bind TFIIIB by mimicking the interaction surface 1995a). A25(0.15) is a 50–150 mM (NH ) SO step fraction; it contains 4 2 4 TFIIIB and has a protein concentration of 0.08 mg/ml. A25(1.0) is a that is presented by TBP alongside BRF (Figure 10). 0.15–1.0 M (NH ) SO step fraction; it contains pol III and has a protein 4 2 4 Clearly, this does not displace TBP and BRF, since our concentration of 0.015 mg/ml. For glycerol gradient fractionation of pull-down and immunoprecipitation assays have shown TFIIIB, PC-B was sedimented for 24 h at 39 000 r.p.m. in an that these polypeptides remain associated with RB. Addi- SW40Ti rotor on a 15–50% glycerol gradient in 50 mM Tris–HCl, pH 7.9, 200 mM KCl, 0.5 mM EDTA, 12.5 mM MgCl , 1 mM DTT, tional interactions between subunits may hold the inactive 0.1 mM PMSF. Fractions were then dialysed into LDB. For heparin complex together in the presence of RB. gradient chromatography, PC-B was applied to heparin–Sepharose CL- 6B in PC buffer (20 mM HEPES–KOH, pH 7.9, 20% glycerol, 0.2 mM Regulation of TAF activity by RB may provide a EDTA, 0.5 mM DTT) containing 100 mM KCl. After extensive washing, common mechanism for the cell cycle control of the bound protein was eluted using a linear gradient up to 1.2 M KCl. Fractions were dialysed into LDB. Recombinant TBP, affinity-purified transcription by pols II and III TFIIIB TAFs (B-TAF), and the corresponding control fraction (CON) We have presented evidence that RB is able to regulate were prepared as previously (White et al., 1995a). pol III transcription by interacting with the TFIIIB com- The CHep-1.0 fraction was prepared by chromatography of PC-C on plex. This finding is reminiscent of recent data which heparin–Sepharose CL-6B, as previously (White et al., 1995a). CHep- show that RB can bind to the TAF 250 subunit of 1.0 contained TFIIIC and pol III, and had a protein concentration of II 0.6 mg/ml. Affinity-purified TFIIIC was prepared as previously (White the pol II TFIID complex (Shao et al., 1995). Thus, et al., 1995a) and had a protein concentration of 0.093 mg/ml. association with class-specific TAFs appears to provide The SS700 fraction was prepared by chromatography of PC-D one mechanism by which RB is able to regulate gene fractions on S-Sepharose. PC-D was applied to S-Sepharose in AM expression. In contrast to the situation with TFIID and buffer (20 mM Tris–HCl, pH 7.9, 0.1 mM EDTA, 20% glycerol, 5 mM MgCl , 1 mM DTT, 1 mM PMSF) plus 100 mM KCl. After washing TFIIIB, we detected no RB in fractions containing the 2 with this buffer, the column was eluted with AM buffer plus 320 mM pol I-specific TBP-containing complex SL1 (Figure 6A). KCl to generate the SS320 fraction, and then with AM buffer plus Instead, RB is reported to control pol I transcription by 700 mM KCl to generate the SS700 fraction. Peak fractions were binding to the class-specific regulator UBF (Cavanaugh dialysed into LDB. The SS700 fraction contained SL1 and had a protein concentration of 0.26 mg/ml. et al., 1995). Recombinant GST and GST–RB fusion proteins were prepared as Both genetic and biochemical data have implicated previously (White et al., 1996). Significant batch variations were noted TAF 250 in regulating the passage through G phase II 1 in the specific activities of different GST–RB preparations. The reason (Sekiguchi et al., 1988, 1991; Hisatake et al., 1993; for this is unknown. Ruppert et al., 1993; Wang and Tjian, 1994). It remains Transcription and band shift assays to be determined to what extent RB is involved in this Transcription and band shift assays were carried out as described effect. We have shown previously that the activity of a previously (White et al., 1989), except that transcription was for1hat TAF component of TFIIIB increases significantly when 30°C and band-shifts were run in 0.25 TBE. The B-block and MSV cells progress from early G into S phase (White et al., 1 oligonucleotides have been described previously (White et al., 1989). 1995b). Since RB represses TFIIIB and becomes in- Transcription reactions that were reconstituted using fractionated 2069 C.G.C.Larminie et al. factors contained 1 μl each of 0.38M-TFIIIB (0.45 μg) and 0.48M- Hisatake,K., Hasegawa,S., Takada,R., Nakatani,Y., Horikoshi,M. and TFIIIB (0.11 μg) and 2 μl of CHep-1.0 (1.2 μg) fractions. Roeder,R.G. (1993) The p250 subunit of native TATA box-binding factor TFIID is the cell-cycle regulatory protein CCG1. Nature, 362, Pull-down assays 179–181. PC-C or PC-B fractions (25 μl) were incubated at 4°C on an orbital Hoeffler,W.K. and Roeder,R.G. (1985) Enhancement of RNA polymerase shaker with 25 μl of glutathione–agarose beads containing equivalent III transcription by the E1A gene product of adenovirus. Cell, 41, amounts (as estimated by Coomassie blue staining) of immobilized GST 955–963. or GST–RB (379–928). After 10 min, the beads were pelleted and 4 μl Jacks,T., Fazeli,A., Schmitt,E.M., Bronson,R.T., Goodell,M.A. and aliquots of supernatant were removed for transcription assays. The Weinberg,R.A. (1992) Effects of an Rb mutation in the mouse. Nature, remainder of the samples were incubated at 4°C on the orbital shaker 359, 295–300. for a further 3 h. Samples were then pelleted, supernatants removed and Kassavetis,G.A., Nguyen,S.T., Kobayashi,R., Kumar,A., Geiduschek,E.P. the beads were washed five times with 700 μl of LDB. The bound and Pisano,M. (1995) Cloning, expression, and function of TFC5, the material was analysed by Western blotting. gene encoding the B’ component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB. Proc. Natl Acad. Sci. Western blotting USA, 92, 9786–9790. Western immunoblotting was carried out as previously (White et al., Khoo,B., Brophy,B. and Jackson,S.P. (1994) Conserved functional 1995a). The antibodies used for immunoblotting were G3-245 domains of the RNA polymerase III general transcription factor BRF. (Pharminogen) against RB, SL30 against TBP and α-CSH409 against Genes Dev., 8, 2879–2890. BRF. Lassar,A.B., Martin,P.L. and Roeder,R.G. (1983) Transcription of class III genes: formation of preinitiation complexes. Science, 222, 740–748. Immunoprecipitation Lobo,S.M., Tanaka,M., Sullivan,M.L. and Hernandez,N. (1992) A TBP PC-B fractions (50 μl) were incubated at 4°C for 6 h on an orbital complex essential for transcription from TATA-less but not TATA- shaker with 25 μl of protein A–Sepharose beads containing prebound containing RNA polymerase III promoters is part of the TFIIIB antibodies. Samples were then pelleted, supernatants removed and the fraction. Cell, 71, 1029–1040. beads were washed four times with 700 μl of LDB and once with 700 μl Loeken,M., Bikel,I., Livingston,D.M. and Brady,J. (1988) Trans- of LDB containing 200 mM KCl and 0.1% NP-40. The bound material activation of RNA polymerase II and III promoters by SV40 small t was analysed by Western blotting. The antibodies used for immuno- antigen. Cell, 55, 1171–1177. precipitation were G99-549 (Pharminogen) against RB, MTBP-6 against Lopez-de-Leon,A., Librizzi,M., Tuglia,K. and Willis,I. (1992) PCF4 TBP and IC6 against Sp1. encodes an RNA polymerase III transcription factor with homology to TFIIB. Cell, 71, 211–220. Primary embryonic fibroblasts –/– / Mital,R., Kobayashi,R. and Hernandez,N. (1996) RNA polymerase III Early passage primary cells from Rb and Rb mice (Jacks et al., transcription from the human U6 and adenovirus type 2 VAI promoters 1992) were grown in Dulbecco’s modified Eagle’s medium supplemented has different requirements for human BRF, a subunit of human TFIIIB. with 20% fetal calf serum. Whole-cell extracts were prepared as Mol. Cell. Biol., 16, 7031–7042. previously (White et al., 1995a). Nasmyth,K. (1996) Another role rolls in. Nature, 382, 28–29. Patel,G. and Jones,N.C. (1990) Activation in vitro of RNA polymerase Acknowledgements II and III directed transcription by baculovirus produced E1A protein. Nucleic Acids Res., 18, 2909–2915. We are grateful to James Millner-White for advice concerning sequence Pines,J. (1995) Cyclins, CDKs and cancer. Semin. Cancer Biol., 6, 63–72. homology, Bart Williams and George Mulligan for primary embryonic Qin,X., Chittenden,T., Livingston,D.M. and Kaelin,W.G. (1992) Identi- fibroblasts, Sibylle Mittnacht for Sf9-expressed RB, Jane Flint for fication of a growth suppression domain within the retinoblastoma antibody MTBP-6 and Nouria Hernandez for antibodies SL30 and α- gene product. Genes Dev., 6, 953–964. CSH409. This work was funded by the following grants: SP2314/0101 Rigby,P.W.J. (1993) Three in one and one in three: it all depends on to R.J.W. and SP2143/0101 to S.P.J. from the Cancer Research Campaign, TBP. Cell, 72, 7–10. CO5766 to R.J.W. from the Biotechnology and Biological Sciences Roberts,S., Miller,S.J., Lane,W.S., Lee,S. and Hahn,S. (1996) Cloning Research Council, SCI/180/94/365/G to R.J.W. from the and functional characterization of the gene encoding the TFIIIB90 Nuffield Foundation and NIH R01GM38810 to Nouria Hernandez. subunit of RNA polymerase III transcription factor TFIIIB. J. Biol. R.J.W. is a Jenner Fellow of the Lister Institute of Preventive Medicine. Chem., 271, 14903–14909. Ruppert,S., Wang,E.H. and Tjian,R. (1993) Cloning and expression of human TAF 250: a TBP-associated factor implicated in cell-cycle References II regulation. Nature, 362, 175–179. Buratowski,S. and Zhou,H. (1992) A suppressor of TBP mutations Ruth,J., Conesa,C., Dieci,G., Lefebvre,O., Dusterhoft,A., Ottonello,S. encodes an RNA polymerase III transcription factor with homology and Sentenac,A. (1996) A suppressor of mutations in the class III to TFIIB. Cell, 71, 221–230. transcription system encodes a component of yeast TFIIIB. EMBO J., Cavanaugh,A.H., Hempel,W.M., Taylor,L.J., Rogalsky,V., Todorov,G. 15, 1941–1949. and Rothblum,L.I. (1995) Activity of RNA polymerase I transcription Segall,J., Matsui,T. and Roeder,R.G. (1980) Multiple factors are required factor UBF blocked by Rb gene product. Nature, 374, 177–180. for the accurate transcription of purified genes by RNA polymerase Chiang,C.-M., Ge,H., Wang,Z., Hoffmann,A. and Roeder,R.G. (1993) III. J. Biol. Chem., 255, 11986–11991. Unique TATA-binding protein-containing complexes and cofactors Sekiguchi,T., Miyata,T. and Nishimoto,T. (1988) Molecular cloning of involved in transcription by RNA polymerases II and III. EMBO J., the cDNA of human X chromosomal gene (CCG1) which complements 12, 2749–2762. the temperature-sensitive G mutants, tsBN462 and ts13, of the BHK Colbert,T. and Hahn,S. (1992) A yeast TFIIB-related factor involved in cell line. EMBO J., 7, 1683–1687. RNA polymerase III transcription. Genes Dev., 6, 1940–1949. Sekiguchi,T., Nohiro,Y., Nakamura,Y., Hisamoto,N. and Nishimoto,T. Dignam,J.D., Lebovitz,R.M. and Roeder,R.G. (1983) Accurate (1991) The human CCG1 gene, essential for progression of the G1 transcription initiation by RNA polymerase II in a soluble extract phase, encodes a 210-kilodalton nuclear DNA-binding protein. Mol. from isolated mammalian nuclei. Nucleic Acids Res., 11, 1475–1489. Cell. Biol., 11, 3317–3325. Geiduschek,E.P. and Kassavetis,G.A. (1995) Comparing transcriptional Shao,Z., Ruppert,S. and Robbins,P.D. (1995) The retinoblastoma- initiation by RNA polymerases I and III. Curr. Opin. Cell Biol., 7, susceptibility gene product binds directly to the human TATA-binding 344–351. protein-associated factor TAF 250. Proc. Natl Acad. Sci. USA, 92, Hagemeier,C., Bannister,A.J., Cook,A. and Kouzarides,T. (1993) The II 3115–3119. activation domain of transcription factor PU.1 binds the retinoblastoma Sinn,E., Wang,Z., Kovelman,R. and Roeder,R.G. (1995) Cloning and (RB) protein and the transcription factor TFIID in vitro: RB shows characterization of a TFIIIC2 subunit (TFIIICβ) whose presence sequence similarity to TFIID and TFIIB. Proc. Natl Acad. Sci. USA, correlates with activation of RNA polymerase III-mediated 90, 1580–1584. Hernandez,N. (1993) TBP, a universal eukaryotic transcription factor? transcription by adenovirus E1A expression and serum factors. Genes Genes Dev., 7, 1291–1308. Dev., 9, 675–685. 2070 RB represses TFIIIB Taggart,A.K.P., Fisher,T.S. and Pugh,B.F. (1992) The TATA-binding protein and associated factors are components of pol III transcription factor TFIIIB. Cell, 71, 1015–1028. Teichmann,M. and Seifart,K.H. (1995) Physical separation of two different forms of human TFIIIB active in the transcription of the U6 or the VAI gene in vitro. EMBO J., 14, 5974–5983. Wang,E. and Tjian,R. (1994) Promoter-selective transcriptional defect in cell cycle mutant ts13 rescued by hTAF 250. Science, 263, 811–814. II Wang,Z. and Roeder,R.G. (1995) Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains. Proc. Natl Acad. Sci. USA, 92, 7026–7030. Weinberg,R.A. (1995) The retinoblastoma protein and cell cycle control. Cell, 81, 323–330. White,R.J. (1994) RNA Polymerase III Transcription. R.G.Landes Company, Austin, TX. White,R.J. (1997) Regulation of RNA polymerases I and III by the retinoblastoma protein: a mechanism for growth control? Trends Biochem. Sci., 22, 77–80. White,R.J. and Jackson,S.P. (1992) Mechanism of TATA-binding protein recruitment to a TATA-less class III promoter. Cell, 71, 1041–1053. White,R.J., Stott,D. and Rigby,P.W.J. (1989) Regulation of RNA polymerase III transcription in response to F9 embryonal carcinoma stem cell differentiation. Cell, 59, 1081–1092. White,R.J., Gottlieb,T.M., Downes,C.S. and Jackson,S.P. (1995a) Mitotic regulation of a TATA-binding-protein-containing complex. Mol. Cell. Biol., 15, 1983–1992. White,R.J., Gottlieb,T.M., Downes,C.S. and Jackson,S.P. (1995b) Cell cycle regulation of RNA polymerase III transcription. Mol. Cell. Biol., 15, 6653–6662. White,R.J., Trouche,D., Martin,K., Jackson,S.P. and Kouzarides,T. (1996) Repression of RNA polymerase III transcription by the retinoblastoma protein. Nature, 382, 88–90. Whyte,P. (1995) The retinoblastoma protein and its relatives. Semin. Cancer Biol., 6, 83–90. Willis,I.M. (1993) RNA polymerase III. Genes, factors and transcriptional specificity. Eur. J. Biochem., 212, 1–11. Wolffe,A.P. and Brown,D.D. (1986) DNA replication in vitro erases a Xenopus 5S RNA gene transcription complex. Cell, 47, 217–227. Yoshinaga,S.K., Dean,N., Han,M. and Berk,A.J. (1986) Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration. EMBO J., 5, 343–354. Received on July 5, 1996; revised on January 7, 1997 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein

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10.1093/emboj/16.8.2061
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Abstract

The EMBO Journal Vol.16 No.8 pp.2061–2071, 1997 Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein 1 1 We have demonstrated recently a novel function for RB Christopher G.C.Larminie , Carol A.Cairns , 2 3 in controlling RNA polymerase (pol III) transcription Renu Mital , Klaus Martin , 3 3 (White et al., 1996). Overexpression of RB represses pol Tony Kouzarides , Stephen P.Jackson and 1,3,4 III templates in transfected cells, whereas control pol II Robert J.White promoters are unaffected. Furthermore, purified recombin- Institute of Biomedical and Life Sciences, Division of Biochemistry ant RB inhibits pol III transcription when added to and Molecular Biology, Davidson Building, University of Glasgow, a system reconstituted with fractionated factors. These Glasgow, G12 8QQ, UK, Cold Spring Harbor Laboratory, experiments demonstrate that RB can repress pol III when Cold Spring Harbor, NY 11724, USA and Wellcome/CRC Institute, it is overexpressed. To determine whether endogenous Tennis Court Road, Cambridge CB2 1QR and Departments of Zoology and Pathology, University of Cambridge, Cambridge, UK levels of RB can control pol III under physiological circumstances, we investigated the expression of class III Corresponding author genes in cells that have lost RB function (White et al., 1996). When two human osteosarcoma lines were com- The tumour suppressor protein RB restricts cellular pared, RB-deficient SAOS2 cells were found to have a growth. This may involve inhibiting the synthesis of more active pol III transcription apparatus than RB- tRNA and 5S rRNA by RNA polymerase (pol) III. We positive U2OS cells. In addition, primary fibroblasts from have shown previously that RB can repress pol III RB-knockout mice were shown to have much higher pol transcription when overexpressed either in vitro or III activity than primary fibroblasts from wild-type mice. in vivo. We also demonstrated that pol III activity is No general increase in pol II activity was seen when elevated substantially in primary fibroblasts from SAOS2 cells were compared with U2OS cells or when RB-deficient mice. Here we address the molecular –/– / Rb fibroblasts were compared with Rb fibroblasts. mechanism of this regulation. RB is shown to repress These experiments established that endogenous RB plays all types of pol III promoter. It can do this even if an important role in suppressing pol III transcription. added after transcription complex assembly. Func- The minimal region of RB that is required for growth tional assays demonstrate that RB targets specifically suppression comprises amino acid residues 379–928 (Qin the general pol III factor TFIIIB. A physical interaction et al., 1992). The same sequences are sufficient to inhibit between TFIIIB and RB is indicated by fractionation, pol III transcription (White et al., 1996). Various deletions pull-down and immunoprecipitation data. We show that remove segments from within this region abolish the that TFIIIB activity is elevated in primary fibroblasts ability of RB to regulate pol III (White et al., 1996). from RB-deficient mice. TFIIIB is a multisubunit Several naturally occurring mutations that prevent RB complex that includes the TATA-binding protein (TBP) from functioning as a tumour suppressor also inhibit its and a TFIIB-related factor called BRF. We show that ability to repress pol III (White et al., 1996). This raises RB itself contains regions of homology to both TBP the possibility that regulation of pol III contributes to and BRF and propose a model in which RB disrupts the tumour suppressive activity of RB (Nasmyth, 1996; TFIIIB by mimicking these two components. Keywords: RB/RNA polymerase III/TBP/TFIIIB/ White, 1997). transcription The viral oncoproteins SV40 large T antigen and adenovirus E1A can bind to RB and neutralize its function (Whyte, 1995). Both T antigen and E1A can also activate pol III transcription (Loeken et al., 1988; Patel and Jones, Introduction 1990; White et al., 1996). One way in which they may achieve this is by overcoming the repression of pol III The retinoblastoma protein (RB) is a 105 kDa nuclear transcription by RB (White et al., 1996). The ability of phosphoprotein that is encoded by an important tumour these oncoproteins to activate pol III by relieving the suppressor gene (reviewed by Weinberg, 1995; Whyte, physiological constraint that is normally provided by RB 1995). In normal cells, RB is involved in constraining may contribute to their transforming capability. growth and proliferation; in its absence, the ability of cells Transcription by pol III involves at least two general to shut down these functions is compromised (Weinberg, factors, named TFIIIB and TFIIIC (reviewed by Willis, 1995; Whyte, 1995). RB is mutated in a variety of tumours, 1993; White, 1994; Geiduschek and Kassavetis, 1995). including retinoblastomas, small-cell lung carcinomas, TFIIIB is a multisubunit complex that contains the TATA- sarcomas and bladder carcinomas (Weinberg, 1995). In many other human malignancies, the function of RB is binding protein TBP (Hernandez, 1993; Rigby, 1993). lost due to the disruption of upstream control pathways One of the TBP-associated factors (TAFs) in TFIIIB is (Pines, 1995; Weinberg, 1995). It is therefore of consider- structurally and functionally related to the pol II general able importance to understand fully the ways in which factor TFIIB (Buratowski and Zhou, 1992; Colbert and RB is able to influence cellular activity. Hahn, 1992; Lopez-de-Leon et al., 1992; Khoo et al., © Oxford University Press 2061 C.G.C.Larminie et al. 1994; Wang and Roeder, 1995; Mital et al., 1996). This to be sufficient to allow VA to recruit TFIIIB into a TAF has been variously named TDS4, PCF4, BRF and stable complex and thereby exclude expression of the TFIIIB90, but we shall refer to it as BRF. Although second gene. yeast TFIIIB has been reconstituted from recombinant Having established that these conditions allow the stable components (Kassavetis et al., 1995; Roberts et al., 1996; assembly of TFIIIC and TFIIIB onto the VA promoter, Ruth et al., 1996), the mammalian factor is much less we then tested whether RB is able to disrupt the preformed well characterized (reviewed by Hernandez, 1993; Rigby, complex (Figure 1C). The pol III factors were mixed with 1993). This partly reflects a tendency for TFIIIB to either glutathione S-transferase (GST) or a GST fusion dissociate during purification (Lobo et al., 1992; Taggart protein containing residues 379–928 of RB. The recombin- et al., 1992; Chiang et al., 1993; Teichmann and Seifart, ant proteins were added either 15 min prior to the addition 1995). Several groups have identified polypeptides as of VA DNA (Figure 1C, lanes 1 and 2), simultaneously candidate TAFs for human TFIIIB (Lobo et al., 1992; with the addition of VA (lanes 3 and 4) or 15 min after Taggart et al., 1992; Chiang et al., 1993; Teichmann and the factors were mixed with VA DNA (lanes 5 and 6). Seifart, 1995; Wang and Roeder, 1995; Mital et al., 1996), Nucleotides were then added to allow transcription. RB but there is little consensus and BRF is the only subunit was found to repress transcription to a similar extent which has been cloned and had its function established whether it was added before, during or after initiation categorically (Wang and Roeder, 1995; Mital et al., 1996). complex assembly. Most pol III templates lack a TATA box and so are not recognized directly by TFIIIB; in these cases, TFIIIB is RB represses all types of pol III promoter recruited via protein–protein interactions with promoter- Three distinct types of promoter structure are utilized by bound TFIIIC (reviewed by Willis, 1993; White, 1994; pol III, and this diversity reflects clear differences in Geiduschek and Kassavetis, 1995). transcription factor requirements: type 1 promoters have In the current work, we have investigated the mechan- internal A and C blocks and are unique to 5S rRNA istic basis of pol III regulation by RB. We demonstrate genes; type 2 promoters have internal A and B blocks and that RB is able to disrupt the activity of a preassembled are utilized by most pol III templates, including tRNA pol III transcription complex. We also show that it can and VA genes; type 3 promoters are located entirely inhibit expression from all types of pol III promoter. upstream of the transcription unit, include a TATA box, Functional studies implicate TFIIIB as a specific target and are found in U6 and 7SK genes (reviewed by Willis, for repression by RB. In support of this, we find that a 1993; White 1994; Geiduschek and Kassavetis, 1995). We population of RB molecules consistently co-fractionates tested the ability of RB to regulate transcription directed with cellular TFIIIB. Furthermore, immunoprecipitation by each of these promoter types. Increasing quantities of and pull-down experiments demonstrate an association RB(379–928) were added to reactions containing 5S (type between RB and TFIIIB. RB itself contains a region of 1), VA (type 2) or U6 (type 3) templates (Figure 2). As homology to TBP that is followed by a region of homology controls, we added an equal amount of GST (Figure 2B) to BRF. We present a model in which RB inactivates or a GST fusion protein containing residues 612–711 of TFIIIB by mimicking TBP and BRF in order to disrupt RB (Figures 2A and C). We have demonstrated previously TFIIIB. that GST–RB(612–711) is unable to regulate VA transcrip- tion (White et al., 1996). With each template, transcription was strongly repressed by the GST–RB(379–928) protein, Results whereas an equal amount of the control protein had little RB can disrupt the activity of a preformed pol III or no effect. We conclude that RB is effective in repressing transcription complex transcription from all categories of pol III promoter. Fully formed pol III transcription complexes are extremely stable and support multiple rounds of transcription without RB inactivates TFIIIB specifically dissociating from the template (Lassar et al., 1983; Since RB can regulate each of these promoter types, it reviewed by White, 1994). We tested whether RB is able seemed likely that it acts upon a general component of to repress transcription from a preinitiation complex that the pol III transcription apparatus that is utilized by all had already assembled on the adenovirus VA gene (Figure of these templates. We therefore investigated its ability to 1). Initially, we used template commitment assays to inactivate the pol III enzyme and the general pol III factors establish that, under the conditions of our experiments, TFIIIB and TFIIIC. If RB represses transcription by a stable transcription complex had assembled on VA inhibiting a specific component, then it should be possible following a 15 min incubation. Figure 1A demonstrates to restore expression in the presence of RB by adding more that 15 min is sufficient for VA to recruit factors and of that component. We carried out add-back experiments to thereby exclude the expression of a second template that test whether this is the case. was added subsequently (lanes 4 and 5). However, pre- VA transcription was reconstituted using partially puri- emptive complex formation did not require the presence fied factors (Figure 3A). Recombinant RB repressed of TFIIIB (lanes 5 and 7). We therefore carried out expression in this system, as previously (Figure 3A, lanes additional assays to confirm that TFIIIB is recruited to 1 and 2). Inclusion of additional pol III or TFIIIC fractions the complex within the 15 min period. In these experiments did not restore transcription (lanes 3 and 4). In contrast, (Figure 1B), VA was again preincubated with a full set a fraction containing partially purified TFIIIB produced a of factors but was then challenged by a second template substantial reversal in the level of inhibition (lane 5). that had been preincubated separately with TFIIIC in the Figure 3B demonstrates that repression by RB can be absence of TFIIIB. The 15 min preincubation was found reversed completely in a dose-dependent manner using 2062 RB represses TFIIIB Fig. 1. RB inactivates pol III transcription whether added before, during or after initiation complex assembly. (A) Template 1 (500 ng), as indicated, was preincubated for 15 min at 30°C with either a full set of fractionated factors (all lanes except 5 and 7) or with 2 μl of CHep-1.0 alone (lanes 5 and 7). Template 2 (500 ng) was then added, together with 1 μl each of 0.38M-TFIIIB and 0.48M-TFIIIB in the case of lanes 5 and 7. Nucleotides were added immediately to initiate transcription. The VA template was pVA and the tRNA template was Mcet1. The short transcript that runs just below the tRNA in lanes 2–5 is derived from pVA .(B) Template 1 (500 ng), as indicated, was preincubated for 15 min at 30°C with a full set of fractionated factors. Template 2 (500 ng) was preincubated separately with 2 μl of CHep-1.0. The reactions were then mixed and nucleotides were added immediately to initiate transcription. The VA template was pVA and the B2 template was pAG38. (C) Fractionated factors were preincubated for 15 min at 30°C before the addition of pVA (250 ng); after a further 15 min at 30°C, nucleotides were added and transcription was allowed to proceed. Reactions were supplemented with 250 ng of GST or GST–RB(379–928), as indicated, which were added at the indicated times. Fig. 2. RB represses transcription of 5S, VA and U6 genes. (A) Transcription of pHu5S3.1 (250 ng) using nuclear extract (10 μg) preincubated (15 min at 30°C) with no addition (lanes 1 and 5) or with 100, 200 or 300 ng of GST–RB(379–928) (lanes 2, 3 and 4, respectively) or 100, 200 or 300 ng of GST–RB(612–711) (lanes 6, 7 and 8, respectively). (B) Transcription of pVA (250 ng) using fractionated factors preincubated (15 min at 30°C) with no addition (lanes 1 and 5) or with 75, 150 or 300 ng of GST–RB(379–928) (lanes 2, 3 and 4, respectively) or 75, 150 or 300 ng of GST (lanes 6, 7 and 8, respectively). (C) Transcription of pU6/Hae/RA.2 (500 ng) using nuclear extract (10 μg) preincubated (15 min at 30°C) with no addition (lanes 1 and 5) or with 100, 200 or 300 ng of GST–RB(379–928) (lanes 2, 3 and 4, respectively) or 100, 200 or 300 ng of GST–RB(612–711) (lanes 6, 7 and 8, respectively). the TFIIIB fraction (lanes 1–5). In contrast, the same Thus, TFIIIB activity is in excess in the absence of fraction has little or no stimulatory effect when added in recombinant RB, but becomes limiting when RB is added. the absence of recombinant RB (lanes 6–9). This is This demonstrates that RB is able to bring about a specific because TFIIIC is limiting and TFIIIB is in relative excess decrease in TFIIIB activity such that it becomes limiting in the reconstituted system, as shown by the ability of the for transcription. The fact that a TFIIIB fraction alone TFIIIC fraction to increase transcription (lanes 10–12). is sufficient to overcome repression indicates that the 2063 C.G.C.Larminie et al. Fig. 4. Repression by RB can be relieved using affinity-purified TFIIIB TAFs but not using TBP. Transcription of pVA (250 ng) using fractionated factors preincubated (15 min, 30°C) with 250 ng of GST (lane 1) or GST–RB (lanes 2–9), and 1, 3 or 6 μl of TBP (lanes 3–5, respectively), 3 or 6 μl of B-TAF fraction (lanes 6 and 7, respectively) or 3 or 6 μl of control fraction (lanes 8 and 9, respectively). the level of inhibition. In contrast, the affinity-purified B-TAF fraction produced a significant, dose-dependent reversal of repression. That this response is due to the presence of TBP-associated factors in the B-TAF fraction is demonstrated by the inability of the control fraction to relieve inhibition. This experiment suggests that a TAF component of TFIIIB is the specific target for repression Fig. 3. (A) TFIIIB relieves repression by recombinant RB whereas TFIIIC and pol III do not. Transcription of pVA (250 ng) using of pol III transcription by RB. fractionated factors preincubated (15 min, 30°C) with 250 ng of GST (lane 1) or GST–RB(379–928) (lanes 2–5) and 3 μl of A25(1.0) TFIIIB interacts with immobilized recombinant RB fraction containing pol III (lane 3), CHep-1.0 fraction containing We carried out pull-down experiments to test for a physical TFIIIC (lane 4) or A25(0.15) fraction containing TFIIIB (lane 5). (B) TFIIIB is limiting for transcription in the presence of recombinant interaction between RB and pol III factors (Figure 5). RB, whereas in the absence of recombinant RB TFIIIC is limiting and Crude phosphocellulose (PC) fractions were incubated TFIIIB is in excess. Transcription of pVA (250 ng) using fractionated on a vibrating platform with glutathione–agarose beads factors preincubated (15 min, 30°C) with 250 ng of GST (lanes 1 and carrying immobilized GST or GST–RB, and the unbound 6–12) or GST–RB(379–928) (lanes 2–5) and 1 μl (lanes 3 and 7), 2 μl material was then assayed for transcription factor activity. (lanes 4 and 8) or 4 μl (lanes 5 and 9) of A25(0.15) fraction containing TFIIIB or 1 μl (lane 10), 2 μl (lane 11) or 4 μl (lane 12) of When PC-C fractions were treated in this way, we detected CHep-1.0 fraction containing TFIIIC. no depletion of TFIIIC activity (Figure 5A). In contrast, TFIIIB activity was partially depleted from PC-B fractions activities of TFIIIC and pol III are not compromised by using GST–RB (Figure 5B). Depletion under these condi- RB. We conclude that RB inhibits TFIIIB specifically. tions was relatively inefficient and we were unable to improve it by extending the incubation period because A TBP-associated component of TFIIIB is prolonged treatment in this way resulted in a general inactivated by RB loss of transcriptional activity. Nevertheless, these results In order to determine which part of TFIIIB is the target suggest that the RB portion of GST–RB interacts with for repression by RB, we carried out add-back experiments TFIIIB. We confirmed this by Western blotting the material using TBP alone or the TAF component of the complex that remained bound to the beads after extensive washing. (Figure 4). TFIIIB TAFs (B-TAFs) were affinity-purified Figure 5C shows that both TBP and BRF were detected by chromatography on a column containing immobilized in association with the GST–RB beads, but were not TBP, as described previously (White and Jackson, 1992; bound to GST alone. These data provide evidence for a White et al., 1995a). We also prepared a control fraction physical association between RB and TFIIIB. by the same procedure but using a column without TBP. Whereas the B-TAF fraction can reconstitute pol III A population of RB molecules co-fractionates with transcription in the presence of TBP, TFIIIC and pol III, TFIIIB the control fraction is inactive (White et al., 1995a). The results above indicate a physical and functional Recombinant RB was used to repress VA transcription interaction between TFIIIB and RB when the latter is in a system reconstituted with fractionated factors (Figure overexpressed as a recombinant protein. A more important 4). Addition of recombinant TBP made no difference to issue is whether these factors interact with each other 2064 RB represses TFIIIB Fig. 5. Immobilized RB binds TFIIIB but not TFIIIC. (A) Transcription of pVA (250 ng) using 2 μl of PC-B (all lanes) and 2 μl of PC-C (lane 1), 2 μl of LDB (lane 2), 4 μl of supernatant from PC-C that had been preincubated with GST beads (lane 3) or 4 μl of supernatant from PC-C that had been preincubated with GST–RB beads (lane 4). (B) Transcription of pVA (250 ng) using 2 μl of CHep-1.0 (all lanes) and 2 μl of PC-B (lane 1), 2 μl of LDB (lane 2), 4 μl of supernatant from PC-B that had been preincubated with GST beads (lane 3) or 4 μl of supernatant from PC-B that had been preincubated with GST–RB beads (lane 4). (C) Proteins from PC-B that remained bound to GST–RB (lanes 1 and 4) or GST (lanes 2 and 5) beads after extensive washing were resolved on an SDS–7.8% polyacrylamide gel and then analysed by Western immunoblotting with antibodies against TBP (lanes 1–3) or BRF (lanes 4–6). Lane 3 contains 10 ng of recombinant TBP. Lane 6 contains 15 μl of PC-B. when they are present at physiological concentrations. To Endogenous RB associates with TFIIIB address this, we assayed for the presence of RB in TFIIIB We carried out immunoprecipitation assays as an fractions by immunoblotting fractionated factors and additional test of whether cellular RB associates with probing with an anti-RB antibody. Consistent co- TFIIIB (Figure 7). Fractions containing TFIIIB were purification would be suggestive of a stable interaction immunoprecipitated using a monoclonal antibody that between these two factors. specifically recognizes the hypophosphorylated (active) Figure 6A demonstrates that RB is readily detectable form of RB. After extensive washing in 200 mM KCl and in TFIIIB fractions that have been purified through 0.1% NP-40, the precipitated material was immunoblotted phosphocellulose and DEAE–Sephadex (lane 3) or phos- and probed with antibodies against TBP and BRF. We phocellulose and a glycerol gradient (lane 4). In contrast, found that both these TFIIIB subunits were co-precipitated little or no RB was detected in fractions containing TFIIIC with RB (Figure 7A, lanes 4 and 8). Neither TBP nor (lane 1), pol III (lane 2), or the TBP-containing complex BRF was immunoprecipitated using a control antibody SL1 (lane 5). These data support the contention that RB that recognizes Sp1 (Figure 7B, lanes 5 and 10). As an interacts specifically with TFIIIB. additional test of specificity, we carried out immuno- To provide an additional test for an association between precipitations with the anti-RB antibody in the presence RB and TFIIIB, we examined whether these factors co- of blocking agents. A short GST–RB polypeptide con- fractionate during gradient chromatography on heparin– taining the region of RB that is recognized by this antibody Sepharose. A phosphocellulose fraction containing RB was able to block the co-precipitation of TBP and BRF and TFIIIB was applied to a heparin–Sepharose column (Figure 7B, lanes 3 and 8). In contrast, an equal amount and the bound proteins were eluted with a linear salt of GST protein did not prevent the immunoprecipitation gradient. Individual fractions were assayed for their content of these subunits (Figure 7B, lanes 2 and 7). These of TFIIIB (Figure 6B, upper panel) and RB (Figure 6B, experiments provide direct evidence that endogenous RB lower panel). Neither TFIIIB nor RB was detectable before associates with TFIIIB at physiological concentrations. fraction 49; both factors began to elute in fraction 50, peaked in fractions 51 and 52, and then tailed off gradually Extracts of cells from RB-knockout mice have in the next few fractions. Thus, RB co-fractionates closely elevated TFIIIB activity with TFIIIB on a heparin–Sepharose salt gradient. We have shown previously that endogenous RB represses These data demonstrate that a population of RB mole- pol III transcription in primary fibroblasts from embryonic cules co-purifies with TFIIIB on phosphocellulose, DEAE– mice (White et al., 1996). If this effect is achieved through Sephadex and heparin–Sepharose columns, and on glycerol the repression of TFIIIB, then we would expect to find gradients. We have also found RB in TFIIIB fractions that an increase in TFIIIB activity in these cells following were prepared using Q-Sepharose, Mono Q and Macro- disruption of the Rb gene. To test this, we prepared Prep CM resins (data not shown). Such consistent co- extracts from primary cells that had been isolated from fractionation is suggestive of a stable interaction. In either wild-type or RB-knockout mice (Jacks et al., 1992). contrast, little or no RB was detected in fractions con- The RB-negative extracts supported higher rates of pol taining pol III, TFIIIC or the TBP-containing complex III transcription than the RB-positive extracts (Figure 8A). SL1 (Figure 6A). Together, these results suggest that there As a control for factor recovery, we compared the levels is a specific physical and functional interaction between of TFIIIC in these extracts and detected little or no endogenous RB and TFIIIB. difference (Figure 8B). In contrast, when TFIIIB activity 2065 C.G.C.Larminie et al. TFIIIB is required for the expression of all class III genes, its targeting by RB provides an explanation for the general increase in pol III transcription that is observed in RB- knockout mice (White et al., 1996). RB can repress transcription from a preformed pol III initiation complex The ability of RB to regulate transcription is believed to be restricted to a defined time window during the cell cycle. Thus, RB is active during early G phase, is inactivated at the G /S transition, and then remains inactive until the end of mitosis (reviewed by Weinberg, 1995; Whyte, 1995). Since DNA replication erases initiation complexes from class III genes (Wolffe and Brown, 1986), complexes must reform during S and G phases, when RB is believed to be inactive. For RB to repress TFIIIB during the subsequent G phase, it may need to regulate transcription complexes that had assembled during the previous cycle. We have confirmed that RB is indeed capable of inactivating a preassembled pol III preinitiation complex. Alternatively, it is possible that the pol III preinitiation complex is disrupted during mitosis; in this case RB would inhibit complexes that reform during the early part of G . Fig. 6. A population of RB molecules co-fractionates with TFIIIB. (A) RB is readily detectable in fractions containing TFIIIB but not in RB represses pol III transcription by inactivating fractions containing pol III, TFIIIC or SL1. Fractionated factors (15 μl) as indicated were resolved on an SDS–7.8% polyacrylamide TFIIIB gel and then analysed by Western immunoblotting with anti-RB We have presented several independent lines of evidence antibody G3-245. The TFIIIC fraction in lane 1 was affinity-purified that implicate TFIIIB as the component of the pol III and has a protein concentration of 93 ng/μl. The pol III fraction in transcription apparatus that is targeted by RB. (i) Addition lane 2 was A25(1.0) and has a protein concentration of 15 ng/μl. The of recombinant RB to reconstituted transcription reactions TFIIIB fraction in lane 3 was prepared by chromatography on phosphocellulose followed by glycerol gradient sedimentation; it has a causes a specific decrease in the activity of TFIIIB protein concentration of 200 ng/μl. The TFIIIB fraction in lane 4 was such that this factor becomes limiting for transcription. A25(0.15) and has a protein concentration of 80 ng/μl. The SL1 Repression by RB is overcome by the addition of more fraction in lane 5 was SS700 and has a protein concentration of TFIIIB, but not by TFIIIC or pol III. (ii) Recombinant RB 260 ng/μl. (B) RB co-fractionates with TFIIIB during gradient chromatography on heparin–Sepharose. The upper panel shows TFIIIB that has been immobilized on beads depletes fractions of activity of individual fractions and the lower panel shows the RB TFIIIB, but not TFIIIC activity. The BRF and TBP content of the same fractions. In each case lane 1 contains buffer, lane subunits of TFIIIB were shown to bind to the immobilized 2 contains input material (PC-B), lane 3 contains flow-through, and RB. (iii) A population of RB molecules co-purifies with lanes 4–18 contain fractions 46–60, respectively. TFIIIB activity was TFIIIB during a variety of distinct fractionation pro- assayed using 4 μl of the indicated fraction, 2 μl of PC-C and 500 ng of pVA ; after 15 min incubation at 30°C, nucleotides were added to I cedures. In contrast, little or no RB is detected in fractions assay transcription. RB content was assayed using 14 μlofthe containing TFIIIC, pol III or SL1. Although we cannot indicated fractions which were resolved on an SDS–7.8% exclude the possibility that the co-purification of TFIIIB polyacrylamide gel and then analysed by Western immunoblotting with and RB is fortuitous, we consider that this is unlikely anti-RB antibody G3-245. given the consistency with which it is observed under a variety of distinct chromatographic conditions. We have also fractionated TFIIIB from SAOS2 cells, which contain was measured by complementation assays, it was found inactive mutant RB. We find that TFIIIB can be readily to be significantly elevated in the RB-negative extracts separated from SAOS2 RB (data not shown), which (Figure 8C). We conclude that TFIIIB is a physiological supports the idea that co-fractionation of TFIIIB and RB target for repression by RB in mouse embryonic fibroblasts. reflects a functional interaction. In addition, we have examined the fractionation properties of RB that has been expressed in Sf9 cells. This recombinant RB displayed Discussion chromatographic behaviour that was distinct from that of We have shown previously that endogenous RB plays an TFIIIB (data not shown). It is therefore unlikely that the important role in suppressing the level of pol III transcrip- co-fractionation of TFIIIB with a population of cellular tion in primary murine fibroblasts (White et al., 1996). In RB molecules is due to these factors possessing identical this study, we have addressed the molecular mechanism chromatographic properties. The most plausible explana- of this regulation. We have demonstrated that RB is able tion of the co-purification data is that a proportion of to disrupt the function of a preformed pol III transcription endogenous TFIIIB exists in association with RB. (iv) complex. We have also shown that it can regulate the TFIIIB subunits (TBP and BRF) co-immunoprecipitate activity of each type of pol III promoter. This appears to with RB. This effect is specific, since it is not seen using be achieved by the specific inactivation of TFIIIB. Since control antibodies and it can be blocked using an excess 2066 RB represses TFIIIB Fig. 7. TBP and BRF co-immunoprecipitate with RB. (A) Proteins from PC-B that were immunoprecipitated using anti-TBP antibody bound to protein A–Sepharose (lanes 2 and 6), protein A–Sepharose alone (lanes 3 and 7) or anti-RB antibody bound to protein A–Sepharose (lanes 4 and 8) were resolved on an SDS–7.8% polyacrylamide gel and then analysed by Western immunoblotting with antibodies against TBP (lanes 1–4) or BRF (lanes 5–8). Lane 1 contains 10 ng of recombinant TBP. Lane 5 contains 15 μl of PC-B. (B) Proteins from PC-B that were immunoprecipitated using anti-RB antibody (lanes 2, 3, 7 and 8), anti-TBP antibody (lanes 4 and 9) or anti-Sp1 antibody (lanes 5 and 10) were resolved on an SDS–7.8% polyacrylamide gel and then analysed by Western immunoblotting with antibodies against TBP (lanes 1–5) or BRF (lanes 6–10). The reactions in lanes 2 and 7 contained 1.5 μg of GST. The reactions in lanes 3 and 8 contained 1.5 μg of GST–RB(379–611). Lane 1 contains 10 ng of recombinant TBP. Lane 6 contains 15 μl of PC-B. Fig. 8. Primary fibroblasts from RB-knockout mice have elevated TFIIIB activity. (A) Transcription of pVA (250 ng) using whole-cell extract / –/– (24 μg) prepared from RB (lanes 1 and 3) or RB (lanes 2 and 4) primary fibroblasts. (B) Band shift assay using 4 ng of radiolabelled B-block –/– / oligonucleotide probe, 500 ng of poly(dI-dC), no protein (lane 1), 12.7 μgofRB extract (lane 2) or 12.7 μgofRB extract (lanes 3–5) and 200 ng of B-block (lane 4) or MSV (lane 5) oligonucleotide competitor. (C) Transcription of pVA (500 ng) using 4 μl of PC-C and 5 ng of recombinant / –/– TBP (all lanes) and 25 μg of heat-treated RB extract (lane 1), 25 μg of heat-treated RB extract (lane 2), no addition (lane 3) or 3 μl of heat-treated Mono Q-purified TFIIIB (lane 4). of the epitope. In addition to detecting TBP and BRF in shown). (v) TFIIIB activity is elevated in extracts immunoprecipitates from an anti-RB antibody, we have prepared from RB-knockout fibroblasts. This provides also co-precipitated RB using antibodies against TBP and genetic evidence for a functional interaction between BRF (data not shown). These data provide direct evidence TFIIIB and RB. We consider that these independent for an interaction between RB and TFIIIB. We were lines of evidence together provide a compelling case for unable to detect this interaction using a TFIIIB fraction believing that RB regulates pol III transcription by target- from SAOS2 cells, in which RB is inactive (data not ing the general factor TFIIIB. 2067 C.G.C.Larminie et al. Fig. 9. Sequence homology between RB, TBP and BRF. The regions of RB that are related to TBP and BRF are indicated and the alignments are shown underneath. Sequence identities are indicated with black boxes and sequence similarities (using highly stringent criteria) are indicated with grey boxes. Adenovirus infection RB activity is high (in those experiments it was added in Although E1A can overcome the repressive effects of RB, excess) (White et al., 1996). In contrast to the results in there is little or no increase in TFIIIB activity when HeLa HeLa cells, we would predict that TFIIIB activity will cells are infected by adenovirus (Hoeffler and Roeder, increase significantly during adenovirus infection of cells 1985; Yoshinaga et al., 1986). Instead, the activation of such as fibroblasts that have high RB activity. pol III transcription that accompanies adenoviral infection is mediated by changes in TFIIIC (Hoeffler and Roeder, RB may bind TFIIIB by mimicking TBP and BRF 1985; Yoshinaga et al., 1986; Sinn et al., 1995). We Our results indicate that TFIIIB is subject to repression believe that the explanation for this apparent paradox lies by RB, but do not determine which of its subunits is in the fact that RB activity is very low in HeLa cells. targeted. The fact that repression is overcome using Although accurate quantitation is extremely difficult, our affinity-purified TFIIIB TAFs but not TBP alone suggests best estimate is that 5–10% of TFIIIB is associated with that RB targets one or more of the pol III TAFs. However, RB in extracts made from uninfected asynchronous HeLa the possibility remains that an unidentified intermediate cells (data not shown). Even if E1A displaces all of this component is required for the association between RB RB during adenovirus infection, the overall effect on total and TFIIIB. Proof of direct binding will require interaction TFIIIB activity is unlikely to be detectable. In order to studies with purified and separated subunits of TFIIIB. stimulate pol III transcription significantly, adenovirus Such analyses are hampered at present by the uncertainty must employ an additional mechanism. The activation of concerning the precise composition of human TFIIIB. TFIIIC is the decisive step in stimulating pol III transcrip- Much of the function of RB is carried out by a region tion during infection of HeLa cells (Hoeffler and Roeder, called the ‘pocket’ that stretches between residues 379 1985; Yoshinaga et al., 1986). and 792 (Weinberg, 1995; Whyte, 1995). The pocket is The relatively small proportion of TFIIIB that associates composed of two essential regions, A and B, that are with RB in HeLa cells is consistent with the high pol III separated by a spacer, the sequence of which is not activity of this line. It is likely that HPV E7 protein that required for function (Weinberg, 1995; Whyte, 1995). is expressed in these cells may help overcome repression Disruption of either the A domain or the B domain can by RB, although we have yet to confirm this for pol III. prevent RB from repressing pol III (White et al., 1996). In other cell types, RB appears to play a much more It has been observed previously that the A domain of the significant role in repressing pol III. This is particularly RB pocket bears 21% identity and 38% similarity to a obvious in mouse embryo fibroblasts, where deletion of 151 amino acid region from the C-terminus of TBP RB causes a 5-fold increase in pol III transcription (White (Hagemeier et al., 1993). The C-terminal domain of TBP et al., 1996). We have shown previously that E1A can that encompasses this homology is sufficient to support overcome repression by RB under conditions in which pol III transcription (White and Jackson, 1992). We found 2068 RB represses TFIIIB activated at the G /S transition, it seems likely that the increase in pol III transcription that accompanies progression into S phase may reflect, at least in part, the release of TFIIIB from inhibition by RB. We are currently investigating this possibility. The existing data suggest that a common mechanism for achieving cell cycle control of transcription by pols II and III may involve the regulation of TAF activity by RB. Fig. 10. Model proposing that RB may bind and inactivate TFIIIB by Materials and methods mimicking TBP and BRF. Plasmids The plasmids used in this work have all been described previously that an extended 230 amino acid region of BRF (residues (White et al., 1989, 1995a). 96–326) displays 15% identity and 33% similarity to the B domain and C-terminal region (residues 665–910) of Protein fractions RB (Figure 9). This homology was assessed using very Nuclear extracts were prepared from proliferating HeLa cells by the stringent criteria for similarity (R/K, E/D, S/T, F/Y and method of Dignam et al. (1983). The extracts were then chromato- L/I/V/M). Thus, RB bears homology to two of the subunits graphed on phosphocellulose according to the method of Segall et al. (1980). Direct assay confirmed that PC-B fractions (0.1–0.35 M KCl of the TFIIIB complex that it is able to regulate. step) contained TFIIIB and pol III, PC-C fractions (0.35–0.6 M KCl Because the level of homology between RB, TBP and step) contained TFIIIC and pol III, and PC-D fractions (0.6–1.0 M KCl BRF is relatively low, one cannot be certain that it is step) contained SL1. significant. However, we are struck by the fact that adjacent The 0.38M-TFIIIB and 0.48M-TFIIIB fractions were prepared from PC-B by Mono Q gradient chromatography, as described by Lobo et al. regions of RB bear a resemblance to two polypeptides that (1992). Fractions were dialysed into LDB buffer (20 mM HEPES–KOH, interact within TFIIIB. This apparent resemblance could pH 7.9, 17% glycerol, 100 mM KCl, 12 mM MgCl , 0.1 mM EDTA, provide the key to the mechanism of repression. The 2 mM DTT). The 0.38M-TFIIIB fraction eluted at ~340 mM KCl and adjacent homologous regions may allow RB to present a had a protein concentration of 0.45 mg/ml. The 0.48M-TFIIIB fraction eluted at ~450 mM KCl and had a protein concentration of 0.11 mg/ml. molecular surface that resembles TBP alongside BRF. Western immunoblotting analysis revealed that TBP and BRF are present This could enable it to associate with another TAF subunit in the 0.38M-TFIIIB but not in the 0.48M-TFIIIB (Mital et al., 1996). through a similar set of interactions to those that occur in The A25(0.15) and A25(1.0) fractions were prepared by chromatography intact TFIIIB. We therefore propose a model in which RB of PC-B fractions on DEAE–Sephadex, as previously (White et al., is able to bind TFIIIB by mimicking the interaction surface 1995a). A25(0.15) is a 50–150 mM (NH ) SO step fraction; it contains 4 2 4 TFIIIB and has a protein concentration of 0.08 mg/ml. A25(1.0) is a that is presented by TBP alongside BRF (Figure 10). 0.15–1.0 M (NH ) SO step fraction; it contains pol III and has a protein 4 2 4 Clearly, this does not displace TBP and BRF, since our concentration of 0.015 mg/ml. For glycerol gradient fractionation of pull-down and immunoprecipitation assays have shown TFIIIB, PC-B was sedimented for 24 h at 39 000 r.p.m. in an that these polypeptides remain associated with RB. Addi- SW40Ti rotor on a 15–50% glycerol gradient in 50 mM Tris–HCl, pH 7.9, 200 mM KCl, 0.5 mM EDTA, 12.5 mM MgCl , 1 mM DTT, tional interactions between subunits may hold the inactive 0.1 mM PMSF. Fractions were then dialysed into LDB. For heparin complex together in the presence of RB. gradient chromatography, PC-B was applied to heparin–Sepharose CL- 6B in PC buffer (20 mM HEPES–KOH, pH 7.9, 20% glycerol, 0.2 mM Regulation of TAF activity by RB may provide a EDTA, 0.5 mM DTT) containing 100 mM KCl. After extensive washing, common mechanism for the cell cycle control of the bound protein was eluted using a linear gradient up to 1.2 M KCl. Fractions were dialysed into LDB. Recombinant TBP, affinity-purified transcription by pols II and III TFIIIB TAFs (B-TAF), and the corresponding control fraction (CON) We have presented evidence that RB is able to regulate were prepared as previously (White et al., 1995a). pol III transcription by interacting with the TFIIIB com- The CHep-1.0 fraction was prepared by chromatography of PC-C on plex. This finding is reminiscent of recent data which heparin–Sepharose CL-6B, as previously (White et al., 1995a). CHep- show that RB can bind to the TAF 250 subunit of 1.0 contained TFIIIC and pol III, and had a protein concentration of II 0.6 mg/ml. Affinity-purified TFIIIC was prepared as previously (White the pol II TFIID complex (Shao et al., 1995). Thus, et al., 1995a) and had a protein concentration of 0.093 mg/ml. association with class-specific TAFs appears to provide The SS700 fraction was prepared by chromatography of PC-D one mechanism by which RB is able to regulate gene fractions on S-Sepharose. PC-D was applied to S-Sepharose in AM expression. In contrast to the situation with TFIID and buffer (20 mM Tris–HCl, pH 7.9, 0.1 mM EDTA, 20% glycerol, 5 mM MgCl , 1 mM DTT, 1 mM PMSF) plus 100 mM KCl. After washing TFIIIB, we detected no RB in fractions containing the 2 with this buffer, the column was eluted with AM buffer plus 320 mM pol I-specific TBP-containing complex SL1 (Figure 6A). KCl to generate the SS320 fraction, and then with AM buffer plus Instead, RB is reported to control pol I transcription by 700 mM KCl to generate the SS700 fraction. Peak fractions were binding to the class-specific regulator UBF (Cavanaugh dialysed into LDB. The SS700 fraction contained SL1 and had a protein concentration of 0.26 mg/ml. et al., 1995). Recombinant GST and GST–RB fusion proteins were prepared as Both genetic and biochemical data have implicated previously (White et al., 1996). Significant batch variations were noted TAF 250 in regulating the passage through G phase II 1 in the specific activities of different GST–RB preparations. The reason (Sekiguchi et al., 1988, 1991; Hisatake et al., 1993; for this is unknown. Ruppert et al., 1993; Wang and Tjian, 1994). It remains Transcription and band shift assays to be determined to what extent RB is involved in this Transcription and band shift assays were carried out as described effect. We have shown previously that the activity of a previously (White et al., 1989), except that transcription was for1hat TAF component of TFIIIB increases significantly when 30°C and band-shifts were run in 0.25 TBE. The B-block and MSV cells progress from early G into S phase (White et al., 1 oligonucleotides have been described previously (White et al., 1989). 1995b). Since RB represses TFIIIB and becomes in- Transcription reactions that were reconstituted using fractionated 2069 C.G.C.Larminie et al. factors contained 1 μl each of 0.38M-TFIIIB (0.45 μg) and 0.48M- Hisatake,K., Hasegawa,S., Takada,R., Nakatani,Y., Horikoshi,M. and TFIIIB (0.11 μg) and 2 μl of CHep-1.0 (1.2 μg) fractions. Roeder,R.G. (1993) The p250 subunit of native TATA box-binding factor TFIID is the cell-cycle regulatory protein CCG1. Nature, 362, Pull-down assays 179–181. PC-C or PC-B fractions (25 μl) were incubated at 4°C on an orbital Hoeffler,W.K. and Roeder,R.G. (1985) Enhancement of RNA polymerase shaker with 25 μl of glutathione–agarose beads containing equivalent III transcription by the E1A gene product of adenovirus. Cell, 41, amounts (as estimated by Coomassie blue staining) of immobilized GST 955–963. or GST–RB (379–928). After 10 min, the beads were pelleted and 4 μl Jacks,T., Fazeli,A., Schmitt,E.M., Bronson,R.T., Goodell,M.A. and aliquots of supernatant were removed for transcription assays. The Weinberg,R.A. (1992) Effects of an Rb mutation in the mouse. Nature, remainder of the samples were incubated at 4°C on the orbital shaker 359, 295–300. for a further 3 h. Samples were then pelleted, supernatants removed and Kassavetis,G.A., Nguyen,S.T., Kobayashi,R., Kumar,A., Geiduschek,E.P. the beads were washed five times with 700 μl of LDB. The bound and Pisano,M. (1995) Cloning, expression, and function of TFC5, the material was analysed by Western blotting. gene encoding the B’ component of the Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIB. Proc. Natl Acad. Sci. Western blotting USA, 92, 9786–9790. Western immunoblotting was carried out as previously (White et al., Khoo,B., Brophy,B. and Jackson,S.P. (1994) Conserved functional 1995a). The antibodies used for immunoblotting were G3-245 domains of the RNA polymerase III general transcription factor BRF. (Pharminogen) against RB, SL30 against TBP and α-CSH409 against Genes Dev., 8, 2879–2890. BRF. Lassar,A.B., Martin,P.L. and Roeder,R.G. (1983) Transcription of class III genes: formation of preinitiation complexes. Science, 222, 740–748. Immunoprecipitation Lobo,S.M., Tanaka,M., Sullivan,M.L. and Hernandez,N. (1992) A TBP PC-B fractions (50 μl) were incubated at 4°C for 6 h on an orbital complex essential for transcription from TATA-less but not TATA- shaker with 25 μl of protein A–Sepharose beads containing prebound containing RNA polymerase III promoters is part of the TFIIIB antibodies. Samples were then pelleted, supernatants removed and the fraction. Cell, 71, 1029–1040. beads were washed four times with 700 μl of LDB and once with 700 μl Loeken,M., Bikel,I., Livingston,D.M. and Brady,J. (1988) Trans- of LDB containing 200 mM KCl and 0.1% NP-40. The bound material activation of RNA polymerase II and III promoters by SV40 small t was analysed by Western blotting. The antibodies used for immuno- antigen. Cell, 55, 1171–1177. precipitation were G99-549 (Pharminogen) against RB, MTBP-6 against Lopez-de-Leon,A., Librizzi,M., Tuglia,K. and Willis,I. (1992) PCF4 TBP and IC6 against Sp1. encodes an RNA polymerase III transcription factor with homology to TFIIB. Cell, 71, 211–220. Primary embryonic fibroblasts –/– / Mital,R., Kobayashi,R. and Hernandez,N. (1996) RNA polymerase III Early passage primary cells from Rb and Rb mice (Jacks et al., transcription from the human U6 and adenovirus type 2 VAI promoters 1992) were grown in Dulbecco’s modified Eagle’s medium supplemented has different requirements for human BRF, a subunit of human TFIIIB. with 20% fetal calf serum. Whole-cell extracts were prepared as Mol. Cell. Biol., 16, 7031–7042. previously (White et al., 1995a). Nasmyth,K. (1996) Another role rolls in. Nature, 382, 28–29. Patel,G. and Jones,N.C. (1990) Activation in vitro of RNA polymerase Acknowledgements II and III directed transcription by baculovirus produced E1A protein. Nucleic Acids Res., 18, 2909–2915. We are grateful to James Millner-White for advice concerning sequence Pines,J. (1995) Cyclins, CDKs and cancer. Semin. Cancer Biol., 6, 63–72. homology, Bart Williams and George Mulligan for primary embryonic Qin,X., Chittenden,T., Livingston,D.M. and Kaelin,W.G. (1992) Identi- fibroblasts, Sibylle Mittnacht for Sf9-expressed RB, Jane Flint for fication of a growth suppression domain within the retinoblastoma antibody MTBP-6 and Nouria Hernandez for antibodies SL30 and α- gene product. Genes Dev., 6, 953–964. CSH409. This work was funded by the following grants: SP2314/0101 Rigby,P.W.J. (1993) Three in one and one in three: it all depends on to R.J.W. and SP2143/0101 to S.P.J. from the Cancer Research Campaign, TBP. Cell, 72, 7–10. CO5766 to R.J.W. from the Biotechnology and Biological Sciences Roberts,S., Miller,S.J., Lane,W.S., Lee,S. and Hahn,S. (1996) Cloning Research Council, SCI/180/94/365/G to R.J.W. from the and functional characterization of the gene encoding the TFIIIB90 Nuffield Foundation and NIH R01GM38810 to Nouria Hernandez. subunit of RNA polymerase III transcription factor TFIIIB. J. Biol. R.J.W. is a Jenner Fellow of the Lister Institute of Preventive Medicine. Chem., 271, 14903–14909. Ruppert,S., Wang,E.H. and Tjian,R. (1993) Cloning and expression of human TAF 250: a TBP-associated factor implicated in cell-cycle References II regulation. Nature, 362, 175–179. Buratowski,S. and Zhou,H. (1992) A suppressor of TBP mutations Ruth,J., Conesa,C., Dieci,G., Lefebvre,O., Dusterhoft,A., Ottonello,S. encodes an RNA polymerase III transcription factor with homology and Sentenac,A. (1996) A suppressor of mutations in the class III to TFIIB. Cell, 71, 221–230. transcription system encodes a component of yeast TFIIIB. EMBO J., Cavanaugh,A.H., Hempel,W.M., Taylor,L.J., Rogalsky,V., Todorov,G. 15, 1941–1949. and Rothblum,L.I. (1995) Activity of RNA polymerase I transcription Segall,J., Matsui,T. and Roeder,R.G. (1980) Multiple factors are required factor UBF blocked by Rb gene product. Nature, 374, 177–180. for the accurate transcription of purified genes by RNA polymerase Chiang,C.-M., Ge,H., Wang,Z., Hoffmann,A. and Roeder,R.G. (1993) III. J. Biol. Chem., 255, 11986–11991. Unique TATA-binding protein-containing complexes and cofactors Sekiguchi,T., Miyata,T. and Nishimoto,T. (1988) Molecular cloning of involved in transcription by RNA polymerases II and III. EMBO J., the cDNA of human X chromosomal gene (CCG1) which complements 12, 2749–2762. the temperature-sensitive G mutants, tsBN462 and ts13, of the BHK Colbert,T. and Hahn,S. (1992) A yeast TFIIB-related factor involved in cell line. EMBO J., 7, 1683–1687. RNA polymerase III transcription. Genes Dev., 6, 1940–1949. Sekiguchi,T., Nohiro,Y., Nakamura,Y., Hisamoto,N. and Nishimoto,T. Dignam,J.D., Lebovitz,R.M. and Roeder,R.G. (1983) Accurate (1991) The human CCG1 gene, essential for progression of the G1 transcription initiation by RNA polymerase II in a soluble extract phase, encodes a 210-kilodalton nuclear DNA-binding protein. Mol. from isolated mammalian nuclei. Nucleic Acids Res., 11, 1475–1489. Cell. Biol., 11, 3317–3325. Geiduschek,E.P. and Kassavetis,G.A. (1995) Comparing transcriptional Shao,Z., Ruppert,S. and Robbins,P.D. (1995) The retinoblastoma- initiation by RNA polymerases I and III. Curr. Opin. Cell Biol., 7, susceptibility gene product binds directly to the human TATA-binding 344–351. protein-associated factor TAF 250. Proc. Natl Acad. Sci. USA, 92, Hagemeier,C., Bannister,A.J., Cook,A. and Kouzarides,T. (1993) The II 3115–3119. activation domain of transcription factor PU.1 binds the retinoblastoma Sinn,E., Wang,Z., Kovelman,R. and Roeder,R.G. (1995) Cloning and (RB) protein and the transcription factor TFIID in vitro: RB shows characterization of a TFIIIC2 subunit (TFIIICβ) whose presence sequence similarity to TFIID and TFIIB. Proc. Natl Acad. Sci. USA, correlates with activation of RNA polymerase III-mediated 90, 1580–1584. Hernandez,N. (1993) TBP, a universal eukaryotic transcription factor? transcription by adenovirus E1A expression and serum factors. Genes Genes Dev., 7, 1291–1308. Dev., 9, 675–685. 2070 RB represses TFIIIB Taggart,A.K.P., Fisher,T.S. and Pugh,B.F. (1992) The TATA-binding protein and associated factors are components of pol III transcription factor TFIIIB. Cell, 71, 1015–1028. Teichmann,M. and Seifart,K.H. (1995) Physical separation of two different forms of human TFIIIB active in the transcription of the U6 or the VAI gene in vitro. EMBO J., 14, 5974–5983. Wang,E. and Tjian,R. (1994) Promoter-selective transcriptional defect in cell cycle mutant ts13 rescued by hTAF 250. Science, 263, 811–814. II Wang,Z. and Roeder,R.G. (1995) Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains. Proc. Natl Acad. Sci. USA, 92, 7026–7030. Weinberg,R.A. (1995) The retinoblastoma protein and cell cycle control. Cell, 81, 323–330. White,R.J. (1994) RNA Polymerase III Transcription. R.G.Landes Company, Austin, TX. White,R.J. (1997) Regulation of RNA polymerases I and III by the retinoblastoma protein: a mechanism for growth control? Trends Biochem. Sci., 22, 77–80. White,R.J. and Jackson,S.P. (1992) Mechanism of TATA-binding protein recruitment to a TATA-less class III promoter. Cell, 71, 1041–1053. White,R.J., Stott,D. and Rigby,P.W.J. (1989) Regulation of RNA polymerase III transcription in response to F9 embryonal carcinoma stem cell differentiation. Cell, 59, 1081–1092. White,R.J., Gottlieb,T.M., Downes,C.S. and Jackson,S.P. (1995a) Mitotic regulation of a TATA-binding-protein-containing complex. Mol. Cell. Biol., 15, 1983–1992. White,R.J., Gottlieb,T.M., Downes,C.S. and Jackson,S.P. (1995b) Cell cycle regulation of RNA polymerase III transcription. Mol. Cell. Biol., 15, 6653–6662. White,R.J., Trouche,D., Martin,K., Jackson,S.P. and Kouzarides,T. (1996) Repression of RNA polymerase III transcription by the retinoblastoma protein. Nature, 382, 88–90. Whyte,P. (1995) The retinoblastoma protein and its relatives. Semin. Cancer Biol., 6, 83–90. Willis,I.M. (1993) RNA polymerase III. Genes, factors and transcriptional specificity. Eur. J. Biochem., 212, 1–11. Wolffe,A.P. and Brown,D.D. (1986) DNA replication in vitro erases a Xenopus 5S RNA gene transcription complex. Cell, 47, 217–227. Yoshinaga,S.K., Dean,N., Han,M. and Berk,A.J. (1986) Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration. EMBO J., 5, 343–354. Received on July 5, 1996; revised on January 7, 1997

Journal

The EMBO Journal – Springer Journals

Published: Apr 15, 1997

Keywords: RB; RNA polymerase III; TBP; TFIIIB; transcription

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