Developmental Chromatin Restriction of Pro‐Growth Gene Networks Acts as an Epigenetic Barrier to Axon Regeneration in Cortical Neurons

Abstract

Axon regeneration in the central nervous system is prevented in part by a developmental decline in the intrinsic regenerative ability of maturing neurons. This loss of axon growth ability likely reflects widespread changes in gene expression, but the mechanisms that drive this shift remain unclear. Chromatin accessibility has emerged as a key regulatory mechanism in other cellular contexts, raising the possibility that chromatin structure may contribute to the age-dependent loss of regenerative potential. Here we establish an integrated bioinformatic pipeline that combines analysis of developmentally dynamic gene networks with transcription factor regulation and genome-wide maps of chromatin accessibility. When applied to the developing cortex, this pipeline detected overall closure of chromatin in sub-networks of genes associated with axon growth. We next analyzed mature CNS neurons that were supplied with various pro-regenerative transcription factors. Unlike prior results with SOX11 and KLF7, here we found that neither JUN nor an activated form of STAT3 promoted substantial corticospinal tract regeneration. Correspondingly, chromatin accessibility in JUN or STAT3 target genes was substantially lower than in predicted targets of SOX11 and KLF7. Finally, we used the pipeline to predict pioneer factors that could potentially relieve chromatin constraints at growth-associated loci. Overall this integrated analysis substantiates the hypothesis that dynamic chromatin accessibility contributes to the developmental decline in axon growth ability and influences the efficacy of pro-regenerative interventions in the adult, while also pointing toward selected pioneer factors as high-priority candidates for future combinatorial experiments. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000-000, 2018.

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

“…To identify potential Klf6 co-occupiers, we first assembled a list of genes that decline in expression as cortical neurons mature, using data from corticospinal motor neurons (CSMNs) in vivo and cortical neurons ages in vitro 23 , 24 (Fig. 1a ).…”

“…2a ). Using a well-established screening platform, postnatal cortical neurons received candidate genes by plasmid electroporation and were cultured at low density on laminin substrates, followed 2 days later by automated tracing to quantify neurite outgrowth 7 , 12 , 24 , 28 – 31 . Nuclear-localized enhanced green fluorescent protein (EGFP) served to mark transfected neurons and βIII tubulin immunohistochemistry labeled neuronal processes for automated tracing (Fig.…”

“… 23 - GSE2039; In vitro primary neurons aged in culture by ref. 24 , SRP151916). In vitro RNA-Seq datasets were processed using the updated Tuxedo suite of tools 24 .…”

“… 24 , SRP151916). In vitro RNA-Seq datasets were processed using the updated Tuxedo suite of tools 24 . Trimmed reads were mapped to the rat reference genome [UCSC, Rat genome assembly: Rnor_6.0] using HISAT2 aligner software (unspliced mode along with–qc filter argument to remove low-quality reads prior to transcript assembly) 59 .…”

UTX/KDM6A deletion promotes the recovery of spinal cord injury by epigenetically triggering intrinsic neural regeneration

“…Phenotypic transition is the result of progressive changes in the chromatin profile that ensure the stable expression of underlying genes. 33 However, the developmental chromatin status becomes an epigenetic barrier to mature CNS neuron axonal regeneration. Thus, it is crucial to remove the epigenetic restriction of injured neurons to reboot the regenerative transcriptional program and allow regrowth.…”