Research hypothesis:

Quiescent hematopoietic stem c ells (HSCs) are protected from DNA damage due to low metabolic activity. However, if DNA damage happens, the dormancy of HSCs leads to preferential use of NHEJ, the error-prone repair mechanism. Recent study by Dr. Weissman group demonstrated that strand breaks accrue in quiescent HSCs and are repaired after induction of cell cycling. Independently, we found that deficiency of heme oxygenase-1 (HO-1) in HSCs leads to increase in DNA breaks, and attenuates their repair despite induction of cell cycling. Heme oxygenase-1 is a cytoplasmic enzyme which acts as antioxidant factor. Our results indicate, however, that in HSCs the HO-1 is located preferentially in the nucleus, where it forms foci and can supposedly interact with proteins involved in DNA repair. We hypothesize that HO-1 plays a unique role in HSCs, preserving their dormancy, facilitating DNA repair, and supporting genome fidelity. We want elucidate the role of HO-1 in NHEJ and HR, and clarify whether its action is universal or cell type specific – distinct in HSCs and in downstream hematopoietic progenitors.

Research project methodology: Experiments will be performed in highly purified HSCs (Lin-/cKit+/Sca1+/CD150+/CD48-
/CD34-), MPPs (multipotent progenitor cells, Lin-/cKit+/Sca1+/CD150-/CD48+/CD34+), and GMPs (granulocyte-macrophage progenitors Lin-/cKit+/Sca1-/CD150-/CD48+/CD34+/CD32+), collected from the bone marrow of HO-1 knock-out (KO) and wild-type (WT) mice. We will analyze DNA breaks and oxidative damage, expression of DNA repair genes, localization and activity of proteins involved in DNA damage response. HSCs, MPPs, and GMPs will be assayed in vitro immediately after isolation or after activation in the cytokine rich medium with/without irradiation. We will check significance of putative interactions between HO-1 and DNA repair proteins, and role of two HO-1 products: CO and iron. To clarify significance of nuclear or cytoplasmic localization we will rescue the HO-1 KO phenotype by lentiviral transduction of HSCs with nuclear and cytoplasmic forms of HO-1. We will also transplant HO-1 KO HSCs (control or transduced with nuclear/cytoplasmic forms of HO-1) to the WT recipients to create hybrid mice, with HSCs supported by HO-1 competent niche. In such animals we will check the maintaining of repopulation potential of HSCs and assay the irradiation-induced DNA damage and repair parameters. Finally we will employ the comparative genome hybridization
and exome-seq assay to analyze effect of HO-1 deficiency in HO-1 deficient and HO-1 competent niche on long-term genome integrity after irradiation.

Impact of the research project on the development of science: The project will be the first investigation of nuclear HO-1 in hematopoiesis and first study on role of HO-1 in maintaining genome integrity. It will be also one of a very few research comparing the specific mechanisms of DNA repair in highly purified HSCs and downstream hematopoietic
progenitors, the fundamental biological phenomena. Deregulation of these processes may result in premature HSC depletion or preleukemic progression of HSC-derived hematopoietic progenitors. Understanding the role of HO-1 can also be clinically relevant, as efficacy of HO-1 pathway is variable in human population. Results can be interesting for hematologists, oncologists and researchers working on cell biology and oxidative stress.