Lnk deficiency enhances translesion synthesis to alleviate replication stress and promote hematopoietic stem cell fitness
Brijendra Singh, Md Akram Hossain, Xiao Hua Liang, Jeremie Fages, Carlo Salas Salinas, Roger A. Greenberg, Wei Tong
Brijendra Singh, Md Akram Hossain, Xiao Hua Liang, Jeremie Fages, Carlo Salas Salinas, Roger A. Greenberg, Wei Tong
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Research Article Development Hematology

Lnk deficiency enhances translesion synthesis to alleviate replication stress and promote hematopoietic stem cell fitness

Abstract

The adaptor protein LNK/SH2B3 negatively regulates hematopoietic stem cell (HSC) homeostasis. Lnk-deficient mice show marked expansion of HSCs without premature exhaustion. Lnk deficiency largely restores HSC function in Fanconi anemia (FA) mouse models and primary FA patient cells, albeit protective mechanisms remain enigmatic. Here, we uncover a role for LNK in regulating translesion synthesis (TLS) during HSC replication. Lnk deficiency reduced replication stress–associated DNA damage, particularly in the FA background. Lnk deficiency suppressed single-strand DNA breaks, while enhancing replication fork restart in FA-deficient HSCs. Diminished replication-associated damage in Lnk-deficient HSCs occurred commensurate with reduced ATR/p53 checkpoint activation that is linked to HSC attrition. Notably, Lnk deficiency ameliorated HSC attrition in FA mice without exacerbating carcinogenesis during aging. Moreover, we demonstrated that enhanced HSC fitness from Lnk deficiency was associated with increased TLS activity via REV1 and, to a lesser extent, TLS polymerase eta (η). TLS polymerases are specialized to execute DNA replication in the presence of lesions or natural replication fork barriers that stall replicative polymerases. Our findings implicate elevated use of these specialized DNA polymerases as critical to the enhanced HSC function imparted by Lnk deficiency, which has important ramifications for stem cell therapy and regenerative medicine in general.

Authors

Brijendra Singh, Md Akram Hossain, Xiao Hua Liang, Jeremie Fages, Carlo Salas Salinas, Roger A. Greenberg, Wei Tong

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Figure 1

Lnk deficiency reduces DNA damage in Fancd2–/– HSPCs at steady state and upon replication stress.

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Lnk deficiency reduces DNA damage in Fancd2–/– HSPCs at steady state an...
(A and B) Comparison of levels of DNA damage in HSCs and HSPCs at steady state. (A) Quantification of γH2AX (fold change in MFI as normalized to WT) within LSK and SLAM LSK populations analyzed by flow cytometry. (B) Experimental design, representative flow cytometry plots, and quantification of chromatin-bound γH2AX within the HSC population. EdU was injected 2 hours before BM harvest, and percentage γH2AX+ cells and fold change in MFI of γH2AX within the S phase of HSCs as determined by EdU+ cells are shown. (C) Comparison of levels of DNA damage in HSPCs in ex vivo culture. Experimental design for ex vivo LSK culture, representative immunofluorescence images of γH2AX foci, and quantification of fluorescence intensity in day 3 HSPCs are shown. IR, irradiated. Original magnification, ×63. (D) LSK cells were cultured, and γH2AX levels were examined by flow cytometry at the indicated days. Left: Quantification of fold change in MFI of γH2AX in ex vivo–cultured HSPCs. Right: Representative histogram plots for γH2AX in day 5 HSPCs. NIR, non-irradiated. WT cells 2 hours after 2 Gy x-ray irradiation were used as a positive control. (E–G) Comparison of levels of DNA damage in HSCs and HSPCs upon replication stress in vivo. (E) Experimental design for γH2AX examination within donor cells of LSK during transplantation-induced replication stress. (F) Quantification of fold changes in MFI of γH2AX within donor LSK cells, MPPs, and HSCs (SLAM LSK), days 6 and 10 after BMT (short-term hematopoietic stress), and a representative histogram plot for γH2AX within donor LSK cells, day 6 after BMT. (G) Fold changes in MFI of γH2AX within donor LSK cells 16 weeks after BMT (long-term hematopoietic stress). (H) Immunoblots showing the γH2AX level in freshly sorted LK cells treated with HU or CPT for 2 hours. In all relevant panels, each symbol represents an individual mouse; bars indicate mean values; error bars indicate SEM. P values were calculated using 1-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.