Padmanabhan Jaya
Department of Molecular Medicine, USF Health Byrd Alzheimer's Institute, University of South Florida, 4001 E Fletcher Ave, Tampa, 33613, FL, USA,
Methods Mol Biol. 2015;1288:231-44. doi: 10.1007/978-1-4939-2474-5_13.
Post-translational modifications of histones play an important role in regulation of gene expression through condensation and decondensation of chromatin structure. These modifications include acetylation, methylation, phosphorylation and ubiquitination. Phosphorylation on histones is associated with cellular responses such as DNA damage, transcription, chromatin compaction and mitosis or meiosis. One of the most extensively studied modifications of histones is the Serine 10 phosphorylation on histone H3 N-terminal tail. This specific phosphorylation on Histone H3 has been associated with condensation and transcriptional inactivation of mitotic chromosomes, but recent studies have suggested a role for this specific phosphorylation in chromatin relaxation and activation of transcription in interphase cells. Co-immunostaining analysis of cells using antibodies specific to serine 10P-Histone H3 together with those to cell cycle specific markers will allow us to determine the nature of phosphorylation in a cell cycle-specific manner. In a complex system, such as tissue specimens, analysis using P-Histone H3 and a cell type specific antibody will allow identification of specific cells that are affected by this histone modification. This is of particular interest in the field of cancer biology or neurobiology where identification or quantification of the transcriptionally active or mitotic cells will enable one to evaluate the progression of the disease development. The protocol described here provides details on how co-immunostaining and analysis can be performed in tissue cultured cells or tissue sections.
组蛋白的翻译后修饰通过染色质结构的凝聚和去凝聚在基因表达调控中发挥重要作用。这些修饰包括乙酰化、甲基化、磷酸化和泛素化。组蛋白上的磷酸化与细胞反应相关，如DNA损伤、转录、染色质压缩以及有丝分裂或减数分裂。组蛋白最广泛研究的修饰之一是组蛋白H3 N端尾巴上的丝氨酸10磷酸化。组蛋白H3上的这种特定磷酸化与有丝分裂染色体的凝聚和转录失活有关，但最近的研究表明这种特定磷酸化在间期细胞的染色质松弛和转录激活中也发挥作用。使用针对丝氨酸10P -组蛋白H3的特异性抗体以及细胞周期特异性标记物的抗体对细胞进行共免疫染色分析，将使我们能够以细胞周期特异性方式确定磷酸化的性质。在诸如组织标本这样的复杂系统中，使用磷酸化组蛋白H3和细胞类型特异性抗体进行分析，将能够鉴定受这种组蛋白修饰影响的特定细胞。这在癌症生物学或神经生物学领域特别有意义，在这些领域中，鉴定或量化转录活跃或有丝分裂的细胞将使人们能够评估疾病发展的进程。这里描述的方案提供了关于如何在组织培养细胞或组织切片中进行共免疫染色和分析的详细信息。
