Quantitative Kinetic Analyses of Histone Turnover Using Imaging and Flow Cytometry.

Abstract

Dynamic histone changes occur as a central part of chromatin regulation. Deposition of histone variants and post-translational modifications of histones are strongly associated with properties of chromatin status. Characterizing the kinetics of histone variants allows important insights into transcription regulation, chromatin maintenance and other chromatin properties. Here we provide a protocol of quantitative and sensitive approaches to test the timing of incorporation and dissociation of histones using a two-color SNAP-labeling system, labelling pre-existing and newly-incorporated histones distinctly. Together with cell cycle synchronization methods and cell cycle markers, this approach enables a pulse-chase analysis to determine the turnover of histone variants during the cell cycle, detected using imaging or flow cytometry methods at single cell resolution. As well as testing global histone turnover, cell cycle-dependent cellular localization of histone variants can be also addressed using imaging approaches.