Volume 43, Issue 2, Pages 240-252.e5 (October 2017) PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression  Ying Liu, Jaakko Mattila, Sami Ventelä, Leena Yadav, Wei Zhang, Nicole Lamichane, Jari Sundström, Otto Kauko, Reidar Grénman, Markku Varjosalo, Jukka Westermarck, Ville Hietakangas  Developmental Cell  Volume 43, Issue 2, Pages 240-252.e5 (October 2017) DOI: 10.1016/j.devcel.2017.09.022 Copyright © 2017 Elsevier Inc. Terms and Conditions

Developmental Cell 2017 43, 240-252. e5DOI: (10. 1016/j. devcel. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 PWP1 Regulates Tissue Growth and Proliferation (A) dPWP1 RNAi in the posterior compartment of the developing wing (En-Gal4) leads to reduced compartment size. (B) Quantification of the ratio of posterior (P) (n = 10) and anterior (A) (n = 10) wing areas in (A). (C) Cell density ratio between posterior (P) (n = 10) and anterior (A) (n = 10) compartments in (A). (D) Pupation kinetics of control (n = 5) and dpwp1nclb1/2 (n = 5) larvae. dAEL, days after egg laying. (E) Representative images of control and dpwp1nclb1/2 pupae. (F) Quantification of pupal volumes of control (n = 4) and dpwp1nclb1/2 (n = 4) pupae. (G) Proliferation in Ctrl (Lac dsRNA) (n = 3) and dPWP1-specific dsRNA (n = 3) treated S2 cells. (H) Proliferation of HeLa cells after transfection with non-targeting (Ctrl) (n = 3) or PWP1-specific (n = 3) siRNAs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student's t test). Error bars indicate SDs. See also Figure S1. Developmental Cell 2017 43, 240-252.e5DOI: (10.1016/j.devcel.2017.09.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 PWP1 Regulates Pol I-Mediated Ribosomal Gene Expression (A) dpwp1nclb1/2 mutants display short and thin bristles. (B) Pupation kinetics of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted larvae. dAEL, days after egg laying. (C) Representative images of control and dPWP1 fat-body (Cg-Gal4)-depleted pupae. (D) Quantification of volumes of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted pupae. (E) Representative immunofluorescent images of endogenous dPWP1 localization in comparison with fibrillarin in fat bodies of early third instar larvae. Scale bar, 5 μm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, and 28S rRNA (RNA polymerase I targets) expression in control larvae (n = 3) and dpwp1 mutants (n = 3). cdk7 was used as a reference gene. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student's t test). Error bars indicate SDs. See also Figure S2. Developmental Cell 2017 43, 240-252.e5DOI: (10.1016/j.devcel.2017.09.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 PWP1 Associates with rDNA Chromatin and Promotes Pol I-Mediated Transcription (A) Representative immunofluorescent images of PWP1 and POLR1E localization in HeLa cells. Scale bar, 5 μm. (B) Co-purification of GFP-tagged PWP1 upon pull-down of V5-tagged POLR1E from HeLa cells. Tubulin serves as a loading control. (C) Chromatin immunoprecipitation (ChIP) revealed the enrichment of PWP1 binding across an rDNA repeat unit and promoter (n = 3). (D) Representative immunofluorescent images of endogenous dPWP1 localization in comparison with dCDK7 in fat bodies of early third instar larvae. Scale bar, 5 μm. (E) Gel electrophoresis analysis of total RNA (lower panel, visualized by Midori green staining) and newly transcribed RNA (upper panel, visualized by streptavidin-horseradish peroxidase [HRP] detection) prepared from U2OS cells transfected with indicated siRNAs followed by 30 min 4sU labeling. RNA from non-transfected U2OS cells cultured in the absence of 4sU (No 4sU) was used as a control for streptavidin-HRP signal specificity. (F) ChIP revealed the relative level of H4K12Ac on different regions of rDNA in U2OS cells following PWP1 depletion. Ratio of 1 between control and PWP1 siRNA-treated samples (expected when no change occurs) is indicated by a dashed line. (n = 3). (G) ChIP revealed the relative level of H3K9me2 on different regions of rDNA in U2OS following PWP1 depletion (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student's t test). Error bars indicate SDs. See also Figure S3. Developmental Cell 2017 43, 240-252.e5DOI: (10.1016/j.devcel.2017.09.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 PWP1 Functionally Cooperates with MYBBP1A and Nucleolin (A) Summary of dPWP1 interacting ribosome biogenesis regulators in S2 cells. (B) Summary of PWP1 interacting ribosome biogenesis regulators in HEK293 cells. (C) Co-purification of HA-tagged dPWP1 with dMYBBP1A upon pull-down of V5-tagged dMYBBP1A from S2 cells. Tubulin serves as a loading control. (D) Representative immunofluorescent images of PWP1 and Nucleolin localization in U2OS cells. Scale bar, 5 μm. (E) Representative immunofluorescent images of Nucleolin localization in U2OS cells followed by PWP1 depletion. Scale bar, 5 μm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, 28S, and 5S rRNA expression in control larvae (n = 3) and dmybbp1a mutants (n = 3). cdk7 was used as a reference gene. (G) Representative images of control (w-) and mybbp1a mutant larvae at 96 hr after egg laying. (H) Representative immunofluorescent images of dPWP1 localization in fat bodies of early third instar larvae. Depletion of dMYBBP1A from the fat body (Fb-GAL4) leads to the dissociation of nucleolar dPWP1. Scale bar, 5 μm. (I) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (H). A total of 15 nuclei from 3 independent fat bodies were quantified. (J and K) Gel electrophoresis analysis of total RNA (lower panel, visualized by Midori green or ethidium bromide staining) and newly transcribed RNA (upper panel, visualized by streptavidin-HRP detection) prepared from U2OS cells transfected with indicated siRNAs followed by 30 min of 4sU labeling. RNA from non-transfected U2OS cells cultured in the absence of 4sU (No 4sU) was used as a control for streptavidin-HRP signal specificity. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student's t test). Error bars indicate SDs. See also Figure S4. Developmental Cell 2017 43, 240-252.e5DOI: (10.1016/j.devcel.2017.09.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 mTORC1-Dependent Phosphorylation Regulates Nucleolar PWP1 (A) Representative immunofluorescent images of endogenous dPWP1 localization in fat bodies of early third instar fed and starved (6 hr) larvae. Scale bar, 5 μm. (B) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (A). A total of 15 nuclei from 3 independent fat bodies were quantified. (C) Representative immunofluorescent images of endogenous dPWP1 localization in fat bodies of early third instar starved (4 hr) and re-fed (6 hr) larvae. Scale bar, 5 μm. (D) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (C). A total of 15 nuclei from 3 independent fat bodies were quantified. (E) Representative immunofluorescent images of dPWP1 localization in fat bodies dissected from early third instar larvae fed without or with rapamycin (14 hr). Scale bar, 5 μm. (F) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (E). A total of 15 nuclei from 3 independent fat bodies were quantified. (G) Immunoblot of S2 cell lysates expressing a V5-tagged form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin alone (10 min) or in combination with rapamycin (2 hr). Phospho-dPWP1 species (anti-V5) are indicated by arrowheads. (H) Immunoblot of lysates of S2 cell expressing a V5-tagged form of dPWP1 together with RNAi against LacZ (ctrl), dS6K, or dRaptor resolved on Phos-tag SDS-PAGE. Cells were treated without or with insulin (10 min). (I) Quantification of phospho-dPWP1 species of (H). (J) Immunoblot of S2 cell lysates expressing a V5-tagged wild-type or S384 alanine mutated form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin (10 min). (K) Quantification of phospho-dPWP1 species of (J). (L) Immunofluorescent analysis of fat body expressing the wild-type and S384A form of dPWP1 dissected from early third instar larvae. Scale bar, 5 μm. (M) Quantification of the nucleolus/nucleoplasm localization ratio in (L). A total of 15 nuclei from 3 independent fat bodies were quantified. ∗∗p < 0.01, ∗∗∗p < 0.001 (Student's t test). Error bars indicate SDs. See also Figure S5. Developmental Cell 2017 43, 240-252.e5DOI: (10.1016/j.devcel.2017.09.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 dPWP1 Is Involved in Nutrient-Dependent Growth Control (A) qRT-PCR analysis of dPWP1 mRNA expression in third instar larvae upon starvation (24 hr) (n = 3) or re-feeding with a high-protein diet (20% yeast, 6 hr) (n = 3) or a high-sugar diet (20% sucrose, 6 hr) (n = 3). cdk7 was used as a reference gene. (B) qRT-PCR analysis of dPWP1 mRNA expression in control (w−) (n = 3) and TOR mutant (TORdeIP) (n = 3) second instar larvae on 5% sucrose food (48 hr) and 20% yeast food re-feeding (20 hr). All samples were normalized to the 5% sugar-starved control larvae. cdk7 was used as a reference gene. (C) qRT-PCR analysis of dPWP1 mRNA expression in control (n = 3) and InR overexpressing (n = 3) second instar larvae (48 hr). cdk7 was used as a reference gene. (D) qRT-PCR analysis of dPWP1 mRNA expression in control (n = 3) and Rheb overexpressing (n = 3) second instar larvae (48 hr). rp49 was used as a reference gene. (E) qRT-PCR analysis of dPWP1 mRNA expression in control (n = 3) and Myc RNAi or overexpressing (n = 3) second instar larvae (48 hr). cdk7 was used as a reference gene. (F) dPWP1 RNAi in the posterior compartment of the developing wing (En-Gal4) alone or in combination with insulin-like receptor (InR) overexpression. The ratio of posterior (P) (n = 10) and anterior (A) (n = 10) wing areas was quantified. (G) dPWP1 RNAi in the posterior compartment of the developing wing (En-Gal4) alone or in combination with Rheb or Myc overexpression. The ratio of posterior (P) (n = 12) and anterior (A) (n = 12) wing areas was quantified. (H) Immunofluorescence analysis shows dCDK7 localization in the fat bodies of early third instar larvae with dPWP1 RNAi alone or combination with Rheb overexpression. Scale bar, 5 μm. (I) qRT-PCR analysis of rRNA expression upon yeast re-feeding of control (n = 3) and dpwp1nclb2 null mutant (n = 3) larvae. cdk7 was used as a reference gene. ANOVA showed a significant genotype by feeding interaction in terms of 18S and 28S rRNA expression (5.8S: F1, 8 = 1.44, p = 0.26; 18S: F1, 8 = 7.92, p = 0.02; 28S: F1, 8 = 8.41, p = 0.02). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (I was analyzed by ANOVA, all others by Student's t test). Error bars indicate SDs. See also Figure S6. Developmental Cell 2017 43, 240-252.e5DOI: (10.1016/j.devcel.2017.09.022) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 PWP1 Is Highly Expressed in Aggressive HNSCC Tumors (A) Proliferation of control (n = 3) and PWP1 (n = 3)- or MYBBP1A (n = 3)-depleted HNSCC (UT-SCC-2) cells. (B) Proliferation of control (n = 3) and Nucleolin (n = 3)-depleted HNSCC (UT-SCC-2) cells. (C) Representative immunohistochemistry images of HNSCC tissue sections displaying weak and strong PWP1 expression. Normal tissue devoid of PWP1 signal is indicated by arrowheads. Scale bar, 100 μm. (D) Kaplan-Meier survival curves of patients with HNSCC stratified for weak and strong PWP1 expression. Error bars indicate SDs. Developmental Cell 2017 43, 240-252.e5DOI: (10.1016/j.devcel.2017.09.022) Copyright © 2017 Elsevier Inc. Terms and Conditions