Integrating nuclear receptor mobility in models of gene regulation

Abstract

The mode of action of nuclear receptors in living cells is an actively investigated field but much remains hypothetical due to the lack, until recently, of methods allowing the assessment of molecular mechanisms in vivo. However, these last years, the development of fluorescence microscopy methods has allowed initiating the dissection of the molecular mechanisms underlying gene regulation by nuclear receptors directly in living cells or organisms. Following our analyses on peroxisome proliferator activated receptors (PPARs) in living cells, we discuss here the different models arising from the use of these tools, that attempt to link mobility, DNA binding or chromatin interaction, and transcriptional activity.

Figure 1. Imaging methods to study protein behavior in living cells.

Fluorescence recovery after photobleaching (FRAP). A small region of interest is photobleached by application of a high power laser beam. Fluorescence recovery is then calculated based upon quantification of fluorescence intensity in the region of interest (ROI) as a function of time, normalized to the global fluorescence in the cell to take into account photobleaching outside the ROI and during imaging. If a fraction of the molecules are not mobile, recovery does not reach 100%. The half-recovery time (i.e. the time required to get half of the maximal recovery) is often used to describe the mobility of the fluorescent molecule. Fluorescence correlation spectroscopy (FCS). FCS is a method based on the analysis of fluctuations of fluorescence intensity due to the diffusion of a labeled protein through a very small volume (in the range of 1 μm3) [Brock et al., 1998; Dittrich et al., 2001]. It allows study of the diffusion of mobile molecules at a higher spatial and temporal (microsecond range) resolution than FRAP. A mathematical transformation of the signal allows one to draw an autocorrelation curve G(t), from which the concentration of fluorescent particles and their average diffusion coefficient can be derived. Fluorescence Resonance Energy Transfer (FRET). FRET is the non-radiative transfer of energy between two fluorophores. The energy transfer efficiency decreases proportionally to the sixth power of the distance between the two fluorophores, and hence occurs only when the molecules harboring the fluorophores are in contact or very close proximity. Different methods and programs, such as PixFRET, can be used to calculate and map FRET in a cell or in a cell population [Berney and Danuser, 2003; Feige et al., 2005b]. PixFRET is available for download at http://www.unil.ch/cig/page16989.html.

Figure 2. A new hypothetical model for PPAR action in living cells.

This model is based on the results reported in the present article and in [Feige et al., 2005a; McNally et al., 2000; Phair et al., 2004]. (A) in the absence of ligand, PPAR and RXR are heterodimerized, recruit corepressors, and roam the nucleus where they interact transiently with chromatin, both on genuine PPREs and unspecific binding sites. (B) Upon ligand binding, PPAR mobility is reduced due to its AF-2-dependent binding to cofactors. PPAR/cofactor complexes may transiently bind to "non-specific" sites on chromatin, performing a three dimensional-scanning of the genome, until they encounter a genuine response element in a promoter, at which chromatin remodeling and transcription are initiated.