title: A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2.
abstract: The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors.
authors.affiliation: Department of Molecular Biology, Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
authors.forename: Marti
authors.initials: M
authors.lastname: Borkent
authors.affiliation: Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Bennett
authors.affiliation: Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
authors.forename: Brad
authors.initials: B
authors.lastname: Lackford
authors.affiliation: Department of Molecular Biology, Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
authors.forename: Ori
authors.initials: O
authors.lastname: Bar-Nur
authors.affiliation: Department of Molecular Biology, Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Brumbaugh
authors.affiliation: Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Du
authors.affiliation: Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Fargo
authors.affiliation: Department of Molecular Biology, Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
authors.forename: Effie
authors.initials: E
authors.lastname: Apostolou
authors.affiliation: Department of Molecular Biology, Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
authors.forename: Sihem
authors.initials: S
authors.lastname: Cheloufi
authors.affiliation: Department of Molecular Biology, Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
authors.forename: Nimet
authors.initials: N
authors.lastname: Maherali
authors.affiliation: Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA; Department of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Elledge
authors.affiliation: Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. Electronic address: hug4@niehs.nih.gov.
authors.forename: Guang
authors.initials: G
authors.lastname: Hu
authors.affiliation: Department of Molecular Biology, Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address: khochedlinger@mgh.harvard.edu.
authors.forename: Konrad
authors.initials: K
authors.lastname: Hochedlinger
mesh_terms: D002454:Cell Differentiation / Q000235:genetics*; D065150:Cellular Reprogramming / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics*
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins