Characterization of metabolic safeguarding factors in the context of direct cellular reprogramming

Abstract

Cellular identities are established through the expression of fate-determining transcription factors (TFs). The terminally differentiated cell fates can be maintained by inhibitory mechanisms that suppress alternative TFs. Mis-expression of these fate-determining TFs can enable the reprogramming of the original cell fate into target cell fate. In C. elegans, the zinc-finger TF CHE-1 is essential for specifying the glutamatergic ASE neuronal fate. While ASE fate is normally repressed in non-neuronal tissues, it can be induced through ectopic expression of CHE-1 and knockdown of reprogramming barrier genes that safeguard cell identity. Using RNAi to remove these barriers, ASE neuron fate has been successfully expressed in diverse cell types. We investigated the mechanisms of action of NAD+ dependent mitochondrial isocitrate dehydrogenase 3 (IDH-3), a candidate reprogramming barrier gene. Our findings revealed reliable ASE neuron fate reporter expression in the germline upon ectopic expression of CHE-1 upon RNAi knockdown of alpha subunit (IDHA-1) of IDH3 complex. Furthermore, we identified FDGT-2, a putative membrane glucose transporter, that enhances germline reprogramming when co-depleted with IDHA-1. FDGT-2 was shown to be mainly expressed in regions covering the gut tissue and the germline. We also explored the role of TCA cycle metabolites, identifying α-ketoglutarate (α-KG) and fumarate as consistent enhancers of reprogramming efficiency. Additionally, we investigated the role of glutamine anaplerosis pathway in replenishment of α-KG levels depleted in the idha-1RNAi phenotype. The TCA cycle emerges as a central hub, linking energy metabolism to the proliferative and developmental processes essential for life. Examining male- and female-specific germline components, we observed that reprogramming overlaps with sperm and spermatheca-associated regions in closer proximity to vulva. Depletion of male-specific genes spe-8 and spe-27 reduced idha-1 mediated reprogramming rate, and genes required for gametogenesis and germline development were also necessary for reprogramming. Our study aimed to uncover the molecular mechanisms by which mitochondrial signals feedback to the nucleus, driving germ cell conversion into ASE neurons. These findings highlight the critical role played by metabolism in cell fate determination and reprogramming.