Real-time visualization of chromatin modification in isolated nuclei
| Autor: | Svetlana Khakhina, Weam Elbezanti, Zachary Klase, Chris Janetopoulos, Hari Shroff, Luca Sardo, Lucas Beckman, Harshad D. Vishwasrao, Tara Jaison, Angel Lin, Luis Ricon |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Transcription Genetic Biology Time-Lapse Imaging Histone Deacetylases Histones 03 medical and health sciences Imaging Three-Dimensional Transcription (biology) Computer Systems Nucleic Acids Organelle medicine Histone code Humans Microscopy Nuclear Lamina 030102 biochemistry & molecular biology Lysine Nuclear Proteins Acetylation Cell Biology Chromatin Cell biology Tools and Resources 030104 developmental biology medicine.anatomical_structure Histone biology.protein RNA tat Gene Products Human Immunodeficiency Virus Nucleus Immunostaining HeLa Cells |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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