We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.
Abstract
Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur,
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding,
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP).
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.
Protocol
1. Sciatic & dorsal column nerve injury The animal is placed on a surgical towel and underneath it a thermopad is present throughout the procedure keeping the body temperature of the mouse at 37°C. All animals are anesthetized for surgery with a continuous isoflurane/O2 administration. Surgical instruments are autoclaved before the procedure. For sciatic injury, both hindquarters are carefully shaved, and depilation is completed with generic hair remover prior to cleansing skin wi…
Discussion
This protocol provides a method to directly ask about the chromatin environment during axonal regeneration in the adult
nervous system following axonal injury. It incorporates the DRG injury model with chromatin immunoprecipitation to probe the transcriptional and
epigenetic environment subsequent to injury to either the PNS or CNS. It is particularly useful for investigators who would like to characterize
putative binding sites for their favorite transcription factor, and to determine whether the occupancy of these s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Andrea Tedeschi for help in setting the initial ChIP experiments in the laboratory and Ricco Lindner
for his contribution to fine tune the conditions for ChIP. This work was supported by the Hertie Foundation; the Fortune Grant, University of
Tuebingen, and the DFG DI 1497/1-1 grants (all granted to Simone Di Giovanni).
Floriddia, E., Nguyen, T., Di Giovanni, S. Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury. J. Vis. Exp. (53), e2803, doi:10.3791/2803 (2011).