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

Abstract

Heterochromatin 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 HP1 α and HP1 β 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 HP1 α/ 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 t res ≤ 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.