Hematopoietic stem cells (HSCs) generate blood cells over a lifetime, remaining largely quiescent until replenishment is required. Mechanisms governing HSC function and quiescence are unknown however, precluding improvements in HSC-based therapies, functional ex vivo expansion, and experimental applications. Mitochondria and their diverse functions serve as a critical regulatory node in HSC physiology and function. While HSCs preferentially utilize glycolysis for ATP production, they contain high mitochondrial mass, indicative of an alternative purpose of HSC mitochondrial content that could resolve elusive mechanisms. Mitofusin 2 (Mfn2), a multifunctional, membrane-bound mitochondrial and endoplasmic reticulum (ER) protein, is an essential regulator of HSC biology, promoting HSC lymphoid potential by mediating intracellular calcium buffering through ER-mitochondrial tethering (Luchsinger et al., Nature 2016) and potentially preventing premature HSC aging. Despite these findings, Mfn2 may have additional impacts on HSC physiology, as Mfn2 has diverse roles in mediating mitochondrial dynamics and regulating intracellular processes.

Furthermore, Mfn2 still remains only partially characterized in HSCs, as its potential effects on quiescence, overall aging, and additional physiological properties remain unknown. The goal of this thesis then is to investigate the potential additional roles of Mfn2 in HSC biology and lifelong homoeostasis through its diverse regulatory functions. Through targeted deletion in the murine hematopoietic system, we first found Mfn2 was required for HSC quiescence but dispensable for self-renewal, an unusual phenotype unlinking dormancy and functional potential. We then showed Mfn2 represses type I interferon (IFN) signaling in HSCs, a novel regulatory role independent of the immune signaling adapter mitochondrial antiviral signaling protein (Mavs). We further found Mfn2 inhibits both HSC cycling and type I IFN signaling by negatively regulating signal transducer and activator of transcription 1 (Stat1), an IFN-response transcription factor. However, Stat1 and Mfn2 also exhibited alternative regulatory roles in HSC function and lineage potential, indicative of complex regulatory interplay between Mfn2, mitochondria, and IFN signaling.

Additionally, Mfn2 was shown to attenuate age-related changes in HSCs, connecting mitochondrial function and lifelong HSC homeostasis. Overall, these findings advance the fundamental role the mitochondrion has in HSC physiology and function, further resolving Mfn2 as an indispensable regulator of HSC biology.