Uncovering the role of NF-kB2 in embryonic stem cell pluripotency and differentiation

Project summary (maximum of 4000 characters including spaces/returns) NF-kB describes a signalling network that is critical for cell-fate decisions. NF-kB has been implicated in the regulation of early embryonic cell-fate decisions in species such as drosophila, xenopus, and zebrafish, however its role in mammalian development is less well understood. To probe the role of NF-kB in early mammalian embryonic development, we took a mouse embryonic stem cell (mESC) approach and discovered an unusual localisation of NF-kB2 in pluripotency to large, discrete, rod/ring/figure of eight structures in the cytoplasm which dissipated during multilineage differentiation. NF-kB2 is synthesised as a p100 precursor that is processed to form the active p52 subunit, and our data suggests that it is the p52 form within these structures. We consequently found that these NF-kB2 structures were enriched for inositol monophosphate dehydrogenase (IMPDH2), an enzyme important in nucleotide biosynthesis, and co-immunoprecipitation revealed NF-kB2 and IMPDH2 interact. Interestingly, although these 'IMPDH2' filaments have been shown before, they have never been associated with NF-kB. Disrupting NF-kB2 either by blocking nuclear export with leptomycinB, or targeting NF-kB2 with siRNA, prevented or reduced (respectively) IMPDH2 filament formation, resulting in cytoplasmic IMPDH2. Furthermore, induction of IMPDH2 filaments with the antiviral ribavirin during lineage-specific differentiation resulted in maintenance of both NF-kB2:IMPDH2 filaments and pluripotency markers. These observations suggest that there's a critical functional link between NF-kB2, IMPDH2, and cell fate that regulates the cell-fate decisions during early lineage segregation at gastrulation.
The aim of this proposal is to understand the nature of NF-kB2 signalling in early development, the regulation of its processing, the nature of its interaction with IMPDH2, and the physiological function of this interaction. We will examine three hypotheses: (1) that NF-kB2 and IMPDH2 physically interact, and that the dynamics of this process is critical for IMPDH2 filament formation; (2) the NF-kB2:IMPDH2 association controls the processing of NF-kB2 (p100) to its active, shorter form (p52), and that this is a functional requirement for pluripotency/differentiation; (3) the single-cell nucleocytoplasmic dynamics of NF-kB2 can tune the balance between pluripotency and differentiation. We will take a mESC approach, coupling fixed immunofluorescence with live imaging of NF-kB2/IMPDH2 and bulk cell assays.