Lig4 Is Essential for Maintaining HSC Homeostasis

Blood cells of all lineages are generated from small pools of long-term hematopoietic stem cells (LT-HSCs) that continually replenish throughout life. LT-HSCs regulate the balanced turnover of all mature blood lineages by switching between self-renewal, differentiation and quiescence, thereby maintaining hematopoietic homeostasis in steady state and in response to injury. In any given cell, some DNA damage may remain despite the action of DNA repair processes, including in LT-HSCs. Over time, HSCs lose their long-term capability to self-renew due to misrepair of DNA breaks and increased accumulation of DNA damage, resulting in loss of regenerative plasticity and immune fitness. The accrual of DNA damage is the principal factor that contributes to functional decline in HSC renewal and in the immune system during ageing. In recent studies, the capacity of aging HSCs to self-renew is shown to be dependent on DNA repair pathways, with non-homologous end-joining (NHEJ) as the principle pathway implicated in DNA repair in quiescent HSCs from ex-vivo cell-based assays. Although NHEJ in particular has been implicated in this process in LT-HSCs, there has so far been very little evidence of this activity in vivo.

DNA Ligase IV (Lig4), which catalyzes the end-ligation of broken DNA ends mediated by NHEJ, has no known functions outside of NHEJ. Because a deficiency in Lig4 in mice is embryonic lethal, here we assessed the role of Lig4 in HSC homeostasis by assaying HSC functions in a knockin mouse model of a hypomorphic homozygous R278H mutation in Lig4 that had been identified in the first DNA Lig4 Syndrome patient. The R278H mutation significantly impairs the end-ligation function of the Lig4 protein, and mice homozygous for the R278H mutation (Lig4^R/R) showed diminished DSB repair capacity and age-dependent lymphopenia that implicated potential HSC defects.

Consistent with a defect in NHEJ, we show the Lig4 R278H mutation severely limited HSC self-renewal. Lig4^R/R HSC reconstitutions were skewed towards the myeloid lineage and resulted in severely reduced chimerism, confirming the capacity of HSCs to self-renew requires functional DNA repair. Next, we examined if there is increased DNA damage with/without ionizing irradiation (IR). Lig4^R/R LT-HSCs showed an increase in reactive oxygen species (ROS), abnormal cycling and increased apoptosis from accumulated DNA damage in steady state and slow DNA double strand breaks (DSBs) repair kinetics in response to low dose IR because of improper Lig4 function. This led us to check the LT-HSC pool more carefully. It has been shown that the HSC pool is intact and phenotypically increased with age. Strikingly, we found that the HSCs in both young and old Lig4^R/R mice are markedly reduced to 20% of wild-type levels. The severe LT-HSC reduction and lethality of disease in Lig4^R/Rmice was completely rescued by transplantation with wild type bone marrow. These evidences support the notion of a critical role for Lig4 in maintenance of the LT-HSC pool.

In a recent study, it was reported that the steady state pool of murine adult LT-HSCs can be further distinguished into quiescent (~20%) and variably cycling (~80%) populations. Since LT-HSCs in young Lig4^R/R mice are maintained in steady state at 20% of WT HSCs, we hypothesized that the reduced pool of LT-HSCs in the Lig4^R/R mice is caused by the loss of cycling LT-HSCs that continually replenish blood lineages during aging. To compare these populations, microarray analysis was done on the pool of WT and Lig4^R/R LT-HSCs, versus sorted populations of quiescent and variably cycling LT-HSCs. Microarray analysis clearly showed that the Lig4^R/RLT-HSCs correlated with the quiescent LT-HSCs, indicating NHEJ regulates the homeostasis of the faster cycling LT-HSC pool.

Our study suggest that the slowest cycling LT-HSCs serve to replenish the overall LT-HSC pool and HSC homeostasis is maintained by capacity of faster cycling LT-HSC pool to revert to quiescence in response to stress/injury. Additionally, defective NHEJ depletes the faster cycling LT-HSC pool and underlies early HSC exhaustion in Lig4^R/R mice. Our findings demonstrate for the first time a physiological role for Lig4 in the maintenance of HSC homeostasis.