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• W1990647583 abstract "Increased rates of RNA polymerase (pol) III transcription constitute a central feature of the mitogenic response, but little is known about the mechanism(s) responsible. We demonstrate that the retinoblastoma protein RB plays a major role in suppressing pol III transcription in growth-arrested fibroblasts. RB knockout cells are compromised in their ability to down-regulate pol III following serum withdrawal. RB binds and represses the pol III-specific transcription factor TFIIIB during G0 and early G1, but this interaction decreases as cells approach S phase. Full induction of pol III coincides with mid- to late G1 phase, when RB becomes phosphorylated by cyclin D- and E-dependent kinases. TFIIIB only associates with the underphosphorylated form of RB, and overexpression of cyclins D and E stimulates pol III transcription in vivo. The RB-related protein p130 also contributes to the repression of TFIIIB in growth-arrested fibroblasts. These observations provide insight into the mechanisms responsible for controlling pol III transcription during the switch between growth and quiescence. Increased rates of RNA polymerase (pol) III transcription constitute a central feature of the mitogenic response, but little is known about the mechanism(s) responsible. We demonstrate that the retinoblastoma protein RB plays a major role in suppressing pol III transcription in growth-arrested fibroblasts. RB knockout cells are compromised in their ability to down-regulate pol III following serum withdrawal. RB binds and represses the pol III-specific transcription factor TFIIIB during G0 and early G1, but this interaction decreases as cells approach S phase. Full induction of pol III coincides with mid- to late G1 phase, when RB becomes phosphorylated by cyclin D- and E-dependent kinases. TFIIIB only associates with the underphosphorylated form of RB, and overexpression of cyclins D and E stimulates pol III transcription in vivo. The RB-related protein p130 also contributes to the repression of TFIIIB in growth-arrested fibroblasts. These observations provide insight into the mechanisms responsible for controlling pol III transcription during the switch between growth and quiescence. RNA polymerase acidic ribosomal phosphoprotein P0 cyclin-dependent kinase Dulbecco's modified Eagle's medium fetal calf serum transcription factor target of rapamycin cytomegalovirus chloramphenicol acetyltransferase polymerase chain reaction TATA-binding protein The retinoblastoma protein RB is a highly abundant tumor suppressor that can bind and regulate a variety of transcription factors (reviewed in Refs. 1Herwig S. Strauss M. Eur. J. Biochem. 1997; 246: 581-601Crossref PubMed Scopus (217) Google Scholar, 2Taya Y. Trends Biochem. Sci. 1997; 22: 14-17Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 3Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar, 4Mulligan G. Jacks T. Trends Genet. 1998; 14: 223-229Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). One example that has been added recently to the growing list of RB-binding proteins is the RNA polymerase (pol)1III-specific factor TFIIIB (5Chu W.-M. Wang Z. Roeder R.G. Schmid C.W. J. Biol. Chem. 1997; 272: 14755-14761Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Larminie C.G.C. Cairns C.A. Mital R. Martin K. Kouzarides T. Jackson S.P. White R.J. EMBO J. 1997; 16: 2061-2071Crossref PubMed Scopus (83) Google Scholar). Recombinant RB was shown to bind to TFIIIB in vitro and repress its activity (5Chu W.-M. Wang Z. Roeder R.G. Schmid C.W. J. Biol. Chem. 1997; 272: 14755-14761Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Larminie C.G.C. Cairns C.A. Mital R. Martin K. Kouzarides T. Jackson S.P. White R.J. EMBO J. 1997; 16: 2061-2071Crossref PubMed Scopus (83) Google Scholar). Furthermore, coimmunoprecipitation and cofractionation experiments demonstrated a stable association between endogenous cellular RB and TFIIIB (6Larminie C.G.C. Cairns C.A. Mital R. Martin K. Kouzarides T. Jackson S.P. White R.J. EMBO J. 1997; 16: 2061-2071Crossref PubMed Scopus (83) Google Scholar). The functional significance of this interaction was shown in studies of RB knockout mice, since primary fibroblasts from Rb−/− mice display elevated TFIIIB activity relative to fibroblasts derived from their wild-type siblings (6Larminie C.G.C. Cairns C.A. Mital R. Martin K. Kouzarides T. Jackson S.P. White R.J. EMBO J. 1997; 16: 2061-2071Crossref PubMed Scopus (83) Google Scholar). These results establish TFIIIB as a bona fide target for repression by RB. Similar approaches have shown that TFIIIB is also bound and repressed by the RB-related proteins p107 and p130 (7Sutcliffe J.E. Cairns C.A. McLees A. Allison S.J. Tosh K. White R.J. Mol. Cell. Biol. 1999; 19: 4255-4261Crossref PubMed Scopus (52) Google Scholar). TFIIIB is required for the expression of all pol III templates (reviewed in Refs. 8Paule M.R. White R.J. Nucleic Acids Res. 2000; 28: 1283-1298Crossref PubMed Google Scholar and 9White R.J. RNA Polymerase III Transcription. Springer-Verlag New York Inc., New York1998Crossref Google Scholar). It serves to recruit the polymerase to a promoter and position it over the transcription start site (10Kassavetis G.A. Braun B.R. Nguyen L.H. Geiduschek E.P. Cell. 1990; 60: 235-245Abstract Full Text PDF PubMed Scopus (360) Google Scholar). By interacting with this general factor, RB appears able to regulate the expression of all pol III-transcribed genes, including tRNA, 5 S rRNA, U6 small nuclear RNA, VA1, and Alu genes (5Chu W.-M. Wang Z. Roeder R.G. Schmid C.W. J. Biol. Chem. 1997; 272: 14755-14761Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 6Larminie C.G.C. Cairns C.A. Mital R. Martin K. Kouzarides T. Jackson S.P. White R.J. EMBO J. 1997; 16: 2061-2071Crossref PubMed Scopus (83) Google Scholar, 11White R.J. Trouche D. Martin K. Jackson S.P. Kouzarides T. Nature. 1996; 382: 88-90Crossref PubMed Scopus (184) Google Scholar). Since a high rate of tRNA and rRNA synthesis is required to sustain rapid growth, it has been speculated that the inhibition of pol III transcription may contribute to the growth suppression capacity of RB (12Nasmyth K. Nature. 1996; 382: 28-29Crossref PubMed Scopus (42) Google Scholar, 13White R.J. Trends Biochem. Sci. 1997; 22: 77-80Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 14Larminie 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). RB function is regulated by cyclin-dependent kinases (reviewed in Refs. 2Taya Y. Trends Biochem. Sci. 1997; 22: 14-17Abstract Full Text PDF PubMed Scopus (234) Google Scholar and 3Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar and Ref. 15Mittnacht S. Curr. Opin. Genet. Dev. 1998; 8: 21-27Crossref PubMed Scopus (334) Google Scholar). The cyclin D-dependent kinases CDK4 and CDK6 phosphorylate RB partially and the process is completed by cyclin E-CDK2 (16Zarkowska T. Mittnacht S. J. Biol. Chem. 1997; 272: 12738-12746Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 17Lundberg A.S. Weinberg R.A. Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (856) Google Scholar). The action of cyclin E-CDK2 appears to depend on prior phosphorylation by the cyclin D-dependent kinases (17Lundberg A.S. Weinberg R.A. Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (856) Google Scholar). At least 10 serine and threonine residues can become phosphorylated in RB (2Taya Y. Trends Biochem. Sci. 1997; 22: 14-17Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 16Zarkowska T. Mittnacht S. J. Biol. Chem. 1997; 272: 12738-12746Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). Once hyperphosphorylated, RB loses its ability to bind to many of its targets and function as a growth suppressor (2Taya Y. Trends Biochem. Sci. 1997; 22: 14-17Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 15Mittnacht S. Curr. Opin. Genet. Dev. 1998; 8: 21-27Crossref PubMed Scopus (334) Google Scholar). This occurs at the G1/S phase transition, in parallel with the synthesis of cyclins D and E (2Taya Y. Trends Biochem. Sci. 1997; 22: 14-17Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 15Mittnacht S. Curr. Opin. Genet. Dev. 1998; 8: 21-27Crossref PubMed Scopus (334) Google Scholar). The cyclin D-dependent kinases become active in mid- to late G1, at a stage called the restriction, or R, point when cells lose their serum dependence (18Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2590) Google Scholar). Cyclin E-CDK2 is activated shortly afterward, as cells leave G1 phase (18Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2590) Google Scholar). RB is then maintained in the hyperphosphorylated state throughout S, G2, and M phases, until it is dephosphorylated by protein phosphatase 1 at the end of mitosis (15Mittnacht S. Curr. Opin. Genet. Dev. 1998; 8: 21-27Crossref PubMed Scopus (334) Google Scholar). In cycling cells, therefore, the underphosphorylated form of RB is only present during the early period of G1. However, it is also found in resting G0 cells, which do not express significant levels of cyclins D and E (3Grana X. Garriga J. Mayol X. Oncogene. 1998; 17: 3365-3383Crossref PubMed Scopus (283) Google Scholar). The level of pol III transcription decreases significantly when growing fibroblasts are deprived of serum (19Johnson L.F. Abelson H.T. Green H. Penman S. Cell. 1974; 1: 95-100Abstract Full Text PDF Scopus (178) Google Scholar, 20Mauck J.C. Green H. Cell. 1974; 3: 171-177Abstract Full Text PDF PubMed Scopus (45) Google Scholar). This is likely to reflect a diminished requirement for protein production. Although the switch between G0 and G1 phases is the principle determinant of proliferation rate in mammalian cells, the molecular mechanism(s) responsible for regulating pol III activity during this transition are largely uncharacterized. This constitutes an important gap in our current understanding, since the rate of pol III transcription will undoubtedly have a major influence on the growth and proliferation of cells. One study concluded that a specific reduction in TFIIIB activity was responsible for down-regulating pol III transcription in growth-arrested cells, although the molecular details were not determined (21Tower J. Sollner-Webb B. Mol. Cell. Biol. 1988; 8: 1001-1005Crossref PubMed Scopus (36) Google Scholar). In contrast, another laboratory demonstrated that HeLa cells down-regulate pol III transcription when grown in low serum due to a decrease in the activity of TFIIIC2 (22Hoeffler W.K. Kovelman R. Roeder R.G. Cell. 1988; 53: 907-920Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 23Sinn E. Wang Z. Kovelman R. Roeder R.G. Genes Dev. 1995; 9: 675-685Crossref PubMed Scopus (56) Google Scholar). This is associated with a specific reduction in the levels of an essential subunit called TFIIICβ (23Sinn E. Wang Z. Kovelman R. Roeder R.G. Genes Dev. 1995; 9: 675-685Crossref PubMed Scopus (56) Google Scholar). However, HeLa cells continue to grow actively under the low serum conditions used in these studies (22Hoeffler W.K. Kovelman R. Roeder R.G. Cell. 1988; 53: 907-920Abstract Full Text PDF PubMed Scopus (98) Google Scholar) and may not provide a clear indication of how pol III is regulated during exit from the cell cycle. We have therefore investigated the regulation of pol III transcription during the transition between resting and growing states in untransformed fibroblasts. When such cells are stimulated to resume cycling, the major increase in tRNA synthesis occurs during the G1/S transition (19Johnson L.F. Abelson H.T. Green H. Penman S. Cell. 1974; 1: 95-100Abstract Full Text PDF Scopus (178) Google Scholar, 20Mauck J.C. Green H. Cell. 1974; 3: 171-177Abstract Full Text PDF PubMed Scopus (45) Google Scholar). Since this coincides with the hyperphosphorylation of RB by cyclin-dependent kinases, we examined the possibility that the increase in pol III transcription that accompanies cell cycle reentry involves a release of TFIIIB from interaction with RB. Immunoprecipitation analyses provide evidence that this is the case; RB associates with TFIIIB during G0 and early G1phases, but this interaction is substantially diminished after cells have passed the R point. The dissociation of RB from TFIIIB coincides with an increase in pol III activity. Only the underphosphorylated form of RB associates with TFIIIB. This suggests that TFIIIB is released from repression by RB at the G1/S transition due to hyperphosphorylation of the latter by the cyclin-dependent kinases. Indeed, overexpression of cyclins D and E activates pol III transcription. We also demonstrate that RB knockout cells are compromised in their ability to down-regulate pol III following serum deprivation. In addition, the RB-related pocket protein p130 is shown to interact with TFIIIB during G0 and early G1phase and contributes to the repression of pol III in serum-starved cells. We conclude that RB, p130, and the cyclin-dependent kinases play a major role in controlling pol III transcription during the switch between growth and quiescence. Balb/c 3T3 (A31), SV3T3 (Cl38), and mouse embryonic fibroblast cells were all grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.) supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 μg/ml streptomycin and were harvested when subconfluent. An insulin-transferrin-selenium supplement (Life Technologies) was added to the medium used to grow mouse embryonic fibroblasts. Unless otherwise specified, cell growth was arrested by reducing the serum concentration to 0.5%; mitogenic stimulation was then induced with 20% serum. Cells to be analyzed by flow cytometry were harvested in dissociation buffer (Sigma) and fixed in phosphate-buffered saline/ethanol (1:1, v/v). Propidium iodide (40 μg/ml) was added, and the DNA content of cell samples was measured using a Becton Dickinson FACScan (10,000 events/sample). Data were analyzed using Cell Quest software. [3H]Thymidine (0.1 μCi/ml) was added to serum-stimulated or quiescent cells 3 h prior to harvesting. Cells were then washed twice in phosphate-buffered saline, three times in 5% trichloroacetic acid, and twice in ethanol. Samples were solubilized in 0.3 m NaOH, and the incorporation of [3H]thymidine into DNA was then measured by liquid scintillation counting. Subconfluent Balb/c 3T3 cells were cultured for 24 h in DMEM containing 0.5% FCS. They were then incubated for 15 h in phosphate-free DMEM containing 100 μCi/ml [32P]orthophosphate either in the absence of FCS, to give G0 phase cells, or the presence of 10% FCS, to give S phase cells; early G1 phase cells were generated by adding 10% FCS for the final 3 h of the incubation. Cells were then lysed in RIPA buffer (64 mm Hepes, pH 7.4, 150 mm NaCl, 50 mm NaF, 10 mm EDTA, 1.2% Triton X-100, 0.64% sodium deoxycholate, 0.128% SDS, 10 mm β-glycerophosphate, 1 mm sodium orthovanadate, 10 mm sodium phosphate, 1 mmphenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 1.0 μg/ml trypsin inhibitor, 0.5 μg/ml aprotinin, 40 μg/ml bestatin), solubilized by incubation for 1 h at 4 °C, and centrifuged for 15 min at 4 °C. Total cellular RNA was extracted using TRI reagent (Sigma), according to the manufacturer's instructions. Agarose gel electrophoresis, Northern transfer, and hybridization were carried out as previously (24Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (157) Google Scholar). The B2 gene probe was a 240-base pair EcoRI–PstI fragment from pTB14 (25White R.J. Stott D. Rigby P.W.J. Cell. 1989; 59: 1081-1092Abstract Full Text PDF PubMed Scopus (90) Google Scholar). The tRNALeu gene was a 240-base pair EcoRI–HindIII fragment from pLeu (25White R.J. Stott D. Rigby P.W.J. Cell. 1989; 59: 1081-1092Abstract Full Text PDF PubMed Scopus (90) Google Scholar). The acidic ribosomal phosphoprotein P0 (ARPP P0) probe was a 1-kilobase pair EcoRI–HindIII fragment from the mouse cDNA (26Hurford R.K. Cobrinik D. Lee M.-H. Dyson N. Genes Dev. 1997; 11: 1447-1463Crossref PubMed Scopus (382) Google Scholar). Nuclear run-on assays were carried out as previously (11White R.J. Trouche D. Martin K. Jackson S.P. Kouzarides T. Nature. 1996; 382: 88-90Crossref PubMed Scopus (184) Google Scholar). Transient transfections used the calcium phosphate precipitation method. DNA precipitates were left on the plates overnight, and then the cells were washed with phosphate-buffered saline and cultured for 24 h before harvesting. Total RNA was extracted using TRI reagent (Sigma), according to the manufacturer's instructions. It was then analyzed by primer extension using primers for VA1 (5′-CACGCGGGCGGTAACCGCATG-3′) and CAT (5′-CGATGCCATTGGGATATATCA-3′), as described previously (11White R.J. Trouche D. Martin K. Jackson S.P. Kouzarides T. Nature. 1996; 382: 88-90Crossref PubMed Scopus (184) Google Scholar). The pVA1 plasmid contains the adenovirus VA1 gene (27Dean N. Berk A.J. Mol. Cell. Biol. 1988; 8: 3017-3025Crossref PubMed Scopus (44) Google Scholar). pHu5S3.1, pLeu, and pU6/Hae/RA.2 contain human 5 S rRNA, tRNALeu, and U6 gene promoters, respectively (25White R.J. Stott D. Rigby P.W.J. Cell. 1989; 59: 1081-1092Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 31White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). Expression vectors Rc-CDK2, Rc-CDK4, Rc-cycD1, and Rc-cycE contain CDK2, CDK4, cyclin D1, and cyclin E cDNAs, respectively, cloned into the pRc-CMV vector (Invitrogen) downstream of the CMV immediate early promoter (28Hinds P.W. Mittnacht S. Dulic V. Arnold A. Reed S.I. Weinberg R.A. Cell. 1992; 70: 993-1006Abstract Full Text PDF PubMed Scopus (876) Google Scholar). pCMVp16 contains the p16 cDNA fused to a CMV polyadenylation signal and cloned downstream of the CMV immediate early promoter (29Sandig V. Brand K. Herwig S. Lukas J. Bartek J. Strauss M. Nat. Med. 1997; 3: 313-319Crossref PubMed Scopus (221) Google Scholar). Rz 89-12 contains a ribozyme against murine p16 mRNA subcloned into the pX expression vector (30Nylandsted J. Rohde M. Bartek J. Strauss M. FEBS Lett. 1998; 436: 41-45Crossref PubMed Scopus (7) Google Scholar). pCAT (Promega) contains the CAT gene driven by the SV40 promoter and enhancer. Whole-cell extracts were prepared using a freeze-thaw procedure described previously (31White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). HeLa nuclear extracts were purchased from the Computer Cell Culture Center (Mons). PC-B and PC-C phosphocellulose step fractions were prepared as previously (31White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). A25(0.15) fraction containing TFIIIB was prepared by chromatography on phosphocellulose and DEAE-Sephadex, as previously (31White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). CHep-1.0 fraction containing TFIIIC and pol III was prepared by sequential chromatography on phosphocellulose and heparin-Sepharose, as previously (31White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). Recombinant TBP was purchased from Promega. Transcription reactions were carried out as previously (25White R.J. Stott D. Rigby P.W.J. Cell. 1989; 59: 1081-1092Abstract Full Text PDF PubMed Scopus (90) Google Scholar), except that pBR322 was not included, and the incubations were for 60 min at 30 °C. Whole cell extract (150 μg) was incubated for 4 h at 4 °C on an orbital shaker with 20 μl of protein A-Sepharose beads carrying equivalent amounts of prebound IgG. Samples were then pelleted, supernatants were removed, and the beads were washed five times with 150 μl of LDB buffer (25White R.J. Stott D. Rigby P.W.J. Cell. 1989; 59: 1081-1092Abstract Full Text PDF PubMed Scopus (90) Google Scholar). The bound material was analyzed by Western blotting. In the experiment shown in Fig. 7 A, reticulocyte lysate (15 μl) containing RB translated in the presence of [35S]Met and [35S]Cys was treated for 10 min at 30 °C with or without a mixture of baculovirus-expressed cyclin D-CDK4, cyclin E-CDK2, and cyclin A-CDK2; it was then incubated for 4 h with whole cell extract (150 μg) during immunoprecipitation. In this case, the precipitated material was analyzed by autoradiography rather than Western blotting. Antibodies used were C-15 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and G3–245 (Pharminogen) against RB, C-20 (Santa Cruz Biotechnology) against p130, monoclonal antibody clone 46 (Transduction Laboratories) against TFIIICβ, SI-1 against TFIIB (Santa Cruz Biotechnology), M-19 (Santa Cruz Biotechnology) against TAFI48, SL30 against TBP (32Lobo S.M. Tanaka M. Sullivan M.L. Hernandez N. Cell. 1992; 71: 1029-1040Abstract Full Text PDF PubMed Scopus (117) Google Scholar), and 128 against BRF (24Cairns C.A. White R.J. EMBO J. 1998; 17: 3112-3123Crossref PubMed Scopus (157) Google Scholar, 33Alzuherri H.M. White R.J. J. Biol. Chem. 1998; 273: 17166-17171Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Antibodies against RB that has been phosphorylated at specific sites (34Boylan J.F. Sharp D.M. Leffet L. Bowers A. Pan W. Exp. Cell Res. 1999; 248: 110-114Crossref PubMed Scopus (30) Google Scholar) were obtained from New England Biolabs. Western immunoblot analysis was performed as described previously (31White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). RNA was extracted using TRI Reagent (Sigma), according to the manufacturer's specifications. Reverse transcription reactions were performed for 1 h at 42 °C using 3 μg of RNA, 200 ng of Random Hexamers (Promega), and 400 units of Superscript II Reverse Transcriptase (Life Technologies) in a total volume of 40 μl of 1× First Strand Buffer (Life Technologies) containing 10 mm dithiothreitol and a 0.5 mmconcentration of each dNTP. PCRs were carried out using a PTC-100 programmable thermal controller (MJ Research Inc). 2 μl of cDNA was amplified with 20 pmol of either TFIIICβ primers (5′-CCAGAAGGGGTCTCAAAAGTCC-3′ and 5′-CTTTCTTCAGAGATGTCAAAGG-3′) to give a 303-base pair product, or ARPP P0 primers (5′-GACCTGGAAGTCCAACTACTTC-3′ and 5′-TGAGGTCCTCCTTGGTGAACAC-3′) to give a 268-base pair product. Amplification reactions contained 0.5 units of Taq DNA Polymerase (Promega) in a total volume of 1× Taq DNA polymerase buffer (Promega) containing 1.5 mmMgCl2 and a 0.2 mm concentration of each dNTP. PCR was performed under the following cycling parameters: 1) TFIIICβ, 94 °C for 3 min, six cycles of 95 °C for 1 min, 66 °C for 40 s, and 72 °C for 40 s; 22 cycles of 95 °C for 1 min, 62 °C for 40 s, and 72 °C for 40 s; 72 °C for 5 min; 2) ARPP P0, 95 °C for 2 min, 25 cycles of 95 °C 1 min, 58 °C for 30 s, and 72 °C for 1 min; 72 °C for 3 min. Reaction products were resolved on a 2% agarose gel and visualized by ethidium bromide staining. Actively growing Balb/c 3T3 cells were made quiescent by serum withdrawal. The majority of cells had arrested in a G0/G1 phase state after 1 day of culture under serum-free conditions, as indicated by flow cytometric analyses of their DNA content (Fig. 1 A, 0 h serum stimulation). This conclusion was supported by measurements of thymidine incorporation into newly synthesized DNA (Fig. 1 B). The abundance of pol III transcripts derived from the B2 middle repetitive gene family was substantially reduced in the growth-arrested cells, as revealed by Northern blotting (Fig.1 C, upper panel, compare lanes 9 and 10). This effect was specific, since levels of a pol II transcript encoding ARPP P0 did not diminish following serum deprivation (Fig. 1 C,lower panel). The growth-arrested fibroblasts were stimulated to reenter the cell cycle by the addition of medium containing 20% serum. Flow cytometric analysis and thymidine incorporation measurements demonstrated that S phase was reached between 12 and 15 h after serum stimulation (Fig. 1, A and B). Northern blotting with a B2 gene probe revealed a slight increase in pol III transcript levels by mid-G1 phase, 6–9 h after the addition of serum, and revealed that near maximal expression was reached by 12 h after mitogenic stimulation, shortly before S phase entry (Fig.1 C). We conclude that Balb/c 3T3 cells undergo growth arrest within 24 h of serum withdrawal and that this is accompanied by a significant reduction in pol III activity; when these fibroblasts resume cycling, pol III activity is restored during late G1phase. These observations are consistent with previous studies of 3T6 and BHK cells (19Johnson L.F. Abelson H.T. Green H. Penman S. Cell. 1974; 1: 95-100Abstract Full Text PDF Scopus (178) Google Scholar, 20Mauck J.C. Green H. Cell. 1974; 3: 171-177Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 35Lania L. Pannuti A. La Mantia G. Basilico C. FEBS Lett. 1987; 219: 400-404Crossref PubMed Scopus (19) Google Scholar). To begin to investigate the mechanism responsible for the growth control of pol III transcription in Balb/c 3T3 fibroblasts, we prepared whole cell extracts after various periods of culture in 10% serum or serum-free medium. Although little or no apoptosis was detected after 24 or 48 h without serum, flow cytometry suggested that a fraction of the cells undergo apoptosis after 72 h in serum-free medium. 2P. H. Scott, unpublished data. Extracts of fibroblasts maintained without serum for 24 h or more were found to transcribe the adenovirus VA1 gene significantly less actively than extracts prepared from proliferating cells that had been cultured in 10% serum (Fig. 2 A). Similar results were obtained with other pol III templates, including tRNA, 5 S rRNA, and U6 small nuclear RNA genes (Fig. 2 A). The extracts therefore mimic the serum-responsiveness of pol III transcription that is observed in vivo. Sinn et al. (23Sinn E. Wang Z. Kovelman R. Roeder R.G. Genes Dev. 1995; 9: 675-685Crossref PubMed Scopus (56) Google Scholar) reported previously that growth of HeLa cells in 0.5% serum results in a specific decrease in the abundance of the β subunit of TFIIIC2. If this is true in untransformed 3T3 cells, then it may account for the down-regulation of pol III transcription under quiescent conditions. To address this possibility, we carried out Western blots with extracts to test whether the decrease in pol III transcription correlated with a down-regulation of TFIIICβ. However, little or no change was detected in the level of TFIIICβ when growing and arrested 3T3 cells were compared (Fig. 2 B). Indeed, TFIIICβ levels were maintained even after culture for 72 h without serum. This result was confirmed using two additional antisera raised against different regions of TFIIICβ. 3H. M. Alzuherri, unpublished data. Reverse transcriptase-PCR analysis was employed to compare the levels of the mRNA encoding TFIIICβ, as an independent method to investigate the expression of this essential component of TFIIIC2. This approach also provided no evidence that TFIIICβ is sensitive to serum availability (Fig. 2 C). We conclude that changes in the abundance of TFIIICβ are unlikely to be responsible for regulating pol III transcription in growth-arrested Balb/c 3T3 cells. Add-back experiments were carried out to determine which factor is limiting for pol III transcription in extracts of 3T3 cells. A fraction containing partially purified TFIIIB was found to stimulate transcription when titrated into extracts of either growing or serum-starved cells (Fig. 3 A). This effect was highly specific, since little or no stimulation was observed in response to a fraction containing TFIIIC and pol III. The activity of all fractions was confirmed using complementation assays. 4R. J. White, unpublished data. The data suggest that under the conditions used, TFIIIB is limiting, while TFIIIC and pol III are in relative excess. This implies that the rate of pol III transcription in 3T3 cells may be dictated by the availability of active TFIIIB. We therefore carried out complementation assays to compare directly the activity of TFIIIB in extracts prepared from cells harvested either before or after serum withdrawal. In these assays, the extracts are subjected to mild heat treatment, which selectively inactivates endogenous TFIIIC; they are then tested for their ability to support transcription when mixed with a complementing system containing excess TFIIIC, TBP, and pol III (31White R.J. Gottlieb T.M. Downes C.S. Jackson S.P. Mol. Cell. Biol. 1995; 15: 1983-1992Crossref PubMed Scopus (96) Google Scholar). Extracts of growing 3T3 cells were found to contain sufficient TFIIIB activity to allow robust transcription in this assay. In contrast, equal amounts of extract from serum-starved cells gave little or no expression above background (Fig. 3 B). We conclude that TFIIIB activity is low and limiting in extracts of serum-deprived 3T3 cells. We investigated whether the abundance of TFIIIB changes in response to serum. Western blotting showed that 72 h of serum deprivation resulted in little or no change in the level of the BRF subunit of TFIIIB (Fig. 4 A). Levels of the TFIIIB subunit TBP are also maintained after extended periods without serum (Fig. 4 B). As an internal control, we also monitored the pol II-specific factor TFIIB in these extracts and found that this too remained unchanged (Fig. 4 C). Although we cannot exclude the possibility that unidentified" @default.
• W1990647583 created "2016-06-24" @default.
• W1990647583 creator A5007470711 @default.
• W1990647583 creator A5024647268 @default.
• W1990647583 creator A5033134863 @default.
• W1990647583 creator A5036724322 @default.
• W1990647583 creator A5051115438 @default.
• W1990647583 creator A5053983471 @default.
• W1990647583 creator A5058700110 @default.
• W1990647583 date "2001-01-01" @default.
• W1990647583 modified "2023-09-26" @default.
• W1990647583 title "Regulation of RNA Polymerase III Transcription during Cell Cycle Entry" @default.
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