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Related Experiment Videos

Chromatin immunoprecipitation in postmortem brain.

Hsien-Sung Huang1, Anouch Matevossian, Yan Jiang

  • 1Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA 01604, USA. hsien-sung@umassmed.edu

Journal of Neuroscience Methods
|April 1, 2006
PubMed
Summary
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Profiling histone methylation in postmortem human brain is challenging. This study demonstrates micrococcal nuclease digestion of unfixed tissue enables effective chromatin immunoprecipitation for studying epigenetic gene regulation.

Area of Science:

  • Epigenetics
  • Neuroscience
  • Molecular Biology

Background:

  • Histone modifications regulate chromatin structure and gene expression.
  • Understanding histone modifications in the human brain, especially postmortem, is limited.
  • Existing chromatin immunoprecipitation protocols are not optimized for postmortem tissues.

Purpose of the Study:

  • To develop and validate a chromatin immunoprecipitation (ChIP) protocol for profiling histone methylation in postmortem human brain.
  • To assess the impact of autolysis time and tissue pH on ChIP assay effectiveness.
  • To compare different tissue preparation methods for ChIP.

Main Methods:

  • Cerebral cortex from human postmortem brain and mice were processed using micrococcal nuclease (MNase) digestion or formaldehyde fixation followed by sonication.

Related Experiment Videos

  • ChIP assays were performed using antibodies against methylated histones.
  • Nucleosomal DNA integrity and histone-DNA association were assessed over time postmortem.
  • The influence of autolysis time and tissue pH on histone methylation profiling was evaluated.

    • Main Results:

    • Nucleosomal DNA remains associated with histones for at least 30 hours postmortem.
    • MNase digestion of unfixed tissue yielded at least 10-fold higher efficiency for methylated histone immunoprecipitation compared to fixation/sonication.
    • Histone methylation patterns at specific genomic loci were preserved across a broad range of autolysis times and tissue pH.
    • The developed method allows for profiling histone methylation at defined genomic loci in postmortem brain tissue.

    Conclusions:

    • Micrococcal nuclease digestion of unfixed postmortem brain tissue is a feasible and effective method for chromatin immunoprecipitation.
    • This approach overcomes limitations of existing protocols, enabling robust analysis of histone methylation in postmortem human brain.
    • The findings facilitate the study of epigenetic regulation in neurological disorders using postmortem brain samples.