Macrophages (MPs) are immune cells which are crucial for tissue repair. In skeletal muscle regeneration, pro-inflammatory cells first infiltrate to promote myogenic cell proliferation, then they switch into an anti-inflammatory phenotype to sustain myogenic cells differentiation and myofiber formation. This phenotypical switch is induced by dead cell phagocytosis. We previously demonstrated that the transcription factor Nfix, a member of the nuclear factor I (Nfi) family, plays a pivotal role during muscle development, regeneration and in the progression of muscular dystrophies. Here, we show that Nfix is mainly expressed by anti-inflammatory macrophages. Upon acute injury, mice deleted for Nfix in myeloid line displayed a significant defect in the process of muscle regeneration. Indeed, Nfix is involved in the macrophage phenotypical switch and macrophages lacking Nfix failed to adopt an anti-inflammatory phenotype and interact with myogenic cells. Moreover, we demonstrated that phagocytosis induced by the inhibition of the RhoA-ROCK1 pathway leads to Nfix expression and, consequently, to acquisition of the anti-inflammatory phenotype. Our study identified Nfix as a link between RhoA-ROCK1-dependent phagocytosis and the MP phenotypical switch, thus establishing a new role for Nfix in macrophage biology for the resolution of inflammation and tissue repair.

My PhD project focuses on Nuclear Factor I X (NFIX), which is a transcription factor belonging to the NFI DNAbinding proteins family. NFIX plays an essential role in the development of several organs, most importantly skeletal muscle. It has been shown that upon NFIX inhibition in a dystrophic mice model there is an amelioration in the pathological features of the muscular dystrophy. Thus, these data demonstrated that the absence of NFIX protects from the progression of the disease in vivo. This study shed a light on the possible use of NFIX as a genetic tool to slow down the progress of muscular dystrophy, which is still incurable.On these bases, my project aims of purifying and solving the novel structure of NFIX alone and in complex with its palindromic DNA consensus sequence. The goal of the study is to find small molecules able to inhibit NFIX transcriptional function.During my PhD, I managed to find an acceptable protocol for expression and purification of truncated constructs of NFIX via a prokaryotic expression system. I was also able to find crystallization conditions suitable for NFIX-DNA complex, but I still have to work on crystals optimization. Moreover, I performed several biophysical analysis to better characterize the binding of NFIX to the target DNA and the protein stability.