Kimura Hiroshi, Hayashi-Takanaka Yoko, Goto Yuji, Takizawa Nanako, Nozaki Naohito
Nuclear Function and Dynamics Unit, HMRO, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Cell Struct Funct. 2008;33(1):61-73. doi: 10.1247/csf.07035. Epub 2008 Jan 28.
Histone modifications play critical roles in the epigenetic regulation of gene expression and in the maintenance of genome integrity. Acetylation and methylation of histone H3 are particularly important in gene activation and silencing. We generated and characterized a panel of mouse monoclonal antibodies that specifically recognize different modifications on K4, K9, and K27 residues on histone H3. By using these antibodies for chromatin immunoprecipitation and immunoblotting, we analyzed the relationship between different modifications in nearby nucleosomes in human cells. Within a few nucleosome neighbors, trimethyl-K4 was associated with acetyl-K27, rather than with dimethyl-K4 and acetyl-K9, consistent with their co-localization on active promoters. Furthermore, simultaneous immunofluorescence using directly-labeled antibodies revealed that di- and tri-methylation on K4 was diminished during replicative senescence. These highly-reliable and fully-characterized monoclonal antibodies may facilitate future epigenomic studies on healthy and diseased cells.
组蛋白修饰在基因表达的表观遗传调控以及基因组完整性的维持中发挥着关键作用。组蛋白H3的乙酰化和甲基化在基因激活和沉默中尤为重要。我们制备并鉴定了一组小鼠单克隆抗体，它们能特异性识别组蛋白H3上K4、K9和K27残基的不同修饰。通过使用这些抗体进行染色质免疫沉淀和免疫印迹，我们分析了人类细胞中相邻核小体不同修饰之间的关系。在几个相邻核小体中，三甲基化的K4与乙酰化的K27相关联，而不是与二甲基化的K4和乙酰化的K9相关联，这与它们在活性启动子上的共定位一致。此外，使用直接标记抗体的同步免疫荧光显示，在复制性衰老过程中，K4上的二甲基化和三甲基化减少。这些高度可靠且经过充分表征的单克隆抗体可能会促进未来对健康和患病细胞的表观基因组研究。
