Volume 3, Issue 3, Pages 651-660 (March 2013) Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State  Federico González, Daniela Georgieva, Fabio Vanoli, Zhong-Dong Shi, Matthias Stadtfeld, Thomas Ludwig, Maria Jasin, Danwei Huangfu  Cell Reports  Volume 3, Issue 3, Pages 651-660 (March 2013) DOI: 10.1016/j.celrep.2013.02.005 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 3, 651-660DOI: (10.1016/j.celrep.2013.02.005) Copyright © 2013 The Authors Terms and Conditions

Figure 1 Reprogramming Induces DSBs and Apoptosis (A) Representative fluorescence-activated cell sorting (FACS) plots of 4F- and 3F-infected wild-type MEFs stained for γH2AX, SSEA1, and Annexin V after cells were cultured with or without doxycycline (DOX) for 5 days. Numbers indicate percentages of positive cells. PI, propidium iodide; DAPI, 4′6-diamidino-2-phenylindole; L, alive; EA, early apoptotic; LA, late apoptotic; N, necrotic. (B) Quantification of the percentage of γH2AX+ cells in wild-type MEFs infected with reprogramming genes in combination or individually. OS, Oct4-Sox2; O, Oct4; S, Sox2; K, Klf4; M, c-Myc. (C and D) Quantification of the percentage of SSEA1+ (C) and Annexin V+ (D) cells in wild-type MEFs transduced with 4F and 3F. Apoptotic cells are the sum of EA and LA cells. (E) Time-lapse flow cytometric quantification of γH2AX+ cells present in reprogrammable MEFs with or without DOX treatment; cells were separated based on expression of SSEA1. In all column graphs of this study, error bars indicate SEM and p values by two-tailed Student’s t test. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001, respectively. Cell Reports 2013 3, 651-660DOI: (10.1016/j.celrep.2013.02.005) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Reprogramming Is Impaired in Brca1 and Brca2 Mutant MEFs (A) Schematics of all virus-mediated reprogramming experiments in this study. MEFs were infected with 4F or 3F 1 day after plating and replated in 12-well dishes the next day on irradiated MEFs at densities specified (indicated below the AP-staining pictures for all figures in this study). AP and Nanog staining was performed after 3 weeks (unless otherwise noted). (B–D) Representative AP staining (B) and quantification of AP+ (C) and Nanog+ (D) colonies generated with 4F reprogramming from Brca1Tr/Tr, Brca1S1598F/S1598F, and Brca2Δ27/Δ27 MEFs in comparison to wild-type (WT) MEFs from littermate controls. (E–G) Representative AP staining (E) and quantification of AP+ (F) and Nanog+ (G) colonies generated with 3F reprogramming. (H) Quantification of the percentage of γH2AX+ cells in 4F- and 3F-infected, Brca1Tr/Tr mutant, and control wild-type MEFs after 5 days of DOX treatment. See also Figure S1. Cell Reports 2013 3, 651-660DOI: (10.1016/j.celrep.2013.02.005) Copyright © 2013 The Authors Terms and Conditions

Figure 3 HR Genes Are Directly Required during Reprogramming (A–C) Representative AP staining (A) and quantification of AP+ (B) and Nanog+ (C) colonies generated with 4F reprogramming and a panel of shRNAs targeting Brca1 (shBrca1-a, shBrca1-b, and shBrca1-c), Brca2 (shBrca1-a, shBrca1-b, and shBrca1-c), and Rad51 (shRad51-a, shRad51-b, and shRad51-c) compared to the shRNA control vector (shCtrl). Lower case letters refer to individual shRNAs targeting each HR gene. shBrca1-c, shBrca2-b, and shRad51-b were used for further experiments. (D) The upper panel shows representative fluorescence images of Oct4-GFP+ colonies generated with 4F and shRNAs targeting HR genes. The lower panel shows representative AP staining images from reprogrammable (Rep.) MEFs infected with shRNAs against HR genes. (E) Quantification of Oct4-GFP+ colonies from experiments with 4F-infected Oct4-GFP MEFs and acute HR-gene knockdown. (F and G) Quantification of AP+ (F) and Nanog+ (G) colonies from experiments using reprogrammable MEFs and acute HR-gene knockdown. See also Figures S2 and S3. Cell Reports 2013 3, 651-660DOI: (10.1016/j.celrep.2013.02.005) Copyright © 2013 The Authors Terms and Conditions

Figure 4 Downregulating p53 Rescues the Reprogramming Phenotype of HR-Defective MEFs (A–C) Representative AP staining (A) and quantification of AP+ (B) and Nanog+ (C) colonies generated with 4F reprogramming from Brca2Δ27 homozygous mutant and wild-type MEFs infected with an shRNA targeting p53 (shp53) or vector control (shCtrl). (D–F) Representative AP staining (D) and quantification of AP+ (E) and Nanog+ (F) colonies generated with 4F reprogramming from p53−/− and wild-type MEFs under acute HR-gene knockdown. All staining were performed 16 days after infected cells were replated. (G and H) Quantification of the percentage of Phospho-Histone H3+ (PH3+) (G) and Cleaved Caspase-3+ (CSP3+) (H) cells 6 days postinfection of 4F and HR gene knockdown in p53−/− mutant and wild-type MEFs. (I) Our results support a critical role of the HR pathway for efficient reprogramming. We propose a model in which reprogramming increases the level of DNA damage, which is responsible for the genetic aberrations observed in iPSC lines (indicated by a lightly shaded box). A defective HR pathway may lead to increased genetic aberration (indicated by dark shaded boxes) or the elimination of abnormal cells through p53-mediated cell-cycle arrest or apoptosis. See also Figure S4. Cell Reports 2013 3, 651-660DOI: (10.1016/j.celrep.2013.02.005) Copyright © 2013 The Authors Terms and Conditions

Figure S1 Characterization of Brca1 and Brca2 Reprogrammed and Partially Reprogrammed Lines, Related to Figure 2 (A) Quantification of Nanog+ iPSC lines generated from wild-type, Brca1Tr/Tr and Brca2Δ27/Δ27 MEFs. 10 colonies were picked for each condition. (B and C) qRT-PCR analysis of wild-type, and Brca1Tr/Tr and Brca2Δ27/Δ27 lines using primers detecting endogenous expression of Nanog, Oct4 and Col6a2 (B), the Oct4-2A-Sox2 (OS) bicistronic transcript (C), upper panel, and the Klf4-2A-cMyc (KM) bicistronic transcript (C), lower panel. B4, C4, D3, F3, G1 and H5 are different reprogrammed and partially reprogrammed lines, and genotypes are indicated in brackets. Wild-type MEFs, ESCs, and MEFs collected 5 days postinfection with 4F (inf. MEFs) were included controls. (D) Representative AP staining of wild-type, Brca1Tr/Tr and Brca2Δ27/Δ27 mutant colonies, and immunohistochemical analysis of pluripotency marker expression: Nanog, Oct4, Sox2, SSEA1. Transgene expression could contribute to the Oct4 and Sox2 immunostaining in partially reprogrammed lines. All analyses were performed between passages 5 and 9. Scale bar is indicated below the picture panel. (E and F) Representative fluorescence images (E) and quantification (F) of Phospho-Histone H3+ cells (green) in Brca2Δ27/Δ27 mutant and control wild-type iPSC lines (two from each). Nuclei were stained with DAPI (blue). (G and H) Representative FACS plots (G) and quantification of apoptotic cells (H) in Brca2Δ27/Δ27 mutant and control wild-type iPSC lines (two from each) stained for SSEA1 and Annexin V. Numbers indicate percentages of positive cells found in the SSEA1+ population. Apoptotic cells are the sum of early apoptotic (EA) and late apoptotic (LA) cells. Cell Reports 2013 3, 651-660DOI: (10.1016/j.celrep.2013.02.005) Copyright © 2013 The Authors Terms and Conditions

Figure S2 Transduction Efficiency in Brca1, Brca2 Mutant, and HR-Gene Knockdown Cells, Related to Figure 3 (A and B) Representative fluorescence images and quantification of the percentage of GFP+ cells in Brca1S1598F/S1598F, Brca2Δ27/Δ27 and wild-type MEFs 3 days after infection with a pMXs-GFP retroviral vector (A) or an FUW-TetO-GFP lentiviral vector (B). (C) Representative fluorescence images and quantification of WT GFP infected MEFs, 3 days after transduction of pMXs-GFP and shRNAs targeting Brca1, Brca2 or Rad51 (shBrca1-c, shBrca2-b, shRad51-b). (D) Representative ArrayScan VTI fluorescence images and quantification of WT 4F-infected MEFs, 3 days posttransduction with 4F and the same shRNAs described above. Nuclei were stained with DAPI (blue) and antibodies recognizing Oct4 (Green) and Klf4 (Red) were used to identify cells expressing respectively the Oct4-2A-Sox2 (OS) or the Klf4-2A-cMyc (KM) bicistronic cassettes. Cell Reports 2013 3, 651-660DOI: (10.1016/j.celrep.2013.02.005) Copyright © 2013 The Authors Terms and Conditions

Figure S3 Reprogramming with Acute HR-Gene Knockdown, Related to Figure 3 (A–C) Representative AP staining (A), and quantification of AP+ (B) and Nanog+ (C) colonies generated with 3F-reprogramming and a panel of shRNAs targeting Brca1 (shBrca1-a, b, c), Brca2 (shBrca2-a, b, c) and Rad51 (shRad51-a, b, c) compared to the shRNA control vector (shCtrl). (D and E) qRT-PCR assessment of HR-gene knockdown efficiency during 4F- (D) or 3F-reprogramming (E) using shRNAs targeting Brca1, Brca2, and Rad51 compared to the shRNA control vector. (F) Quantification of AP+ colonies in 4F-reprogramming and acute HR-gene knockdown using Oct4-GFP MEFs. (G) qRT-PCR assessment of HR-gene knockdown efficiency during 4F-reprogramming of Oct4-GFP MEFs. (H) qRT-PCR assessment of HR-gene knockdown during reprogramming of reprogrammable MEFs. Cell Reports 2013 3, 651-660DOI: (10.1016/j.celrep.2013.02.005) Copyright © 2013 The Authors Terms and Conditions

Figure S4 Assessment of Gene Knockdown, Proliferation and Apoptosis in p53 Experiments, Related to Figure 4 (A) qRT-PCR assessment of p53 knockdown efficiency during 4F-reprogramming of Brca2Δ27/Δ27 mutant and wild-type control MEFs. (B) qRT-PCR assessment of HR-gene knockdown efficiency during 4F-reprogramming and acute HR-gene knockdown in p53−/− and wild-type control MEFs. (C and D) Representative ArrayScan VTI fluorescence images of p53−/− and wild-type MEFs 6 days postinfection with 4F and shRNAs targeting Brca1, Brca2 and Rad51. Nuclei were stained with DAPI (Blue) and an antibody recognizing the mitotic marker Phospho-Histone H3 (Green) (C) or the apoptotic marker cleaved Caspase-3 (Green) (D). The last two columns show zoomed-in images to illustrate identification of DAPI+ nuclei (orange circles) and PH3+ nuclei (green circles) (C), or CSP+ cytoplasms (green circles) (D) respectively using the Target Activation.V4 or Compartmental AnalysisV4 BioApplications. Cell Reports 2013 3, 651-660DOI: (10.1016/j.celrep.2013.02.005) Copyright © 2013 The Authors Terms and Conditions