Co-occupancy analysis reveals novel transcriptional synergies for axon growth

Abstract

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 novel bioinformatics pipeline, centered on TF co-occupancy of regulatory DNA, and used it to predict factors that improve axon growth in corticospinal tract (CST) axons when combined with a known pro-regenerative TF, Klf6. Assays of neurite outgrowth confirmed cooperative activity by 12 candidates, and in vivo testing showed strong promotion of CST growth upon combined expression of Klf6 and Nr5a2. Transcriptional profiling of CST neurons identified Klf6/Nr5a2-responsive gene networks involved in macromolecule biosynthesis and DNA repair. These data identify novel TF combinations that promote enhanced CST growth, clarify the transcriptional correlates, and provide a bioinformatics roadmap to detect TF synergy. Introduction :As they mature, neurons in the central nervous system (CNS) decline in their capacity for robust axon growth, which broadly limits recovery from injury 1-4 . Axon growth depends on the transcription of large networks of regeneration-associated genes (RAGs), and coaxing activation of these networks in adult CNS neurons is a major unmet goal 2,5 . During periods of developmental axon growth, RAG expression is supported by pro-growth transcription factors (TFs) that bind to relevant promoter and enhancer regions to activate transcription 3,6 . One factor that limits RAG expression in mature neurons is the developmental downregulation of these pro-growth TFs 2,5 . Thus identifying TFs that act developmentally to enable axon growth and supplying them to mature neurons is a promising approach to improve regenerative outcomes in the injured nervous system.An ongoing challenge, however, is to decode the optimal set of factors for axon growth. To date, progress has centered on identifying individual TFs whose ectopic expression in mature neurons leads to improved axon growth. For example, we showed previously that the TF Klf6 is developmentally downregulated and that viral re-expression drives improved axon growth in mature corticospinal neurons, which are critical mediators of fine movement 7 . The restoration of axon growth from this and other single-factor studies remains partial, however, indicating the need for additional intervention. 7-12 . One likely possibility is that because multiple TFs generally act in a coordinated manner to regulate transcription, a more complete restoration of axon growth will depend on multiple TFs. Consistent with this, regeneration competent neurons recruit groups of interacting TFs in the hours to days post-injury, which results in transcriptional remodeling leading to reactivation of growth gene networks and functional recovery [13][14][15] . The need for multi-TF interventions in CNS neurons is recognized conceptually 5,16 , but a major challenge has been to develop a systematic pipeline ...

Inositol Polyphosphate-5-Phosphatase K (Inpp5k) Enhances Sprouting of Corticospinal Tract Axons after CNS Trauma

“…Approaches to identify growth modulators include screening the efficacy of developmentally regulated genes (Blackmore et al, 2010), phosphatases (Zou et al, 2015), kinases (Buchser et al, 2010), and genome-wide siRNA libraries (Sekine et al, 2018) to stimulate or repress neurite outgrowth in dissociated embryonic neurons in vitro. Additional approaches use transcriptional profiling of CNS neurons harvested in vivo during postnatal development (Wang et al, 2007;Moore et al, 2009;Venkatesh et al, 2018Venkatesh et al, , 2021, or during injury-induced axon growth (Fink et al, 2017;Tran et al, 2019;Lindborg et al, 2021). Together, these approaches have identified Klf6, Nr5a2, sac2, Lppr1 and Rab27b, Myl10, Airn, Prg2, and Il-22, which stimulate axon growth in vitro and in vivo.…”

Inositol Polyphosphate-5-phosphatase K (Inpp5k) enhances sprouting of corticospinal tract axons after CNS trauma

“…Disadvantages of in vitro screens include the embryonic age of the neurons and the simplicity of the in vitro growth environment, both of which poorly reflect the adult CNS. To overcome these challenges, two in vivo approaches have emerged to identify novel cell autonomous axon growth activators: identifying factors that drive the postdevelopmental growth of CNS neurons (Venkatesh et al, 2018;Venkatesh et al, 2021), and factors that support plasticity of intact CNS neurons after partial SCI (Fink et al, 2017). Recently we exploited the latter approach and profiled intact adult corticospinal tract neurons (CSNs) undergoing functional plasticity after unilateral corticospinal tract (CST) lesion (unilateral pyramidotomy, uPyX).…”