Co-occupancy identifies transcription factor co-operation for axon growth.

Transcription factors (TFs) act as powerful levers to regulate neural physiology and can be targeted to improve cellular responses to injury or disease. Because TFs often depend on cooperative activity, a major challenge is to identify and deploy optimal sets. Here we developed a bioinformatics pipeline, centered on TF co-occupancy of regulatory DNA, and used it to predict factors that potentiate the effects of pro-regenerative Klf6 in vitro. High content screens of neurite outgrowth identified cooperative activity by 12 candidates, and systematic testing in a mouse model of corticospinal tract (CST) damage substantiated three novel instances of pairwise cooperation. Combined Klf6 and Nr5a2 drove the strongest growth, and transcriptional profiling of CST neurons identified Klf6/Nr5a2-responsive gene networks involved in macromolecule biosynthesis and DNA repair. These data identify TF combinations that promote enhanced CST growth, clarify the transcriptional correlates, and provide a bioinformatics approach to detect TF cooperation.

KLF6 and STAT3 co-occupy regulatory DNA and functionally synergize to promote axon growth in CNS neurons.

The failure of axon regeneration in the CNS limits recovery from damage and disease. Members of the KLF family of transcription factors can exert both positive and negative effects on axon regeneration, but the underlying mechanisms are unclear. Here we show that forced expression of KLF6 promotes axon regeneration by corticospinal tract neurons in the injured spinal cord. RNA sequencing identified 454 genes whose expression changed upon forced KLF6 expression in vitro, including sub-networks that were highly enriched for functions relevant to axon extension including cytoskeleton remodeling, lipid synthesis, and bioenergetics. In addition, promoter analysis predicted a functional interaction between KLF6 and a second transcription factor, STAT3, and genome-wide footprinting using ATAC-Seq data confirmed frequent co-occupancy. Co-expression of the two factors yielded a synergistic elevation of neurite growth in vitro. These data clarify the transcriptional control of axon growth and point the way toward novel interventions to promote CNS regeneration.