title: Differential role of nonhomologous end joining factors in the generation, DNA damage response, and myeloid differentiation of human induced pluripotent stem cells.
abstract: Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation, microcephaly, and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4), Artemis, and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders, we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity, but not Artemis deficiency, were associated with markedly reduced reprogramming efficiency, which could be partially rescued by genetic complementation. Moreover, we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest, particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-, but not Artemis- or DNA-PK-mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming, with a major role for LIG4 and DNA-PKcs and a minor, if any, for Artemis.
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115;
authors.forename: Kerstin
authors.initials: K
authors.lastname: Felgentreff
authors.affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, SE 14186 Stockholm, Sweden;
authors.forename: Likun
authors.initials: L
authors.lastname: Du
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115;Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115;
authors.forename: Katja G
authors.initials: KG
authors.lastname: Weinacht
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115;
authors.forename: Kerry
authors.initials: K
authors.lastname: Dobbs
authors.affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, SE 14186 Stockholm, Sweden;
authors.forename: Margarita
authors.initials: M
authors.lastname: Bartish
authors.affiliation: A. Nocivelli Institute for Molecular Medicine, Pediatric Clinic, University of Brescia, Spedali Civili, 25123 Brescia, Italy;
authors.forename: Silvia
authors.initials: S
authors.lastname: Giliani
authors.affiliation: Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115;
authors.forename: Thorsten
authors.initials: T
authors.lastname: Schlaeger
authors.affiliation: Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115;
authors.forename: Alexander
authors.initials: A
authors.lastname: DeVine
authors.affiliation: Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115;Institute of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany;
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, United Kingdom;
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Woodbine
authors.affiliation: Centre for Immunodeficiency, Great Ormond Street Hospital and Institute of Child Health, London WC1N 3JH, United Kingdom;
authors.forename: Graham
authors.initials: G
authors.lastname: Davies
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115;
authors.forename: Sachin N
authors.initials: SN
authors.lastname: Baxi
authors.affiliation: Department of Immunology, Erasmus Medical Center, 3015 GE, Rotterdam, The Netherlands;
authors.forename: Mirjam
authors.initials: M
authors.lastname: van der Burg
authors.affiliation: Division of Bone Marrow Transplant and Immunodeficiency, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229;
authors.forename: Jack
authors.initials: J
authors.lastname: Bleesing
authors.affiliation: Institute of Cellular Medicine, Pediatric Immunology, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE1 4LP, United Kingdom;
authors.forename: Andrew
authors.initials: A
authors.lastname: Gennery
authors.affiliation: Department of Transfusion Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115; and.
authors.forename: John
authors.initials: J
authors.lastname: Manis
authors.affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, SE 14186 Stockholm, Sweden;
authors.forename: Qiang
authors.initials: Q
authors.lastname: Pan-Hammarström
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115;Harvard Stem Cell Institute, Cambridge, MA 02138 Luigi.Notarangelo@childrens.harvard.edu.
authors.forename: Luigi D
authors.initials: LD
authors.lastname: Notarangelo
mesh_terms: D002384:Catalysis; D002453:Cell Cycle; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000235:genetics; D004268:DNA-Binding Proteins; D004720:Endonucleases; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010641:Phenotype
chemical_list: D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004720:Endonucleases; D011088:DNA Ligases; D000072481:DNA Ligase ATP