1Laboratory for NeuroRegeneration and Repair, Department of Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, 2Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen
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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).
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.
1. Sciatic & dorsal column nerve injury
NOTE: The same incision is made to expose the sciatic nerve, but the wound is sutured closed leaving the nerve intact for the sham injury.
NOTE: For most experiments, the laminectomy is considered the Sham injury.
CRITICAL STEP. Use fresh, molecular biology grade formaldehyde.
3. Nuclei preparation and chromatin shearing
CRITICAL STEP. Lysis and disruption of the tissue are vital for a good yield of crosslinked chromatin.
CAUTION. The shearing of the chromatin must be optimized for your particular sonication set up and proper fragmentation of the chromatin should be checked before running the actual IP experiment (see section 3).
NOTE. Fragmentation of the chromatin could also be performed by micrococcal nuclease (MNase) digestion. As a rule of thumb, fragmentation by sonication is preferred for fixed tissue, while MNase digestion is favored for native tissue chromatin immunoprecipitation. Cross-linked, sheared chromatin can be stored at -80° C for up to 2 months, but avoid repeated freeze/thaw cycles.
4. Analysis of chromatin digestion (recommended)
CAUTION. Over-fragmentation of chromatin may lead to decreased signal, while incomplete fragmentation will lead to diminished resolution and increased background.
NOTE. When determining the number of immunoprecipitation, a positive control (e.g. Histone H3 antibody) and a negative control (Normal IgG) should be considered. The quantity of antibody used for each IP varies between antibodies and must be empirically determined. However, it is usually within a range of 2-10 ?g/immunoprecipitation.
7. Elution and reversal of cross-linking
NOTE. The elution step can be performed at room temperature, too, but it might be not as efficient.
8. DNA recovery
CAUTION. The addition of a carrier, such as the glycogen, is needed to enhance the precipitation of relatively small sized DNA fragments.
NOTE. The precipitation step can also be performed overnight at -20°C.
9. Representative Results
A representative result of a ChIP experiment in DRGs following sciatic lesion is shown (Figures 1 and 2). First, we show fragmented DNA to a length of approximately 200-1000 bp (Figure 1). Second, we demonstrate a PCR signal following ChIP from the proximal promoter of the growth associated protein-43 (GAP-43) only upon sciatic nerve injury (lane 4, Figure 2, 5'). No PCR signal is present when the animal receives a sham injury only (lane 3), and when using normal IgG serum for the IP (lane 5 and 6). To test for the specificity of the antibody, we analyzed the same DNA samples, but used a control primer set which detects a region in the 3'-UTR of our gene of interest where no occupancy is expected. PCR signals are absent from all lanes (lanes 3-6, Figure 2, 3'), except for the input signal, representing non-IP DNA samples as standard PCR controls (lane 1-2, Figure 2). This ChIP procedure can be performed following either sciatic or spinal dorsal column injury as summarized in a schematic (Figure 3).
Figure 1. Agarose gel of reversed cross-linked DNA that has been sheared by sonication to the proper range of fragment lengths.
Figure 2. Semi-quantitative PCR results from DRG tissue following sciatic nerve lesion showing that acetylated p53 binds to the GAP-43 proximal promoter region 48 hours after injury to the sciatic nerve. Control PCR from the same DRG tissue show that acetylated p53 does not bind to a control region of DNA located in the 3’-untranslated region (UTR) of the GAP-43 gene (S = Sham, I = Injured).
Figure 3. A general diagram showing the location of the lesion sites on the sciatic nerve, and the dorsal column.
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 sites occurs in response to injury. Epigenetic modification of histones and DNA at these sites can also be simultaneously monitored. A similar protocol can be performed following facial nerve lesion, where the facial nerve nuclei can be dissected from the brainstem and processed by ChIP as we previously reported (Tedeschi et al., 2009).
As typical for chromatin immunoprecipitation assays, two critical steps in the protocol are, (1) efficient fragmentation and solubilization of the DNA-protein complex, and (2) the availability of a good immunoprecipitation antibody for your protein of interest. One limitation that might confound these two steps is the low level of starting material. Compared to other structures such as the brain or spinal cord, DRG provide a relatively small amount of tissue. In addition, DRG consists of a mix population of neurons as well as glial cells, all of which might have different chromatin environments. This may lead to heterogeneous IP signals; however this caveat is present in most samples taken from the nervous system. A potential solution is to perform ChIP after fluorescent activated cell sorting of transgenically fluorescently labeled DRG neurons, (see for example YFP-H mice, Bogdan et al., 2004), or immuno-isolation of neurons via magnetic beads (Lee et al., 2005). However, these two approaches need to be validated for ChIP in DRGs and will likely need an increased amount of starting material.
We have successfully used this method to identify binding sites for several transcription factors and coactivators at the promoter of known regeneration associated genes, and we have used both semi-quantitative and quantitative PCR methods to detect the immunoprecipitated DNA. One future addition to this protocol could be the use of tiled microarrays following ChIP (ChIP-on-chip), as means to detect the final DNA signal. ChIP-on-chip would greatly increase the number of identified transcription sites via a high throughput unbiased approach. It could also allow for the study of how two or more transcription factor might cooperatively interact on a genomic level.
No conflicts of interest declared.
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).
|10x ChIP Buffer||Cell Signaling Technology||7008|
|2x ChIP Elution Buffer||Cell Signaling Technology||7009|
|ChIP Grade Protein G Magnetic Beads||Cell Signaling Technology||9006|
|Magna Grip Rack (8 well)||EMD Millipore||20-400|
|Chloroform||Merck & Co., Inc.||UN 1888|
|37% Formaldehyde||Carl Roth Gmbh||CP10.1|
|10x Glycine Solution||Cell Signaling Technology||7005|
|10x HBSS||GIBCO, by Life Technologies||14185|
|Histone H3 antibody (rabbit)||Cell Signaling Technology||2650|
|Normal Rabbit IgG||Cell Signaling Technology||2729|
|Phenol/Chloroform/Isoamyl Alcohol||Carl Roth Gmbh||A156.1|
|Protease Inhibitors Cocktail Tablets||Roche Group||04 693 116 001|
|Proteinase K (20 mg/ml)||Cell Signaling Technology||10012|
|SDS Lysis Buffer||Upstate, Millipore||20-163|