Research project objectives/Research hypothesis:

Heme oxygenases (Hmox1 andHmox2) degrade the prooxidative heme to biliverdin, iron and carbon monoxide. Hmox1 is a stress-inducible enzyme that may translocate from the cytoplasm to the nucleus. Cytoplasmic Hmox2 is a constitutive form. The role of nuclear Hmox1 is still unknown. In cells, in which Hmox1 constitutively localizes to the nucleus, we found that Hmox1 deficiency leads to increased cell cycling, replication stress, and DNA double strand breaks. Unexpectedly, we also found a strong upregulation of genes responsible for resolving of guanine (G) quadruplexes (G4) accompanied by enhanced G4 immunostaining in Hmox1 deficient cells. Moreover, proximity ligation assays performed in wild type cells suggest a direct co-localization of Hmox1 protein with G4 structures. Physicochemical studies have demonstrated that G4-folds, formed by oligonucleotides in permissive buffers, are stabilized by heme. Such interaction has never been analyzed in biological systems. We hypothesize that heme can be an endogenous stabilizer of G4 and that Hmox1 facilitates the destabilization and resolving of G4 structures due to the removal of heme. We suppose that Hmox1 may constitute a part of G4 processing complex. The aim of our proposal is to verify this hypothesis.

Research project methodology:

Experiments will be performed using primary murine embryonic fibroblasts (MEF) isolated from the wild type (WT) and Hmox1-/- mice. In WT and Hmox1-/- fibroblasts we will knock-out Hmox2 gene using CRISPR/Cas9 strategy. Such cells will be used to discriminate the role of Hmox1 and Hmox2 in the regulation of heme metabolism and stabilization of G-quadruplexes. Additionally, to the cells deficient in both endogenous heme oxygenases, we will lentivirally introduce the modified Hmox1 transgenes, coding for native (enzymatically active) or H25A mutated (enzymatically inactive) forms of Hmox1, either with NLS (nuclear localization signal) or NES (nuclear export signal). These cells will allow to discriminate the heme-degrading and non-enzymatic functions of Hmox1 and determine the meaning of its nuclear localization.
We will answer four questions: i) what is a role of Hmox1 and Hmox2 in controlling the cytoplasmic and nuclear free heme pool; ii) do heme oxygenases prevent the accumulation of G-quadruplexes; iii) is Hmox1 required for proper DNA replication and maintaining genome stability, the processes that are impaired by unresolved G-quadruplexes; iv) how to obtain subcellular resolution and direct detection of G4 in cells.

Expected impact of the research project on the development of science:

Answers to those questions can point to the unknown mechanism of heme toxicity, especially in the induction of oxidative damage and mutagenesis. This may be important in cancer studies, as G4 motifs were found at multiple rearrangement sites e.g. in lymphoma. Confirmation of the hypothesized role Hmox1 would provide a common mechanism to explain a variety of Hmox1 deficiency effects described earlier. We plan to optimize techniques for analysis of free heme at subcellular level and for detection of G4 in living cells. These methods would be useful in biochemistry and cell biology.