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

Felgentreff, Kerstin; Du, Likun; Weinacht, Katja G.; Dobbs, Kerry; Bartish, Margarita; Giliani, Silvia; Schlaeger, Thorsten M.; DeVine, Alexander L.; Schambach, Axel; Woodbine, Lisa; Davies, Graham; Baxi, Sachin N.; Burg, Mirjam van der; Bleesing, Jack J.; Gennery, Andrew R.; Manis, John P.; Pan‐Hammarström, Qiang; Notarangelo, Luigi D.

doi:10.1073/pnas.1323649111

Cited by 37 publications

(28 citation statements)

References 42 publications

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“…Upon irradiation of peripheral blood mononuclear cells (PBMC) with 10Gy, increased DNA damage and delayed kinetics of DNA repair, as shown by elevated levels of phosphorylated H2AX (γH2X) (Fig. S2), were consistent with what we had previously demonstrated for various LIG4-deficient cell lines [16], and were therefore suggestive of LIG4 syndrome.…”

Section: Resultssupporting

confidence: 87%

“…The two LIG4 mutations in P2 have been reported previously [16]. For sequencing of the other siblings, the regions around the mutations were amplified using Choice Taq ™ DNA polymerase (Denville Scientific Inc., South Plainfield, NJ, USA); primer sequences are reported in the Supplementary Material.…”

Section: Methodsmentioning

confidence: 99%

“…Patients with LIG4 deficiency manifest short stature, microcephaly, variable degrees of CID, developmental delay, pancytopenia, and severe cellular radiosensitivity [10–15]. LIG4 deficiency is also associated with chromosomal instability [16], and an increased risk of malignancies [11, 13, 17–19]. The fact that some patients have a more severe immunodeficiency, consistent with SCID or Omenn syndrome (OS) [17, 18, 20, 21], and present severe neurological abnormalities [22], while others have less pronounced immune defects and develop normally[19, 23], is illustrative of the variable clinical expressivity of LIG4 syndrome.…”

Section: Introductionmentioning

confidence: 99%

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Ligase-4 Deficiency Causes Distinctive Immune Abnormalities in Asymptomatic Individuals

et al. 2016

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Purpose DNA Ligase 4 (LIG4) is a key factor in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway needed for V(D)J recombination and the generation of the T receptor and immunoglobulin molecules. Defects in LIG4 result in a variable syndrome of growth retardation, pancytopenia, combined immunodeficiency, cellular radiosensitivity, and developmental delay. Methods We diagnosed a patient with LIG4 syndrome by radiosensitivity testing on peripheral blood cells, and established that two of her four healthy siblings carried the same compound heterozygous LIG4 mutations. An extensive analysis of the immune phenotype, cellular radiosensitivity, telomere length, and T and B cell antigen receptor repertoire was performed in all siblings. Results In the three genotypically affected individuals, variable severities of radiosensitivity, alterations of T and B cell counts with an increased percentage of memory cells, and hypogammaglobulinemia, were noticed. Analysis of T and B cell antigen receptor repertoires demonstrated increased usage of alternative microhomology-mediated end-joining (MHMEJ) repair, leading to diminished N nucleotide addition and shorter CDR3 length. However, overall repertoire diversity was preserved. Conclusions We demonstrate that LIG4 syndrome presents with high clinical variability even within the same family, and that distinctive immunologic abnormalities may be observed also in yet asymptomatic individuals.

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Section: Resultssupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Ligase-4 Deficiency Causes Distinctive Immune Abnormalities in Asymptomatic Individuals

et al. 2016

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“…This is also in agreement with a recent publication showing that the cells from a mouse model, expressing a kinase-dead DNA-PKcs protein, exhibited severe defects in direct end-ligation during repair of V(D)J coding and signal ends, similar to XRCC4-and Lig4-deficient cells (46). However, because DNA-PKcs expression is normal in P1 but dramatically diminished in P2, the more severe or additional alteration in the repair pattern during CSR/NHEJ or in the efficiency of pluripotency reprogramming (47) in P2 could also be due to a combination of lost protein expression and kinase activity. Our results also indicate that, although the c-NHEJ machinery is involved in both IgA and IgG switching, deficiency of a NHEJ factor does not always lead to the same alterations in the Sm-Sa and Sm-Sg recombination patterns (increased longer MH for Sm-Sa and increased sequential switching or small insertions for Sm-Sg), suggesting that different backup mechanisms or A-EJ pathways might be operating.…”

Section: Discussionmentioning

confidence: 98%

“…Cell lines derived from Lig4-deficient patients F07614 lig4 (homozygous A3V, T9I, and R278H), HYGM022 (compound heterozygous R814X and K449Q), 411BR (homozygous A3V, T9I, and R278H), and SCID072 (compound heterozygous Q558P and K424fs) (47). **p , 0.01, ***p , 0.001 versus control, x 2 test.…”

Section: Discussionmentioning

confidence: 99%

DNA-PKcs Is Involved in Ig Class Switch Recombination in Human B Cells

Björkman

Felgentreff

et al. 2015

The Journal of Immunology

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Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break repair pathways in mammalian cells and is required for both V(D)J recombination and class switch recombination (CSR), two Ig gene–diversification processes occurring during B cell development. DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is a component of the classical NHEJ machinery and has a critical function during V(D)J recombination. However, its role in CSR has been controversial. In this study, we examined the pattern of recombination junctions from in vivo–switched B cells from two DNA-PKcs–deficient patients. One of them harbored mutations that did not affect DNA-PKcs kinase activity but caused impaired Artemis activation; the second patient had mutations resulting in diminished DNA-PKcs protein expression and kinase activity. These results were compared with those from DNA-PKcs–deficient mouse B cells. A shift toward the microhomology-based alternative end-joining at the recombination junctions was observed in both human and mouse B cells, suggesting that the classical NHEJ pathway is impaired during CSR when DNA-PKcs is defective. Furthermore, cells from the second patient showed additional or more severe alterations in CSR and/or NHEJ, which may suggest that DNA-PKcs and/or its kinase activity have additional, Artemis-independent functions during these processes.

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Comparison of DNA stability and its related genes of neurons derived from induced pluripotent stem cells and primary retinal neurons

Qiu

et al. 2022

Cell Biology International

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Maintaining DNA stability in induced pluripotent stem cells (iPSCs) and iPSCs‐derived neurons is a challenge in their clinical application. In the present study, we compared DNA stability between primary retinal neurons and differentiated neurons. We found that the basal level of γ‐H2AX phosphorylation, a specific marker of DNA breaks, was notably higher (~26‐folds) in human iPSCs compared to iPSCs‐derived neurons. However, iPSCs‐derived neurons are more sensitive to UV treatment compared to primary rat retinal neurons (postnatal Day 1). UV treatment induced a significantly decreasing in the cell viability of iPSCs‐derived neurons by ~76.1%, whereas ~20.8% in primary retinal neurons. After analyzing the expression levels of genes involved in DNA stability, such as Brca1, Ligase IV, Ku80, and Mre11, we found that Ku80 and its heterodimeric partner, Ku70 were positive in iPSCs‐derived neurons. However, both Ku80 and Ku70 are not expressed in primary retinal neurons and cerebellar neurons. Similarly, both Ku80 and Ku70 are also expressed in 3D retinal organoids from human embryonic stem cells (ESCs), except for a few Map2‐negative cells and the hyaloid vessels of mice E12.5 retinas. Hence, Ku80, and Ku70 are specifically expressed in stem cell‐derived neurons. Moreover, using the Ku80 inhibitor Compound L, our data showed that Ku80 promotes the DNA stability and cell viability of iPSCs‐derived neurons. Thus, our results demonstrated that iPSCs‐, ESCs‐derived neurons have specific characteristics of DNA stability. This study provides new insights into the neural differentiation of stem cells but might also warrant the future clinical application of stem cells in neurodegenerative diseases.

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

Cited by 37 publications

References 42 publications

Ligase-4 Deficiency Causes Distinctive Immune Abnormalities in Asymptomatic Individuals

Ligase-4 Deficiency Causes Distinctive Immune Abnormalities in Asymptomatic Individuals

DNA-PKcs Is Involved in Ig Class Switch Recombination in Human B Cells

Comparison of DNA stability and its related genes of neurons derived from induced pluripotent stem cells and primary retinal neurons

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