Isolation of neuronal chromatin from brain tissue

BMC Neurosci. 2008 Apr 28;9:42. doi: 10.1186/1471-2202-9-42.

Abstract

Background: DNA-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.

Results: Here, 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.

Conclusion: With 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Animals
  • Antigens, Nuclear / analysis
  • Antigens, Nuclear / metabolism
  • Brain / cytology
  • Brain / metabolism*
  • Cell Nucleus / chemistry
  • Cell Nucleus / metabolism
  • Child
  • Chromatin / genetics
  • Chromatin / isolation & purification*
  • Chromatin Immunoprecipitation / methods*
  • DNA Methylation
  • Flow Cytometry
  • Histones / analysis
  • Histones / chemistry
  • Histones / metabolism
  • Humans
  • Mice
  • Middle Aged
  • Molecular Biology / methods*
  • Nerve Tissue Proteins / analysis
  • Nerve Tissue Proteins / metabolism
  • Neurochemistry / methods*
  • Neurons / chemistry*
  • Neurons / metabolism
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / metabolism

Substances

  • Antigens, Nuclear
  • Chromatin
  • Histones
  • Nerve Tissue Proteins
  • Recombinant Fusion Proteins
  • neuronal nuclear antigen NeuN, human