decondensation, coactivator recruitment and maximal reporter mRNA accumulation. Conversely, treatment with the anti- estrogen 4-hydroxy-tamoxifen (4HT) induces large-scale chroma- tin condensation, abrogates coactivator recruitment, concomitant with a marked repression of reporter gene transcription. PRL- HeLa can be used to simultaneously examine several mechanistic aspects of ER transcription regulation at early (minutes) or late (hours) stages. ER is an important regulator of pituitary function, and the expression of the prolactin gene is also responsive to other factors, including EGF [20]. Accordingly, we sought to compare indirect (E2)- and indirect (EGF)-responsive regulation of ER-mediated transcription using our PRL-HeLa model system. Using quanti- tative automated imaging [21], our studies reveal differential recruitment of GFP-ER to the PRL array, sustained, maximum chromatin decondensation over 24 hours in E2 treated cells, accompanied by cyclic levels of reporter mRNA accumulation at the PRL-array. In contrast, EGF treatment induces a single pulse of ER-dependent chromatin decondensation and mRNA accu- mulation. These studies indicate a previously unknown difference between ligand-dependent and -independent control of chromatin decondensation by ER, coincident with different transcriptional responses. Results EGF-dependent ER promoter targeting PRL-HeLa cells transiently transfected with a GFP-ER expression vector were maintained for 48 hours in a hormone- free medium, and were then treated with 10 nM E2, 100 ng/ml EGF or 10 nM 4HT. PRL-arrays were visualized as bright foci of nuclear fluorescence in GFP-ER-expressing cells (Figure 1A, arrows), These foci co-localize with the integrated PRL reporter chromatin by DNA and RNA fluorescence in situ hybridization (FISH) [19]. Transient transfection results in a range of protein expression [19,21], and analyses of individual cells require careful selection based upon fluorescence (GFP-ER) level [21]. To avoid overexpression issues, array responses were assayed in cells expressing GFP-ER at levels of ,1.5 fold compared to endogenous ER in MCF-7 cells [19]. Relative to vehicle control, ERa Function by Image Analyses both E2 and 4HT treatments result in an increase in the percentage of cells with visible PRL arrays in GFP-ER expressing cells (60% versus 100% and 100%, respectively, Figure 1B). EGF treatment (indirect activation) resulted in 78% of GFP-expressing cells demonstrating visible arrays, which is statistically greater than vehicle control, but lower in percentage than E2-treated (direct activation). To determine the functional domains of ER required for EGF- mediated array responses, we expressed previously described deletion mutants (Figure 2 and [19]). Consistent with previous observations [19], expression of mutated ER in which the AF1 and DNA binding domain (GFP-ER251–595), or the ligand-binding domain (GFP-ER1–282) are deleted resulted in diffuse nuclear fluorescence (no visible foci) regardless of treatment (Figure 2 and Table 1). Expression of receptor in which the AF-1 domain (GFP- ER179–595) is removed demonstrated PRL-array targeting in the presence of E2, but not EGF (Figure 2). Therefore, the activity of the AF1 transactivation domain in the ER is necessary, but not sufficient, for optimal EGF-induced association with the PRL- array. Expression of GFP-ER1–534 (deleted for the F-domain and helix 12 of the E domain) resulted in PRL-array targeting with E2 treatment, but not EGF (Figure 2 and Table 1). Consistent with previous observations [19], the PRL-array remained condensed in cells treated with E2 (Figure 2), confirming that helix 12 is necessary for E2- induced large-scale chromatin decondensation. EGF treatment of cells expressing GFP-ER1–554 (deleted for the F- domain) resulted in array targeting, which was indistinguishable from wild type ER (data not shown). These data exclude the involvement of the F domain in the observed differential recruitment to the PRL-array. To summarize these mapping experiments, both the AF1 and AF2 domains (helix 12) appear necessary for ER interactions with the PRL array. Based on these differential responses, we next assayed coregulator recruitment in each setting. E2 and EGF each induced recruitment of p160 coregulators SRC-1/SRC-3, the chromatin remodeling protein BRG1, and both CDK7 and cyclin D1 (each involved in transcriptional elongation) to the PRL-array in cells expressing wild type GFP-ER (Figure S1). Confirming the specificity of the immunostaining reagents, cells Figure 1. Estrogen receptor promoter binding in PRL-HeLa cells. GFP-ER was transiently expressed in PRL-HeLa cells and then treated with ethanolic vehicle, 17-b estradiol, EGF or 4-hydroxytamoxifen for 2 hours prior to fixation. Each ligand concentration was 10 nM except for EGF, which was 100 ng/ml. A. Representative images of PRL-HeLa cells treated with EGF. Arrowheads point to visible arrays in two cells. One of the cells has two fluorescent foci/arrays, while the other has one larger focus. B The percentage of each cell population that has visible accumulation at the array in response to each treatment has been calculated and graphed (n.200). Bars indicate Standard Deviations from 3 different experiments. Student t-test was performed for each bar compared to vehicle treatment. *p,0.05. doi:10.1371/journal.pone.0002286.g001 PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2286