Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, USA.
J Mol Cell Cardiol. 2012 Oct;53(4):552-8. doi: 10.1016/j.yjmcc.2012.07.009. Epub 2012 Jul 28.
Despite the extensive knowledge of the functional unit of chromatin-the nucleosome-for which structural information exists at the atomic level, little is known about the endogenous structure of eukaryotic genomes. Chromosomal capture techniques and genome-wide chromatin immunoprecipitation and next generation sequencing have provided complementary insight into global features of chromatin structure, but these methods do not directly measure structural features of the genome in situ. This lack of insight is particularly troublesome in terminally differentiated cells which must reorganize their genomes for large scale gene expression changes in the absence of cell division. For example, cardiomyocytes, which are fully committed and reside in interphase, are capable of massive gene expression changes in response to physiological stimuli, but the global changes in chromatin structure that enable such transcriptional changes are unknown. The present study addressed this problem utilizing super-resolution stimulated emission depletion (STED) microscopy to directly measure chromatin features in mammalian cells. We demonstrate that immunolabeling of histone H3 coupled with STED imaging reveals chromatin domains on a scale of 40-70 nm, several folds better than the resolution of conventional confocal microscopy. An analytical workflow is established to detect changes in chromatin structure following acute stimuli and used to investigate rearrangements in cardiomyocyte genomes following agonists that induce cellular hypertrophy. This approach is readily adaptable to investigation of other nuclear features using a similar antibody-based labeling technique and enables direct measurements of chromatin domain changes in response to physiological stimuli.
尽管人们对染色质的功能单位——核小体有了广泛的了解，其结构信息在原子水平上已经存在，但人们对真核生物基因组的内源性结构知之甚少。染色体捕获技术和全基因组染色质免疫沉淀和下一代测序为染色质结构的整体特征提供了补充性的见解，但这些方法不能直接测量基因组在原位的结构特征。在终末分化的细胞中，这种缺乏洞察力尤其成问题，因为这些细胞必须在没有细胞分裂的情况下重新组织其基因组，以实现大规模的基因表达变化。例如，心肌细胞是完全分化的，处于间期中，能够对生理刺激做出大量的基因表达变化，但使这种转录变化成为可能的染色质结构的全局变化尚不清楚。本研究利用超分辨率受激辐射耗尽（STED）显微镜直接测量哺乳动物细胞中的染色质特征来解决这个问题。我们证明，与 STED 成像相结合的组蛋白 H3 免疫标记揭示了 40-70nm 尺度上的染色质结构域，其分辨率比传统共聚焦显微镜的分辨率高出几个数量级。建立了一个分析工作流程来检测急性刺激后染色质结构的变化，并将其用于研究诱导细胞肥大的激动剂后心肌细胞基因组的重排。这种方法很容易适应使用类似抗体标记技术研究其他核特征，并能够直接测量对生理刺激的染色质结构域变化。
