Multimodal optical nanoscopy to study chromatin organization and epigenome remodelling sustaining adipose tissue differentiation and neuroblastoma cancer

The main goal of my three-years PhD project was to explore the chromatin remodeling pro-cesses together with nuclear architecture and epigenome changes associated with cell differ-entiation and malignant transformation, both of which require the precise coordination of mul-tiple regulatory signaling pathways. To achieve this, two in vitro models were utilized: adipo-cyte maturation and hypertrophy to mimic adipogenesis and obesity, and neuroblastoma retro-transformation to mimic neoplastic transformation. The research employed an integrated mul-timodal approach, combining high-resolution fluorescence microscopy techniques such as con-focal and stimulated emission depletion (STED) microscopy, alongside immunoassays and mo-lecular biology techniques, to investigate the potential alterations in the nuclear organization and the chromatin epigenome during these cellular processes. In the context of adipocyte differentiation, the study primarily focused on how the 3D nuclear morphology might correlate with key epigenetic modifications such as DNA methylation (5-mC) and histone H3 lysine 9 acetylation (H3K9Ac), which are essential for regulating gene ex-pression during adipogenesis and hypertrophy. The redistribution of chromatin domains, par-ticularly the isoconcentric distribution of H3K9-acetylated regions, was also investigated. Ad-ditionally, the roles of KAT2A, an enzyme supporting H3K9 acetylation, and BrD2, a protein interacting with acetylated histones, were explored for their contribution to chromatin structure and function. The second topic of the study focused on chromatin remodeling in a neuroblastoma model, in-vestigating the reprogramming of malignant neuroblastoma cells towards a neuron-like pheno-type induced by overexpression of the non-coding RNA Neuroblastoma Differentiation Marker 29 (NDM29). The results showed as this process led to significant changes in nuclear morphol-ogy, including elongation and volume, alongside epigenetic modifications such as redistribu-tion of histone modifications (H3K9Me3 and H3K9Ac) and global DNA methylation changes. These chromatin remodeling events were linked to the transition from a malignant to a less ag-gressive, neuron-like state. Furthermore, NF-κB-regulated genes and KAT2A expression were analyzed. In conclusion, these studies demonstrate that a marked chromatin remodeling, includ-ing changes in nuclear morphology and epigenetic modifications, occurs in both physiological and pathological events of the cell life, therefore representing a crucial mechanism to regulate the cellular fate.