Fig. 1: Schematic diagram summarizing the overall experimental flow integrating iterative bio-informatics and experimental validation.

Multiple independent functional genomics analyses of distinct injury models were analyzed to computationally identify upstream TFs associated with CNS regeneration. In the first set of analysis (a, left), we performed a mutual information-based network analysis using ARACNe to characterize the transcriptional regulatory network formed by regeneration-associated TFs in multiple independent data sets from spinal cord and peripheral nerve injury. The hierarchical structure of the TF regulatory network was further characterized so as to identify potential upstream regulators. This step-wise analysis predicted REST, a transcriptional repressor, as an upstream negative regulator inhibiting the core pro-regenerative TFs to drive the expression of regeneration-associated genes (RAGs). In parallel (a, right), we performed an additional TF screen in another CNS tissue, optic nerve, under pro-growth and native conditions to identify TF regulators of regeneration. Among the ~1000 TF-target gene sets tested via Gene Set Enrichment Analysis, REST was ranked as the top negative regulator of the RGC regeneration state-associated gene set. Multiple independent bio-informatic analyses of external data sets confirmed and converged on our model b, by which REST is activated by CNS injury and acts as a potential upstream negative regulator of the core regenerative TFs. To test this, we performed gene expression analysis in the injured CNS with REST and after REST depletion, showing REST increases following CNS injury, while the core pro-regenerative TFs and genes remain suppressed. Depleting REST activates a core molecular program driven by a tightly controlled TF network similar to the one activated during regeneration. These results predicted that REST depletion would improve regeneration, which we directly tested in two well-established models of regeneration in vivo c, confirming REST’s functional effect as a suppressor of regeneration. In the case of optic nerve injury, REST depletion or inhibition enhanced both RGC regeneration and survival. These analyses identify a role for REST as an upstream suppressor of the intrinsic regenerative program in the CNS and demonstrate the power of a systems biology approach involving integrative genomics analysis to predict key regulators of CNS repair.