Volume 38, Issue 4, Pages 539-550 (May 2010) The ARF Tumor Suppressor Controls Ribosome Biogenesis by Regulating the RNA Polymerase I Transcription Factor TTF-I  Frédéric Lessard, Françoise Morin, Stacey Ivanchuk, Frédéric Langlois, Victor Stefanovsky, James Rutka, Tom Moss  Molecular Cell  Volume 38, Issue 4, Pages 539-550 (May 2010) DOI: 10.1016/j.molcel.2010.03.015 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 38, 539-550DOI: (10.1016/j.molcel.2010.03.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Exogenous and Endogenous Mouse TTF-I and p19ARF Interact In Vitro and In Vivo (A) The organization of mouse and human ARF proteins showing the MDM2/HDM2 interaction sites. (B) Pull-down assay of in vitro-translated 35S-labeled 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. Molecular Cell 2010 38, 539-550DOI: (10.1016/j.molcel.2010.03.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 mARF Induction in MT-ARF and Wild-Type MEFs Causes the Displacement of mTTF and Corresponds with Downregulation of rRNA Synthesis and Processing (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 T1-binding 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). Molecular Cell 2010 38, 539-550DOI: (10.1016/j.molcel.2010.03.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

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 anti-YFP immunoprecipitates from whole-cell protein extracts were analyzed for HA-NPM coprecipitation. The NPM interaction site mapped to the C-terminal DNA-binding domain of mTTF. (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. Molecular Cell 2010 38, 539-550DOI: (10.1016/j.molcel.2010.03.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Conditional Knockdown of mTTF Recapitulates ARF Induction (A) Time course of doxycyclin withdrawal to induce shRNA expression (Induction) in the A10 NIH 3T3 cell clone carrying a tet-off regulated anti-mTTF shRNAmir transgene in the nonexpressing B10 control clone (Cntrl) and in the A10 clone carrying a tet-off regulated mTTF transgene (A10+FLAG-mTTF, Rescue). Cells were analyzed for de novo synthesis of rRNA using the standard 3 hr [3H]-uridine pulse labeling and for endogenous mTTF and FLAG-mTTF protein levels in comparison with fibrillarin (FIB). (B) Quantitation of total de novo rRNA synthesis normalized to day 1 of induction. (C) The corresponding ratios of 45S:28S and 45S:18S rRNA de novo labeling. (B and C) Data were derived from three independent pulse-labeling analyses, and error bars indicate the standard errors. Molecular Cell 2010 38, 539-550DOI: (10.1016/j.molcel.2010.03.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2010 38, 539-550DOI: (10.1016/j.molcel.2010.03.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 ARF Prevents the Nucleolar Localization of TTF by Inhibiting NoLS Function (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 anti-YFP 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 ARF-BS1 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 HA-ARF 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. Molecular Cell 2010 38, 539-550DOI: (10.1016/j.molcel.2010.03.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2010 38, 539-550DOI: (10.1016/j.molcel.2010.03.015) Copyright © 2010 Elsevier Inc. Terms and Conditions