Dynamics and spatial organization of transcription factors in living cells by a fluorescence microscopy approach

STORTZ, M.; BRUNO, L.; ANNIBALE, P.; GRATTON, E.; PECCI A; LEVI, V.

Barcelona

Congreso; 29th Annual Symposium of the Protein Society; 2015

Protein Society

The glucocorticoid receptor (GR) is a ligand-activated transcription factor and plays a relevant role in physiology, with a great variety of effects. GR can be directly recruited to specific response elements or can also interact with other transcription factors finally inducing or repressing gene expression.The activity of GR is modulated by different co-regulators, e.g. TIF2/GRIP1. GR and TIF2 do not distribute homogeneously within the nucleus but accumulate in distinctive clusters. The functional role of this particular intranuclear organization is still unknown.We observed that TIF2 forms few large clusters in the nucleus that redistribute upon GR activation by dexamethasone (DEX), leading to small TIF2 clusters that co-localize with GR foci.In order to study the dynamics of these two proteins in the nucleus and the relevance of their spatial distribution, we performed fluorescence correlation spectroscopy (FCS) measurements in newborn hamster kidney (BHK) cells transiently expressing GR and/or TIF2 fused to fluorescent proteins. Obtained data could be fitted with a model that considers that TIF2 and GR diffuse in the nucleus and bind to fixed targets. Both GR and TIF2 autocorrelation curves reveal an increase in the bound population upon hormonal GR activation. A cross-correlation analysis showed that DEX-stimulus increases GR-TIF2 interaction, as expected, and especially on the binding component.Finally, we analyzed the dynamics of these molecules at the nuclear clusters performing line-scanning FCS experiments. A positive cross-correlation in the intensity fluctuations of TIF2-GR+DEX clusters, between both channels, indicates that GR and TIF2 interact at these foci and dissociate from them as a hetero-complex.