A Role for TAF3B2 in the Repression of Human RNA Polymerase III Transcription in Nonproliferating Cells
2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês
10.1074/jbc.m102295200
ISSN1083-351X
AutoresKnut Eichhorn, Stephen P. Jackson,
Tópico(s)Genomics and Chromatin Dynamics
ResumoRNA polymerase III (Pol III) synthesizes various small RNA species, including the tRNAs and the 5 S ribosomal RNA, which are involved in protein synthesis. Here, we describe the regulation of human Pol III transcription in response to sustained cell cycle arrest. The experimental system used is a cell line in which cell cycle arrest is induced by the regulated expression of the tumor suppressor protein p53. We show that the capacity of cells to carry out Pol III transcription from various promoter types, when tested in vitro, is severely reduced in response to sustained p53-mediated cell cycle arrest. Furthermore, this effect does not appear to be due to direct inhibition by p53. By using complementation assays, we demonstrate that a subcomponent of the Pol III transcription factor IIIB, which contains the proteins TATA-binding protein and TAF3B2, is the target of repression. Moreover, we reveal that TAF3B2 levels are markedly reduced in extracts from cell cycle-arrested cells because of a decrease in TAF3B2 protein stability. These findings provide a novel mechanism of Pol III regulation and yield insights into how cellular biosynthetic capacity and growth status can be coordinated. RNA polymerase III (Pol III) synthesizes various small RNA species, including the tRNAs and the 5 S ribosomal RNA, which are involved in protein synthesis. Here, we describe the regulation of human Pol III transcription in response to sustained cell cycle arrest. The experimental system used is a cell line in which cell cycle arrest is induced by the regulated expression of the tumor suppressor protein p53. We show that the capacity of cells to carry out Pol III transcription from various promoter types, when tested in vitro, is severely reduced in response to sustained p53-mediated cell cycle arrest. Furthermore, this effect does not appear to be due to direct inhibition by p53. By using complementation assays, we demonstrate that a subcomponent of the Pol III transcription factor IIIB, which contains the proteins TATA-binding protein and TAF3B2, is the target of repression. Moreover, we reveal that TAF3B2 levels are markedly reduced in extracts from cell cycle-arrested cells because of a decrease in TAF3B2 protein stability. These findings provide a novel mechanism of Pol III regulation and yield insights into how cellular biosynthetic capacity and growth status can be coordinated. polymerase transcription factor III TATA-binding protein TBP-associated factor N-acetyl-Leu-Leu-norleucinal phosphocellulose fraction Cellular growth and proliferation are two distinct processes. Proliferation is defined as the increase in the number of cells, which occurs when cells progress through a new cell division cycle. Cell growth, on the other hand, is defined as the increase in mass of an individual cell. Growth is related to the biosynthetic capacity of a cell, a reliable indicator of which is the level of protein synthesis (1Larminie C.G.C. Alzuherri H.M. Cairns C.A. McLees A. White R.J. J. Mol. Med. 1998; 76: 94-103Crossref PubMed Scopus (31) Google Scholar). Conceptually, it seems requisite that the processes of growth and proliferation are linked and that sensitive fine tuning exists between them. At present, however, very little is known about the molecular mechanisms that ensure that this crucial cross-regulation is achieved.After neoplastic transformation of cells, uncontrolled proliferation is only sustainable if it is linked to deregulated cell growth (2Rosenwald I.B. BioEssays. 1996; 18: 243-250Crossref PubMed Scopus (45) Google Scholar). Major determinants of protein synthesis are the ribosomes and the machinery that delivers activated amino acids to the ribosomes during translation. Growth is therefore dependent on the availability of adequate supplies of rRNAs and tRNAs. It has been shown that, as cells enter quiescence, existing ribosomes disaggregate into their subunits, the levels of rRNAs and tRNAs decrease, and net protein synthesis is down-regulated. These events are reversed upon mitogenic stimulation (2Rosenwald I.B. BioEssays. 1996; 18: 243-250Crossref PubMed Scopus (45) Google Scholar). But how are regulation of growth and regulation of proliferation jointly accomplished? The prevailing model is that tumor suppressor proteins play a crucial role in eliciting this control (1Larminie C.G.C. Alzuherri H.M. Cairns C.A. McLees A. White R.J. J. Mol. Med. 1998; 76: 94-103Crossref PubMed Scopus (31) Google Scholar, 3White R.J. Int. J. Oncol. 1998; 12: 741-748PubMed Google Scholar, 4White R.J. Gene Ther. Mol. Biol. 1998; 1: 613-628Google Scholar). For example, the retinoblastoma protein regulates entry into S phase by controlling E2F, a transcription factor involved in the expression of S phase-inducing genes (5Kouzarides T. Semin. Cancer Biol. 1995; 6: 91-98Crossref PubMed Scopus (64) Google Scholar, 6Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4295) Google Scholar). It may also affect cell growth potential by repressing the activities of RNA polymerases I (7Cavanaugh A.H. Hempel W.M. Taylor L.J. Rogalsky V. Todorov G. Rothblum L.I. Nature. 1995; 374: 177-180Crossref PubMed Scopus (290) Google Scholar) and III (8White R.J. Trouche D. Martin K. Jackson S.P. Kouzarides T. Nature. 1996; 382: 88-90Crossref PubMed Scopus (183) Google Scholar), which synthesize rRNAs and tRNAs, respectively. Multi-functional proteins such as retinoblastoma protein may therefore be vital for adjusting cellular growth potential to match proliferative activity. Conversely, as a consequence of losing retinoblastoma protein function, cells may lose control over both proliferation and growth and be put on a fast track toward neoplasia (1Larminie C.G.C. Alzuherri H.M. Cairns C.A. McLees A. White R.J. J. Mol. Med. 1998; 76: 94-103Crossref PubMed Scopus (31) Google Scholar).RNA polymerase (Pol)1 III synthesizes a number of small RNA species, including the 5 S rRNA, tRNAs, the spliceosomal U6 small nuclear RNA, the 7SL RNA of the signal recognition particle, and the adenovirus VAI RNA (9White R.J. RNA Polymerase III Transcription. R. G. Landes Company, Austin, TX1994Google Scholar). The promoters of genes transcribed by Pol III are subdivided into three groups, type 1, type 2, and type 3 promoters, based on promoter structure and their requirements for basal Pol III transcription factors (10Willis I.M. Eur. J. Biochem. 1993; 212: 1-11Crossref PubMed Scopus (191) Google Scholar). TFIIIA is a monomeric protein factor specific for the promoters of the 5 S rRNA genes (type 1 promoters) (11Engelke D.R. Ng S.-Y. Shastry B.S. Roeder R.G. Cell. 1980; 19: 717-728Abstract Full Text PDF PubMed Scopus (428) Google Scholar, 12Ginsberg A.M. King B.O. Roeder R.G. Cell. 1984; 39: 479-489Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 13Moorefield B. Roeder R.G. J. Biol. Chem. 1994; 269: 20857-20865Abstract Full Text PDF PubMed Google Scholar). TFIIIC is a multimeric protein required for both type 1 and type 2 promoters. Human TFIIIC consists of at least nine polypeptides (14Wang Z. Roeder R.G. Mol. Cell. 1998; 1: 749-757Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and can be split into two subcomponents, TFIIIC1 and TFIIIC2 (15Yoshinaga S.K. Boulanger P.A. Berk A.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3585-3589Crossref PubMed Scopus (93) Google Scholar). For type 3 promoters, only the C1 component of TFIIIC is required as an initiation factor (16Yoon J.-B. Murphy S. Bai L. Wang Z. Roeder R.G. Mol. Cell. Biol. 1995; 15: 2019-2027Crossref PubMed Scopus (116) Google Scholar). Like TFIIIC, TFIIIB is an important Pol III transcription factor involved in initiating transcription on type 1 and type 2 promoters. According to the sequential recruitment model, TFIIIB is assembled into the preinitiation complex by TFIIIC on type 2 promoters or by the TFIIIC-TFIIIA complex on type 1 promoters and is positioned upstream of the transcription start site (17Kassavetis G.A. Burkhard R.B. Lam H.N. Geiduschek E.P. Cell. 1990; 60: 235-245Abstract Full Text PDF PubMed Scopus (359) Google Scholar). In vitrostudies have shown that TFIIIB then recruits Pol III to the promoter to direct accurate initiation of transcription and can do so even after TFIIIC has been stripped off the promoter (18Chédin S. Ferri M.L. Peyroche G. Andrau J.C. Jourdain S. Lefebvre O. Werner M. Carles C. Sentenac A. Cold Spring Harbor Symp. Quant. Biol. 1998; LXIII: 381-389Crossref Scopus (67) Google Scholar). TFIIIB contains the TATA-binding protein (TBP) and various TBP-associated factors (TAFs) (19Buratowski S. Zhou H. Cell. 1992; 71: 221-230Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 20Lobo S.M. Tanaka M. Sullivan M.L. Hernandez N. Cell. 1992; 71: 1029-1040Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 21Taggart A.K.P. Fisher T.S. Pugh B.F. Cell. 1992; 71: 1015-1028Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 22White R.J. Jackson S.P. Cell. 1992; 71: 1041-1053Abstract Full Text PDF PubMed Scopus (82) Google Scholar). Although the number and identity of TAFs in human TFIIIB have not been fully established, a TAF that has been characterized in some detail is TAF3B2 (previously named BRF or hTFIIIB90) (23Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar, 24Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 6: 7031-7042Crossref Scopus (67) Google Scholar). Recently, two additional TAFs that form part of TFIIIB have been identified: hB“, which is the human homologue of the yeast TFIIIB component, B”, and hBRFU/TFIIIB50 (25Schramm L. Pendergrast P.S. Sun Y.L. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar, 26Teichmann M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14200-14205Crossref PubMed Scopus (67) Google Scholar).The aim of the work presented here was to investigate how Pol III transcription is regulated during changes in cell growth and proliferation. Toward this end, we used the human fibroblast cell line TR9–7, derived from a patient with Li-Fraumeni syndrome, in which both copies of the endogenous gene for p53 are inactivated (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). TR9–7 cells are stably transfected with a recombinant construct that allows for the inducible expression of p53 from a tetracycline-controlled promoter. Thus, whereas p53 expression is barely detectable in TR9–7 cells grown in the presence of 1 μg/ml of tetracycline, decreasing the tetracycline concentration in the growth medium results in an increase in both p53 expression and function, as determined by the induction of the p53-responsive p21/Waf1 gene (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). Furthermore, the levels of p53 expression in TR9–7 cells upon tetracycline withdrawal are comparable with those induced in a wild-type cell line in response to DNA damage (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). This degree of p53 induction in the TR9–7 cells results in a slow onset, reversible cell cycle arrest in both the G1 and G2/M stages of the cell cycle. Complete cell cycle arrest is achieved 4–6 days after the onset of p53 induction. TR9–7 cells can be maintained in this arrested state for up to 20 days and yet can still return to a normal cycling state afterward (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). The TR9–7 cell line therefore provides a versatile experimental system in which cell cycle arrest can be manipulated. As discussed below, by use of this system, we have gained insights into the mechanisms by which sustained cell cycle arrest leads to a reduced capacity for Pol III transcription.DISCUSSIONThe aim of this study was to investigate whether RNA Pol III transcription is regulated in response to sustained cell cycle arrest. RNA Pol III transcription is responsible for the synthesis of various small RNA species involved in maintaining cellular biosynthetic capacity. Under conditions of prolonged cell cycle arrest, when cells do not require high levels of protein synthesis, it might therefore be assumed that cells can afford to down-regulate Pol III transcriptional activity. We tested this hypothesis in the human TR9–7 tissue culture cell line in which cell cycle arrest can be initiated by the inducible expression of p53 (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). These studies revealed that Pol III capacity for a range of type 1 and type 2 Pol III promoters is indeed reduced greatly as a consequence of sustained cell cycle arrest. Furthermore, we showed that this was not the result of decreased activity of the RNA polymerase III enzyme itself nor decreased TFIIIC activity. Instead, we showed that TFIIIB activity was the target for repression and that it was the 0.38M-TFIIIB component but not the 0.48M-TFIIIB component, of this factor that was specifically repressed. Finally, we revealed that this repression was accompanied by a dramatic destabilization of the 0.38M-TFIIIB component TAF3B2 and showed that reversing this destabilization by treating cells with proteasome inhibitors led to restored Pol III transcriptional capacity. Because TAF3B2 is essential for Pol III transcription (23Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar, 24Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 6: 7031-7042Crossref Scopus (67) Google Scholar), we conclude that its destabilization is likely to play a major role in effecting the loss of Pol III capacity in the TR9–7 system. However, it should be noted that not all human TFIIIB subunits have so far been cloned; it therefore remains possible that down-regulation of another TFIIIB component may also contribute to the reduced Pol III transcription that we observe. It will clearly be of great interest to determine the features of TAF3B2 that allow it be targeted for degradation upon sustained cell cycle arrest and to identify and characterize thetrans-acting factors that mediate this control.Two previous studies reported that p53 can act as a direct repressor of Pol III transcription through inhibitory interactions with TFIIIB (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar,47Chesnokov I. Chu W.-M. Botchan M.R. Schmid C.W. Mol. Cell. Biol. 1996; 16: 7084-7088Crossref PubMed Scopus (88) Google Scholar). Furthermore, the promoters we used in our study, namely those of the human 5 S rRNA gene, two different tRNA genes, and the adenoviral VAI gene, were inhibited directly by p53 in the study of Cairns and White (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar). In light of these findings, we considered the possibility that the repressive effects on Pol III transcription that we observed in TR9–7 cells could be mediated directly by p53. However, several lines of evidence indicate that this is not the case. First, the kinetics of Pol III repression in TR9–7 cells correlated best with the onset of cell cycle arrest, not the kinetics of p53 induction. Second, the addition of recombinant p53 to extracts of proliferating TR9–7 cells, in amounts similar to those used by Cairns and White (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar), did not reproduce the repression observed in extracts from cell cycle-arrested cells. The amount of exogenous p53 added in these experiments was at least 100-fold higher than the amounts present endogenously in extracts of cell cycle-arrested cells. Third, transcriptional repression was sustained after removing p53 selectively from extracts of cell cycle-arrested cells by immunodepletion. Finally, and most compelling, we showed that p53 protein was maintained at high levels when cell cycle-arrested cells were treated with a proteasome inhibitor, yet there was full recovery of Pol III transcriptional activity. It therefore seems clear that p53 is not directly responsible for repression of Pol III transcription capacity following sustained cell cycle arrest in the TR9–7 cell system.The data we present here are in agreement with the model that there is an intimate linkage between cellular biosynthetic capacity and the activity of RNA Pol III transcription apparatus (1Larminie C.G.C. Alzuherri H.M. Cairns C.A. McLees A. White R.J. J. Mol. Med. 1998; 76: 94-103Crossref PubMed Scopus (31) Google Scholar). We describe a novel pathway by which Pol III transcription may be negatively regulated during times when cells do not require elevated levels of active protein synthesis, such as during sustained cell cycle arrest. Taken together with other work, our results suggest that down-regulation of Pol III transcription can be elicited by multiple mechanisms: direct inhibitory interactions between the retinoblastoma protein and TFIIIB (8White R.J. Trouche D. Martin K. Jackson S.P. Kouzarides T. Nature. 1996; 382: 88-90Crossref PubMed Scopus (183) Google Scholar), direct inhibition of TFIIIB by the p53 protein (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar), and destabilization of TAF3B2. Remarkably, all three pathways converge on TFIIIB, which therefore seems to have evolved as a crucial target in the regulation of Pol III transcription (3White R.J. Int. J. Oncol. 1998; 12: 741-748PubMed Google Scholar). It is tempting to speculate that these mechanisms represent distinct but complementary pathways to bring about regulation of Pol III activity in response to changes in cellular growth potential and proliferative status. These pathways could operate in different biological contexts or, at least in some circumstances, could co-operate to reinforce the level of transcriptional control. The unraveling of the molecular details of TAF3B2 destabilization upon sustained cell cycle arrest may provide further insights into how this important cross-regulation is achieved, and this might eventually lead to a better understanding of tissue growth under both physiological and pathological conditions. Cellular growth and proliferation are two distinct processes. Proliferation is defined as the increase in the number of cells, which occurs when cells progress through a new cell division cycle. Cell growth, on the other hand, is defined as the increase in mass of an individual cell. Growth is related to the biosynthetic capacity of a cell, a reliable indicator of which is the level of protein synthesis (1Larminie C.G.C. Alzuherri H.M. Cairns C.A. McLees A. White R.J. J. Mol. Med. 1998; 76: 94-103Crossref PubMed Scopus (31) Google Scholar). Conceptually, it seems requisite that the processes of growth and proliferation are linked and that sensitive fine tuning exists between them. At present, however, very little is known about the molecular mechanisms that ensure that this crucial cross-regulation is achieved. After neoplastic transformation of cells, uncontrolled proliferation is only sustainable if it is linked to deregulated cell growth (2Rosenwald I.B. BioEssays. 1996; 18: 243-250Crossref PubMed Scopus (45) Google Scholar). Major determinants of protein synthesis are the ribosomes and the machinery that delivers activated amino acids to the ribosomes during translation. Growth is therefore dependent on the availability of adequate supplies of rRNAs and tRNAs. It has been shown that, as cells enter quiescence, existing ribosomes disaggregate into their subunits, the levels of rRNAs and tRNAs decrease, and net protein synthesis is down-regulated. These events are reversed upon mitogenic stimulation (2Rosenwald I.B. BioEssays. 1996; 18: 243-250Crossref PubMed Scopus (45) Google Scholar). But how are regulation of growth and regulation of proliferation jointly accomplished? The prevailing model is that tumor suppressor proteins play a crucial role in eliciting this control (1Larminie C.G.C. Alzuherri H.M. Cairns C.A. McLees A. White R.J. J. Mol. Med. 1998; 76: 94-103Crossref PubMed Scopus (31) Google Scholar, 3White R.J. Int. J. Oncol. 1998; 12: 741-748PubMed Google Scholar, 4White R.J. Gene Ther. Mol. Biol. 1998; 1: 613-628Google Scholar). For example, the retinoblastoma protein regulates entry into S phase by controlling E2F, a transcription factor involved in the expression of S phase-inducing genes (5Kouzarides T. Semin. Cancer Biol. 1995; 6: 91-98Crossref PubMed Scopus (64) Google Scholar, 6Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4295) Google Scholar). It may also affect cell growth potential by repressing the activities of RNA polymerases I (7Cavanaugh A.H. Hempel W.M. Taylor L.J. Rogalsky V. Todorov G. Rothblum L.I. Nature. 1995; 374: 177-180Crossref PubMed Scopus (290) Google Scholar) and III (8White R.J. Trouche D. Martin K. Jackson S.P. Kouzarides T. Nature. 1996; 382: 88-90Crossref PubMed Scopus (183) Google Scholar), which synthesize rRNAs and tRNAs, respectively. Multi-functional proteins such as retinoblastoma protein may therefore be vital for adjusting cellular growth potential to match proliferative activity. Conversely, as a consequence of losing retinoblastoma protein function, cells may lose control over both proliferation and growth and be put on a fast track toward neoplasia (1Larminie C.G.C. Alzuherri H.M. Cairns C.A. McLees A. White R.J. J. Mol. Med. 1998; 76: 94-103Crossref PubMed Scopus (31) Google Scholar). RNA polymerase (Pol)1 III synthesizes a number of small RNA species, including the 5 S rRNA, tRNAs, the spliceosomal U6 small nuclear RNA, the 7SL RNA of the signal recognition particle, and the adenovirus VAI RNA (9White R.J. RNA Polymerase III Transcription. R. G. Landes Company, Austin, TX1994Google Scholar). The promoters of genes transcribed by Pol III are subdivided into three groups, type 1, type 2, and type 3 promoters, based on promoter structure and their requirements for basal Pol III transcription factors (10Willis I.M. Eur. J. Biochem. 1993; 212: 1-11Crossref PubMed Scopus (191) Google Scholar). TFIIIA is a monomeric protein factor specific for the promoters of the 5 S rRNA genes (type 1 promoters) (11Engelke D.R. Ng S.-Y. Shastry B.S. Roeder R.G. Cell. 1980; 19: 717-728Abstract Full Text PDF PubMed Scopus (428) Google Scholar, 12Ginsberg A.M. King B.O. Roeder R.G. Cell. 1984; 39: 479-489Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 13Moorefield B. Roeder R.G. J. Biol. Chem. 1994; 269: 20857-20865Abstract Full Text PDF PubMed Google Scholar). TFIIIC is a multimeric protein required for both type 1 and type 2 promoters. Human TFIIIC consists of at least nine polypeptides (14Wang Z. Roeder R.G. Mol. Cell. 1998; 1: 749-757Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and can be split into two subcomponents, TFIIIC1 and TFIIIC2 (15Yoshinaga S.K. Boulanger P.A. Berk A.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3585-3589Crossref PubMed Scopus (93) Google Scholar). For type 3 promoters, only the C1 component of TFIIIC is required as an initiation factor (16Yoon J.-B. Murphy S. Bai L. Wang Z. Roeder R.G. Mol. Cell. Biol. 1995; 15: 2019-2027Crossref PubMed Scopus (116) Google Scholar). Like TFIIIC, TFIIIB is an important Pol III transcription factor involved in initiating transcription on type 1 and type 2 promoters. According to the sequential recruitment model, TFIIIB is assembled into the preinitiation complex by TFIIIC on type 2 promoters or by the TFIIIC-TFIIIA complex on type 1 promoters and is positioned upstream of the transcription start site (17Kassavetis G.A. Burkhard R.B. Lam H.N. Geiduschek E.P. Cell. 1990; 60: 235-245Abstract Full Text PDF PubMed Scopus (359) Google Scholar). In vitrostudies have shown that TFIIIB then recruits Pol III to the promoter to direct accurate initiation of transcription and can do so even after TFIIIC has been stripped off the promoter (18Chédin S. Ferri M.L. Peyroche G. Andrau J.C. Jourdain S. Lefebvre O. Werner M. Carles C. Sentenac A. Cold Spring Harbor Symp. Quant. Biol. 1998; LXIII: 381-389Crossref Scopus (67) Google Scholar). TFIIIB contains the TATA-binding protein (TBP) and various TBP-associated factors (TAFs) (19Buratowski S. Zhou H. Cell. 1992; 71: 221-230Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 20Lobo S.M. Tanaka M. Sullivan M.L. Hernandez N. Cell. 1992; 71: 1029-1040Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 21Taggart A.K.P. Fisher T.S. Pugh B.F. Cell. 1992; 71: 1015-1028Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 22White R.J. Jackson S.P. Cell. 1992; 71: 1041-1053Abstract Full Text PDF PubMed Scopus (82) Google Scholar). Although the number and identity of TAFs in human TFIIIB have not been fully established, a TAF that has been characterized in some detail is TAF3B2 (previously named BRF or hTFIIIB90) (23Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar, 24Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 6: 7031-7042Crossref Scopus (67) Google Scholar). Recently, two additional TAFs that form part of TFIIIB have been identified: hB“, which is the human homologue of the yeast TFIIIB component, B”, and hBRFU/TFIIIB50 (25Schramm L. Pendergrast P.S. Sun Y.L. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (109) Google Scholar, 26Teichmann M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14200-14205Crossref PubMed Scopus (67) Google Scholar). The aim of the work presented here was to investigate how Pol III transcription is regulated during changes in cell growth and proliferation. Toward this end, we used the human fibroblast cell line TR9–7, derived from a patient with Li-Fraumeni syndrome, in which both copies of the endogenous gene for p53 are inactivated (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). TR9–7 cells are stably transfected with a recombinant construct that allows for the inducible expression of p53 from a tetracycline-controlled promoter. Thus, whereas p53 expression is barely detectable in TR9–7 cells grown in the presence of 1 μg/ml of tetracycline, decreasing the tetracycline concentration in the growth medium results in an increase in both p53 expression and function, as determined by the induction of the p53-responsive p21/Waf1 gene (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). Furthermore, the levels of p53 expression in TR9–7 cells upon tetracycline withdrawal are comparable with those induced in a wild-type cell line in response to DNA damage (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). This degree of p53 induction in the TR9–7 cells results in a slow onset, reversible cell cycle arrest in both the G1 and G2/M stages of the cell cycle. Complete cell cycle arrest is achieved 4–6 days after the onset of p53 induction. TR9–7 cells can be maintained in this arrested state for up to 20 days and yet can still return to a normal cycling state afterward (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). The TR9–7 cell line therefore provides a versatile experimental system in which cell cycle arrest can be manipulated. As discussed below, by use of this system, we have gained insights into the mechanisms by which sustained cell cycle arrest leads to a reduced capacity for Pol III transcription. DISCUSSIONThe aim of this study was to investigate whether RNA Pol III transcription is regulated in response to sustained cell cycle arrest. RNA Pol III transcription is responsible for the synthesis of various small RNA species involved in maintaining cellular biosynthetic capacity. Under conditions of prolonged cell cycle arrest, when cells do not require high levels of protein synthesis, it might therefore be assumed that cells can afford to down-regulate Pol III transcriptional activity. We tested this hypothesis in the human TR9–7 tissue culture cell line in which cell cycle arrest can be initiated by the inducible expression of p53 (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). These studies revealed that Pol III capacity for a range of type 1 and type 2 Pol III promoters is indeed reduced greatly as a consequence of sustained cell cycle arrest. Furthermore, we showed that this was not the result of decreased activity of the RNA polymerase III enzyme itself nor decreased TFIIIC activity. Instead, we showed that TFIIIB activity was the target for repression and that it was the 0.38M-TFIIIB component but not the 0.48M-TFIIIB component, of this factor that was specifically repressed. Finally, we revealed that this repression was accompanied by a dramatic destabilization of the 0.38M-TFIIIB component TAF3B2 and showed that reversing this destabilization by treating cells with proteasome inhibitors led to restored Pol III transcriptional capacity. Because TAF3B2 is essential for Pol III transcription (23Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar, 24Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 6: 7031-7042Crossref Scopus (67) Google Scholar), we conclude that its destabilization is likely to play a major role in effecting the loss of Pol III capacity in the TR9–7 system. However, it should be noted that not all human TFIIIB subunits have so far been cloned; it therefore remains possible that down-regulation of another TFIIIB component may also contribute to the reduced Pol III transcription that we observe. It will clearly be of great interest to determine the features of TAF3B2 that allow it be targeted for degradation upon sustained cell cycle arrest and to identify and characterize thetrans-acting factors that mediate this control.Two previous studies reported that p53 can act as a direct repressor of Pol III transcription through inhibitory interactions with TFIIIB (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar,47Chesnokov I. Chu W.-M. Botchan M.R. Schmid C.W. Mol. Cell. Biol. 1996; 16: 7084-7088Crossref PubMed Scopus (88) Google Scholar). Furthermore, the promoters we used in our study, namely those of the human 5 S rRNA gene, two different tRNA genes, and the adenoviral VAI gene, were inhibited directly by p53 in the study of Cairns and White (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar). In light of these findings, we considered the possibility that the repressive effects on Pol III transcription that we observed in TR9–7 cells could be mediated directly by p53. However, several lines of evidence indicate that this is not the case. First, the kinetics of Pol III repression in TR9–7 cells correlated best with the onset of cell cycle arrest, not the kinetics of p53 induction. Second, the addition of recombinant p53 to extracts of proliferating TR9–7 cells, in amounts similar to those used by Cairns and White (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar), did not reproduce the repression observed in extracts from cell cycle-arrested cells. The amount of exogenous p53 added in these experiments was at least 100-fold higher than the amounts present endogenously in extracts of cell cycle-arrested cells. Third, transcriptional repression was sustained after removing p53 selectively from extracts of cell cycle-arrested cells by immunodepletion. Finally, and most compelling, we showed that p53 protein was maintained at high levels when cell cycle-arrested cells were treated with a proteasome inhibitor, yet there was full recovery of Pol III transcriptional activity. It therefore seems clear that p53 is not directly responsible for repression of Pol III transcription capacity following sustained cell cycle arrest in the TR9–7 cell system.The data we present here are in agreement with the model that there is an intimate linkage between cellular biosynthetic capacity and the activity of RNA Pol III transcription apparatus (1Larminie C.G.C. Alzuherri H.M. Cairns C.A. McLees A. White R.J. J. Mol. Med. 1998; 76: 94-103Crossref PubMed Scopus (31) Google Scholar). We describe a novel pathway by which Pol III transcription may be negatively regulated during times when cells do not require elevated levels of active protein synthesis, such as during sustained cell cycle arrest. Taken together with other work, our results suggest that down-regulation of Pol III transcription can be elicited by multiple mechanisms: direct inhibitory interactions between the retinoblastoma protein and TFIIIB (8White R.J. Trouche D. Martin K. Jackson S.P. Kouzarides T. Nature. 1996; 382: 88-90Crossref PubMed Scopus (183) Google Scholar), direct inhibition of TFIIIB by the p53 protein (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar), and destabilization of TAF3B2. Remarkably, all three pathways converge on TFIIIB, which therefore seems to have evolved as a crucial target in the regulation of Pol III transcription (3White R.J. Int. J. Oncol. 1998; 12: 741-748PubMed Google Scholar). It is tempting to speculate that these mechanisms represent distinct but complementary pathways to bring about regulation of Pol III activity in response to changes in cellular growth potential and proliferative status. These pathways could operate in different biological contexts or, at least in some circumstances, could co-operate to reinforce the level of transcriptional control. The unraveling of the molecular details of TAF3B2 destabilization upon sustained cell cycle arrest may provide further insights into how this important cross-regulation is achieved, and this might eventually lead to a better understanding of tissue growth under both physiological and pathological conditions. The aim of this study was to investigate whether RNA Pol III transcription is regulated in response to sustained cell cycle arrest. RNA Pol III transcription is responsible for the synthesis of various small RNA species involved in maintaining cellular biosynthetic capacity. Under conditions of prolonged cell cycle arrest, when cells do not require high levels of protein synthesis, it might therefore be assumed that cells can afford to down-regulate Pol III transcriptional activity. We tested this hypothesis in the human TR9–7 tissue culture cell line in which cell cycle arrest can be initiated by the inducible expression of p53 (27Agarwal M.L. Agarwal A. Taylor W.R. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8493-8497Crossref PubMed Scopus (792) Google Scholar). These studies revealed that Pol III capacity for a range of type 1 and type 2 Pol III promoters is indeed reduced greatly as a consequence of sustained cell cycle arrest. Furthermore, we showed that this was not the result of decreased activity of the RNA polymerase III enzyme itself nor decreased TFIIIC activity. Instead, we showed that TFIIIB activity was the target for repression and that it was the 0.38M-TFIIIB component but not the 0.48M-TFIIIB component, of this factor that was specifically repressed. Finally, we revealed that this repression was accompanied by a dramatic destabilization of the 0.38M-TFIIIB component TAF3B2 and showed that reversing this destabilization by treating cells with proteasome inhibitors led to restored Pol III transcriptional capacity. Because TAF3B2 is essential for Pol III transcription (23Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar, 24Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 6: 7031-7042Crossref Scopus (67) Google Scholar), we conclude that its destabilization is likely to play a major role in effecting the loss of Pol III capacity in the TR9–7 system. However, it should be noted that not all human TFIIIB subunits have so far been cloned; it therefore remains possible that down-regulation of another TFIIIB component may also contribute to the reduced Pol III transcription that we observe. It will clearly be of great interest to determine the features of TAF3B2 that allow it be targeted for degradation upon sustained cell cycle arrest and to identify and characterize thetrans-acting factors that mediate this control. Two previous studies reported that p53 can act as a direct repressor of Pol III transcription through inhibitory interactions with TFIIIB (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar,47Chesnokov I. Chu W.-M. Botchan M.R. Schmid C.W. Mol. Cell. Biol. 1996; 16: 7084-7088Crossref PubMed Scopus (88) Google Scholar). Furthermore, the promoters we used in our study, namely those of the human 5 S rRNA gene, two different tRNA genes, and the adenoviral VAI gene, were inhibited directly by p53 in the study of Cairns and White (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar). In light of these findings, we considered the possibility that the repressive effects on Pol III transcription that we observed in TR9–7 cells could be mediated directly by p53. However, several lines of evidence indicate that this is not the case. First, the kinetics of Pol III repression in TR9–7 cells correlated best with the onset of cell cycle arrest, not the kinetics of p53 induction. Second, the addition of recombinant p53 to extracts of proliferating TR9–7 cells, in amounts similar to those used by Cairns and White (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar), did not reproduce the repression observed in extracts from cell cycle-arrested cells. The amount of exogenous p53 added in these experiments was at least 100-fold higher than the amounts present endogenously in extracts of cell cycle-arrested cells. Third, transcriptional repression was sustained after removing p53 selectively from extracts of cell cycle-arrested cells by immunodepletion. Finally, and most compelling, we showed that p53 protein was maintained at high levels when cell cycle-arrested cells were treated with a proteasome inhibitor, yet there was full recovery of Pol III transcriptional activity. It therefore seems clear that p53 is not directly responsible for repression of Pol III transcription capacity following sustained cell cycle arrest in the TR9–7 cell system. The data we present here are in agreement with the model that there is an intimate linkage between cellular biosynthetic capacity and the activity of RNA Pol III transcription apparatus (1Larminie C.G.C. Alzuherri H.M. Cairns C.A. McLees A. White R.J. J. Mol. Med. 1998; 76: 94-103Crossref PubMed Scopus (31) Google Scholar). We describe a novel pathway by which Pol III transcription may be negatively regulated during times when cells do not require elevated levels of active protein synthesis, such as during sustained cell cycle arrest. Taken together with other work, our results suggest that down-regulation of Pol III transcription can be elicited by multiple mechanisms: direct inhibitory interactions between the retinoblastoma protein and TFIIIB (8White R.J. Trouche D. Martin K. Jackson S.P. Kouzarides T. Nature. 1996; 382: 88-90Crossref PubMed Scopus (183) Google Scholar), direct inhibition of TFIIIB by the p53 protein (43Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (155) Google Scholar), and destabilization of TAF3B2. Remarkably, all three pathways converge on TFIIIB, which therefore seems to have evolved as a crucial target in the regulation of Pol III transcription (3White R.J. Int. J. Oncol. 1998; 12: 741-748PubMed Google Scholar). It is tempting to speculate that these mechanisms represent distinct but complementary pathways to bring about regulation of Pol III activity in response to changes in cellular growth potential and proliferative status. These pathways could operate in different biological contexts or, at least in some circumstances, could co-operate to reinforce the level of transcriptional control. The unraveling of the molecular details of TAF3B2 destabilization upon sustained cell cycle arrest may provide further insights into how this important cross-regulation is achieved, and this might eventually lead to a better understanding of tissue growth under both physiological and pathological conditions. We thank Jane Bradbury and Steve Bell for valuable scientific input. We are also extremely grateful to George R. Stark for allowing us to use the TR9–7 cell line, to Nouria Hernandez for antibodies against TAF3B2, and to S. J. Flint for the MBP-6 cell line and TBP antibodies.
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