Carrillo, Federica (2021) Relevance of NCOA4 functions in tissue regeneration. [Tesi di dottorato]

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Iron is an essential microelement for DNA replication since it acts as a cofactor for several enzymes involved in DNA metabolism such as DNA polymerases, DNA primases and Ribonucleotide Reductase. Indeed, cells tightly control intracellular iron levels and upon low iron condition they arrest cell cycle avoiding S phase entry and DNA replication. The Nuclear Receptor CoActivator 4 (NCOA4) has been described as a novel regulator of iron homeostasis that upon low iron conditions controls intracellular iron levels by promoting lysosomal ferritin degradation (Ferritinophagy). Moreover, NCOA4 also acts as a negative regulator of DNA replication by inhibiting DNA replication origin activation via its interaction with MCM7 protein, a component of the MCM2-7 complex which represents the processive helicase of the replication fork. We demonstrated that NCOA4 couples the control of DNA replication activation to intracellular iron levels. Indeed, NCOA4 acts as iron sensing protein which upon low iron conditions increases its binding onto chromatin to block activation of DNA replication origins. Loss of NCOA4 in cells promotes unscheduled entry in S phase, replication stress and DNA damage with decreased cell viability. In this thesis, we demonstrated that the function of NCOA4 in coupling iron levels to DNA replication is relevant for tissue regeneration. Mice with genetic inactivation of NCOA4 (NCOA4 KO) displayed impaired intestinal regeneration after epithelial injury induced by Sodium Dextran Sulfate compared to WT animals, due to accumulation of DNA damage, activation of apoptosis and reduction of intestinal stem cell pool. Furthermore, we demonstrated that NCOA4 activity also affected the proliferation of bone marrow derived cells. BM-derived cells from NCOA4 KO mice exhibited a significant self-renewal reduction after 5-FU treatment compared to WT ones. Since NCOA4 was involved both in controlling iron availability and DNA replication activation we investigated which of NCOA4 functions mainly affected proliferation. We treated mice with iron dextran injections to supply iron availability to sustain tissue regeneration during DSS treatment. We confirmed that, despite iron supply, NCOA4 KO mice showed reduced intestinal regenerative response and presence of DNA damage after DSS treatment. Additionally, we used HeLa cells stably silenced for NCOA4 to evaluate Fe-S clusters enzymes expression in silenced cells at the steady state and upon iron chelation with Deferoxamine. In this condition, the expression of Fe-S clusters enzymes was not affected by the absence of NCOA4. All together our findings indicated a crucial role for NCOA4 in tissue regeneration and cellular proliferation. Particularly, it is the NCOA4 control of DNA replication origins activation, more than the ferritin degradation, to be essential for an appropriate tissue regeneration.

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