Real-time visualization of chromatin modification in isolated nuclei.
Chromatin modification is traditionally assessed in biochemical assays that provide average measurements of static events given that the analysis requires components from many cells. Microscopy can visualize single cells, but the cell body and organelles can hamper staining and visualization of the nucleus. Normally, chromatin is visualized by immunostaining a fixed sample or by expressing exogenous fluorescently tagged proteins in a live cell. Alternative microscopy tools to observe changes of endogenous chromatin in real-time are needed. Here, we isolated transcriptionally competent nuclei from cells and used antibody staining without fixation to visualize changes in endogenous chromatin. This method allows the real-time addition of drugs and fluorescent probes to one or more nuclei while under microscopy observation. A high-resolution map of 11 endogenous nuclear markers of the histone code, transcription machinery and architecture was obtained in transcriptionally active nuclei by performing confocal and structured illumination microscopy. We detected changes in chromatin modification and localization at the single-nucleus level after inhibition of histone deacetylation. Applications in the study of RNA transcription, viral protein function and nuclear architecture are presented. This article has an associated First Person interview with the first author of the paper.
['Sardo|Luca|L|', 'Lin|Angel|A|', 'Khakhina|Svetlana|S|', 'Beckman|Lucas|L|', 'Ricon|Luis|L|', 'Elbezanti|Weam|W|', 'Jaison|Tara|T|', 'Vishwasrao|Harshad|H|', 'Shroff|Hari|H|', 'Janetopoulos|Christopher|C|', 'Klase|Zachary A|ZA|http://orcid.org/0000-0002-3957-6416']
D000107:Acetylation D002843:Chromatin D003199:Computer Systems D006367:HeLa Cells D006655:Histone Deacetylases D006657:Histones D006801:Humans D021621:Imaging, Three-Dimensional D008239:Lysine D008853:Microscopy D034881:Nuclear Lamina D009687:Nuclear Proteins D009696:Nucleic Acids D012313:RNA D059008:Time-Lapse Imaging D014158:Transcription, Genetic D054322:tat Gene Products, Human Immunodeficiency Virus
Chromatin Microscopy Nucleus Histone Acetylation