The EMBO Journal vol.9 no.9 pp.2857-2863, 1990
A growth-dependent transcription initiation factor (TIF-IA) interacting with RNA polymerase I regulates mouse ribosomal RNA synthesis Andreas Schnapp, Christa Pfleiderer, Horst Rosenbauer and Ingrid Grummt Institut ftir Biochemie, Rontgenring 11, 8700 Wurzburg, FRG Communicated by I.Grummt
Control of mouse ribosomal RNA synthesis in response to extracellular signals is mediated by TIF-IA, a regulatory factor whose amount or activity correlates with cell proliferation. Factor TIF-IA interacts with RNA polymerase I (pol I), thus converting it into a transcriptionally active holoenzyme, which is able to initiate specifically at the rDNA promoter in the presence of the other auxilary transcription initiation factors, designated TIF-IB, TIF-IC and UBF. With regard to several criteria, the growth-dependent factor TIF-IA behaves like a bacterial sigma factor: (i) it associates physically with pol I, (ii) it is required for initiation of transcription, (iii) it is present in limiting amounts and (iv) under certain salt conditions, it is chromatographically separable from the polymerase. In addition, evidence is presented that dephosphorylation of pol I abolishes in vitro transcription initiation from the ribosomal gene promoter without
significantly affecting the polymerizing activity of the
enzyme at nonspecific templates. The involvement of both a regulatory factor and post-translational modification of the transcribing enzyme provides an efficient and versatile mechanism of rDNA transcription regulation which enables the cell to adapt ribosome synthesis rapidly to a variety of extracellular signals. Key words: cell-free transcription system/protein phosphorylation/ribosomal genes/transcription factor/tran-
scription regulation
Introduction Regulation of cell proliferation is a complex process that involves both positively and negatively acting signals including differentiation inducers, cell cycle regulators, mitogenic agents or viral infections. The chain of events by which extracellular signals are transmitted from the cell surface to the nucleus is not yet known. The elucidation of the complex transcriptional program which a cell initiates in response to mitogens and differentiating agents is crucial to the final understanding of gene activity and growth control. Recent experiments from a number of laboratories suggest that signal transduction pathways change genetic programs by the modulation of either the abundance or activity of specific transcription factors (reviewed in McKnight and Tjian, 1986; Maniatis et al., 1987; Ptashne, 1988; Wasylyk et al., 1988). These changes in activity may be due to changes in either the rate of synthesis or turnover of individual transcription factors or may be brought about by the covalent attachment of chemical groups which affects Oxford University Press
the structure of the protein, its solubility, its binding to substrate or its interaction with other macromolecules. Changes in cell growth are accompanied by drastic alterations of the rate of rRNA synthesis. Interestingly, the in vitro rDNA transcriptional activity of extracts mirrors the in vivo activity of the cells from which they were prepared (Grummt, 1981; Cavanaugh et al., 1984; Paule et al., 1984; Buttgereit et al., 1985; Gokal et al., 1986; Tower and Sollner-Webb, 1987). We have shown previously that this growth-related regulation of ribosomal gene transcription is mediated by an essential transcription initiation factor (TIFIA) whose level or activity fluctuates in response to the physiological state of the cells (Buttgereit et al., 1985). Extracts from growth-arrested cells do not contain detectable levels of TIF-IA activity, and are transcriptionally inactive. These inactive extracts can be complemented by addition of TIF-IA preparations that have been purified from exponentially growing cells. Interestingly, TIF-IA co-purifies with RNA polymerase I on several chromatographic matrices, suggesting that it is associated with the transcribing enzyme. We have proposed that only those RNA polymerase I molecules that are associated with TIF-IA are capable of assembling into transcription initiation complexes (Buttgereit et al., 1985). Similarly, Tower and Sollner-Webb (1987) have purified this growth-related activity and have postulated that TIF-IA (their factor C) represents a specifically modified form of RNA polymerase I that is functionally distinct from the vast majority of RNA polymerase I which catalyzes nonspecific RNA synthesis. In an attempt to elucidate the pleiotropic control mechanisms which regulate rRNA synthesis according to the physiological state of the cells, we have partially purified factor TIF-IA and studied its mode of action. In contrast to a previous report (Tower and Sollner-Webb, 1987), we find that this regulatory activity is not an 'activated' RNA polymerase I but a positive-acting, growth-dependent protein factor which interacts physically with pol I. Furthermore, we demonstrate that pol I has to be specifically phosphorylated in order to initiate at the rDNA promoter. This combination of two different regulatory pathways may enable the cell to adapt the rate of rRNA synthesis rapidly to a variety of physiological conditions.
Results Variations in rRNA synthesis activity are mediated by transcription factor TIF-IA
We have previously established a cell-free system that faithfully reflects growth rate-dependent regulation of ribosomal gene transcription (Grummt, 1981). Extracts prepared from exponentially growing cells support high levels of rDNA transcription (Figure IA, lane 1) whereas extracts derived from starved or stationary cells exhibit an extremely low activity (lane 5). To analyze the molecular mechanisms which mediate the growth rate-dependent 2857
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Fig. 1. Complementation of extracts from starved cells by partially purified factor TIF-IA. (A) Transcriptional activity of S-100 extracts prepared from logarithmically growing and starved cells, measured in the absence of additional factors (lanes 1 and 5), or in the presence of partially purified factor TIF-IA (lanes 2 and 6), TIF-IB (lanes 3 and 7) and TIF-IC (lanes 4 and 8). Each assay contained 10 pi of extract and 5 ulA of factor fractions. (B) Stimulation of stationary phase and cycloheximide-treated cell extracts by factor TIF-IA. 10 1u of S-100 extracts prepared from control cells (G), quiescent cells from stationary cultures (Q) and from cells treated for 90 min with 100 ig/ml of cycloheximide (CX), were assayed for their transcriptional capacity in the absence (lanes 1-3) and presence of 5 Al partially purified factor TIF-IA (lanes 4-6).
fluctuations of rDNA transcription, we have fractionated extracts from cultured Ehrlich ascites cells according to the purification procedure described in Materials and methods. The individual factors were separated by chromatography on DEAE-Sepharose and Heparnn Ultrogel. Three fractions (H-200, H400 and H-600) were eluted from the heparin column, none of which alone or in combination with any other was able to support specific transcription. If, however, all three fractions were combined, specific run-off transcripts were synthesized, indicating that at least three protein factors are required for transcription initiation. At this stage of purification the fourth essential factor, UBF (Jantzen et al., 1990) is contained both within the pol I and TIF-IB fraction eluting at 400 and 600 mM KCI, respectively (H.Rosenbauer, unpublished results). Further purification of the individual factor activities present in the heparin fractions yielded three transcription initiation factors which were designated TIFIA, TIF-IB and TIF-IC, respectively (Buttgereit et al., 1985; Clos et al., 1986; Schnapp et al., 1990). To investigate which of these factors is missing or inactive in extracts from starved cells, we added partially purified TIF fractions to extracts prepared from either growing or quiescent cells and tested which of these fractions would restore transcriptional activity. As shown in Figure IA, addition of the individual factor fractions to active extracts hardly affected transcription (lanes 1-4). Similarly, addition of TIF-IB and TIF-IC to extracts from quiescent cells did not restore transcription (lanes 7 and 8). However, this inactive extract was stimulated to control levels after complementation with factor TIF-IA (lane 6) indicating that this factor is missing or inactive in the growth-arrested cells. A decline in rRNA synthesis is observed after arrest of cell proliferation by starvation, density inhibition and administration of inhibitors of cellular protein synthesis (Grummt et al., 1976; Gokal et al., 1986; Tower and Sollner-Webb, 1987). To investigate whether this downregulation of rDNA transcription is due to reduced levels of TIF-IA in each case, extracts were prepared from expo-
2858
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Fig. 2. Separation of bulk and transcriptionally active RNA polymerase I by chromatography on DNA cellulose. A pool of pol I-containing fractions eluted from the heparin column (H-400 fraction) were adsorbed to commercial double-stranded calf thymus DNA cellulose (Sigma, 2 mg DNA/g cellulose), washed with buffer AM-100, and the bound proteins were step-eluted with 300 mM KCI. Flow-through and salt-step fractions were assayed both for nonspecific pol I and TIF-IA activity. Specific transcription was assayed in a 25 Al reaction containing 50 ng template DNA pMr 600/EcoRI, 6 Id of inactive extract derived from growth-arrested cells (E) and 9 Al of the flowthrough fractions (FT,, FT2), or 9 1l of individual fractions (numbers 8-15) eluted at 300 mM KCI from the DNA column.
nentially growing Ehrlich ascites cells (Figure iB, lanes 1 and 4), stationary cells (lanes 2 and 5), or cells that had been treated for 90 min with cycloheximide (lanes 3 and 6), and their rDNA transcriptional activity was assayed in the absence (lanes 1-3) and presence (lanes 4-6) of the partially purified TIF-IA fraction. All three extracts contained similar amounts of RNA polymerase I when measured on nicked calf thymus DNA in the presence of high concentrations of c-amanitin, but greatly differed in their ability to transcribe cloned rDNA. Both the extracts from stationary and cycloheximide-treated cultures were virtually inactive (lanes 2 and 3). When they were mixed with extracts from growing cells, no inhibition of transcription was observed. This finding indicates that the low activity of extracts from growth-arrested cells is not due to the presence of diffusible inhibitors but is rather caused by lack of an essential transcription factor (Grummt, 1981; Buttgereit et al., 1985; Tower and Sollner-Webb, 1987). If the different extracts were supplemented with partially purified TIF-IA, the transcriptional activity of all three extracts was approximately the same. This result implies that (i) the changes in cellular rDNA transcription caused by extracellular signals are caused by fluctuations in the amount or activity of factor TIF-IA, and (ii) the other auxiliary rDNA transcription factors are not affected by changes in the physiological state of the cells. Two functionally different forms of RNA polymerase I are separated by chromatography on DNA cellulose To characterize the growth-regulated factor TIF-IA in more detail, we attempted to purify this activity from mid-log
phase cells using conventional chromatographic procedures. TIF-IA activity was monitored by its ability to stimulate transcription of inactive extracts derived from stationary or starved cells. In parallel, each fraction was assayed for pol I activity by measuring a-amanitin-resistant RNA synthesis using nicked calf thymus DNA as template. When the DEAE-eluate was dialyzed against a buffer containing 100 mM KCl and 5 mM MgCl2 before application to the
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Fig. 3. Immunoprecipitation of TIF-IA activity by RNA polymerase I antibodies. (A) The immunoprecipitation of RNA polymerase I and TIF-IA activity was performed with 3 different sera from scleroderma patients (S18, S124 and Duncan) and with a control serum from a healthy individual (NHS). An H-400 fraction from active extracts containing both pol I and TIF-IA activity (method 1, see Figure 4) was incubated for 1 h with an equal volume of antibodies immobilized on protein A-Sepharose. After the beads were pelleted by centrifugation, the supematants were incubated again with matrix-bound IgGs to achieve quantitative removal of the pol I activity. TIF-IA activity was determined by supplementing 5 1l of an inactive extract from growth-arrested cells (lane 13) with either 4 jtl (lanes 1, 4, 7 and 10), 7 ul (lanes 2, 5, 8 and 11) or 10 ul (lanes 3, 6, 9 and 12) of the supernatants. The open bars in the diagram show the nonspecific pol I activity (c.p.m. x 103) and the dark bars represent TIF-IA activity (quantified by densitometric scanning of the autoradiographic bands and expressed in arbitrary units). (B) Differential reconstitution of pol I-depleted extracts by the two forms of pol I separated on calf thymus DNA cellulose. An active extract from exponentially growing cells (lane 1) was treated with a control serum (NHS, lane 2) or with serum from a scleroderma patient containing antibodies against pol I (S18, lanes 3-5). Equal amounts (in terms of catalytic activity) of the two forms of pol I that have been separated on calf thymus DNA cellulose were added to the pol 1-depleted extract. 5 pJ of the 300 mM KCI fraction (lane 4) or 5 y1 of the flow-through fraction (lane 5) were used to complement 10 ul of the depleted extract.
heparin column (method 1, see Figure 4), TIF-IA eluted together with RNA polymerase I at 400 mM KCI. Attempts to separate these two enzymatic activities by further purification on several different chromatographic residues (including gradient elution from Mono Q-, Mono S-, and DEAE-FPLC columns) have so far failed. A partial separation of pol I and TIF-IA activity was obtained by affinity chromatography on DNA cellulose (Figure 2). When the TIF-IA-containing H-400 fraction was applied to double-stranded DNA cellulose, the majority of RNA polymerase I activity (70-80%) did not bind to the column and was found in the flow-through fraction. However, a significant part (20-30%) of cellular RNA polymerase I activity was bound to the immobilized DNA and was eluted at 300 mM KCl. Interestingly, all detectable TIF-IA activity was contained within the fractions eluting at this salt step. When extracts from growth-arrested cells were fractionated according to the same procedure, practically no pol I activity was bound to the column (data not shown). This result suggests that two structurally and functionally different forms of pol I exist, the relative amounts of which depend on the physiological state of the cells.
Co-precipitation of TIF-IA and RNA polymerase I by antibodies against pol I The separation of two forms of RNA polymerase I by chromatography on calf thymus DNA cellulose is consistent with any of the following possibilities: (i) the different chromatographic behavior reflects the presence of different subforms of RNA polymerase I which differ in their ability
to bind to DNA and to assemble into transcription initiation complexes; (ii) TIF-IA, which is present in limiting amounts, associates with part of the RNA polymerase I molecules, thus changing the chromatographic properties of the enzyme; or (iii) the co-purification of TIF-IA with part of the pol I is by pure chance. If TIF-IA represents a modified subpopulation of pol I or a pol I-associated factor, then it should be immunoprecipitated by antibodies directed against RNA polymerase I. The antibodies used (designated S18, S124, Duncan) were present in sera from patients with the autoimmune disorder scleroderma (Reimer et al., 1987), and it has been shown by Western blot analysis that they preferentially react with the largest subunit of RNA polymerase I. The antibodies were coupled to protein A -Sepharose beads and mixed with extracts from exponentially growing cells. The Sepharose beads were pelleted by centrifugation and the resulting supernatant was assayed for either TIF-IA or pol I activity. As shown in Figure 3A, all three sera tested reduced both pol I and TIF-IA activity to the same extent which indicates that both activities are precipitated by the same antibodies. The data imply that the two forms of pol I separated on calf thymus DNA cellulose represent functionally different enzyme moieties, the active one being associated with TIFIA. We therefore tested the ability of the two pol I fractions present in the flow-through and the 300 mM KCI eluate, respectively, to complement extracts that were depleted for both pol I and TIF-IA by treatment with a-pol I antibodies. As shown in Figure 3B, equal amounts of both forms of pol I exhibited different abilities to restore the transcriptional activity of extracts which have been treated with the pol I
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antibodies. As expected, the weak transcriptional activity of the depleted extract (lane 3) was stimulated by pol I that was bound to the column (lane 4), but not by pol I present in the flow-through fraction (lane 5). Actually, we reproducibly observe an inhibition of transcription by the non-bound polymerase in this assay system which may be due to sequestration of essential factor(s) by these enzyme molecules. This implies that two structurally and functionally distinct forms of RNA polymerase I exist, the bulk or core pol I which is active at nonspecific template DNA, but unable to initiate specifically at the rDNA promoter, and a transcriptionally active form, the holoenzyme, which is associated with the growth-related factor TIF-IA. Only the holoenzyme appears to be able to assemble into transcription complexes and to initiate specifically in the presence of the other auxiliary factors. TIF-IA activity can be separated from RNA polymerase I The results presented so far are consistent with the hypothesis that TIF-IA activity represents a subpopulation of RNA polymerase I as suggested by Tower and Sollner-Webb (1987). However, the following experiment demonstrates that TIF-IA is a separate entity distinct from the 'activated' pol I. Under certain salt conditions, TIF-IA activity can be separated from pol I at an early step in the purification procedure. Figure 4A shows the fractionation on Heparin Ultrogel of the proteins eluted from DEAE by two slightly different procedures. In method 1, the DEAE fractions were dialyzed against buffer AM-100 before application to the heparin column, in method 2, the Mg2+-free DEAE fractions were loaded directly onto the heparin column and eluted with AM-buffer containing 200, 400 and 600 mM KCl, respectively. The pooled fractions were assayed for TIF-IA and pol I activity, respectively (Figure 4B). It turned out that irrespective of the method used, TIF-IC was found in the H-200 fraction, pol I in the H-400 fraction, and TIF-IB in the H-600 fraction. However, depending on the KCl and MgCl2 concentrations at which the DEAE eluate was applied to the heparin column, TIF-IA activity eluted either at 400 mM KCl together with pol I (method 1), or at 200 mM KCl together with TIF-IC (method 2). The TIFIA preparation obtained by method 2 does not contain significant amounts of pol I activity and therefore very probably represents pol I-free TIF-IA which can be separated from TIF-IC in subsequent chromatographic steps (data not shown). To prove unequivocally that TIF-IA is not a constituent of pol I, a pol I-free TIF-IA preparation was treated with ca-pol I antibodies and the supernatant was assayed for TIFIA activity. Free TIF-IA was not precipitated by the pol Ispecific antibodies as shown by the ability of the supematant to stimulate transcription of an inactive extract (Figure SA, lanes 2 and 4). However, when TIF-IA was mixed with pol I before antibody treatment, the stimulatory activity was completely lost (lane 5) indicating that TIF-IA was bound to pol I. In an analogous experiment, we tested whether TIF-IA and pol I could functionally complement each other, or whether both proteins need to act together. Active extracts from mid-log phase cells were treated with pol I antibodies and the depleted extract (Figure SB, lane 2) was supplemented with either polI (lane 3), free factor TIF-IA (lane
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Fig. 4. Chromatographic separation of TIF-IA activity from RNA polymerase I. (A) Schematic outline of the fractionation procedure used to separate pol I and TIF-IA. (B) Identification of TIF-IA and pol I activity in different fractions step-eluted from the heparin column. Active fractions from the DEAE-cellulose column were chromatographed on Heparin-Ultrogel according to method I or 2 (see above). 8 jil of the fractions eluted at 200 mM KCI (H-200, lanes 2 and 5), 400 mM KCI (H-400, lanes 3 and 6), or 600 mM KCI (H-600, lanes 4 and 7) were added to 7 izl of inactive extract derived from stationary cells (lane 1) and assayed for specific transcription in the run-off assay. Nonspecific pol I activity of the same fractions was tested on nicked calf thymus DNA. Fractions H-400 from each column contained 36 and 37 x 103 c.p.m./10 1u, respectively (lanes 3 and 5), whereas none of the other fractions contained any detectable pol I activity.
4), or a mixture of both activities (lane 5). It turned out that neither the pol I nor the TIF-IA fraction could on their own significantly reconstitute the transcriptional activity of the pol I-depleted extract. However, specific transcription was restored when both fractions were added together. This result confirms that TIF-IA is an essential regulatory protein which interacts physically with pol I and thus enables the enzyme to initiate at the rDNA promoter.
Specific transcriptional activity of RNA polymerase I is dependent on phosphorylation The rapid changes of TIF-IA in response to extracellular signals suggest that the activity of this regulatory factor may be modulated by a post-translational modification mechanism. Since several pol fl-specific transcription factors have been shown to be modified by phosphorylation, which in turn alters DNA binding or transcriptional activity, we studied whether the activity of TIF-IA or the other two pol I factors is controlled by phosphorylation. Partially purified protein fractions were incubated with agarose-bound calf intestine alkaline phosphatase, and then assayed for transcriptional activity. As seen in Figure 6A, incubation with phosphatase did not significantly affect the activity of any of the three initiation factors. Each of the individual fractions was active after phosphatase treatment when assayed in the presence of the other two factors and pol I. This finding implies that either the phosphorylation state of the factors
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Fig. 5. TIF-IA and pol I are different proteins. (A) TIF-IA activity is precipitated by antibodies against pol I only in the presence of RNA polymerase I. 20 pI protein A-Sepharose beads were loaded with control serum (NHS) or S18 antiserum and incubated with TIF-IA alone or together with pol I, respectively. Immunoprecipitation was performed as described in Materials and methods. TIF-IA activity was assayed by complementing 7 ul of inactive extract with 8 ytl of the supematants of the individual reactions. Lane 1: control reaction showing the activity of the extract used for complementation, lane 2: complementation of the inactive extract with 6 1u of TIF-IA, lane 3: 8 yd of TIF-IA treated with control antibodies (NHS) were added to the inactive extract, lane 4: same as lane 3 except that TIF-IA was treated with the S18 antibodies, lane 5: TIF-IA and pol I were mixed together before S18 antibody treatment and assaying 8 tl of the supernatant for TIF-IA activity, lane 6: addition of pol I to the inactive extract, lane 7: same as lane 5 but with control antibodies (NHS). (B) Both pol I and TIF-IA are required for reconstitution of specific transcription of pol I-depleted extracts. Extract prepared from mid log phase cells (lane 1) was depleted of pol I (lane 2) by treatment with S18 antiserum as described in Materials and methods. The ability of 'free' TIF-IA and pol I to reconstitute transcription alone or in combination with each other was tested by complementing 7 tl of the depleted extract with 8 u1 of pol I (lane 3), 8 ul of TIF-IA (lane 4), or 4 Al each of pol I and TIF-IA (lane 5).
does not affect their biological activity, or the still rather crude factor preparations contain protein kinases which rapidly rephosphorylate the polypeptide(s). A different result was obtained when pol I was dephosphorylated. Treatment with phosphatase for 10 or 15 min, respectively, progressively inactivated the specific transcription activity (Figure 6B, lanes 2 and 3), whereas nonspecific transcription on denatured calf thymus DNA was not affected (data not shown). To prove that the activity responsible for the inactivation of pol I is the phosphatase bound to the agarose beads and not some other contaminant present in the commercial preparation of the matrix-bound enzyme, we included sodium vanadate in the reaction which inhibits the activity of alkaline phosphatase (Lopez et al., 1976; Seargeant and Stinson, 1979). In the presence of vanadate, the decrease in transcriptional activity was not observed (lanes 4 and 5), indicating that the inactivation of specific transcription was indeed caused by dephosphorylation of pol Apparently, the dephosphorylated enzyme is not able to interact with the essential transcriptional factors and, therefore, does not assemble into the preinitiation complex. l.
Discussion Cellular rRNA synthesis fluctuates in response to the physiological state of the cells and has been shown to be accompanied by changes in the ratio of template-bound to free RNA polymerase I, indicating that the control of rRNA
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Fig. 6. Phosphatase treatment of transcription factors and RNA polymerase I. (A) Transcriptional activity of factors TIF-IA, TIF-IB, TIF-IC is not affected by phosphatase treatment. Partially purified initiation factors were incubated with matrix-bound alkaline phosphatase and aliquots of the supernatants were tested for transcriptional activity in the presence of pol I and the other two factors. Lane 1: control reaction containing 3 tl of TIF-IA, 3 yd of TIF-IB, 3 itl of TIF-IC and 6 yd of pol I. None of the fractions were treated with phosphatase. Lanes 2-5: incubation of factor TIF-IB with calf intestinal phosphatase (CIP) for 10 min (lanes 2 and 4) or 15 min (lanes 3 and 5) in the absence (lanes 1-3) or presence (lanes 4 and 5) of 100 &M sodium orthovanadate. Lanes 6-9 are identical to lanes 2-5 except that a mixture of factors TIF-IA and TIF-IC (H-200 fraction prepared according to method 2, see Figure 5) was treated with the phosphatase. (B) Inactivation of specific transcription by dephosphorylation of pol I. Partially purified pol I was incubated with (lanes 2-5) or without (lane 1) phosphatase for 10 and 15 min, respectively, and then assayed for transcriptional activity in the presence of factors TIF-IA, TIF-IB and TIF-IC. As a control, 100 jM sodium orthovanadate was included in the dephosphorylation reaction (lanes 4 and 5) which inhibits the phosphatase activity.
synthesis occurs at the level of transcription initiation (Yu and Feigelson, 1972; Grummt et al., 1976; Grummt and Grummt, 1976). Interestingly, this transcriptional control is reflected in cell-free transcription systems containing cloned mouse rDNA and extracts from cultured mouse cells (Grummt, 1981). Therefore, this in vitro system proves to be a convenient tool for investigating the underlying molecular mechanisms of this transcriptional control. In an attempt to understand the basic mechanism and the principles of regulation of rRNA synthesis, we have manipulated the physiological state of cultured mouse cells by changing either the density or the nutritional state of the cells, or by inhibiting cellular protein synthesis by cycloheximide. Extracts prepared from these cells were used to isolate and functionally characterize the protein factors involved in rDNA transcription. We have identified three essential factors which are required in addition to RNA polymerase I to reconstitute faithful and efficient transcription initiation (Schnapp et al., 1990). These factors, termed TIF-IA, TIFIB and TIF-IC, as well as a fourth factor, UBF, assemble together with pol I into a preinitiation complex at the rDNA promoter via specific DNA-protein and protein-protein interactions (reviewed in Grummt, 1989). For two of these auxiliary factors, TIF-IB and TIF-IC, there is no indication that they play a regulatory role in the transcription process. Both activities were recovered with about the same yield from exponentially growing or growth-arrested cells (unpublished data). Also, the overall amount and activity of pol I as measured in the nonspecific transcription assay on nicked calf thymus DNA is not affected by the growth rate of the cells. Instead, the regulation of rRNA synthesis in response to extracellular signals seems to be mediated by
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the protein termed TIF-IA (Buttgereit et al., 1985). Transcriptionally inactive extracts from stationary, amino acid-starved, or cycloheximide-treated cells possess extremely low levels of TIF-IA activity and, therefore, are virtually inactive. Supplementation of such inactive extracts with partially purified factor TIF-IA strongly stimulates transcription indicating that TIF-IA was either missing or functionally inactive. TIF-IA physically interacts with pol I as shown by co-purification of both activities on a number of different chromatographic residues and by co-precipitation of pol I and TIF-IA by antibodies against pol I. Association of TIF-IA with pol I slightly changes its chromatographic properties which in turn results in a partial separation of two subpopulations of pol I on a nonspecific DNA column and on different FPLC columns (unpublished data). The bulk of cellular pol I was not associated with TIF-IA and was therefore not capable of supporting specific transcription. These data are compatible with the view that TIF-IA represents a modified form of pol I, as proposed by Tower and Sollner-Webb (1987). These investigators have also identified and fractionated the growth rate-related activity (termed factor C) and could not separate it from pol l. They concluded that the regulatory activity represents a structurally altered, activated form of pol I which is functionally distinct from bulk pol l. Similarly, two functionally different forms of pol I have first been observed during encystment of Acanthamoeba. Pol I prepared from growing or encysted Acanthamoeba shows equal activity in nonspecific assays, but only pol I isolated from vegetative cells can support rDNA transcription when supplemented with an auxiliary transcription initiation factor (TIF) derived from either cysts or growing cells (Paule et al., 1984; Bateman and Paule, 1986). It has been suggested that pol I itself is the major target in regulation of rRNA synthesis during encystment. The results presented in this paper imply that an essential regulatory factor which is distinct from pol I and a structural modification of pol I by phosphorylation may be targets for growth-dependent regulation of rDNA transcription initiation. Although we have not yet successfully purified TIFIA to homogeneity, and are therefore still ignorant of its size and physical properties, we were able to separate it from pol I at an early step in the fractionation procedure. Dissociation of this protein from pol I results in loss of specific initiation. If pol I-free TIF-IA was added back to either bulk polymerase or to pol I from growth-arrested cells, transcriptional activity was restored. This situation is reminiscent of transcriptional regulation in bacteria, which is controlled by the sigma factor. We consider the bulk pol I to be functionally analogous to the bacterial core enzyme which has the ability to synthesize RNA on a nonspecific DNA template, but cannot initiate at the proper sites. Association of the sigma factor converts the core into the holoenzyme which is able to initiate specifically. The present experiments did not address the question of whether the growth-dependent fluctuations in TIF-IA activity are achieved by either quantitative changes in its amount or by qualitative changes in its activity. We also do not know whether there is a causal relationship between TIF-IA activity and the degree of phosphorylation of pol I. The fact that after phosphatase treatment pol I loses its ability to initiate at the rDNA promoter suggests that specific phosphorylation is a prerequisite for proper initiation. Future work has to identify the sites within the multisubunit enzyme which are
2862
involved in this functionally important modification, and to discover at which step of the initiation reaction (i.e. promoter recognition, assembly into stable transcription complexes or transition into the elongation process) specific phosphorylation is required. One attractive hypothesis, which links phosphorylation of pol I with TIF-IA activity, is that TIFIA itself is a specific protein kinase whose activity is growthregulated. Alternatively, TIF-IA may be the substrate for a growth-regulated kinase. In this scenario, either pol I or TIF-IA is the final target in the sequence of events by which extracellular signals are transferred into the nucleolus and specific protein kinase(s) play(s) a key role in regulating this signal transduction pathway. The probable involvement of specific protein phosphorylation in rDNA transcription regulation is favored for the following reasons: (i) on several chromatographic columns, co-purification of TIF-IA and a spermine-stimulated protein kinase activity which uses casein as substrate is observed (unpublished observation), and (ii) there is a strict correlation between variations in rDNA transcriptional activity and the size of cellular ATP and GTP pools (Grummt and Grummt, 1976). When cells were starved of essential amino acids, both rDNA transcription and purine nucleotide pools declined with similar kinetics. These concomitant alterations of both nucleolar activity and intracellular purine nucleotide concentrations suggested that growth-dependent fluctuation in ATP and GTP levels may be one of the limiting factors which determine the rate of rRNA synthesis. The data presented in this paper may bridge the gap between the observed changes in cellular ATP and GTP levels and the control of ribosomal gene transcription. We postulate that alterations of TIF-IA activity in response to growth factors and mitogens are mediated by a specific phosphorylation reaction. A likely candidate for the enzymatic activity that modifies TIF-IA during a variety of normal regulatory responses is casein kinase NIl. This enzyme has been shown to accumulate in the nucleolus (Pfaff and Anderer, 1988) and to phosphorylate a number of nucleolar proteins, such as pol I, topoisomerase I, and nucleolin (see Edelman 1987, for review). Interestingly, casein kinase NII appears to be associated with pol I (Rose et al., 1981) and its activity flucutates according to the growth rate of the cells at a similar degree to rDNA transcription (Belenguer et al., 1989). This intriguing correlation between the proliferation rate of the cells, the rate of rDNA transcription, the activity of TIF-IA and the level of kinase NII suggests that this kinase or a related enzyme plays a central role in growth-dependent regulation of rRNA synthesis.
Materials and methods Cultivation of cells and extract preparation
Ehrlich ascites cells were cultured in RPMI medium containing 5% new-
born calf serum for 20-40 h. Transcriptionally active extracts were obtained from logarithmically growing cells (9 x I05 cells/ml). Transcriptionally
inactive extracts were prepared from stationary phase cells, which were taken either directly from the body cavity of mice, or were grown to maximal density (1.5-2 x 106 cells/ml) in culture medium and incubated for an additional 24 h before harvesting. For nutritional starvation logarithmically growing cells were transferred into histidine- and isoleucine-free RPMI medium containing 5 % dialyzed calf serum and cultured overnight. Cells to be treated with cycloheximide were incubated with 100 jg/ml of the drug for 90 min prior to extract preparation. S-100 extracts were prepared according to Weil et al. (1979) and nuclear extracts according to Dignam et al. (1983).
Control of rRNA synthesis In vitro transcription assays The soluble cell-free transcription system and the analysis of the RNA synthesized have been described before (Grummt, 1981; Clos et al., 1986). Usually, 50- 100 ng of pMr600 (Wandelt and Grummt, 1983) truncated with EcoRI were incubated with 15 A1 of a mixture of S-100 and nuclear extracts in a total volume of 25 AI containing 12 mM HEPES pH 7.9, 0.1 mM EDTA, 0.5 mM DTE, 5 mM MgCl2, 75 mM KCl, 10 mM creatine phosphate, 12 % glycerol (v/v), 0.66 mM each of ATP, CTP and UTP, 0.01 mM GTP and 1.5 ACi of [a-32P]GTP (400 Ci/mmol). After incubation for 60 min at 30°C, the nucleic acids were extracted, precipitated and analyzed on non-denaturing 5% polyacrylamide gels. Total pol I activity was assayed in a 25 Al reaction containing 6 mM Tris-HCI (pH 7.9), 0.1 mM EDTA, 5 mM MgCl2, 80 mM KCI, 6% glycerol, 0.5 mM DTE, 0.66 mM each of ATP, CTP and GTP, 3.6 AM [3H]UTP (5.5 Ci/mmol), 7.5 jg of calf thymus DNA and S jg of caamanitin. After incubation for 30 miin at 37°C, the reaction was stopped by addition of 0.2 ml of saturated Na4P207 containing 50 Ag carrier RNA and precipitated with 5 % trichloroacetic acid. The precipitates were collected on glass fiber filters and quantified by scintillation counting.
Purification of transcription factors A typical factor preparation was started from 100-200 ml of a mixture of nuclear and cytoplasmic extracts from cultured Ehrlich ascites cells. All buffers used contained 0.5 mM DTE and 0.5 mM PMSF added immediately prior to use. Extract proteins were applied onto a DEAE -Sepharose CL-6B column, washed with buffer A (20 mM Tris-HCI, pH 7.9,0.1 mM EDTA, 20% glycerol) containing 100 mM KCI and step eluted at 280 mM KCI. This fraction which contains pol I and the transcription factors TIF-IA, TIFIB and TIF-IC was either dialyzed against buffer AM-100 (same as buffer A-100 but with 5 mM MgCI2, method 1, see Figure 4) or loaded directly (at 200 mM KCI) onto a Heparin-Ultrogel A4-R column (method 2) and eluted with 200, 400 and 600 mM KCI, yielding fractions H-200, H-400 and H-600, respectively. Factor TIF-IC present in the H-200 fraction was further purified by chromatography on Q-Sepharose and on a Mono Q-FPLC column (H.Rosenbauer, unpublished data). RNA polymerase I was present in the H-400 fraction (Buttgereit et al., 1985). Depending on the salt concentration at which the DEAE eluate was applied to the Heparin Ultrogel (method 1 or 2, see above), it was either associated with the growth-rate related activity TIF-IA (method 1) or it contained very little if any TIF-IA activity (method 2) which in these salt conditions was present mainly in the H-200 fraction together with TIF-IC. Further purification of pol I (whether or not it was associated with TIF-IA) involved chromatography on calf thymus DNA cellulose, Mono Q-FPLC, and Mono S-FPLC. TIFIB was recovered in the H-600 fraction and purified further by chromatography on CM-Sepharose and a Mono S-FPLC column (Schnapp etal., 1990). Alkaline phosphatase treatment of transcription factors Partially purified transcription factors and RNA polymerase I were incubated on ice for 10 or 15 min with agarose-bound calf intestinal phosphatase (Sigma, 2500 U/ml) in buffer AM-100 containing the protease inhibitors aprotinin (5 jig/ml), pepstatin (5 Ag/ml), leupeptin (5 jig/ml), benzamidinhydrochloride (2 mM), antipain (2 Ag/lml) and chymostatin (2 jg/ml). The phosphatase was removed by a brief centrifugation at 5000 r.p.m. in a microfuge. The supernatant was centrifuged again and was assayed for activity.
Acknowledgements are indebted to Dr G. Reimer and Dr Luderschmidt for generously providing the human autoimmune ci-RNA polymerase I sera and control IgGs. We thank W.Hadelt for help in cell cultivation, extract preparation and factor purification. This work was supported by the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie.
We
References Bateman,E. and Paule,M.R. (1986) Cell, 47, 445-450. Belenguer,P., Baldin,V., Mathieu,C., Prats,H., Bensaid,M., Bouche,G. and Amalric,F. (1989) Nucleic Acids Res., 17, 6625-6636. Buttgereit,D., Pflugfelder,G. and Grummt,I. (1985) Nucleic Acids Res.,
13, 8165-8179. Cavanaugh,A.H. and Thompson,E.A. (1984) Proc. Natl. Acad. Sci. USA, 81, 718-721. Clos,J., Buttgereit,D. and Grummt,I. (1986) Proc. Natl. Acad. Sci. USA, 83, 604-608. Dignam,J.D., Lobowitz,R.M. and Roeder,R.G. (1983) Nucleic Acids Res., 11, 1475-1489. Edelman,A. (1987) Annu. Rev. Biochem., 57, 567-613. Gokal,P.K., Cavanaugh,A.H. and Thompson,E.A., Jr. (1986) J. Bio. Chem., 261, 2536-2541. Grummt,I. (1981) Proc. Natl. Acad. Sci. USA, 78, 727-731. Grummt,I. (1989) In: Eckstein,F. and Lilley,D.M.J. (eds), Nucleic Acids and Molecular Biology. Springer-Verlag, Berlin. Vol. 3, pp. 148-163. Grummt,I. and Grummt,F. (1976) Cell, 7, 447-453. Grummt,I., Smith,V.A. and Grummt,F. (1976) Cell, 7, 439-445. Jantzen,H.-M., Admon,A., Bell,S. and Tjian,R. (1990) Nature, 344, 830-836. Lopez,V., Stevens,T. and Lindquist,R.N. (1976) Arch. Biochem. Biophys., 175, 31-38. McKnight,S. and Tjian,R. (1986) Cell, 46, 795-805. Maniatis,T., Goodborn,S. and Fischer,J.A. (1987) Science, 236, 1237-1245. Paule,M.R., Iida,C.T., Perna,P.J., Harris,G.H., Knoll,D.A. and D'Alessio,J.M. (1984) Nucleic Acids Res., 12, 8161-8180. Pfaff,M. and Anderer,F.A. (1988) Biochim. Biophys. Acta, 969, 100-109. Ptashne,M. (1988) Nature, 325, 683-689. Reimer,G., Rose,K., Scheer,U. and Tan,E. (1987) J. Clin. Invest., 79,
65-72. Rose,K., Stettler,D.A. and Jacob,S.T. (1981) Proc. Natl. Acad. Sci. USA,
78, 2833-2837. Schnapp,A., Hadelt,W., Clos,J., Schreck,R., Cvekl,A. and Grummt,I. (1990) Nucleic Acids Res., 18, 1385-1393. Seargeant,L.E. and Stinson,R.A. (1979) Biochem. J., 182, 247-250. Tower,J. and Sollner-Webb,B. (1987) Cell, 50, 873-883. Wandelt,C. and Grummt,I. (1983) Nucleic Acids Res., 11, 3795-3809. Wasylyk,C., Imler,J. and Wasylyk,B. (1988) EMBO J., 7, 2475 -2483. Weil,P.A., Luse,D.S., Segall,J. and Roeder,R.G. (1979) Cell, 18, 469-484. Yu,F.L. and Feigelson,P. (1972) Proc. Natl. Acad. Sci. USA, 69, 2833-2837.
Received
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April 5, 1990; revised
on
May 17, 1990
Precipitation of Pol I by RNA polymerase I antibodies Sera from scleroderma patients containing antibodies directed against pol I (Reimer et al., 1987) and control sera were provided by Dr Georg Reimer (Augsburg) and Dr Luderschmidt (Munich). Purified rabbit anti-RNA polymerase IgG was a generous gift from Dr Kathleen Rose (Rose et al., 1981). The antibodies were bound to protein A-Sepharose (Sigma) by incubating 10 Jl of serum for 10 h at 4°C with 100 ul of a 10% suspension of protein A-Sepharose beads (v/v) in buffer B (25 mM Tris-HCI, pH 7.9, 100 mM KCI, 5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTE and the protease inhibitors aprotinin, pepstatin, leupeptin, benzamidinhydrochloride, antipain and chymostatin at the concentrations indicated above) which has been supplemented with 1 mg/ml bovine serum albumin. The beads were washed 5 times with buffer B and twice with buffer AM-100. For immunoprecipitation, the Sepharose beads were incubated with 5-6 volumes of either cell extract, partially purified factor TIF-IA or pol I for 1 h on ice with occasional shaking. After centrifugation the resulting supernatants were assayed for RNA polymerase I activity and for their ability to stimulate transcription of extracts derived from growth arrested cells.
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