The ARF Tumor Suppressor Controls Ribosome Biogenesis by Regulating the RNA Polymerase I Transcription Factor TTF-I

Molecular Cell

Article The ARF Tumor Suppressor Controls Ribosome Biogenesis by Regulating the RNA Polymerase I Transcription Factor TTF-I Fre´de´ric Lessard,1 Franc¸oise Morin,1 Stacey Ivanchuk,2,3 Fre´de´ric Langlois,1 Victor Stefanovsky,1 James Rutka,2,3 and Tom Moss1,* 1Cancer Research Centre and Department of Molecular Biology, Medical Biochemistry, and Pathology of Laval University, CHUQ Research Centre, Pavillon St. Patrick, 9 rue McMahon, Que´bec, G1R 3S3 Que´bec, Canada 2Division of Neurosurgery, The University of Toronto, Suite 1504, 555 University Avenue, Toronto, M5G 1X8 Ontario, Canada 3The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, TMDT 11-701, 101 College Street, Toronto, M5G 1L7 Ontario, Canada *Correspondence: [email protected] DOI 10.1016/j.molcel.2010.03.015

SUMMARY

The p14/p19ARF (ARF) product of the CDKN2A gene displays tumor suppressor activity both in the presence and absence of p53/TP53. In p53-negative cells, ARF arrests cell proliferation, at least in part, by suppressing ribosomal RNA synthesis. We show that ARF does this by controlling the subnuclear localization of the RNA polymerase I transcription termination factor, TTF-I. TTF-I shuttles between nucleoplasm and nucleolus with the aid of the chaperone NPM/B23 and a nucleolar localization sequence within its N-terminal regulatory domain. ARF inhibits nucleolar import of TTF-I by binding to this nucleolar localization sequence, causing the accumulation of TTF-I in the nucleoplasm. Depletion of TTF-I recapitulates the effects of ARF on ribosomal RNA synthesis and is rescued by the introduction of a TTF-I transgene. Thus, our data delineate the pathway by which ARF regulates ribosomal RNA synthesis and provide a compelling explanation for the role of NPM.

INTRODUCTION ARF (p19ARF in mouse, p14ARF in human) and p53 (TP53) are key components of a major human tumor suppressor pathway that is responsible for arresting cell-cycle progression and inducing cell death in response to DNA damage and oncogenic stress (Hahn and Weinberg, 2002). Loss or mutation of p53 occurs in nearly 50% of human cancers, and loss of ARF is very nearly as common, occurring in as many as 40% of human cancers (Levine et al., 2006; Sharpless, 2005; Vousden and Lu, 2002). In proliferating cells, p53 is targeted for degradation by the ubiquitin E3 ligase MDM2 (HDM2 in human). ARF binds to MDM2, inhibits its activity, and sequesters it in the nucleolus, thus stabilizing p53 (Sherr, 2006). Consistent with ARF acting upstream of p53, inactivation of either the ARF or p53 genes induces a similar,

though not identical, range of spontaneous tumors (Kamijo et al., 1999). However, the combined inactivation of both the p53 and ARF genes leads to a broader range of tumors and to enhanced tumor growth than inactivation of the p53 gene alone (Kelly-Spratt et al., 2004; Sharpless, 2005; Weber et al., 2000). Further, ARF was shown to arrest cell-cycle progression in cells lacking p53, strongly suggesting that it is a tumor suppressor in its own right (Carnero et al., 2000; Weber et al., 2000). Various attempts to understand the mechanism of p53-independent tumor suppression by ARF have identified more than 30 potential interactor proteins, including E2F1 and Myc. However, the role of these proteins in ARF tumor suppression remains controversial (Sherr, 2006). A potential explanation for ARF’s p53-independent activity was revealed when it was shown to repress ribosomal RNA (rRNA) processing (Sugimoto et al., 2003) and hence ribosome biogenesis, an essential function for cell and tumor growth (Moss et al., 2007). In subsequent work, repression of ribosome biogenesis was suggested to be due to the ability of ARF to interact with nucleophosmin (NPM, NPM-1, and B23) and to ubiquitinylate and degrade it (Bertwistle et al., 2004; Itahana et al., 2003; Lindstro¨m and Zhang, 2006; Zhang, 2004). NPM is a highly abundant protein of the nucleoplasmin protein chaperone family (NPM1 to 3) that shuttles between the nucleolus, nucleoplasm, and cytoplasm (Szebeni et al., 2003). NPM is an essential gene in mouse (Colombo et al., 2005; Grisendi et al., 2005), and its overexpression or mutation has been associated with a broad range of human cancers (Grisendi et al., 2006; Lim and Wang, 2006; Naoe et al., 2006). It has been implicated in a plethora of cellular processes, including genome stability, centrosome duplication, cell-cycle progression, and response to stress, as well as mRNA polyadenylation and nuclear and nucleolar import and export (Grisendi et al., 2006; Palaniswamy et al., 2006). Its inactivation could, therefore, affect ribosome biogenesis in any one of a number of ways, both direct and indirect. Further, the nucleolar localization and the stability of the ARF protein depend on NPM (Colombo et al., 2005; Rodway et al., 2004), and a common mutation of NPM in acute myeloid leukemia (AML) is responsible for both downregulating ARF and relocalizing it to the cytoplasm of leukemic blasts (Colombo et al., 2006).

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Figure 1. Exogenous and Endogenous Mouse TTF-I and p19ARF Interact In Vitro and In Vivo

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(A) The organization of mouse and human ARF proteins showing the MDM2/HDM2 interaction sites. (B) Pull-down assay of in vitro-translated 35Slabeled full-length mTTF on the recombinant GST-ARF fusion protein isolated from E. coli. (C) Coimmunoprecipitation (I.P.) of FLAG-mTTF with HA-mARF expressed by transient transfection in HEK293T cells. Whole-cell lysates and corresponding immunoprecipitates were immunoblotted (I.B.) using commercial anti-HA and anti-FLAG antibodies. (D) Endogenous (endo) mTTF was immunoprecipitated from Con3 p53/ MEFs using affinity-purified antibody (anti-TTF#3) or preimmune serum and was immunoblotted to detect endogenous mTTF, endogenous mARF, and endogenous fibrillarin (mFIB). ARF and TTF protein levels in both immunoprecipitate and lysate were estimated on common gel analyses using multiple exposures. It was found that 30% of total endogenous TTF was immunoprecipitated with our anti-TTF antibody #3, and 2% of total endogenous ARF was coimmunoprecipitated with this TTF. We therefore estimate that around 7% of ARF is associated with TTF within these cells.

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While screening for ARF interaction partners, we identified the RNA polymerase I (RPI) transcription termination factor TTF-I. RPI is uniquely responsible for the synthesis of the major rRNAs; hence, this interaction provided a potential explanation for ARF’s ability to inhibit ribosome biogenesis. We subsequently found that ARF inhibits the nucleolar localization of TTF-I and prevents its binding to the rRNA genes, whereas NPM is required for the constitutive localization of TTF-I in the nucleolus. Further, depletion of TTF-I recapitulated the effects of ARF on both rRNA synthesis and processing. Thus, ARF represses ribosome biogenesis both directly and indirectly by controlling TTF-I function on the rRNA genes. RESULTS A genome-wide yeast two-hybrid screen for partners of the human p14ARF (hARF) tumor suppressor identified a clone encoding the human TTF-I (hTTF). TTF-I is the likely ortholog of yeast Reb1p, and both have been implicated in regulating the rRNA genes (Evers et al., 1995; Mason et al., 1997). Given that human and mouse ARF had been implicated in regulating the rRNA genes (Itahana et al., 2003; Sugimoto et al., 2003), an interaction with TTF-I was of potential physiological significance. ARF and TTF-I Interact Both In Vitro and In Vivo We first determined whether the ARF-TTF interaction was direct and evolutionarily conserved. Recombinant mouse p19ARF (mARF) (Figure 1A) effectively pulled down full-length, in vitrotranslated mouse TTF-I (mTTF) (859 amino acids [aa]) (Hu and

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Rothblum, 2000) (Figure 1B). Epitope-tagged mARF and mTTF coexpressed in HEK293T were also found to coimmunoprecipitate (Figure 1C). hTTF also interacted with hARF both in vitro and in vivo (S.I., unpublished data). Most importantly, when endogenous mTTF was immunoprecipitated from mouse embryonic fibroblasts (Con3 p53/ MEFs) (Liu et al., 1998) using an affinity-purified antibody (anti-TTF#3), endogenous mARF was coimmunoprecipitated (Figure 1D). Thus, the interaction of ARF with TTF occurs both in vitro and in vivo and is conserved between mouse and human. ARF Induction Displaces TTF from the Nucleolus To investigate the biological significance of the ARF-TTF interaction, we took advantage of the NIH 3T3-derived MT-ARF cell line in which mARF can be conditionally expressed under control of a Zn2+ responsive promoter (Kuo et al., 2003). ARF induction caused a striking displacement of endogenous TTF from the nucleoli of MT-ARF cells (Figure 2A). In the absence of ARF, all MT-ARF cells displayed TTF positive nucleoli, whereas 85% of ARF-positive cells displayed no detectable nucleolar TTF (Figure 2B), and nucleolar TTF was significantly reduced in the remaining 15% of ARF-positive cells (e.g., see 12 and 24 hr ARF induction in Figure S1A available online). In contrast, nucleolar levels of the rRNA gene-specific factor UBF and the large subunit of RPI (RPA194) were unaffected in ARF-positive nucleoli (Figures 2A and S1E). ARF expression did not affect cellular levels of endogenous TTF protein (see immunoblotting panel in Figure S1B); hence, the disappearance of TTF from the nucleolus was not the result of degradation. Induction of endogenous

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0.2 0.4 0.6 0.8 1 Nucleolar mARF (mean pixel intensity (mpi) per cell) hARF by exogenous E2F1 expression also displaced endogenous hTTF from nucleoli in human diploid fibroblasts (Figure S1F), showing that this activity of ARF was conserved in human. TTF binds to sites T0 and T1 to T5 flanking the 45S pre-rRNA transcribed region of the rRNA genes, and binding to these sites was significantly reduced after ARF induction (Figure 2C). ARF induction was previously shown to inhibit rRNA processing (Sugimoto et al., 2003). In agreement, we observed that ARF accumulation led to a slowing of 45S and 32S precursor pro-

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(A) Localization of mARF (HA-ARF) and endogenous mTTF (anti-TTF#3) and mUBF (anti-UBF#8) before and after induction of the MT-ARF (NIH 3T3) cell line. Both UBF and TTF appeared as discrete nucleolar staining before ARF induction, but TTF displayed dispersed/speckled nucleoplasmic staining after ARF induction. See Figures S1A and S1E for further examples and Figure S1E for parallel detection of endogenous UBF and the large subunit of RPI (RPA194), both of which were unaffected by ARF expression. (B) Statistical analysis of mTTF displacement in induced MT-ARF cells. Cell nucleoli were scored for HA-mARF and for endogenous mTTF. All ARF-negative (ARF) cells displayed TTF-positive (TTF+) nucleoli, whereas only 15% of ARF+ cells displayed detectable nucleolar TTF, and this was at significantly reduced levels (see also time course of ARF induction in Figure S1A). (C) mTTF occupancy of the major T0- and T1binding sites on the rRNA genes was determined using ChIP and QPCR at the indicated times after ARF induction in MT-ARF cells. Data are from three assays analyzed in triplicate, and standard errors are indicated. The approximate locations of the amplicons used to analyze occupancy of the T0 and T1 sites and a control amplicon within the IGS are indicated diagrammatically on the right; see Experimental Procedures for amplicon coordinates. (D) Analysis of the onset of endogenous mARF protein expression during passaging (P1–P6) of wild-type MEFs in comparison with mTTF, MDM2, fibrillarin (FIB), and histone H3. (E) Examples of immunofluorescence staining of endogenous mARF and mTTF in P3 MEFs. (F) Quantitative analysis of nucleolar levels of mARF and mTTF in P3 and P6 MEFs. The mean pixel intensity (mpi) per cell of mTTF nucleolar fluorescence is shown plotted against mARF nucleolar fluorescence for 34 randomly chosen cells. An exponential curve fit to the data is shown, and error bars indicate an estimated experimental error of ± 5% in the measurement of mpi. The displacement of TTF from nucleoli by induction of the mARF transgene in MT-ARF cells and during the natural accumulation of endogenous mARF in late-passage wild-type MEFs corresponded to reductions in rRNA synthesis and processing (see Figures S1B–S1D and S1G).

cessing, and this correlated with the nucleolar displacement of TTF (Figures S1A–S1C). Production of 5S rRNA by RPIII was repressed only at later times of ARF induction, consistent with indirect regulation. We also observed that ARF repressed RPI transcription (total rRNA synthesis in Figure S1D). In this context, the posttranscriptional labeling of the rRNAs with [3H]-methylmethionine used previously to study the effects of ARF on rRNA synthesis (Itahana et al., 2003; Sugimoto et al., 2003) may be less sensitive to changes in de novo rRNA synthesis

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Figure 3. Nucleolar Localization of mTTF Requires NPM/B23 (A) Endogenous NPM or TTF proteins were depleted by siRNA transfection in NIH 3T3 cells. The first three tracks from the left show levels of NPM, TTF, and fibrillarin (FIB) in cells transfected with the control siRNA (Ctrl). 25%, 50% and 100% indicate levels of total protein extract loaded. The last two tracks show that TTF and NPM abundance was less than 25% of normal levels after transfection of the respective siRNA. (B) NIH 3T3 cells transfected with the anti-NPM siRNA were analyzed for endogenous nucleolar NPM and TTF by immunofluorescence. Left and right panels show two examples of cell fields. The white arrows indicate cells in which NPM was not detected, and the gray arrows indicate where it was significantly depleted. The table below the image panels shows scoring for nucleolar TTF in cells displaying either near wild-type or undetectable levels of NPM. Note the absence of nucleolar TTF in cells lacking NPM. In contrast, depletion of endogenous TTF had no effect on the nucleolar localization of NPM (Figure S2A). (C) Exogenous mTTF and NPM interact in vivo. FLAG-mTTF and HA-NPM were transiently expressed in HEK293T cells, TTF immunoprecipitated (I.P.) with an anti-FLAG antibody, and immunoblotted for both FLAG-mTTF and HA-NPM. (D) Endogenous TTF coimmunoprecipitates with endogenous NPM from wild-type MEFs. Whole-cell protein extracts from wild-type MEFs were immunoprecipitated with an NPM-specific or control nonspecific antibodies (IgG1), and immunoprecipitates were probed for both NPM and TTF. (E) YFP-mTTF fusion proteins containing either full-length mTTF or mTTF subdomains were transiently coexpressed with HA-NPM in HEK293T cells, and antiYFP immunoprecipitates from whole-cell protein extracts were analyzed for HA-NPM coprecipitation. The NPM interaction site mapped to the C-terminal DNAbinding domain of mTTF.

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Molecular Cell ARF Represses rRNA Synthesis by Controlling TTF-I

rates than the cotranscriptional incorporation of [3H]-uridine used here. Endogenous ARF Displaces TTF from the Nucleoli of Wild-Type MEFs during Passaging During their passaging in vitro, wild-type MEFs accumulate endogenous ARF and arrest their proliferation. This natural accumulation of ARF did not affect cellular TTF levels (Figure 2D) but did inversely correlate with the nucleolar levels of TTF (Figures 2E and 2F) and with de novo rRNA synthesis (Figure S1G). Thus, induction of ARF expression in primary wild-type MEFs as well as in MT-ARF cells and human diploid fibroblasts led to the nucleolar displacement of TTF and the repression of rRNA transcription. NPM/B23 Is an Essential Chaperone in the Nucleolar Localization of TTF A previous study showed that ARF targeted NPM/B23 for degradation and that NPM depletion repressed rRNA processing (Itahana et al., 2003). However, given the various chaperone activities of NPM, it was possible that this protein was also required for the nucleolar localization of TTF-I. Depletion of NPM from NIH 3T3 cells had no effect on the cellular levels of TTF (Figure 3A). However, cells depleted of NPM displayed little or no nucleolar TTF (Figures 3B and S2A). In contrast, a similar depletion of TTF had no effect on the nucleolar localization of NPM (Figures 3A and S2A). Further, depletion of NPM also reduced rRNA synthesis in a similar manner to ARF induction (Figures S2B and S2C). NPM (NPM1) has been accredited with a range of chaperone and cotransporter activities (Chen et al., 2009; Le´otoing et al., 2008; Lindstro¨m and Zhang, 2008; Maggi et al., 2008; Szebeni and Olson, 1999). Consistent with such activity, HA-NPM coimmunoprecipitated with coexpressed FLAG-TTF (Figure 3C), and endogenous mTTF also coimmunoprecipitated with endogenous mNPM (Figure 3D). Further, NPM interacted specifically with the C-terminal DNA-binding domain of mTTF (aa 471–859), but not with the central (aa 211–470) or N-terminal (2–210) domains (Figure 3E). Partial deletion or point mutation of the DNA-binding domain of TTF (YFP-TTF [2–751], YFP-TTF [W713K]) (Evers and Grummt, 1995) caused a significant degree of relocalization to multiple NPM-positive nuclear foci (Figures S2D and S2E). Overexpression of NPM in NIH 3T3 cells also induced a similar relocalization of wild-type TTF (YFP-mTTF) to these NPM-positive nuclear foci (Figure 3F), further suggesting that the interaction of TTF with NPM is a determinant in its subnuclear localization. Thus, our data strongly suggest that NPM plays an essential role of chaperone or cotransporter in the nucleolar localization of TTF. Depletion of TTF Recapitulates the Effects of ARF on rRNA Synthesis and Processing Because both ARF induction and NPM downregulation led to the nucleolar displacement of TTF and the repression of rRNA

synthesis, our data pointed to TTF as a key target in the pathway regulating ribosome biogenesis. To determine the effect of TTF depletion on rRNA synthesis, NIH 3T3 cells were stably transformed with an anti-mTTF shRNA transgene under control of a doxycyclin (dox)-dependent ‘‘tet-off’’ promoter. After cloning, a cell line displaying R 90% depletion of endogenous TTF after shRNA induction was selected for further study (compare the A10 cells and B10 control cells in Figure 4A). Maximal depletion of TTF in the A10 cells occurred on days 3 and 4 of shRNA induction by dox withdrawal and caused R 60% reduction in total rRNA synthesis, whereas dox withdrawal had no effect on rRNA synthesis in the control clone (Figure 4B). Similar to the effects of ARF expression in MT-ARF cells, processing of precursor 45S rRNA to both 18S and 28S rRNAs was also impaired in the A10 cells, such that by day 3 and 4 of shRNA induction, almost no 18S and little 28S labeling was detected (compare 45S:28S and 45S:18S ratios in Figures 4C and S1C). In an attempt to rescue the effects of TTF depletion, the A10 cell line was retransformed with a FLAG-mTTF transgene also under tet-off. Withdrawal of dox from the resultant cells led to an initial reduction in endogenous TTF levels, a 50% reduction in total rRNA synthesis, and repression of rRNA processing (Figures 4A and 4B and 45S:28S and 45S:18S ratios in Figure 4C). However, TTF levels were re-established by day 4 of doxycyclin withdrawal, and total de novo rRNA synthesis and processing returned to near wild-type levels (Figures 4A–4C). Thus, the specific depletion of TTF recapitulated the effects of ARF induction on rRNA synthesis and processing, strongly suggesting that TTF was indeed a downstream target of the ARF pathway. TTF Shuttles between Nucleolus and Nucleoplasm To explore the mechanism by which ARF controlled the nucleolar localization of TTF, we first determined whether TTF was stably anchored in the nucleolus or shuttled between the nucleoplasm and nucleolus. Consistent with shuttling, subcellular fractionation of NIH 3T3 cells revealed TTF to be present in both nucleolus and nucleoplasm (Figure 5A), its nuclear distribution being not unlike that of histone H3 or the pan-nuclear chromatinremodeling complex protein SNF2H, with which TTF is known to interact (Percipalle et al., 2006). In contrast, fibrillarin was exclusively detected in the nucleolar fraction and tubulin predominantly in the cytoplasm. A similar fractionation of uninduced and induced MT-ARF cells showed that ARF was also present in both nucleolus and nucleoplasm (Figure 5B), consistent with its ability to regulate the function of MDM2, most of which was found in the nucleoplasm (Llanos et al., 2001; Lohrum et al., 2000; Weber et al., 1999). To determine whether or not TTF shuttled between the nucleolar and nucleoplasmic pools, a YFP-mTTF fusion protein was expressed in NIH 3T3 cells and analyzed by fluorescence recovery after photobleaching (FRAP) (Carrero et al., 2004). After photobleaching of whole nucleoli, the fluorescence intensity of TTF recovered with a mean half-time (T1/2) of 27 s, indicating

(F) Coexpression of HA-NPM in NIH 3T3 cells induces the localization of YFP-mTTF to nuclear foci. These foci were not visible when YFP-mTTF or its subdomains were expressed alone (Figures 5D and S3A). White arrows indicate nucleoli, and gray arrows indicate nuclear foci. See also Figures S2D and S2E.

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that TTF was actively imported into the nucleolus (nucleolar recovery in Figure 5C). Partial bleaching of a single nucleolus displayed a somewhat faster recovery (T1/2 of 13 s), consistent 544 Molecular Cell 38, 539–550, May 28, 2010 ª2010 Elsevier Inc.

TTF Contains an Intrinsic Nucleolar Localization Sequence Nucleolar localization is often directed by an integral nucleolar localization sequence (NoLS). However, NoLS sequences are highly degenerate and can usually only be identified functionally (Emmott and Hiscox, 2009). We therefore asked whether mTTF contained intrinsic NoLS activity by mapping sequences able to direct nucleolar localization. As expected, full-length YFPand FLAG-tagged mTTF concentrated within the nucleoli, respectively, of NIH 3T3 and HEK293T cells (Figures 5D and S3). Deletion of aa 1–323 of mTTF abrogated nucleolar localization, and neither the central domain (aa 211–470) nor the C-terminal DNA-binding domain (aa 471–859) of TTF was able to localize YFP to the nucleolus, despite all three constructs being predominantly nuclear (Figures 5D, S3A, and S3B). However, aa 2–210 efficiently directed nucleolar localization of YFP, and further resection of this segment showed a functional NoLS lay between aa 121 and 210. Dissection of this subdomain revealed a bipartite NoLS; aa 121–160 directed robust nucleolar localization, whereas aa 161–210 directed YFP to the nucleolus but also displayed a significant level of nucleoplasmic fluorescence (Figure 5D). (NoLS mapping data are summarized in Figure S4B.) NoLS sequences identified to date are usually less than 20 aa long and often highly basic, though bipartite NoLS activity spread over 30 aa or more have also been identified (Emmott and Hiscox, 2009). Inspection of aa 121–160 of TTF revealed a stretch of 11 aa containing 10 basic residues between aa 128 and 139 (Figure S4B). Consistent with its weaker NoLS activity, aa 161–210 contained only a single run of four basic residues between aa 166–169. The C-terminal DNA-binding domain (aa 471–859) of TTF bound NPM but was clearly unable to direct nucleolar localization, whereas aa 121–210 contained the unique NoLS activity but did not interact with NPM (Figures 5D and 3E). It therefore appeared unlikely that the interaction of NPM with TTF was directly required for the function of its NoLS. However, the N- and C-terminal domains of TTF were shown previously to interact, causing an autoinhibition of the DNA-binding domain (Sander et al., 1996). It is then quite possible that this autoinhibition interaction has a reciprocal effect on NoLS function. Consistent with this, extension of the construct YFP-(161–210) by the addition of the C-terminal domain YFP-(161–859) greatly reduced its ability to direct nucleolar localization (Figures 5D and S3A). NPM might then indirectly promote nucleolar localization by unfolding TTF to reveal its NoLS (see Discussion). The ARF-Binding Site on TTF Overlaps Its NoLS Because ARF interacted with TTF (Figures 1B–1D), it was possible that it directly controlled the nucleolar localization of TTF. The ARF-binding site on TTF was mapped to two distinct domains, the N-terminal regulatory domain containing the NoLS (aa 2–210) and the C-terminal DNA- and NPM-binding site (aa 471–859) (Figure S4A). In investigating the juxtaposition of the N-terminal ARF–binding site (ARF-BS1) and NoLS, we

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Figure 5. Nucleolar Localization of TTF Is a Dynamic Process Dependent on an Intrinsic NoLS (A) Endogenous mTTF is present in both nucleoli and nucleoplasm. Cytoplasmic, nuclear, nucleolar, and nucleoplasmic fractions from untreated NIH 3T3 cells were analyzed by immunoblotting for endogenous tubulin, fibrillarin (FIB), histone H3, SNF2H, and mTTF (antibody #3). (B) ARF is also present in both the nucleoli and nucleoplasm of MT-ARF cells. Nucleolar and nucleoplasmic cell fractions were analyzed for HA-ARF by immunoblotting, and endogenous MDM2, fibrillarin (FIB), and histone H3 before and after induction of ARF expression. (C) Nucleolar localization of TTF is dynamic. A YFP-mTTF fusion protein was expressed in NIH 3T3 cells and analyzed by FRAP. Typical examples of photobleaching and fluorescence recovery are shown in the top panels for whole nucleolar bleaching (circle) and in the bottom panels for subnucleolar bleaching (rectangle). The recovery times are given as the mean of ‘‘n’’ experiments, and the standard error is indicated. (D) TTF contains an intrinsic NoLS. YFP fusion proteins corresponding to full-length mTTF (FL) and mTTF subdomains (aa 211–470, 471–859, 121–210, 121–160, and 161–210) were expressed in NIH 3T3 cells. Cells were then fixed and processed for fibrillarin (FIB) immunofluorescence and were DAPI stained before microscopy. Examples of data from the full analysis of mTTF subdomains can be found in Figure S3 and a summary in Figure S4B.

121210

211470

121160

471859

161210

were able to show that the ARF-BS1 lay within aa 121–210 and that it was proximal to the NoLS (Figure 6A). When this fragment was dissected, neither of the NoLS-containing halves (aa 121– 160 and 161–210) was able to bind ARF. Thus, the ARF-BS1 lay between or across the N- and C-terminal segments of the bipartite NoLS (Figures 6B and S4B). ARF Prevents Nucleolar Accumulation of TTF by Inhibiting NoLS Function The proximity of the N-terminal ARF-binding site (ARF-BS1) to the bipartite NoLS (Figure 6B) suggested that ARF might directly prevent nucleolar localization of TTF by inhibiting the function of its NoLS. To determine whether this was the case, YFPTTF(121–210), encompassing the bipartite NoLS and ARF-BS1 (Figure 6C), was expressed in MT-ARF cells, and the localization of YFP was observed before and after induction of the HA-ARF

transgene. Strikingly, we found that less than 40% of the cells positive for nucleolar YFP-TTF(121–210) before ARF induction remained so after ARF induction (Figures 6D and 6E). Those cells that still displayed nucleolar YFP after ARF induction were found to be either ARF negative or to contain very low levels of ARF (Figure 6D). The level of YFPTTF(121–210) expression was found to be independent of ARF induction throughout the experiment (Figure 6C), excluding the trivial explanation that YFP-TTF(121–210) was degraded during ARF induction. Thus, aa 121–210 of mTTF was not only sufficient for nucleolar localization and ARF binding, but also directed the ARF-dependent nucleolar displacement of an unrelated protein. These data strongly support the conclusion that ARF directly regulates the subnuclear location of TTF by inhibiting the function of its NoLS. Further, the proximity of ARF-BS1 to the NoLS suggests that this inhibition occurs via the stereochemical masking of the NoLS of TTF by ARF. DISCUSSION Our data show that the ARF tumor suppressor inhibits the synthesis and processing of the ribosomal RNAs (rRNAs) by

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A

YFP + YFP-mTTF(211-470) YFP-mTTF(121-470) YFP-mTTF(121-210) YFP-mTTF(121-160) YFP-mTTF(161-210) HA-mARF +

+ +

+ +

+ +

+ +

Figure 6. ARF Prevents the Nucleolar Localization of TTF by Inhibiting NoLS Function

+ +

I.B. YFP

I.P. YFP

I.B. HA

IgG

Lysate I.B. HA

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DNA-binding domain Reb1-domain

mTTF: HN 324

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ARF-BS YFP-TTF(121-210): + Zn++ induction, 24h: YFP-TTF(121-210)

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W713K NPM-BS ARF-BS2

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1 121 161 210 NoLS-1/2: ARF-BS1:

E YFP+ nucleoli, % cells

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(A) The N-terminal ARF-binding site (ARF-BS-1) of mTTF maps within aa 121–210, but neither aa 121–160 nor 161–210 are sufficient for ARF binding. YFP fusion proteins were coexpressed with HA-mARF in HEK293T cells. Whole-cell lysates were immunoprecipitated (I.P.) with antiYFP antibodies and immunoblotted (I.B.) for both YFP fusion proteins and mARF (anti-HA). The low-resolution mapping data can be found in Figure S4A. (B) Summary of the functional subdomains’ organization of TTF and the mapping of the bipartite NoLS (NoLS-1/2), the ARF-binding sites ARFBS1 and -BS2, and the NPM-binding site. The N-terminal activation-repression-autoregulatory domain and the sequence-specific DNA-binding domain are indicated, as are the Reb1 and Myb homologies. A detailed summary of the mapping data can be found in Figure S4B. (C–E) MT-ARF cells were transfected with the expression vector for YFP-TTF(121–210) or were mock transfected and analyzed for expression levels and YFP fluorescence before and after induction of HA-ARF expression. (C) Top diagram shows the YFP-TTF(121–210) fusion protein, and the bottom panel shows the immunoblot analysis of whole-cell protein extracts. YFP-TTF(121–210) levels were unaffected by ARF induction. (D and E) Cells were analyzed by immunofluorescence for nucleolar YFP-TTF(121–210) and HAARF before and after ARF induction for 16 and 24 hr and were scored for both nucleolar ARF and YFP. (D) A typical field of cells after 16 hr of ARF induction. Note the near absolute reciprocity of nucleolar YFP or ARF. (E) Statistical analysis of cells displaying nucleolar YFP-TTF(121–210) before ARF induction and 16 and 24 hr after induction. See also Figure S4.

n=53 n=113

20

Overlay

0 0 16 24 Zn++ induction, h

preventing the nucleolar localization of the RPI transcription factor TTF-I. ARF directly interacts with TTF and induces its displacement, but not that of other RPI transcription factors, from the nucleolus and from the rRNA genes of mouse and human cells. Conditional depletion of TTF recapitulates the effects of ARF on rRNA synthesis and processing and can be rescued by the expression of exogenous TTF. Further, we show that depletion of NPM, a target of ARF-dependent degradation, is required for the nucleolar localization of TTF. Thus, our data not only delineate a pathway between ARF and the rRNA genes via TTF, but also provide a very plausible explanation for

546 Molecular Cell 38, 539–550, May 28, 2010 ª2010 Elsevier Inc.

previous data implicating NPM in rRNA synthesis and in the response to ARF. In proliferating cells, TTF shuttles between the nucleolus and nucleoplasm with a T1/2 of residence in the nucleolus of around 30 s. NPM binds to the C-terminal DNA-binding domain of TTF and is required for localization of TTF to its functional sites within the nucleolus (Figure 7A). ARF binds to a site within the N-terminal regulatory domain of TTF proximal to or overlapping its nucleolar localization sequence (NoLS). In so doing, ARF is able to inhibit NoLS function and hence prevent accumulation of TTF within the nucleolus and binding to its sites on the rRNA genes (Figure 7B). NPM is unlikely to transport TTF into the nucleolus because it does not bind the NoLS of TTF nor does it contain an NoLS itself (Lechertier et al., 2007; Li et al., 1996). Rather, we believe the requirement for NPM may be related to autoinhibition of TTF. The N-terminal domain of TTF interacts with and

Molecular Cell ARF Represses rRNA Synthesis by Controlling TTF-I

A

B rRNA genes

rRNA genes STOP TTF

X

TTF

NPM NPM

ARF

TTF

TTF

TTF: NoLS autoinhibited

NoLS functional

NoLS masked by ARF

Figure 7. ARF and NPM in the Control of TTF and rRNA Synthesis (A and B) The darker blue region represents the nucleoplasm, and the lighter blue region represents the nucleolus. (A) Under normal growth conditions, NPM is required for transport of TTF into the nucleolus and may act by unfolding of the autoinhibited state of TTF to reveal its NoLS. Though the diagram shows only the role of NPM in this process, it is likely that accessory proteins are required for NoLS recognition and transport of TTF into the nucleolus. (B) ARF blocks entry of TTF to the nucleolus by binding and inhibiting its NoLS.

inhibits the DNA-binding activity of the C-terminal domain (Sander et al., 1996). It is quite possible that, at the same time, this interaction masks the NoLS of TTF. By binding to the C-terminal domain, NPM may unfold the autoinhibited state of TTF to reveal this NoLS and hence promote nucleolar import (Figure 7A). In this scenario, ARF’s ability to bind the NoLS of TTF would provide a dominant block to nucleolar import. ARFdependent degradation of NPM (Itahana et al., 2003) might further enhance this block by limiting the active import of TTF into the nucleolus. TTF was originally identified as an RPI transcription termination factor and was shown to bind to a group of termination sites (T1–5) lying 30 of the 45S coding region on the rRNA genes (Kuhn and Grummt, 1989). Subsequent work showed that it also binds to an RPI promoter-proximal site (T0), where it regulates gene activity by recruiting nucleosome-remodeling complexes (La¨ngst et al., 1998; Ne´meth et al., 2004; Strohner et al., 2001, 2004). Thus, the finding that TTF is not only required for transcription of the rRNA genes, but also for the subsequent processing of the rRNAs, is unexpected. However, assembly of the 90S preribosomal particle begins cotranscriptionally during the synthesis of the 45S precursor rRNA, and it is likely that transcription and processing are coupled events (Moss et al., 2007; Tschochner and Hurt, 2003). A failure to terminate would necessarily lead to a slowing or arrest of elongation of nascent rRNA transcripts and was shown previously to cause severe rRNA processing defects in yeast (Schneider et al., 2007). Our data show that ARF is able to regulate TTF function directly, but also suggest that it may indirectly regulate TTF by catalyzing NPM degradation. Experimental suppression of

NPM function, either by siRNA depletion or by expression of the inhibitor NPM-3 or dominant-negative NPM constructs, was found to repress both rRNA synthesis and processing (Bertwistle et al., 2004; Huang et al., 2005) (Figures S2B and S2C). In vitro data suggested that NPM could catalyze the endolytic cleavage of the rRNA precursor at a sequence within ITS-2 (Savkur and Olson, 1998), and it was suggested that this might explain its requirement in rRNA production (Itahana et al., 2003). However, such endolytic activity has not been demonstrated to occur in vivo and could anyhow only explain a requirement for NPM in the processing of the 32S rRNA to the 28S rRNA. Our data show that, in fact, NPM functions as an essential cotransporter in the nucleolar localization of TTF, providing a highly plausible explanation of NPM’s role in both rRNA transcription and processing. Clearly, the ARF-catalyzed degradation of NPM would tend to enhance the direct effect of ARF on TTF. However, NPM is required for the stability and subcellular localization of ARF, and its loss or mutation, both experimentally and in acute myeloid leukemia (AML), prevents the accumulation and function of ARF (Colombo et al., 2005, 2006; Rodway et al., 2004). By catalyzing the partial degradation of NPM (Itahana et al., 2003), ARF must then also limit its own accumulation. Thus, the data point to the direct control of TTF as the major pathway of ARF repression of rRNA synthesis and suggest that regulation of NPM is a secondary contributing pathway. But whatever the relative importance of these two pathways, our data clearly identify TTF-I as the key protein target of the p53independent ARF tumor suppressor pathway in its ability to regulate ribosomal RNA synthesis. EXPERIMENTAL PROCEDURES Plasmid Constructs Full-length p19ARF (mARF) aa 1–169 was cloned into pGEX-4T (Amersham Biosciences) and pcDNA3 (Invitrogen). N-terminal FLAG- or YFP-tagged mouse TTF-I (mTTF) and mutants were expressed using pFLAGCMV2 (Invitrogen), pRevTRE (Clontech), or pEYFP-C1 (Clontech). The mTTF-(W713K) DNAbinding mutant was regenerated and tested for DNA binding by gel shift as in Evers et al. (1995). Anti-mTTF-I shRNAmir (OpenBiosystems, RMM176696882425) was subcloned into MSCV-TMP (Open Biosystems) to produce MSCV-TMP-mTTFshRNAmir. The HA-B23.1 vector (Okuwaki et al., 2001) was provided by K. Nagata. Antibodies Rabbit antisera were raised against mTTF-I aa 471–859 expressed in E. coli and affinity purified on the same polypeptide immobilized on CNBr-activated Sepharose (Amersham Pharmacia Biotech). Anti-RPA194 (#5) and anti-UBF (#8) antisera were raised against mouse RPA194 (aa 231–429) and mouse UBF (aa 2–404). Other antibodies were: anti-FLAG (F7425, Sigma-Aldrich), anti-fibrillarin (MMS-581S, Covance), anti-HA (ab9134, Abcam), anti-p19ARF (5-C3-1, Santa Cruz), anti-Tubulin (AA4.3, Developmental Studies Hybridoma Bank, University of Iowa), anti-H3 (ab1791, Abcam), anti-SNF2H (ab3749, Abcam), anti-MDM2 (2A10) (Chen et al., 1993), anti-NPM (Clone FC82291, Sigma), and anti-YFP (632460, Clontech). Cell Lines NIH 3T3, HEK293T, and HeLa were from ATCC, Con3 p53/ MEFs from James L. Sherley and wild-type MEFs from C57BL/6 E14.5 embryos essentially as in Xu (2005). Cells conditionally expressing the anti-mTTF shRNAmir cells were obtained by cotransfecting NIH 3T3 cells with linearized MSCVTMP-mTTFshRNAmir and pTet-OffDNeo. Clone A10 displayed effective doxycyclin-regulated depletion of TTF and clone B10 no depletion. B10 and

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Molecular Cell ARF Represses rRNA Synthesis by Controlling TTF-I

the parent NIH 3T3 cells were, therefore, used as negative controls. The pRevTREFLAG-mTTF transgene was introduced into clone A10 to produce clone A10+FLAG-mTTF. Cell lines and wild-type MEFs were maintained in highglucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Tet-Off clones in the same medium with the addition of 50 mg/ml doxycyclin. Where indicated, MT-ARF cells were treated with 80 mM ZnSO4, and clones A10, B10, and A10+FLAG-mTTF were grown in the absence of doxycylin.

SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and can be found with this article online at doi:10.1016/j.molcel.2010.03.015. ACKNOWLEDGMENTS

Transfection 1.25 3 106 HEK293T cells were seeded onto poly-L-lysine-treated (1 mg.ml1, Sigma-Aldrich) 60 mm culture dishes 24 hr prior to transfection. Transfection used CaPO4/chloroquine (Luthman and Magnusson, 1983) and 8 or 12 mg total DNA per 60 mm culture dish. NIH 3T3 cells were plated at 1.5 3 105 per 35 mm dish (25% confluency) for YFP constructs or 2.5 3 105 per 35 mm dish (40% confluency) for immunofluorescence (IF) using Ex-GEN 500 (Fermentas).

We wish to thank Dr. C.J. Sherr for providing p19ARF constructs and the MTARF cell line; Dr. James L. Sherley for Con3 p53/ cell line; Drs. L.I. Rothblum and I. Grummt for providing TTF-I cDNAs; K. Nagata for HA-B23.1 (HA-NPM-1); and A.J-. Levine for an anti-MDM2 antibody. We also wish to thank Michel G. Tremblay for wild-type MEFs. The work was funded by operating grants from the Cancer Research Society (CRS/SRC) and a six-month Feasibility Grant from the Canadian Cancer Society Research Institute (CCSRI/NCI-C). V.S. is supported, in part, by an operating grant from the Canadian Institutes of Health Research (CIHR). The Research Centre of the CHUQ, in which the Cancer Research Centre is housed, is supported by the FRSQ (Que´bec).

Cell Fractionation Subcellular fractions were prepared as previously described (Muramatsu et al., 1963); see also http://www.lamondlab.com/f7nucleolarprotocol.htm.

Received: January 20, 2010 Revised: February 24, 2010 Accepted: March 25, 2010 Published: May 27, 2010

Immunoprecipitation Cells were scraped into IP buffer (25 mM Tris-HCl [pH 7.5], 1 mM EDTA, 0.1 mM EGTA, 5 mM MgCl2, 150 mM NaCl, 10% glycerol, 1% NP-40 [Igepal. Sigma-Aldrich], 0.1% SDS, 1% Triton X-100, and 1 mg/ml each pepstatin, leupeptin, and aprotinin [Sigma-Aldrich]), kept on ice for 15 min, and then sonicated 4 3 20 s (S-450 Branson Ultrasonics) at maximum power. Cell lysates were cleared (14,000 rpm, 1 min) and incubated with first antibody for 3 hr at 4 C. Immunoprecipitates were recovered on protein A Sepharose (Amersham Biosciences) and washed once in IP buffer and three times in IP buffer less detergents. After fractionation on 8%, 12%, or 5%–15% gradient SDSPAGE, immunoprecipitates and total cell lysates were analyzed by western blotting.

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