Distinctly Different Dynamics and Kinetics of Two Steroid Receptors at the Same Response Elements in Living Cells

<div><p>Closely related transcription factors (TFs) can bind to the same response elements (REs) with similar affinities and activate transcription. However, it is unknown whether transcription is similarly orchestrated by different TFs bound at the same RE. Here we have compared the recovery half time (t_1/2), binding site occupancy and the resulting temporal changes in transcription upon binding of two closely related steroid receptors, the androgen and glucocorticoid receptors (AR and GR), to their common hormone REs (HREs). We show that there are significant differences at all of these levels between AR and GR at the MMTV HRE when activated by their ligands. These data show that two TFs bound at the same RE can have significantly different modes of action that can affect their responses to environmental cues.</p></div

Distinct mobilities of GFP-AR and GFP-GR at the MMTV array in living cells.

<p>(<b>A</b>) GFP-AR and GFP-GR expression in 3108 and 3617 cells, respectively. Cells were treated with agonists R1881 or DEX as indicated. Total cell extracts were subjected to Western analysis using an anti-GFP antibody (upper panels) and an anti-α-tubulin antibody as loading control (lower panels). Relative quantification of band intensities is indicated below the lanes; α-tubulin at each time point was set to 1.0. (<b>B</b>, <b>C</b>) GFP-AR and GFP-GR show distinct FRAP recovery dynamics at the MMTV array. 3108 cells (<b>B</b>) and 3617 cells (<b>C</b>) were treated with agonists R1881 or DEX for the given time periods. GFP fluorescence at the MMTV array was bleached and recovery after bleaching was followed by live cell imaging. FRAP curves were generated, and recovery half times were determined in a semi-quantitiave manner. Error bars represent means ± standard error.</p

Transcription complexes at the MMTV promoter are differentially sensitive to RNA Pol II inhibitors.

<p>(<b>A</b>) DRB and ActD inhibit hormone induced Ras transcription in 3108 or 3617 cells. (<b>B</b>) DRB and ActD have differential effects on GFP-AR and GFP-GR mobility at MMTV LTR. After treatment with agonist R1881 or DEX alone or in combination with inhibitors DRB or ActD, GFP-AR (3108 cells) and GFP-GR (3617 cells) bound to the MMTV-LTR array were subjected to semi-quantitative FRAP analysis. The recovery half time values for ActD treatment were not determined (nd) as the fluorescence recovery was not large enough during the experimental time period. Error bars represent means ± standard error. (<b>C</b>) DRB and ActD decrease GFP-AR and GFP-GR occupancy at the MMTV array. 3108 or 3617 cells were treated as in (<b>A</b>). qPCR was used to validate ChIP analysis performed with an anti-GFP antibody using known binding sites at MMTV LTR. Error bars represent means ± standard deviation. (<b>D</b>) DRB and ActD have distinct effects on elongating Pol II at the MMTV array. 3108 or 3617 cells were treated as indicated in (<b>A</b>). qPCR was used to validate ChIP analysis performed with an anti-Pol II-pSer2 antibody using known binding sites at MMTV promoter. Error bars represent means ± standard deviation.</p

GFP-AR and GFP-GR differentially associate with and activate transcription from the MMTV LTR.

<p>Time course of GFP-AR (<b>A</b>) and GFP-GR (<b>B</b>) transcriptional activity and promoter occupancy at the MMTV LTR. 3108 cells (AR) in (<b>A</b>) or 3617 cells (GR) in (<b>B</b>) were treated with agonists R1881 or DEX for the given time points. MMTV-Ras transcript levels were examined by qPCR (y-axis on the left-hand side). ChIP analysis was performed using an anti-GFP antibody, and receptor occupancy at MMTV LTR was determined by qPCR (y-axis on the right-hand side). Error bars represent means ± standard deviation.</p