Volume 47, Issue 2, Pages 169-182 (July 2012) Oct4 and Nanog Directly Regulate Dnmt1 to Maintain Self-Renewal and Undifferentiated State in Mesenchymal Stem Cells  Chih-Chien Tsai, Pei-Fen Su, Yi-Feng Huang, Tu-Lai Yew, Shih-Chieh Hung  Molecular Cell  Volume 47, Issue 2, Pages 169-182 (July 2012) DOI: 10.1016/j.molcel.2012.06.020 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 169-182DOI: (10.1016/j.molcel.2012.06.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 MSCs of Early Passage, of Hypoxic Culture, or with p21 Knockdown Show Increase in Proliferation and Differentiation Potential, and Decrease in Expression of Developmental Markers and Tissue-Specific Genes Early-passage (E), hypoxic culture (H), and p21 shRNA-overexpressed (p21KD) MSCs were compared with late passage (L), normoxic culture (N), and control scrambled shRNA-overexpressed (Scr) MSCs, respectively. (A) The cell growth rate assayed by MTT over a 7 day period. (B) The population doubling time calculated from total cell numbers at days 0 and 7. (C) The BrdU incorporation rate. (D) Quantification of differentiation into osteogenic, adipogenic, and chondrogenic lineages were assessed using alizarin red S, oil red O, and Alcian blue staining, respectively. (E) Quantitative RT-PCR for the mRNA levels of genes associated with (E) the developmental markers, (F) germline markers, and (G) tissue-specific genes. (Values are mean ± SD; ∗p < 0.05 and ∗∗p < 0.01 indicate significant variance [independent t test] between E and L, H and N, and p21KD and Scr.) Molecular Cell 2012 47, 169-182DOI: (10.1016/j.molcel.2012.06.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 MSCs of Early Passage, of Hypoxic Culture, or with p21 Knockdown Show Increase in the Expression of Oct4 and Nanog Early-passage (E), hypoxic culture (H), and p21 shRNA-overexpressed (p21KD) MSCs were compared with late passage (L), normoxic culture (N), and control scrambled shRNA-overexpressed (Scr) MSCs, respectively. E/H/p21KD MSCs were developed by overexpressing p21 shRNAs in early MSCs followed by culturing the cells under hypoxic conditions. (A and B) (A) Quantitative RT-PCR and (B) western blot analysis for the expression of Oct4A and Nanog. (C) Immunofluorescence for Oct4A and Nanog. Arrows indicate positive cells. The average percentages of positive cells are shown. (D) (Left) Bisulfite sequencing analysis of Oct4 and Nanog (N1, N2, N3) regulatory regions in indicated cell type. Each CpG is represented by a circle in the 5′–3′ orientation; each row represents the methylation state of each CpG in one bacterial clone of PCR product. ○, unmethylated CpG; ●, methylated CpG. (Right) Percentage of CpG demethylation for each genomic region in indicated cell type. (E–G) MSCs were induced to undergo (E) osteogenic, (F) adipogenic, and (G) chondrogenic differentiation for indicated periods, and quantitative RT-PCR was performed. (Values are mean ± SD; ∗p < 0.05 and ∗∗p < 0.01 indicate significant variance [independent t test] between E and L, H and N, and p21KD and Scr.) Molecular Cell 2012 47, 169-182DOI: (10.1016/j.molcel.2012.06.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Knockdown of Oct4 or Nanog in Early-Passage, Hypoxic Culture, or p21 shRNA-Overexpressed MSCs Show Inhibition of Proliferation and Differentiation Potential and Increase in Spontaneous Differentiation Early-passage (E), hypoxic culture (H), and p21 shRNA-overexpressed (p21KD) MSCs were lentivirally transduced with scrambled (Scr) or shRNAs against Oct4 (OKD) or Nanog (NKD). (A) Quantitative RT-PCR (left and middle) and western blot analysis (right) for Oct4A and Nanog. (B) The cell growth rate over a 7 day period. (C) The population doubling time. (D) The BrdU incorporation rate. (E) Quantification of differentiation into osteogenic, adipogenic, and chondrogenic lineages was assessed using alizarin red S, oil red O, and Alcian blue staining, respectively. (F–H) Quantitative RT-PCR for the mRNA levels of genes associated with (F) the developmental markers, (G) germline markers, and (H) tissue-specific genes. (Values are mean ± SD; ∗p < 0.05 and ∗∗p < 0.01 indicate significant variance [independent t test] compared to Scr in the group of E, H, and p21KD). Molecular Cell 2012 47, 169-182DOI: (10.1016/j.molcel.2012.06.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Overexpression of Oct4 and Nanog in Late Passage MSCs Increases Proliferation and Differentiation Potential and Suppresses Spontaneous Differentiation Late passage (L), normoxic culture (N), and control scrambled shRNA-overexpressed (Scr) MSCs were retrovirally transduced with control plasmids (CTR), Oct4 (Oct4), Nanog (Nanog), or both Oct and Nanog (Oct4/Nanog). (A and B) (A) Quantitative RT-PCR and (B) western blot analysis for Oct4A and Nanog. (C) The cell growth rate over a 7 day period. (D) The BrdU incorporation rate. (E) Quantification of differentiation into osteogenic, adipogenic, and chondrogenic lineages was assessed using alizarin red S, oil red O, and Alcian blue staining, respectively. (F–H) Quantitative RT-PCR for the mRNA levels of genes associated with (F) the developmental markers, (G) germline markers, and (H) tissue-specific genes. (Values are mean ± SD; ∗p < 0.05 and ∗∗p < 0.01 indicate significant variance [independent t test] compared to CTR in the group of L, N, and Scr). Molecular Cell 2012 47, 169-182DOI: (10.1016/j.molcel.2012.06.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Oct4 and Nanog Increase Dnmt1 Expression (A and B) The expressions of Dnmts in early-passage (E), hypoxic culture (H), and p21 shRNA-overexpressed (p21KD) MSCs were compared with those in late passage (L), normoxic culture (N), and control scrambled shRNA-overexpressed (Scr) MSCs, respectively. E/H/p21KD MSCs were developed by overexpressing p21 shRNAs in early MSCs followed by culturing the cells under hypoxic conditions. (A and B) (A) Quantitative RT-PCR and (B) western blot analysis. (C) E, H, and p21KD MSCs were lentivirally transduced with scrambled (Scr) or shRNAs against Oct4 (OKD) or Nanog (NKD). Quantitative RT-PCR (left panel) and western blot analysis (right panel) for the expression of Dnmt1. Values are mean ± SD; ∗p<0.05 and ∗∗p<0.01 indicate significant variance (independent t test) compared between E versus L, H versus N, p21KD versus Scr, and to scr or CTR in each group. (D) Late passage MSCs were retrovirally transduced with control plasmids (CTR), Oct4, Nanog, or both Oct4 and Nanog (Oct4/Nanog), quantitative RT-PCR (left panel) and western blot analysis (right panel) for the expression of Dnmt1. Molecular Cell 2012 47, 169-182DOI: (10.1016/j.molcel.2012.06.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 DNA Methylation Mediated by Dnmt1 Is Required for the Maintenance of MSC Properties (A, B, C, G, and H) Early-passage (E), hypoxic culture (H), and p21 shRNA-overexpressed (p21KD) MSCs were treated with or without (CTR) 1 μM of 5 aza-cytidine (5aza) for 4 days and used for the following analyses. (A) The cell growth rate over a 7 day period. (B) Quantifications of differentiation into osteogenic, adipogenic, and chondrogenic lineages were assessed using alizarin red S, oil red O, and Alcian blue staining, respectively. (C) Quantitative RT-PCR for the mRNA levels of p16 and p21. (G) Quantitative RT-PCR for the mRNA levels of development markers and tissue-specific genes. (D–F) Quantitative RT-PCR and western blot analysis for the expression of p16 and p21. (H) Quantitative RT-PCR and western blot analysis for the expression of Oct4 and Nanog. (D) The expressions of p16 and p21 in E, H, and p21KD MSCs were compared with those in late passage (L), normoxic culture (N), and control scrambled shRNA-overexpressed (Scr) MSCs, respectively. (E) Early-passage MSCs were lentivirally transduced with scrambled (Scr) or shRNAs against Oct4 (OKD) or Nanog (NKD). (F) Late passage MSCs were retrovirally transduced with control plasmids (CTR), Oct4, Nanog, or both Oct4 and Nanog (Oct4/Nanog). Values are mean ± SD; ∗p<0.05 and ∗∗p<0.01 indicate significant variance (independent t test) compared between E versus L, H versus N, p21KD versus Scr, and to Scr or CTR in each group. Molecular Cell 2012 47, 169-182DOI: (10.1016/j.molcel.2012.06.020) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Oct4 or Nanog Directly Regulates Dnmt1 Expression by Binding to Its Promoter (A–C) (A) The schematic representation of the pGL2-Dnmt1 reporter construct and the genomic organization of the region flanking the promoter region of human Dnmt1. Transcription start site, TSS. Reporter assays showing that Oct4 and Nanog enhanced the Dnmt1 promoter activity (n = 3) in immortalized MSCs (B) or 293T cells (C). Renilla was used as a control of transfection efficiency. (D) ChIP analysis of early-passage MSCs lentivirally transduced with scrambled shRNAs (Scr) or shRNAs against Oct4 (OKD) or Nanog (NKD). The chromatin was incubated with anti-Oct4A or anti-Nanog antibody or isotype IgG antibody or without antibodies. Fragments containing the O1 (120 bp), O2 (122 bp), and N (133 bp) binding sites in the Dnmt1 promoter were amplified by PCR (left) and were also quantified with quantitative RT-PCR (right). Input, 2% of total lysate. Results are shown as the mean ± SD values. (E) Mutational analysis of O1, O2, and N binding sites in the Dnmt1 promoter in immortalized MSCs. Reporter constructs containing wild-type Dnmt1; each of O1, O2, or N mutations; or double mutations (O1 and N) were generated and used to analyze the importance of these sites in mediating enhancement by overexpression of Oct4, Nanog, or both Oct4 and Nanog (Oct4 + Nanog) (n = 3). (Each ratio was normalized to the control (pGL2 basic vector), and statistical significance was determined by Student’s t test (∗p < 0.05 and ∗∗p < 0.01 versus control). Molecular Cell 2012 47, 169-182DOI: (10.1016/j.molcel.2012.06.020) Copyright © 2012 Elsevier Inc. Terms and Conditions