Chromatin immunoprecipitation in postmortem brain
Department of Psychiatry, Brudnick Neuropsychiatric Research Institute
Medical Subject Headings
Aged; Animals; Autolysis; Brain Chemistry; Cell Nucleus; Chromatin; Cohort Studies; Cross-Linking Reagents; DNA; Female; Histones; Humans; Hydrogen-Ion Concentration; Hydrolysis; Immunohistochemistry; Immunoprecipitation; Male; Methylation; Mice; Micrococcal Nuclease; Middle Aged; Nucleosomes; *Postmortem Changes; Reverse Transcriptase Polymerase Chain Reaction; Ultrasonics
Life Sciences | Medicine and Health Sciences | Neuroscience and Neurobiology
Methylation and other covalent modifications of nucleosome core histones are key regulators of chromatin structure and function, including epigenetic control of gene expression. For the human brain, however, very little is known about the regulation of histone modifications at specific genomic loci. Furthermore, chromatin immunoprecipitation protocols applicable to postmortem tissue are lacking, and the impact of potential confounds such as autolysis time or tissue pH is unknown. We treated cerebral cortex from human postmortem brain and mice by micrococcal nuclease digestion or, alternatively, by formaldehyde-crosslinking and sonication. We show that the bulk of nucleosomal DNA remains attached to histones during the first 30 h after death. Immunoprecipitation with antibodies against methylated histones was at least 10-fold more effective in unfixed, micrococcal nuclease-digested samples, in comparison to extracts prepared by fixation and sonication. Histone methylation differences across various genomic sites were maintained within a wide range of autolysis times and tissue pH. Therefore, immunoprecipitation of micrococcal nuclease-digested tissue extracts is a feasible approach to profile histone methylation at defined genomic loci in postmortem brain.
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Citation: J Neurosci Methods. 2006 Sep 30;156(1-2):284-92. Epub 2006 Mar 30. Link to article on publisher's site