A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors

Abstract

DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final 'end-joining' step. Mutations in DNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJmediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affected per se, the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result in ex vivo expansion of stem cells with intact genomes.

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

“…This process facilitates chromosomal translocations with MH joins at the junctions (36). We observed increased genomic instability, including emergence of structural chromosomal abnormalities, in iPSC lines derived from LIG4-deficient fibroblasts, and similar results have been recently reported by others (19). In particular, several chromosomal abnormalities have been identified in LIG4-mutated iPSCs that were not present in parental fibroblasts.…”

“…Reprogramming-associated DNA DSBs trigger the DNA damage response (DDR) and upregulate the p53 pathway, thereby promoting cell death and senescence and limiting the efficiency of cell reprogramming (16,17). Consistent with this, impaired reprogramming efficiency has been reported for human Fanconi anemia (FA) cells (18), LIG4-and XLF-mutated human fibroblasts (19,20), and Ataxia Telangiectasia Mutated (ATM)-deficient mouse embryonic fibroblasts (MEFs) (21).…”

High-Risk Human Papillomavirus E6 Protein Promotes Reprogramming of Fanconi Anemia Patient Cells through Repression of p53 but Does Not Allow for Sustained Growth of Induced Pluripotent Stem Cells

“…In a recent study, NHEJ-deficient patient cells were reprogrammed, although at decreased efficiency, and iPSC lines were efficiently derived and maintained (52). In comparison with our observations, it appears that iPSC are much more sensitive to the loss of the FA pathway than NHEJ, suggesting that pluripotent cells depend heavily on FA-mediated repair as opposed to NHEJ.…”

Reprogramming and genome integrity: role of non-homologous end joining

“…The study by Tilgner et al 1 investigates the role of nonhomologous end joining (NHEJ) in reprogramming of human somatic cells to induced pluripotent stem cells and in regulation of their differentiation. NHEJ is the major double-strand break (DSB) repair pathway in mammalian cells.…”

“…Tilgner et al 1 have created three iPSC lines from LIG4 patient-derived fibroblasts using conventional reprogramming protocols based on OCT4, SOX2, KLF4 and c-MYC transduction. Although the authors were able to obtain iPSC lines from control and LIG4 fibroblasts, the reprogramming efficiency was much reduced when DNA ligase IV was mutated.…”