The Essential Role of DNA Repair in Hematopoietic Stem Cell Homeostasis and Disease.

Abstract 2328

Increased DNA damage is the principal factor that contributes to the functional decline in hematopoietic stem cell (HSC) renewal and in the immune system during ageing. Non-homologous DNA end-joining (NHEJ) is a major pathway that repairs DNA double strand breaks (DSBs) in mammalian cells. NHEJ is critical for the repair of DSBs generated by V(D)J recombination (VDJ) and IgH class switch recombination (CSR), and general DSBs in all cell types, including those that suppress the capacity of HSCs to self-renew during ageing. In recent studies, NHEJ is implicated in the repair of DNA damage in quiescent long-lived HSCs in ex-vivo cell-based assays. However, whether NHEJ might play a physiological role in the repair of DNA damage required for maintaining HSC homeostasis remains unclear.

DNA Ligase IV (Lig4) which catalyzes the end-ligation of broken DNA ends mediated by NHEJ, is absolutely required for VDJ and CSR, and has no known functions outside of NHEJ. Because a deficiency in Lig4 in mice is embryonic lethal, and there are no known human Lig4-null alleles, here we assess whether Lig4 plays a physiological role in HSC homeostasis by assaying HSC functions in a knock-in mouse model of the human recessive hypomorphic R278H mutation in Lig4 which underlies the rare genetic disease, DNA Lig4 Syndrome. The R278H mutation was previously shown to significantly impair the end-ligation function of the protein in the human R278H patient cell line. We previously also demonstrated that mouse cells homozygous for the R278H mutation to show diminished DSB repair capacity. Also paralleling the disease, the Lig4 R278H knock-in mice predispose to T lymphoma and manifest incomplete defects in VDJ, CSR and age-dependent lymphopenia, the latter which implicated potential defects in HSC functions.

Our studies of the long-lived HSCs in the R278H knock-in mice reveal that whereas these cells are quiescent and are continually replenished in wild-type mice, we find that these HSCs in both young and old R278H mutant knock-in mice are markedly reduced to 20% of wild-type levels, and appear to undergo increased cycling and g-H2AX induced DNA damage, which may explain their age-dependent lymphopenia. Our findings implicate for the first time a potential physiological role for Lig4 in HSC homeostasis. We also find that introducing rearranged IgH and Igk variable gene exons can bypass the B lymphocyte developmental defects in Lig4 R278H mice, to generate increased numbers of total peripheral B lymphocytes that can undergo robust CSR at near normal levels to specific Ig isotypes, which however still contain high levels of IgH specific DNA breaks and translocations. These latter findings support potential impacts of the R278H mutation in the regulation of the DNA damage signaling cascade, which may also underlie the incomplete CSR defects associated with the R278H disease allele. Thus we suggest Lig4 to not only ligate DNA ends, but also to maintain checkpoint arrest during repair and target irreparably damaged cells to be eliminated, functions we propose are likely fundamental to its role in the maintenance of HSC homeostasis and in CSR.