Chromatin immunoprecipitation in postmortem brain.
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.
Huang
Hsien-Sung
HS
Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA 01604, USA. hsien-sung@umassmed.edu
Matevossian
Anouch
A
Jiang
Yan
Y
Akbarian
Schahram
S
D000368:Aged; D000818:Animals; D001329:Autolysis; D001923:Brain Chemistry / Q000502:physiology*; D002467:Cell Nucleus / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D015331:Cohort Studies; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008297:Male; D008745:Methylation; D051379:Mice; D008836:Micrococcal Nuclease / Q000737:chemistry; D008875:Middle Aged; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism; D011180:Postmortem Changes; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014465:Ultrasonics
D002843:Chromatin; D003432:Cross-Linking Reagents; D006657:Histones; D009707:Nucleosomes; D004247:DNA; D008836:Micrococcal Nuclease