Live Cell Analysis of Mobility and Decay Kinetics of the Histone Variant H3.3.

Incorporation of the variant histone H3.3 into the genome occurs in conjunction with gene expression throughout the cell cycle. However, its precise regulatory mechanisms remain unclear. Traditional methods like Chromatin Immunoprecipitation provide static snapshots of H3.3 distribution that do not provide dynamic insights. To understand H3.3 behavior in live cells, we conducted fluorescence recovery after photobleaching (FRAP) to examine H3.3 mobility in mouse embryonic fibroblasts. The SNAP tag system enabled us to study the mobility of both preexisting and newly synthesized H3.3 pools. Our results showed that H3.3 is significantly more mobile than the core histone H3.1 during the 8-hour FRAP assay. Remarkably, H3.3 mobility was abolished under global transcription inhibition. Furthermore, the deletion of histone chaperone HIRA and NSD2 substantially reduced H3.3 mobility. We also investigated the turnover, or decay dynamics, of H3.3 using live-cell imaging over two days. Similar to its mobility, H3.3 decay was significantly delayed when transcription was inhibited and when HIRA and NSD2 were deleted. Our findings reveal that H3.3 dynamics and turnover are driven by ongoing transcription and depend on chaperone mediated H3.3 loading onto chromatin.