Figure 6. MAPK signaling and the role ER-S118 in PRL array responses. A. PRL-HeLa cells transiently expressing GFP-ER were treated with 50 mM PD98589 or 10 mM UO126 for 30 minutes before adding 10 nM E2 or 100 ng/ml EGF for 2 h. The percentage of each cell population that showed visible accumulation at the array in response to treatment was calculated and reported in the graphs (n.200). Both inhibitors dramatically reduced the number of GFP-ER-targeted arrays in transfected cells (EGF treated only) p,0.05. B. Percentage of cells with a visible array in PRL-HeLa cells transiently transfected with GFP-ER or with the two mutants, GFP-ERS118A and GFP-ERS118E, and treated with ethanol, E2 or EGF for 2 hours. Student t-test was performed for each bar compared to GFP-ER (* p,0.05), or to vehicle ({ p,0.05). doi:10.1371/journal.pone.0002286.g006 transcriptional activity has previously limited the focus of these investigations to each of these individually. The recent develop- ment of PRL-HeLa provides a useful tool that facilitates the study of multiple aspects of ER function at the single cell level. This includes spatiotemporal visualization of ER promoter targeting, reporter mRNA accumulation and coregulator association, in either a live or fixed cells [19]. Here, we used the PRL-HeLa model as a tool to discriminate differences in ligand-dependent and –independent regulation of ER including receptor binding to a promoter/enhancer, large-scale chromatin modification, and induction of transcription. Further, using automated image acquisition and analyses, we have specifically examined cells that expressed low levels of transfected protein, avoiding problems inherent to overexpression and bulk averaging of large populations of cells. With these approaches we report a time-dependent ERa Function by Image Analyses signature of large-scale chromatin modification and mRNA accumulation, which suggests an uncoupling of chromatin decondensation and maximal transcription. ER targeting at the site of transcription The ability of a transcription factor to recognize and bind to specific elements in a promoter/enhancer is an essential step in transcription regulation. Initially, we determined that PRL-HeLa cells expressing only low levels of GFP-ER were heterogeneous in terms of unliganded ER promoter binding (60%). The respon- siveness to EGF by ER in PRL-HeLa is less than E2 responses. Similar to that of E2-induced PRL-array targeting, EGF treatment resulted in a marked, but relatively sub-maximal increase in the number of cells with visible arrays (Figure 1). Indeed, compared to E2, EGF treatments resulted in fewer targeted PRL-arrays in GFP-ER-expressing cells. As shown in Figure 4C, EGF can robustly induce a PRL-reporter gene (up to ,80% of mRNA production compared to E2 levels) in the cells showing a visible array (78%), but analyses of FISH signals in transfected cells without a visible array show a transcription level similar to non- transfected cells (data not shown and [19]). Additionally, when we examined a narrow range of GFP-ER low-expressing cells for reporter gene activity by FISH, we still observed variability in gene expression (Figure 4B). This supports the notion that transcrip- tional heterogeneity may be a stochastic process [24,25], although transcription activation tended to peak at 30 minutes after induction (see below). Large-Scale Chromatin Modification and Transcription The fundamental importance of EGF-dependent activation of ER is further supported by its ability to induce both large-scale chromatin modification and transcriptional activity over time. Relative to E2-induced activation, unliganded ER targeting the PRL-array correlates with a stable, but sub-maximal levels of chromatin decondensation and mRNA accumulation (Figure 3A and 4C, and [19]). Our temporal studies indicate that EGF induces large-scale chromatin modification to a similar degree as E2, although at about one half the rate (30 minutes vs. 15 minutes). This result is similar to spatiotemporal changes in GFP-ER nuclear distribution observed in MCF-7 cells (10 minutes for E2 vs. 30 minutes for EGF) [26]. The physiological significance of the nuclear reorganization of GFP-ER is unknown as the concentrated foci are not frequently localized to sites of transcription [27], and have high exchange dynamics as determined by photobleaching studies [28]. Although these EGF-induced nuclear responses are alike, our PRL-array results here are directly linked to a defined transcription site. Interest- ingly, despite the delayed induction of large-scale chromatin modification by EGF-activated GFP-ER as compared to that by E2, the maximal level of decondensation is similar. It will be important to determine if a similar set of chromatin remodeling proteins is involved in each case, and if the recruitment of these proteins is dependent on coactivator specific post-translational modifications of ER, or specific modifications to the recruited coregulators. It will also be important to determine if these large- scale effects translate to smaller scales of chromatin structure. After the initial decondensation, large-scale chromatin mod- ification (as assayed by array size) induced by E2 is sustained for at least 24 hours. In contrast, continuous EGF treatment resulted in PRL-array recondensation to basal levels within a few hours. This differential regulation may be particularly important for endocrine tissues that require rapid and short responses to growth factors, in addition to the strong and sustained E2-dependent transcriptional response. The results of PLoS ONE | www.plosone.org 8 May 2008 | Volume 3 | Issue 5 | e2286