Volume 44, Issue 6, Pages 741-751.e3 (March 2018) Nutritional Control of Stem Cell Division through S-Adenosylmethionine in Drosophila Intestine  Fumiaki Obata, Kayoko Tsuda-Sakurai, Takahiro Yamazaki, Ryo Nishio, Kei Nishimura, Masaki Kimura, Masabumi Funakoshi, Masayuki Miura  Developmental Cell  Volume 44, Issue 6, Pages 741-751.e3 (March 2018) DOI: 10.1016/j.devcel.2018.02.017 Copyright © 2018 Elsevier Inc. Terms and Conditions

Developmental Cell 2018 44, 741-751. e3DOI: (10. 1016/j. devcel. 2018 Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 1 Methionine Depletion Reduces Stem Cell Proliferation in Midgut (A and C) The experimental scheme. (B, D, and E) Number of phospho-histone H3 (PH3)-positive cells in whole gut of w1118 male (B) or female (D and E) flies. SY, sucrose/yeast medium; Met−, holidic medium without methionine (see STAR Methods); Met+, complete holidic medium. (F) Methionine metabolism and methylation. Met, methionine; SAM, S-adenosylmethionine; SamS, SAM synthase; SAH, S-adenosylhomocysteine. (G) Quantification of methionine, SAM, and sarcosine in whole-body homogenate of male flies maintained for 5 days in holidic medium with or without methionine (n = 4). For all graphs, mean and SEM with all data points are shown. Statistics: two-tailed Student's t test (D and G) or one-way ANOVA with Tukey's multiple comparison test (B and E). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. See also Figure S1. Developmental Cell 2018 44, 741-751.e3DOI: (10.1016/j.devcel.2018.02.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 2 SAM Is Essential for Proliferation of Intestinal Stem Cells (A) Four major cell types in Drosophila midgut. Gal4 drivers (in parentheses) are used for cell-type-specific gene manipulation. EC, enterocyte; ISC, intestinal stem cell; EB, enteroblast; EEC, enteroendocrine cell. (B) Experimental scheme and representative images of the EdU-feeding assay. Control, esgts>lacZ-RNAi. Scale bars, 50 μm. (C) Number of EdU-positive cells in posterior midgut (PMG) of male flies with control (lacZ-RNAi) or three SamS-RNAi in progenitors (ISCs and EBs). (D) Number of EdU-positive cells in posterior midgut (PMG) of male flies with control (lacZ-RNAi) or two SamS-RNAi in ISCs. (E) Number of intestinal stem cells (ISCs) and enteroblasts (EBs) in the part of posterior midgut (R4a) of esgts>SamS-RNAiKK male flies. EBs are marked by the reporter Su(H)GBE-lacZ. The number of ISCs was counted by the number of GFP-positive, lacZ-negative cells. (F) Amount of methionine and SAM in whole gut. NP1 and esg drivers were combined with SamS-RNAiKK (n = 3). For all graphs, mean and SEM with all data points are shown. Statistics: two-tailed Student's t test (E and F) or one-way ANOVA with Tukey's multiple comparison test (C and D). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Developmental Cell 2018 44, 741-751.e3DOI: (10.1016/j.devcel.2018.02.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 3 SAM Is Required for Accelerated Proliferation of Intestinal Stem Cells (A) Experimental scheme and representative images of the EdU-feeding assay. Control, esgts>lacZ-RNAi. Scale bars, 50 μm. (B and C) Number of phospho-histone H3 (PH3)-positive cells in whole gut of male (B) or female (C) flies with or without SamS-RNAiKK in the presence or absence of bleomycin. (D and E) Number of EdU-positive cells in posterior midgut (PMG) of male flies: refed (D) or overexpression of constitutive active form of insulin receptor (InRCA) (E) with or without SamS-RNAi. For all graphs, mean and SEM with all data points are shown. Statistics: two-tailed Student's t test (E) or one-way ANOVA with Tukey's multiple comparison test (B–D). ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Developmental Cell 2018 44, 741-751.e3DOI: (10.1016/j.devcel.2018.02.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 4 Genetic Screening for SAM-Dependent Methyltransferases Regulating Stem Cell Division (A) Overview of genetic screening of putative methyltransferases required for ISC division. (B) Schematic view of the functions of “hit genes.” eEF2, eukaryotic elongation factor 2; RF, release factor. (C) Representative images of the EdU-feeding assay in posterior midgut of male flies. Control, esgts>lacZ-RNAi. Scale bars, 50 μm. (D) Number of EdU-positive cells in posterior midgut (PMG) of male flies. Each gene was knocked down specifically in ISCs. (E) Number of ISCs in the part of posterior midgut (R4a) of male flies. For all graphs, mean and SEM with all data points are shown. Statistics: one-way ANOVA with Tukey's multiple comparison test. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figures S1 and S2; Table S1. Developmental Cell 2018 44, 741-751.e3DOI: (10.1016/j.devcel.2018.02.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 5 SAM Controls Protein Synthesis in ISCs (A) Representative image of the male gut from homopropargylglycine (HPG) protein synthesis assay. High translation zone (HTZ) is indicated by an arrow. AMG, anterior midgut; PMG, posterior midgut; HG, hindgut. Scale bars, 100 μm (low magnification) or 20 μm (high magnification). (B) Representative images of the HTZ of female flies. Scale bars, 10 μm. (C) Quantification of HPG intensity in esg-positive cells relative to surrounding cells in HTZ in SamS- or Dph5-RNAi conditions. Mean and SEM with all data points is shown. The number of samples is shown below the graph. Statistics: two-tailed Student's t test. ∗∗∗∗p < 0.0001. (D) Representative images of the HPG assay in HTZ of male flies with or without bleomycin feeding. SamS-RNAiKK or Dph5-RNAiKK was used. Scale bars, 100 μm. (E) Schematic model of SAM-dependent regulation of protein synthesis. See also Figure S3. Developmental Cell 2018 44, 741-751.e3DOI: (10.1016/j.devcel.2018.02.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 6 SAM Depletion in Enterocytes Triggered Upd3-Induced ISC Division (A) Representative images of EdU-feeding assay with or without SamS-RNAi in enterocytes. Control, NP1ts>lacZ-RNAi. SamS-RNAiGD was used. Scale bars, 50 μm. (B) Number of EdU-positive cells in posterior midgut (PMG) of male flies with knockdown SamS, Dph5, or HemK1 in enterocytes. (C) Number of PH3-positive cells in whole midgut of male flies. (D) Number of EdU-positive cells in posterior midgut (PMG) of male flies. SamS-RNAiGD or overexpressing pro-apoptotic gene rpr was used. (E) Representative images of the apoptotic cells in the posterior midgut of male flies. A caspase reporter, CD8-PARP-Venus, was driven by NP1ts. Scale bar, 50 μm. (F) qRT-PCR of ligands and JAK/STAT target socs36E in whole gut of male flies (n = 3). SamS-RNAiGD was used. (G) Number of EdU-positive cells in posterior midgut (PMG) of male flies. SamS-RNAiGD or upd3-RNAiKK was used. For all graphs, mean and SEM with all data points are shown. Statistics: two-tailed Student's t test (C and F) or one-way ANOVA with Tukey's multiple comparison test (B, D, and G). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Table S2. Developmental Cell 2018 44, 741-751.e3DOI: (10.1016/j.devcel.2018.02.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 7 Upd3 Is Induced during Starvation for Rapid Stem Cell Division upon Refeeding (A) Number of EdU-positive cells in posterior midgut (PMG) of w1118 male flies after 4-day feeding of sucrose/yeast (SY), 1% sucrose (Stv), or 1% sucrose plus 3.2 mM methionine (Stv + Met). (B) Quantification of HPG intensity in esg-positive cells relative to surrounding cells in HTZ of male guts. (C) qRT-PCR of pro-mitotic ligands in whole gut of male flies with or without 4 day starvation (1% sucrose) (n = 3). (D) qRT-PCR of JAK/STAT target socs36E in whole gut of control (w1118) or upd3Δ male flies (n = 3). (E) Number of EdU-positive cells in posterior midgut (PMG) of control (w1118) or upd3Δ male flies. (F) Model of SAM-dependent control of ISCs. For all graphs, mean and SEM with all data points are shown. Statistics: one-way ANOVA with Tukey's multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figure S4. Developmental Cell 2018 44, 741-751.e3DOI: (10.1016/j.devcel.2018.02.017) Copyright © 2018 Elsevier Inc. Terms and Conditions