Differential role of nonhomologous end joining factors in the generation, DNA damage response, and myeloid differentiation of human induced pluripotent stem cells

Proc Natl Acad Sci U S A. 2014 Jun 17;111(24):8889-94. doi: 10.1073/pnas.1323649111. Epub 2014 Jun 2.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalysis
  • Cell Cycle
  • Cell Differentiation
  • Cell Line
  • Cell Lineage
  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair*
  • DNA Ligase ATP
  • DNA Ligases / metabolism
  • DNA-Activated Protein Kinase / genetics
  • DNA-Binding Proteins
  • Endonucleases
  • Fibroblasts / metabolism
  • Fibroblasts / pathology
  • Hematopoietic Stem Cells / cytology
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Mutation
  • Nuclear Proteins / metabolism
  • Phenotype

Substances

  • DNA-Binding Proteins
  • LIG4 protein, human
  • Nuclear Proteins
  • DNA-Activated Protein Kinase
  • DCLRE1C protein, human
  • Endonucleases
  • DNA Ligases
  • DNA Ligase ATP