Methods to Study Dynamic Interaction of Androgen Receptor with Chromatin in Living Cells

Androgen receptor (AR) is a ligand-dependent transcription factor that belongs to the nuclear receptor superfamily. In the presence of its specific ligands, AR translocates into the nucleus, interacts with chromatin at hormone response elements (HREs) and recruits a variety of coregulators and basal transcription factors to regulate transcription. Using green fluorescent protein (GFP) labelling and the tandem gene array system of mouse mammary tumour virus (MMTV), the interaction of AR with HREs can be visualized and studied in live cells. The MMTV array in nuclei can be specifically detected by DNA fluorescence in situ hybridization (DNA FISH) and thereby specific binding of GFP-AR to the array can be confirmed in the presence of specific ligands. The transcriptional activity of GFP-AR at the MMTV array can be visualized by RNA FISH in combination with interactions of GFP-AR or its cofactors, or different components of the transcriptional initiation complex, by indirect immunofluorescence (IF). Finally, using fluorescence recovery after photobleaching (FRAP), dynamic interactions of GFP-AR with the chromatin template can be studied. Methods to carry out these experiments are described herein.