INTRANUCLEAR DYNAMICS OF THE GLUCOCORTICOID RECEPTOR AND ITS CO-REGULATOR TIF2/GRIP1

MARTIN STORTZ; LUCIANA BRUNO; PAOLO ANNIBALE; ENRICO GRATTON; ADALI PECCI; VALERIA LEVI

San Carlos de Bariloche

Simposio; Third South American Symposium in Signal Transduction and Molecular Medicine? (SISTAM 2015); 2015

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 thus we decided to study it using advanced fluorescence microscopy techniques. We observed that TIF2 forms few large clusters in the nucleus that redistribute in the presence of GR activated by dexamethasone (DEX) to many 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 TIF2 fused to GFP and/or GR fused to mCherry or GFP. These data could be fitted with a model that considers that TIF2 and GR diffuse in the nucleus and bind to fixed targets. A cross-correlation analysis showed that DEX-stimulus induces GR-TIF2 interaction, as expected, however our preliminary results suggest that this interaction only occurs when both proteins are bound to a fixed target. Also, the autocorrelation function curve of TIF2 reveals a dramatic increase in the bound population upon hormonal GR activation. Finally, we analyzed the dynamics of these molecules within the nuclear clusters performing orbital and line-scanning FCS experiments. We detected binding/unbinding events of TIF2 and GR in the time scale 0.1 - 2 seconds. A positive cross-correlation between both channels in the intensity fluctuations of TIF2-GR+DEX clusters indicates the existence of GR-TIF2 complexes at these foci.