Volume 18, Issue 9, Pages 2148-2161 (February 2017) The SETD8/PR-Set7 Methyltransferase Functions as a Barrier to Prevent Senescence- Associated Metabolic Remodeling  Hiroshi Tanaka, Shin-ichiro Takebayashi, Akihisa Sakamoto, Tomoka Igata, Yuko Nakatsu, Noriko Saitoh, Shinjiro Hino, Mitsuyoshi Nakao  Cell Reports  Volume 18, Issue 9, Pages 2148-2161 (February 2017) DOI: 10.1016/j.celrep.2017.02.021 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 18, 2148-2161DOI: (10.1016/j.celrep.2017.02.021) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 The Loss of SETD8/PR-Set7 Induces Senescence (A) Schematic representation of senescence models used in this study. SA-β-Gal staining of growing, oncogene-induced senescence (OIS), and replicative senescence (RS) cells are shown. Numbers indicate the percentage of SA-β-Gal-positive cells (each n > 300 cells). Scale bars, 100 μm. (B) Western blot analysis of SETD8 and p16INK4A in growing, OIS, and RS cells. (C) Growth curves for Ctr- or SETD8-KD IMR-90 cells. (D) EdU incorporation assay on day 3 of Ctr- or SETD8-KD cells. EdU-positive and EdU-negative cells were distinguished by measuring the fluorescence intensity of EdU-labeled DNA in each cell (each n > 1,600 cells). (E) SA-β-Gal staining on days 3–15 of Ctr- or SETD8-KD (each n > 300 cells). Scale bars, 100 μm. (F) SAHF on days 3–15 of Ctr- or SETD8-KD (each n > 300 cells). Scale bars, 10 μm. (G) Western blot analysis of SETD8, p16INK4A, and p21CDKN1A on days 3 and 6 of Ctr- or SETD8-KD. (H) Growth curves for DMSO-treated or the SETD8 inhibitor UNC0379-treated IMR-90 cells. (I) SA-β-Gal staining on day 6 of DMSO or UNC0379 treatment (each n > 300 cells). Scale bars, 100 μm. (J) SAHF on day 6 of DMSO or UNC0379 treatment (each n > 300 cells). Scale bars, 10 μm. Values are means ± SD (n = 3). p values were calculated using the Student’s t test (∗p < 0.05, ∗∗p < 0.01). Cell Reports 2017 18, 2148-2161DOI: (10.1016/j.celrep.2017.02.021) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Loss of SETD8 Upregulates RP-Encoding and Senescence-Associated Genes (A) Enrichment plot of GSEA of upregulated genes at 24 hr in SETD8-KD IMR-90 cells, compared with Ctr-KD cells. In each panel, nominal (NOM) p values and FDR q values are indicated. (B) Scatterplot of all probes (23,717) obtained from microarray analysis in Ctr- and SETD8-KD cells (left). Relative signal intensity of all probes (gray) and ribosomal protein (RP) gene probes (orange) between Ctr- and SETD8-KD (right). (C) Venn diagram of RP genes, highly expressed genes (signal intensity > 20,000 in Ctr-KD cells, top 1.5% of all probes), and >1.05-fold upregulated gene probes by SETD8-KD. A set of RP genes (70 of 86) were upregulated by SETD8-KD, compared with Ctr-KD. (D and E) qRT-PCR analysis of representative RP genes (D) and senescence-associated genes (E) on day 1 of Ctr- or SETD8-KD. Values are means ± SD (n = 3). p values were calculated using the Student’s t test (∗p < 0.05, ∗∗p < 0.01). Cell Reports 2017 18, 2148-2161DOI: (10.1016/j.celrep.2017.02.021) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 SETD8 Regulates RP-Encoding and Senescence-Associated Genes via H4K20me1 Deposition (A) Venn diagram of regions containing H4K20me1-enriched islands (8,967 regions) in NHDF-Ad cells, RP genes (86 genes), and >1.05-fold upregulated gene probes (5,478 probes) in SETD8-KD IMR-90 cells (Figure 2C). A set of RP genes (71 of 86) were enriched for H4K20me1, and the overlapping genes (59 of 86) were further upregulated by SETD8-KD. The RPL5 gene is shown as a representative RP gene with an H4K20me1-enriched island. (B) Immunofluorescence of H4K20me1 on day 1 of Ctr- or SETD8-KD. Relative fluorescence was calculated by the mean fluorescence of SETD8-KD cells, compared with that of Ctr-KD cells (each n > 2,000 cells). Scale bar, 100 μm. (C–F) ChIP of H4K20me1, H4K20me3, and H3K36me3 at promoter regions and gene bodies of indicated gene loci on day 1 of Ctr- or SETD8-KD. The data for representative RP gene loci (C and D) and the p16INK4A gene locus (E and F) are shown. Values are means ± SD (n = 3). p values were calculated using the Student’s t test (∗p < 0.05, ∗∗p < 0.01). Ig, immunoglobulin. Cell Reports 2017 18, 2148-2161DOI: (10.1016/j.celrep.2017.02.021) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 SETD8 Regulates rRNA Genes and Nucleolar Function via H4K20me1 Deposition (A and B) ChIP of H4K20me1 and H4K20me3 (A) and H3K36me3 (B) at rRNA gene loci on day 1 of Ctr- or SETD8-KD. (C) Quantification of rRNA transcription by measuring the mean fluorescence of EU-labeled RNAs within the nucleolus in Ctr- and SETD8-KD cells (each n > 1,000 cells). (D and E) Morphological analysis of the nucleolus in Ctr- and SETD8-KD cells. The average number (D) and area (E) of the nucleolus per cell was calculated by measuring the number and area of the fluorescence signals of nucleophosmin (B23) in a single cell (each n > 1,000 cells). The proportion of cells containing one or two nucleoli was increased, while cells containing more than three nucleoli were decreased in SETD8-KD cells (D). Scale bar, 10 μm. Values are means ± SD (n = 3). p values were calculated using the Student’s t test (∗p < 0.05, ∗∗p < 0.01). Ig, immunoglobulin. Cell Reports 2017 18, 2148-2161DOI: (10.1016/j.celrep.2017.02.021) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Loss of SETD8 Activates Mitochondrial OXPHOS via RB (A) Oxygen consumption rate (OCR) on day 6 of Ctr- or SETD8-KD. Values are means ± SD (n = 3–4). Respiratory chain inhibitors were serially added to the culture at the indicated time points. Basal OCRs are shown by subtracting the rotenone/antimycin treated value from the initial value. Data are representative of two independent assays. (B) OCR/ECAR is shown by dividing the initial OCR value in (A) by the initial ECAR (extracellular acidification rate) value (see Figure S6A) in Ctr- and SETD8-KD cells. (C) Assessment of mitochondrial mass and membrane potential indicated by the mean fluorescence of JC-1 monomer and J-aggregate, respectively, on day 6 of Ctr- or SETD8-KD. (D) Intracellular ROS levels determined by MitoSOX fluorescence on day 6 of Ctr- or SETD8-KD. (E–G) The effects of simultaneous KD of RB on mitochondrial activities in SETD8-KD cells. OCR (E) and OCR/ECAR (F) on day 6 are shown as means ± SD (n = 5–6). Data are representative of two independent assays. Mitochondrial membrane potential (G) is indicated by the mean fluorescence of JC-1 J-aggregate on day 6. (H) Intracellular ROS levels on day 6 of Ctr, SETD8-KD, or SETD8/RB1-KD. (I) SA-β-Gal staining on day 6 of Ctr, SETD8-KD, or SETD8/RB1-KD (each n > 300 cells). Scale bars, 100 μm. (J) SAHF on day 6 of Ctr, SETD8-KD, or SETD8/RB1-KD (each n > 300 cells). Scale bars, 10 μm. All values except for those in (A), (B), (E), and (F) are means ± SD (n = 3). p values were calculated using the Student’s t test (∗p < 0.05, ∗∗p < 0.01). Cell Reports 2017 18, 2148-2161DOI: (10.1016/j.celrep.2017.02.021) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 H4K20me1 Marks Are Remodeled at SETD8-Target Genes in Senescent Cells (A) The list of consistently upregulated gene sets in OIS, RS, and SETD8-KD cells, based on our microarray analyses. (B) Relative signal intensity of RP gene probes in OIS and RS cells, compared with growing cells. (C) ChIP of H4K20me1 at RP gene loci in growing and OIS cells. Primers used are shown in Figure 3C. (D) ChIP of H4K20me1 at RPL5 gene locus in growing and RS cells. Primers used are shown in Figure 3C. (E) Quantification of rRNA transcription by measuring the mean fluorescence of EU-labeled RNAs within the nucleolus in growing and OIS cells (each n > 800 cells). (F) ChIP of H4K20me1 at rRNA gene loci in growing and OIS cells. Primers used are shown in Figure 4A. (G) qRT-PCR analysis of p16INK4A in growing and OIS cells. (H) ChIP of H4K20me1 at p16INK4A gene locus in growing and OIS cells. Primers used are shown in Figure 3E. Values are means ± SD (n = 3). p values were calculated using the Student’s t test (∗p < 0.05, ∗∗p < 0.01). Cell Reports 2017 18, 2148-2161DOI: (10.1016/j.celrep.2017.02.021) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 Schematic Role of SETD8 in Proliferative and Senescent Cells (A) Representative immunofluorescence images of nucleoli [nucleophosmin (B23)-stained] and mitochondria in SETD8-KD, OIS, and RS cells. Each senescent state exhibits enlarged and merged nucleoli (red) and increased mitochondria (green) in the presence of SAHF formation. DNA was counterstained with DAPI (blue). Scale bars, 20 μm. (B) SETD8 is responsible for H4K20me1 deposition at gene bodies of specific loci and negatively regulates their transcription at basal levels in proliferative cells. During senescence induced by oncogenic or replicative stresses, SETD8 is downregulated and the H4K20me1-based epigenomic landscape is remodeled at RP, rRNA, and p16INK4A gene loci, resulting in transcriptional derepression of these genes. The loss of SETD8 leads to nucleolar-mitochondrial coactivation, which is the hallmark of senescent cells. Cell Reports 2017 18, 2148-2161DOI: (10.1016/j.celrep.2017.02.021) Copyright © 2017 The Author(s) Terms and Conditions