Identifying key regulators in neuronal transdifferentiation by gene regulatory network analysis

We have found that the overexpression of ASCL1, miR9/9 ${ }^{*}$ -124, nPTB shRNA, and p53 shRNA efficiently converted human skin fibroblasts to neurons. To identify key regulators of the transdifferentiation, we analyzed longitudinal RNA-seq data of human skin fibroblasts being converted with various combinations of these reprogramming factors, and constructed gene regulatory network (GRN) models capturing the high-order information important for neuronal conversion. Examination of gene communities and transcription factors (TFs) in the GRNs identified OTX2 and LMX1A as the key regulators of the conversion to neurons, as they had strongest connections to genes functionally associated with neuronal development and differentiation. Indeed, knocking down OTX2 or LMX1A significantly impaired the transdifferentiation of human skin fibroblasts to neurons. We also validated the approach in neuronal conversion of mouse embryonic stem cells. The study demonstrates the effectiveness of using GRN models to identify key regulators of neuronal conversion. The strategy will enhance mechanistic understanding of cellular reprogramming in general.