Multiscale Analysis of Dynamics and Interactions of Heterochromatin Protein 1 by Fluorescence Fluctuation Microscopy

AbstractHeterochromatin protein 1 (HP1) is a central factor in establishing and maintaining the repressive heterochromatin state. To elucidate its mobility and interactions, we conducted a comprehensive analysis on different time and length scales by fluorescence fluctuation microscopy in mouse cell lines. The local mobility of HP1α and HP1β was investigated in densely packed pericentric heterochromatin foci and compared with other bona fide euchromatin regions of the nucleus by fluorescence bleaching and correlation methods. A quantitative description of HP1α/β in terms of its concentration, diffusion coefficient, kinetic binding, and dissociation rate constants was derived. Three distinct classes of chromatin-binding sites with average residence times tres ≤ 0.2 s (class I, dominant in euchromatin), 7 s (class II, dominant in heterochromatin), and ∼2 min (class III, only in heterochromatin) were identified. HP1 was present at low micromolar concentrations at heterochromatin foci, and required histone H3 lysine 9 methylases Suv39h1/2 for two- to fourfold enrichment at these sites. These findings impose a number of constraints for the mechanism by which HP1 is able to maintain a heterochromatin state