Analysis of neuronal injury transcriptional response identifies CTCF and YY1 as co-operating factors regulating axon regeneration

Abstract

Injured sensory neurons activate a transcriptional program necessary for robust axon regeneration and eventual target reinnervation. Understanding the transcriptional regulators that govern this axon regenerative response may guide therapeutic strategies to promote axon regeneration in the injured nervous system. Here, we used cultured dorsal root ganglia neurons to identify pro-regenerative transcription factors. Using RNA sequencing, we first characterized this neuronal culture and determined that embryonic day 13.5 DRG (eDRG) neurons cultured for 7 days are similar to e15.5 DRG neurons in vivo and that all neuronal subtypes are represented. This eDRG neuronal culture does not contain other non-neuronal cell types. Next, we performed RNA sequencing at different time points after in vitro axotomy. Analysis of differentially expressed genes revealed upregulation of known regeneration associated transcription factors, including Jun, Atf3 and Rest, paralleling the axon injury response in vivo. Analysis of transcription factor binding sites in differentially expressed genes revealed other known transcription factors promoting axon regeneration, such as Myc, Hif1α, Pparγ, Ascl1a, Srf, and Ctcf, as well as other transcription factors not yet characterized in axon regeneration. We next tested if overexpression of novel candidate transcription factors alone or in combination promotes axon regeneration in vitro. Our results demonstrate that expression of Ctcf with Yy1 or E2f2 enhances in vitro axon regeneration. Our analysis highlights that transcription factor interaction and chromatin architecture play important roles as a regulator of axon regeneration.

MORA and EnsembleTFpredictor: An ensemble approach to reveal functional transcription factor regulatory networks

“…EnsembleTFpredictor identified five TFs predicted by both algorithms, and two of them, N-myc and Pax6, were shown to form physical complexes with SnoN to regulate granule neuron precursor development. In a second study, MORA, HOMER, and oPossum-3 were used to identify TFs that regulate genes differentially expressed during dorsal root ganglion (DRG) neuron axon regeneration [ 24 ]. EnsembleTFpredictor identified 43 TFs predicted by at least two methods, including known axon-regeneration TFs, as well as TFs not yet characterized in axon regeneration.…”

“…To our knowledge, this is the first tool performing this type of integration. Literature search and our experimental validation [ 23 , 24 ] demonstrated that MORA and EnsembleTFpredictor are highly effective in identifying functional TFs responding to a perturbation and unraveling novel TFs that cooperate with each other to exert synchronized responses. Consequently, MORA and EnsembleTFpredictor serve as easy and cost-effective hypothesis-generating tools to broadly assess TF activity, yielding new biological insights.…”

Three-dimensional chromatin mapping of sensory neurons reveals that cohesin-dependent genomic domains are required for axonal regeneration

“…( 9 , 45 ). Others have shown that co-expression of CTCF with other TFs contributes to promoting axon growth ( 46 ). While we also found that injury-responsive genes were enriched for binding sites for CCCTC-binding factor (CTCF), these were significantly less numerous than the ones identified for cohesin.…”