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 HP1alpha and HP1beta 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 HP1alpha/beta 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) <or= 0.2 s (class I, dominant in euchromatin), 7 s (class II, dominant in heterochromatin), and approximately 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.

Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites

“…3, Tables 2 and 3). For all proteins the FRAP recovery curves could be fitted well with a diffusion model, in which the contribution of transient binding is included into an effective diffusion coefficient (19). Recovery in the FRAP experiments during G1 phase was almost complete with immobile fractions of only 1-4% (Table 2).…”

“…Furthermore, an additional slow population with D slow = 0.03-0.10 μm 2 ·s −1 (τ = 100-600 ms) was present in the nucleus, which reflects chromatin translocations that manifest themselves as slow intensity fluctuations of bound fluorescently labeled proteins. This explanation is inferred from the value of the anomaly parameter α > 1, which is indicative of a confined diffusion behavior in the FCS analysis, and the observation that this contribution is a general feature of proteins that interact transiently with chromatin like heterochromatin protein 1 (19). This interpretation is corroborated by the absence of a corresponding mobility fraction in the FRAP experiments for Snf2H/Snf2L, where a <150 ms residence time and D values of 0.9 ± 0.1 and 1.0 ± 0.1 μm 2 ·s −1 were measured ( Table 2).…”

“…However, details on their mode of operation in living cells are missing. Here, we analyzed the dynamics and interaction behavior of Snf2H, Snf2L, and Snf2L+13 as well as Acf1 in living cells with a multiscale approach that combined FRAP, CP, and FC(C)S experiments (18)(19)(20)(21). In the case of FRAP, the shape of the recovery curve is used to extract dynamic parameters such as the diffusion coefficient and the kinetic rate constants.…”

“…Other constructs were introduced via transient transfection. FRAP, CP, and F(C)CS experiments were conducted as described previously (19) and in SI Materials and Methods. At least 20 cells were measured for a given experimental condition.…”

“…Autofluorescent protein constructs of human Snf2H, Snf2L, Snf2L+13, and Acf1 were investigated in human osteosarcoma and murine fibroblast cell lines. Their intracellular localization as well as their dynamic behavior was analyzed using a multiscale fluorescence fluctuation microscopy approach that comprised noninvasive fluorescence recovery after photobleaching (FRAP), continuous photobleaching (CP), and fluorescence (cross-)correlation spectroscopy (FCS/ FCCS)-based experiments (18)(19)(20)(21). The results from our quantitative analysis are rationalized in terms of a "continuous sampling" mechanism: In G1/2 phase of the cell cycle, all nucleosomes are probed by a given remodeling complex within minutes via transient binding reactions but only a small fraction of these binding events leads to repositioning.…”