Abstract
BackgroundDNA-protein interactions in mature brain are increasingly recognized as key regulators for behavioral plasticity and neuronal dysfunction in chronic neuropsychiatric disease. However, chromatin assays typically lack single cell resolution, and therefore little is known about chromatin regulation of differentiated neuronal nuclei that reside in brain parenchyma intermingled with various types of non-neuronal cells.ResultsHere, we describe a protocol to selectively tag neuronal nuclei from adult brain – either by (anti-NeuN) immunolabeling or transgene-derived histone H2B-GFP fusion protein – for subsequent fluorescence-activated sorting and chromatin immunoprecipitation (ChIP). To illustrate an example, we compared histone H3 lysine 4 and 9 methylation marks at select gene promoters in neuronal, non-neuronal and unsorted chromatin from mouse forebrain and human cerebral cortex, and provide evidence for neuron-specific histone methylation signatures.ConclusionWith the modifications detailed in this protocol, the method can be used to collect nuclei from specific subtypes of neurons from any brain region for subsequent ChIP with native/un-fixed or crosslinked chromatin preparations. Starting with the harvest of brain tissue, ChIP-ready neuronal nuclei can be obtained within one day.
Highlights
DNA-protein interactions in mature brain are increasingly recognized as key regulators for behavioral plasticity and neuronal dysfunction in chronic neuropsychiatric disease
An increasing number of neurodevelopmental and neuropsychiatric disorders are thought to result from defective DNA:protein interactions in neurons; sustained changes in neuronal gene expression and behavior after exposure to certain drugs or stimuli are likely to involve chromatin remodeling, including DNA methylation and histone modification changes [15]
H2B-GFP transgenic mice The promoter of the ? subunit of the Ca2+/calmodulin dependent protein kinase II gene (CAMKII) was used to drive H2B-EGFP expression; as expected, this transgene labeled most of the neuronal populations in the fore- and midbrain, including cortex, striatum, hippocampus, with the notable exception of the GABAergic interneurons in cerebral cortex and hippocampus (Fig. 3)
Summary
DNA-protein interactions in mature brain are increasingly recognized as key regulators for behavioral plasticity and neuronal dysfunction in chronic neuropsychiatric disease. Immunostaining in conjunction with fluorescence-activated cell sorting (FACS) was used successfully to selectively collect neuronal nuclei from human (postmortem) brain tissue for the purposes of retrospective birth dating [8] or assessment of age-related changes in DNA cytosine methylation [9]. These studies utilized the nuclear harvest for highly sensitive radiation and PCR assays, and it remained unclear whether the protocol could be modified for the purposes of chromatin immunoprecipitation and other techniques that require comparatively larger amount of input (for example, 105 107 nuclei). The methods presented here will be important for the study of molecular mechanisms governing epigenetic control of neuronal gene expression and chromatin remodeling in mature brain
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