Volume 54, Issue 3, Pages 526-535 (May 2014) Antagonism between the Master Regulators of Differentiation Ensures the Discreteness and Robustness of Cell Fates  Kazunori Sunadome, Toshiyasu Suzuki, Mai Usui, Yuhei Ashida, Eisuke Nishida  Molecular Cell  Volume 54, Issue 3, Pages 526-535 (May 2014) DOI: 10.1016/j.molcel.2014.03.005 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 54, 526-535DOI: (10.1016/j.molcel.2014.03.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 The MyoD-Driven and PPARγ-Driven Differentiation Programs Are Mutually Exclusive (A) C3H10T1/2 cells were infected with lentivirus containing Myc-MyoD, lentivirus containing HA-PPARγ, or both. The volume of lentivirus containing each gene was kept constant between single and simultaneous expression (MyoD/PPARγ ratio of 1:1). At 4 days after infection, cells were observed by phase-contrast microscopy. White dots indicate lipid droplets. A magnified fluorescent image of a boxed region in the picture of cells coinfected with Myc-MyoD and HA-PPARγ lentiviruses is shown in the right-hand panel. Cells were visualized by the fluorescence of mCherry, which was introduced by lentivirus (red). Nuclei were revealed by Hoechst staining (blue), and lipid droplets formation was assessed with BODIPY (green). (B) Cells were infected with an HA-PPARγ-IRES-Flag-MyoD lentivirus and observed by phase-contrast microscopy 4 days after infection. (C) Simultaneous expression of Myc-MyoD and HA-PPARγ was performed at the indicated MyoD/PPARγ ratios with or without rosiglitazone (1 μM). (D–F) Cells coinfected with Myc-MyoD and HA-PPARγ lentiviruses (MyoD/PPARγ ratio 1:1) were examined for the expression of MyHC (red) and the formation of lipid droplets (green) at 8 days after infection (E), the expression of myoglobin (red) and the formation of lipid droplets (green) (D), or myogenin and C/EBPα (F) at 4 days after infection. The percentage of myogenin-positive, C/EBPα-positive, or both myogenin- and C/EBPα-positive nuclei of all nuclei was quantified (F). At least 300 nuclei were counted for each treatment in each experiment. Values represent means (± SD) in triplicate determinations. (G) Single-cell-derived clones were coinfected with Myc-MyoD and HA-PPARγ lentiviruses (MyoD/PPARγ ratio 1:1) and observed by phase-contrast microscopy 4 days after infection. M, Myc-MyoD. P, HA-PPARγ. Scale bars, 100 μm. See also Figure S1. Molecular Cell 2014 54, 526-535DOI: (10.1016/j.molcel.2014.03.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Although Exogenously Expressed PPARγ Is Not Attenuated in Myotubes, Exogenously Expressed MyoD Is Degraded in Adipocytes (A) C3H10T1/2 cells were infected with Myc-MyoD or HA-PPARγ lentivirus. The generated myotubes and adipocytes were trypsinized and combined, and then fractionated according to the method described in Supplemental Experimental Procedures. The mRNA levels of exogenous MyoD and PPARγ in each fraction were determined by quantitative RT-PCR analysis. Values represent means (± SD) of duplicate determinations. (B and C) Cells were coinfected with Myc-MyoD and HA-PPARγ lentiviruses (MyoD/PPARγ ratio 1:1). At 4 days after infection, the generated mixture of myotubes and adipocytes was fractionated, and the relative mRNA levels (B) or protein levels (C) of the indicated genes were examined. For protein analyses, band intensities obtained by immunoblotting were measured using ImageJ software and normalized by the levels of α-Tubulin (C, right). Values represent means (± SD) of at least triplicate determinations. In both the mRNA and protein analyses, the value of the M fraction was set to one for each myogenic marker gene, and that of the A fraction was set to one for each adipogenic marker gene. Control, lentivirus-infected cells. (D) Cells were coinfected with mCherry, Myc-MyoD, and HA-PPARγ lentiviruses (MyoD/PPARγ ratio 1:1). At 4 days after infection, cells were examined for expression of MyoD and PPARγ by immunostaining with anti-Myc (lower panel) and anti-HA (upper panel) antibodies, respectively. M, Myc-MyoD. P, HA-PPARγ. Scale bars, 100 μm. See also Figure S2. Molecular Cell 2014 54, 526-535DOI: (10.1016/j.molcel.2014.03.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 MyoD Protein Is Degraded in Adipocytes through the Ubiquitin Proteasome System (A and C) C3H10T1/2 cells were infected with Myc-MyoD or both Myc-MyoD and HA-PPARγ lentiviruses (MyoD/PPARγ ratio 1:5). The volume of lentivirus containing each gene was kept constant between single and simultaneous expression. At this ratio of simultaneous expression, almost all cells differentiated into adipocytes. (A) At 4 days after infection, cells were treated with MG132 (25 μM) for 12 hr and then examined for MyoD protein levels. (B) Cells were coinfected with Myc-MyoD and HA-PPARγ lentiviruses (MyoD/PPARγ ratio 1:1). At 4 days after infection, cells were treated with MG132 (25 μM) for 12 hr and then examined for localization of MyoD. (C) At 4 days after infection, cells were treated with MG132 (25 μM) for 12 hr, washed three times, and then treated with cycloheximide (10 μg/ml). Cells were collected at the indicated time points and examined for MyoD protein levels (left panel). Band intensities were measured using ImageJ software and normalized by α-Tubulin protein levels. Values were adjusted so that those at t = 0 were 100%. The log10 of % values were plotted versus time for each time point, and the half-life (t1/2) was calculated as the log of 50% (middle panel). The mean values from three independent experiments are shown in the right panel; p value, unpaired t test. M, Myc-MyoD. P, HA-PPARγ. Scale bars, 100 μm. See also Figure S3. Molecular Cell 2014 54, 526-535DOI: (10.1016/j.molcel.2014.03.005) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Both the Hyperacetylation of the C/EBPα Gene Locus and the Transcriptional Initiation of C/EBPα Are Inhibited in PPARγ-Expressing Myotubes (A) C3H10T1/2 cells were infected with HA-PPARγ or both Myc-MyoD and HA-PPARγ lentiviruses at the indicated MyoD/PPARγ ratios. Cells were examined for PPARγ expression by immunoblotting. The protein levels of PPARγ were quantified as in Figure 2C. (B) C3H10T1/2 cells coinfected with Myc-MyoD and HA-PPARγ lentiviruses (MyoD/PPARγ ratio 1:1) were fractionated and the relative mRNA levels of the indicated genes were examined. Values represent means (± SD) of at least triplicate determinations. Control, lentivirus-infected cells. For each gene, the value of the A fraction was set to one. (C, D, and F) Cells were infected with HA-PPARγ or both Myc-MyoD and HA-PPARγ lentiviruses (MyoD/PPARγ ratio 4:1). The volume of lentivirus containing each gene was kept constant between single and simultaneous expression. After 3 days of infection, cells were collected and ChIP assays were performed using anti-PPARγ, anti-acetylated histone H3, anti-acetylated histone H4 (∗p < 0.05, unpaired t test, P versus M+P); anti-Pol II, anti-phospho serine 5 Pol II CTD, anti-phospho serine 2 Pol II CTD (∗p < 0.05, unpaired t test) (D); and anti-MyoD (F) antibodies. ChIP signals were detected by quantitative RT-PCR using the primers for the indicated regions. The 10,000 bp upstream region from the TSS of C/EBPα and the Nanog promoter served as a negative control. The values of C/EBPα_TSS (D and F) and C/EBPα_DR1_1 (C) of control lentivirus-infected cells (Control) were set to one. Fabp4 C.B. and Sort1 C.B. are C/EBPα-binding regions in each locus. Values represent means (± SD) in at least triplicate determination. (E) The locations and sequences of PPARγ-binding sites and MyoD-binding sites in the C/EBPα gene locus are shown along with ChIP-seq data for C2C12 myogenesis in the mouse ENCODE project, which were obtained from the UCSC genome browser. Gray bars show significantly enriched regions of the MyoD ChIP signal. (G) Binding of MyoD to the putative MyoD-binding site at the C/EBPα promoter was examined by means of an oligonucleotide precipitation assay using a double-stranded, biotinylated wild-type or mutant oligonucleotide sequence corresponding to bases from +251 to +279 of the C/EBPα promoter. In the mutant oligonucleotide, CCACCTG, a putative MyoD-binding site, was mutated to TATAGAC. (H) C3H10T1/2 cells were transfected with the indicated amounts (ng) of MyoD- and PPARγ-expressing plasmids together with a reporter vector in which the C/EBPα locus (0 to +8,000 bp from TSS) was inserted downstream of the luciferase gene, and then harvested 48 hr later for the luciferase assay. The total amounts of transfected plasmids were kept constant using the empty plasmid. Values represent means (± SD) of duplicate determinations. (I) C2C12 cells were infected with adenovirus containing Id1 at the indicated concentration. The inhibitory effect of Id1 on MyoD activity was confirmed by examining myogenin expression (data not shown). Two days after infection, the medium was switched to MIM (high-glucose DMEM containing 3% heat-inactivated horse serum) or AIM (high-glucose DMEM containing 10% FBS, 0.5 mM IBMX, 125 nM indomethacin, 1 μM dexamethasone, 850 nM insulin, 1 nM T3, and 1 μM rosiglitazone). After 2 days, the mRNA levels of C/EBPα were examined. C/EBPα expression was inhibited in an Id1 dose-dependent manner. Values represent means (±SD) of duplicate determinations. M, Myc-MyoD. P, HA-PPARγ. See also Figure S4. Molecular Cell 2014 54, 526-535DOI: (10.1016/j.molcel.2014.03.005) Copyright © 2014 Elsevier Inc. Terms and Conditions