Huang Hsien-Sung, Matevossian Anouch, Jiang Yan, Akbarian Schahram
Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA 01604, USA.
J Neurosci Methods. 2006 Sep 30;156(1-2):284-92. doi: 10.1016/j.jneumeth.2006.02.018. Epub 2006 Mar 30.
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.
核小体核心组蛋白的甲基化及其他共价修饰是染色质结构和功能的关键调节因子，包括基因表达的表观遗传调控。然而，对于人类大脑，我们对特定基因组位点上组蛋白修饰的调控了解甚少。此外，适用于死后组织的染色质免疫沉淀方案尚不完善，诸如自溶时间或组织pH值等潜在混杂因素的影响也不清楚。我们用微球菌核酸酶消化法，或者用甲醛交联和超声破碎法处理人类死后大脑和小鼠的大脑皮层。我们发现，在死亡后的最初30小时内，大部分核小体DNA仍与组蛋白相连。与通过固定和超声破碎制备的提取物相比，用抗甲基化组蛋白抗体进行免疫沉淀在未固定的、经微球菌核酸酶消化的样品中至少有效10倍。在广泛的自溶时间和组织pH范围内，不同基因组位点的组蛋白甲基化差异得以维持。因此，对经微球菌核酸酶消化的组织提取物进行免疫沉淀是一种可行的方法，可用于分析死后大脑中特定基因组位点的组蛋白甲基化情况。
