DNA damage tolerance in hematopoietic stem and progenitor cells in mice

Abstract

DNA damage tolerance (DDT) enables bypassing of DNA lesions during replication, thereby preventing fork stalling, replication stress, and secondary DNA damage related to fork stalling. Three modes of DDT have been documented: translesion synthesis (TLS), template switching (TS), and repriming. TLS and TS depend on sitespecific PCNA K164 monoubiquitination and polyubiquitination, respectively. To investigate the role of DDT in maintaining hematopoietic stem cells (HSCs) and progenitors, we used Pcna K164R/K164R mice as a unique DDT-defective mouse model. Analysis of the composition of HSCs and HSC-derived multipotent progenitors (MPPs) revealed a significantly reduced number of HSCs, likely owing to increased differentiation of HSCs toward myeloid/erythroidassociated MPP2s. This skewing came at the expense of the number of lymphoid-primed MPP4s, which appeared to be compensated for by increased MPP4 proliferation. Furthermore, defective DDT decreased the numbers of MPP-derived common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-macrophage progenitor (GMP) cells, accompanied by increased cell cycle arrest in CMPs. The HSC and MPP phenotypes are reminiscent of premature aging and stressed hematopoiesis, and indeed progressed with age and were exacerbated on cisplatin exposure. Bone marrow transplantations revealed a strong cell intrinsic defect of DDT-deficient HSCs in reconstituting lethally irradiated mice and a strong competitive disadvantage when cotransplanted with wild-type HSCs. These findings indicate a critical role of DDT in maintaining HSCs and progenitor cells, and in preventing premature aging.proliferating cell nuclear antigen K164 ubiquitination | DNA damage tolerance | hematopoietic stem and progenitor cells | translesion synthesis | template switching

Emerging insights into human health and NK cell biology from the study of NK cell deficiencies

“…The neutrophil defect in GINS1 patients was found to be primarily manifested in the periphery, as opposed to at the bone marrow precursor level, suggesting that the overall defect may be a result of dysregulated balance between apoptosis and proliferation in the periphery . Mouse models of the effect of reduced helicase or DNA damage pathway function, however, reveal skewing of multipotent progenitors at the earliest stages, suggesting there may be effects on lineage potential at the earliest stages of hematopoiesis . In addition, it is likely that these may become more damaging as the effects of aging and, potentially, inflammation change the function and kinetics of the stem cell population .…”

“…91 Mouse models of the effect of reduced helicase or DNA damage pathway function, however, reveal skewing of multipotent progenitors at the earliest stages, suggesting there may be effects on lineage potential at the earliest stages of hematopoiesis. 241 In addition, it is likely that these may become more damaging as the effects of aging and, potentially, inflammation change the function and kinetics of the stem cell population. 232,233 It will be of value to determine the changes in gene expression that accompany NK and ILC precursors in patients with helicase mutations throughout development in comparison with healthy donors.…”

A PCNA-K164R mutation impinges on origin activation and mitotic DNA synthesis

“…This discrepancy could be due to differences in mouse and human cells, as viable PCNA K164R mice were generated (70), whereas this mutation has never been identified in human tissues. However, these mice were sterile and exhibited bone marrow (BM) failure, as well as defects in somatic hypermutation and class switch recombination (70,94,95), implying that K164 ubiquitination is not completely dispensable in mice. Interestingly, the infertility and BM phenotype in the PCNA K164R mice are shared phenotypes with FA mouse models (96,97).…”

USP15 deubiquitinase safeguards hematopoiesis and genome integrity in hematopoietic stem cells and leukemia cells

“…These data supported the hypothesis that USP15 depletion would render normal hematopoietic progenitors more sensitive to genotoxic stress in vivo. To test if this is the case, we injected mice with the chemotherapeutic agent cisplatin (0,8 mg/kg, a relative low dose compared to the 6mg/kg maximal tolerable dose used in C57BL6/6J mice (Pilzecker et al, 2017)) i.v or with PBS, and analyzed the BM after 2 days. Upon cisplatin treatment, USP15 knockout BM cells produced significantly fewer CFUs compared to wt ( Fig.…”

“…Cells were permeabilised in PBS/BSA(1%)/Tween20 (0.025%)(PBT) for 15min at RT and harvested in PBT containing 10 µg/mL DAPI for chromatin labeling. Cell cycle analysis was performed as described (Pilzecker et al, 2017).…”