1. Volume 25, Issue 5, Pages 1103-1117.e6 (May 2017)
Hybrid Cellular Metabolism Coordinated by Zic3 and Esrrb Synergistically Enhances Induction of Naive Pluripotency 
Masamitsu Sone, Nobuhiro Morone, Tomonori Nakamura, Akito Tanaka, Keisuke Okita, Knut Woltjen, Masato Nakagawa, John E. Heuser, Yasuhiro Yamada, Shinya Yamanaka, Takuya Yamamoto 
Cell Metabolism 
Volume 25, Issue 5, Pages e6 (May 2017)
DOI: /j.cmet
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2. Cell Metabolism 2017 25, 1103-1117.e6DOI: (10.1016/j.cmet.2017.04.017)
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3. Figure 1
Zic3 and Esrrb Synergistically Enhance Somatic Cell Reprogramming by Acting on the Early and Middle Phases, Respectively
(A) A schematic illustration of the Split Assay, which was used to identify genes that are expressed specifically in cells committed to reprogramming into iPSCs (see STAR Methods for details).
(B) Relative expression levels of the top 10 highest genes specifically expressed in wells with iPSC-committed cells at 6 dpi (n = 39 wells in total, Benjamini-Hochberg q value < 0.05, see also Table S1). The average expression in wells without iPSC-committed cells is set to 1.
(C) FACS analysis of Oct4-GFP MEFs introduced with the indicated combinations of two TFs together with OSK at 12 dpi. The ranks of the TF in the Split Assay are shown in parentheses. EV, empty vector.
(D) Time course analysis of the expression of pluripotency marker genes measured by RT-qPCR (n = 3). The expression levels are normalized to 18S rRNA, and those of v6.5 ESCs are set to 1.
(E) Indicated combinations of 3 to 5 TFs were introduced into three reporter (Nanog-GFP, Oct4-GFP, and Dppa4-Venus) MEFs, and the percentages of fluorescence-positive cells were analyzed by FACS at 10 dpi (n = 3 or 4).
(F) Glucocorticoid receptor (GR)-fused Zic3 or Esrrb were introduced into Oct4-GFP MEFs and activated by adding dexamethasone (Dex) for 2 (left) or 4 (right) continuous days. GFP-positive cells were analyzed at 10 dpi by FACS (n = 3).
All data are presented as the mean ± SD.
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4. Figure 2
Zic3 and Esrrb Activate Gene Networks Required for Acquisition of Pluripotency in Conventional OSKM Reprogramming
(A) Oct4-GFP MEFs were infected with lentivirus expressing Cas9 and sgRNAs targeting Zic3 or Esrrb and retrovirus expressing OSKM. Two different combinations of two sgRNAs were examined for each target (mix1 and mix2). GFP+ colonies were counted at 12 dpi (n = 3). The sequences of sgRNAs are the same with those in the KO experiment of ESCs (Figure 4).
(B) Retroviral vectors expressing negative control (NC) or repressor domain (KRAB or EnR)-fused dominant-negative (DN) Zic3 and Esrrb in two different doses along with OSKM were introduced into Oct4-GFP MEFs. GFP+ colonies were counted at 12 dpi (n = 4).
(C) Single-cell RT-qPCR analysis of OSKM-introduced MEFs. ZTEG2ΔB9 dual-reporter MEFs were infected with retrovirus expressing OSKM and sorted as single cells by FACS at 8 dpi. Expression levels of indicated genes were determined by Fluidigm RT-qPCR. Cells were categorized into 4 groups based on the expression of Zic3 and Esrrb (n = 141 cells in total).
All data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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5. Figure 3
ChIP-seq Analysis of Zic3 and Esrrb Binding Sites during Reprogramming
(A) Schematic representation of ChIP-seq experiments.
(B–D) Venn diagrams of binding sites (BSs) of FLAG-tagged TFs. In (C) and (D), percentages of the sites co-bound with the indicated TFs are shown in the middle panel, and the top 2 enriched de novo motifs and the names of the TFs whose consensus motifs best matched them are indicated in the bottom panel.
(E) Typical examples of genome browser views of Zic3 and Esrrb BSs (arrowheads). Scale bar, 5 kb.
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6. Figure 4
Loss of Zic3 Function in ESCs Leads to Downregulation of Glycolysis-Related Genes
(A) Immunoblotting analyses of ESCs transiently introduced with Cas9 and sgRNA expression vectors.
(B) Microscopic images of ESCs introduced with the indicated sgRNA mixes (scale bars, 50 μm).
(C) Based on microarray datasets, more than 1.5-fold downregulated genes in KO ESCs compared to control were analyzed with the DAVID program. Overrepresented KEGG pathways and p values corrected for multiple tests (Benjamini) are shown.
(D) Gene expressions of glycolysis-related genes downregulated in Zic3 KO ESCs in microarray analysis are shown.
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7. Figure 5
Zic3 and Esrrb Activate Glycolysis Metabolic Pathway in Somatic Cell Reprogramming
(A) ChIP-seq analysis of glycolysis-related genes at 2 dpi. Arrowheads and numbers indicate the sites analyzed in Figure S4A.
(B) Time course analysis of glycolysis-related gene expressions measured by real-time PCR (n = 3). Expressions in v6.5 ESCs are set to 1.
(C) Glycolysis stress test using Seahorse XF96 analyzer at 4 dpi (n = 7 or 8 wells).
(D) Relative concentration of cellular metabolites quantified by CE-MS (n = 3). Concentrations in v6.5 are set to 1. Names of enzymes that catalyze each step are indicated in blue (See also Figure S4B).
All data are presented as the mean ± SD.
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8. Figure 6
Activation of OXPHOS by Esrrb Is Essential for Efficient Somatic Cell Reprogramming
(A) Mitochondria stress test using a Seahorse XF96 analyzer at 6 days after introduction of reprogramming factors (n = 5 wells).
(B) Time course analysis of ETC complex IV gene expressions measured by real-time PCR (n = 3). Expressions in v6.5 ESCs are set to 1.
(C) Gene Set Enrichment Analysis (GSEA) of ZTEG2ΔB9 chimeric MEFs sorted with EGFP+, tdTomato+, and parental gates by FACS at 8 days after introduction of OSKG. Gene set termed “Mitochondrial part” shows higher expression in EGFP+ cells than the cells of parental or tdTomato+ gates (See also Figures S2E and S2F).
(D) Transmission electron microscopy images of mitochondria at 6 dpi (arrowheads). Scale bar, 500 nm.
(E) Boxplot of mitochondrial area (left) and ratios of minor axis to major axis of best-fit ellipses to the mitochondria (right) (n > 222 mitochondria per condition). p values were calculated using the Wilcoxon rank sum test.
(F) FACS analysis of Oct4-GFP MEFs at 11 days after introduction of OSK, Zic3 and empty vector (EV) or TFs that activate OXPHOS (n = 4). Dexamethasone was added at the indicated durations.
All data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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9. Figure 7
Metabolic Shift toward Naive Pluripotency Is Independent of Hif Function
(A) ChIP-seq analysis of Zic3 and Esrrb binding around Hif2α gene locus at 2 dpi. BSs indicated by arrowheads were analyzed by real-time PCR (see Figure S6A).
(B) Time course expression analyses of Hif genes by real-time PCR (n = 3). Expressions in v6.5 ESCs are set to 1.
(C) Expression levels of Hif genes by real-time PCR at 4 days after introduction of OSKZE and mixtures of CRISPRi virus vectors targeting the indicated genes (n = 3).
(D) FACS analysis of GFP-positive cells at 10 days after introduction of OSKZE and indicated CRISPRi virus vectors (n = 3).
(E) Hif1α/2α were introduced with OSK/Z/E into Oct4-GFP MEF, and the proportion of GFP-positive cells in normoxia or hypoxia (5% O2) was analyzed by FACS at 10 dpi (n = 5).
(F) Immunofluorescent images of the EpiSCs transduced with indicated genes and stained with anti-Dppa4 antibody. Scale bar, 250 μm.
(G–I) Relative numbers of Dppa4+ colonies after introduction of single factors (G) or factors combined with Klf4 (H) or with Esrrb (I) in normoxia or hypoxia (n = 3).
(J) Schematic diagram of metabolic regulations that positively or negatively affect reprogramming processes.
All data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <
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