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Quantitative Kinetic Analyses of Histone Turnover Using Imaging and Flow Cytometry
Hanae Sato1,2, Robert H Singer1,3, John M Greally2
1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, USA.
Bio-Protocol
|October 12, 2020
Summary
This study introduces a novel method to track histone variant dynamics. The technique uses distinct labeling to quantify histone turnover and localization during the cell cycle.
Area of Science:
- Molecular Biology
- Epigenetics
- Chromatin Biology
Background:
- Dynamic histone modifications are crucial for chromatin regulation.
- Histone variants and their post-translational modifications influence chromatin properties.
- Understanding histone variant kinetics provides insights into transcription and chromatin maintenance.
Purpose of the Study:
- To develop a quantitative protocol for analyzing histone variant turnover.
- To investigate the timing of histone variant incorporation and dissociation.
- To enable cell cycle-dependent analysis of histone dynamics.
Main Methods:
- Utilized a two-color SNAP-labeling system to differentiate pre-existing and newly incorporated histones.
- Employed cell cycle synchronization and markers for pulse-chase analysis.
- Applied imaging and flow cytometry for single-cell resolution detection.
Main Results:
- Successfully quantified histone variant turnover kinetics during the cell cycle.
- Demonstrated the ability to distinguish incorporation and dissociation timing.
- Enabled assessment of cell cycle-dependent histone variant localization.
Conclusions:
- The developed protocol offers a sensitive approach to study histone variant dynamics.
- This method provides insights into chromatin regulation and maintenance.
- Facilitates detailed analysis of histone turnover and localization across the cell cycle.
